announcement • •AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY

EIGHTEENTH ANNUAL CONFERENCE

Brisbane. Australia

ANNUAL CONFERENCE 198724 ..26 August, \1987

Sydney

31 AUGUST - 3 SEPTEMBER. 1986

PRO G RAM ME

AND

A B S T R ACT S 0 F PAP E R S

Copyright Australian Society for Reproductive Biology, 1986ISSN 0705-6044

CONTENTS

Abstracts should be prepared by May 1987

ASRB Representative on Local Organizing Committee

Gareth Evans (02) 692 3363

Personnel ,AcknowledgementsProgramme GuideProgrammeAuthorsAbstracts

. Satell ite Sympos i um (1985):"Advances in EmbryoTransfer"

Minutes of 17th AG~1 1985Minutes Annual Student

Meeting ASRB (1985)

IIIII

IVV-XIX

XX-XXII1-134

135-143

AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY

September. 1986

OFF ICE B EAR E R S

j

Chairman

Secretary

Treasurer

Committee Members

Postgraduate StudentRepresentative

DR B.M. BINDON

DR J.M. CUMMINS

DR C.C. NANCARROW

DR B.G. MILLER

DR G. EVANS

DR J. RODGER

DR L.A. HINDS

DR A.O. LAWS

PRO G RAM ME COM MIT TEE

Chairman PROFESSOR D.R. LINDSAY

Committee Members DR N.R. ADAMS

DR N. W. BRUCE

DR I.W. PURVIS

DR P. WILLIAMSON

DR D. WILLCOX

L 0 CAL 0 R GA N I SIN G COM MIT TEE

DR J.r~. CUr~HINS

DR P. CHENOWETHDR J. ELKINGTON

,DR B. HIRSCHFIELD

SISTER S. JUBLEDR L. MARTINDR ~1. WATERSDR. M. WILLIAMSON

(II)

THE AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY

wishes to thank the following fortheir support of the 1986 meeting

ANSETT AIRLINES

ORGANON AUSTRALIA

BOEHRINGER MANNHEIM AUST PTY LTD

AMES DIVISION. MILES LABORATORIES AUST PTY LTD

CSL-NOVO PTY LTD

HOECHST PHARMACEUTICAL DIVISION

(Sponsors at 1.7.1986)

The contents of these Proceedings have not been edited by theSociety or the Editor and are reproduced as submitted.Responsibility for accuracy of the communications rests withthe authors.

Material in these Proceedings may not be reproduced withoutpermission of the Society.

Price of Proceedings to non-members. $A15.0D plus postage.

GENE TRANSFER TECHNOLOGY AND THE EVOLUTION

OF ~ERTILIZATION MECHANISMS

A reminder that this symposium will be held in the Abel SmithLecture Theatre on Thursday 4th September. A full program andlist of. speakers will be available at the conference. It willbe possible to register for the symposium if you have notalready done so.

(III)

Session 3: Session 3:Poster Discussion Oral Presentations3(1) GONADS & GAMETES (Female): Chairman, Dr L MartinDr J K Findlay COMPARATIVE REPRODUCTION3(2) UTERUS, FOETUS, PLACENTA &LACTATION: Or N \oJ. Moore

Hour

0830

1000

1030

1200

1300

1400

1530

1600

1750

Monday 1st September

Session 1:Ora1 presentationsCha i rman, Dr J RodgerEARLY EMBRYONIC DEVELOP­MENT & GENETIC MANIPULATION

Abst. 1 - 6

Session 2:Poster Discussion2(1 ) EARLY EMBRYONICDEVELOPMENT: Dr B Mi 11 er2(2) FERTILITY & MANI PULATIONOF REPROD: Dr R J Scaramuzzi

Abst: 7 - 27

GODING LECTURE

William Hansel

Session 3:Poster Discussion3(1) EARLY EMBRYONICDEVELOPMENT: Dr C Nancarrow3(2) FERTILITY & MANIPULATIONOF REPROD: Dr R F Seamark

Abst. 28 - 47

Session 4:Ora1 Presentati onsChairman, Prof T J RobinsonFERTILITY & MANIPULATION OFREPRODUCTION

Abst. 48 - 53

PROGRAr~ME GUIDE

Tuesday 2nd September

Session 1:Oral presentationsChairman, Dr B BindonREPRODUCTIVE ENDOCRINOLOGY

Abst. 54 - 59

TEA

Session 2:Poster Discussion2(1) REPRODUCTIVE ENDOCRINOL­OGY: Prof D de Kretser2(2) COMPARATIVE REPRODUCTION:Dr M Renfree

Abst. 60 - 81

HARRISON LECTURE

Professor M 0 Thorner

LUNCH

Abst. 82 - 97

TEA

Session 4:Oral PresentationsChairman, B P SetchellGONADS AND GAMETES (Male)

Abst. 98 - 103

ASRB AGM

Wednesday 3rd September

Session 1:Oral presentationsCha i rman, Dr P HartmannUTERUS, FOETUS, PLACENTA &LACTATION

Abst. 104 - 109

Session 2:Poster Discussion2(1) UTERUS, FOETUS,PLACENTA & LACTATION:Dr C 0' Neill (Male):2(2) GONADS & GAMETES(Male): Dr A \oJ BlackshawAbst. 110 - 128

Abst. 129 - 134

Junior Scientist AwardPresented

AUSffiALIAN 9X:IE'lY OF REPRCDUCI'IVE BIOIroY

SUnday 31st August

ROOIS1RATICN; The Registration desk in the Student Union(adjacent to the Axon room) will be open from 1500h to 1900h. Itreopen at 0800h on MOnday and remain open during the rest of theconference.

Time: 1800h

Venue: University Union Refractory. Cost included in the registration

Monday 1st September

Session 1: EARLY EMBRYCNIC DEVEI.DlMENI' AND GENETIC MANIPUIATICNCRAL PRESENI'ATICNS

Cha i rman: Dr. J .Rodger

Time:0830-1000

1 Spinks, N R Anmit, A J Saunders, D M O'Neill, CEmbryo-derived platelet activating factor (PAF)kinetics of its production in vitro

2 Baldo, B A SInal, M A Anmitt, A J Saunders, D MO'Ne! II, C

FUrther characterisation of embryo-derived plateletactivating factor (EiJPAF) in mice

Posters located throughout Meeting for viewing and discussion in Schonell Foyer.Oral presentations and Goding and Harrison Lectures in the Schonell Theatre. 3 Gardner, H Kaye, P L

The effect of insulin treatment during preinplants.tioncuI ture on foetal developnent in the mouse

(IV)4 Laws-King, A 0 Sathananthan, A H Tro\IDson, A 0

Perti lization of hunan oocytes following micransnipulationof a single sperm tmder the zona pellucids

5 Thompson, J G E Waugh, E Wales, R GDevelopnent of a superovulatory regime for the collectionof OVUlated sheep oocytes and early embryos

6 Mercer, WR Lee, C S Gogo lin-E.Wens, K J Brandon, M RA sheep pregnancy specific protein

v

Sess ion 2: EARLY lM3RYClilIC DEVEIDIMENr AND GENETIC MANIPULATHN (1)

a:N:VRR.ENr POS1F.R PRESENI'ATICNS

Chainnan: Dr. B. Mi ller

Time 1030-1200

16

17

Smith, D H Walker, S K Seamark, R Fsynchronization of timing of ovulation in the artificialinsemination of sheep

Htmton, J R Maxwell, WM CRyan, J PFerti 1isation of ova in superovulated merino ewes followingAI wi th fresh and frozen - thawed semen

J

7 Khurana, N K Wales, R GEffects of oxygen tension on the metabol ism of glucose bymouse morulae-early blastocysts in vitro

8 Kaye, P LAmino acid transport in the mouse blastocyst

9 Walker, S K Quinn, P Ashman, R J Smith, D H Seamark, R FProtein supplementation for the CUlture of one-cellembryos of sheep

10 Amnit, A J Collier, M Satmders, D M O'Neill, CDevelopnent of a quantitative bioassay for embryo derivedplatelet activating factor (EJ)PAF)

11 O'Neil, C .Amos, EMote, P Gidley-Baird, AHistological and Ultrastructural evidence for plateletactivation in the reproductive tract during early pregnancy

12 Michalska, A Vize, P Quinn, P Wells, J R E Seamark, R FTransgenic mice-growth regulation by two porcine growthhonmone gene constructs

13 Michalska, A Vize, P Ashman, R J Stone, B AQuinn, P Wells, J R E Seamark, R F

Expression of porcine growth hormone cINA in transgenicpigs

14 Annstrong, D T Michalska, A Ashman, R J Vize, PWells, J R E Seamark, R F

Gene transfer in ~ts

15 M1mro, R K Williams, T J Shelton, J NProduction of overt inter-species bovine chimaeras byaggregation of demi-embryos

2: FEFITILITI AND MANlPULATICN OF REP.RCDIX::rICN (1)

a:N:VRR.ENr POSTER PRESFNTATICNS

Chairman: Dr. P. Quinn

Time: 1030-1200

VI

18 Evans, G Jabbour, H N Moore, N WTime of intrauterine insemination of superovulated ewesusing fresh and frozen semen

19 Eppleston, J Maxwell, WM C Battye, K M Roberts, E MEffect of thawed motil ity and intra-uterine dose of motilespenn on fertil i ty in ewes

20 Maxwell, WM C Hunton, J R Ryan, J P Hood, GEffect of GnRH on time of OVUlation in superovulated merinoewes

21 Maxwell, WM CRyan, J P Htmton, J REffect of ovarian response on distribution of ovulations insuperovula ted mer ina ewes

22 MCConnell, S J Ellendorff, FAbsence of nocturnal plasma melatonin surge lmder long andshort photoperiods in the danestic sow

23 Staples, L D' Foote, M Jones, A McPhee, SReeve, J Wilkins, J Williams, A

Attempts to improve reproductive efficiency of early joinedmerino, EML and corriedaleewes with various melatonintreatments

24 McPhee, S Staples, L Foote, M Killeen, DWilkins, J Williams. A

melatonin treatments to inprove early joining ofBorder Leicester'and Ramey ewes

25 Reeve, J Williams, A McPhee"S Peake, R Staples, LThe effect of season on the response of Border Leicester xmerino (BLxM) ewes to subcutaneous implants of melatonin

26 Staples, L D McPhee, SAyton, B Reeve, J Williams, A H, Optimr.m melatonin treatments to improve reproductive

perfolTb8T1ce of merino, Border Leicester x merino and Rameyewes joined in spring and early surmer

27 Wi 11 iams, A H McPhee, S MAyton, B M Staples, L DDose-dependent effects of continuous melatoninadministration on the seasonal pattern of oestrouscyc1icityand ovulation rate in corriedale ewes

VII

JAMES (]J[)IN:} MEMRIAL LECIURE

Time 1200-1300

Sess ion 3: FERriL ITI AND MANIPUlATICN OF REPRCDtX:.TICN (2)a.:N::URRENI' POSTER PRESENTATICNS

Venue: Schonell Theatre Chainnan: Dr. R.F.Seamark

Willimn Hansel: Control of Steroidogenesis in thaeca andgranulosa derived cells of the corpus luteum Time: 1400-1530

Chainnan: Dr C. Nancarrow

Time: 1400-1530

Session 3: EARLY EMBRYCNIC DEVEl:.()RdENI' AND GENETIC MANIPUlATICN (2)

Wong, M S F Cox, R ILow pregnancy rates in merino ewes boosted withtestosterone:HSA post mating

Hinch, G N Roelofs, J H WLupin feeding and insulin infusion during the late lutealphase can increase oVulation rate in sheep

Wilson, P A Cox,. R I Wong, M S F PaUll, DRImproved proI ificacy in merino ewes irrmrmized against acombination of androgens and oestrogens

Bindon, B M Piper, L R Hi lIard, MA Nethery, R DOvulatory response to exogenous FSH by cattle selected fortwin births

Tjondronegoro, S Williamson, P E Sawyer, G J HaWkins, CDAtkinson, S

Effects of progesterone intravaginal devices on oetrussynchronisation in postpartun dairy cows .

46 Armstrong, J R Davis, G H Farquhar, P A OWens, J LEffect of pregnancy nutrition and pre-lamb shearing onlamb b~rth weights in prolIfic sheep

42

44

43

41

39 Cumlins, L J O'Shea, T Bindon, B MIncreased OVUlation rates in cattle vaccinated with apartially purified fraction of ovine follicular fluid

40 Croker, K P Johns, MA Brown, G APerformsnce of crossbred ewes receiving different Feclmdinbooster doses

38 D'OCchio, M J Gifford, DR Cox, R I Weatherly, TSetchell, B P

OVarian responses of beef heifers to a bovine serl.l11albl.l11in-testosterone based immlmogen

36 Davis, I Parr, R Cahill, L PMaintenance of pregnancy in ovariectomised ewes withsynthetic progestagens

37 Parr, R A Davis, I F Fairclough, R J Miles, MAAn interaction between nutrition and progesterone reducesembryonic survival in sheep

A5

VIII

Wilton, L J Trounson, A 0Viability of mouse embryos and blastomeres following biopsyof a single cell

Szell, A Shelton, J NSurvival of ul tra-rapidly frozen mouse embryos as aflmction of glycerol permeation

Yates, C A Thomas, C J Trounson, A 0 de Kretser, D MMale factor patients with antisperm antibodies in IVF

Pope, A K Harrison, K L Breen, T M Wilson, L MC'tmnins, J M Hennessey, J F

U1 troser G as a serun substi tute in embryo cuI ture mediun

Ki rby, C Trounson, AThe viabi Ii ty of mouse embryos cuI tured in hunsn 8.l11l1ioticfluid

Chiappazzo, L Rogers, P Kirby, C Trounson, AThe use of 8.l11l1iotic fluid as an embryo cuI ture mediun forhU118I1 IVF

30

29 Harrison, K L Wilson, L M Breen, T M Pope, A KCUTmins, J M Hennessey, J F

Fertilization of human oocytes in relation to delay beforeinsemination

34

32

33

28 Yovich, J L Matson, P L Yovich, J M Bootsma; B Spittle, JThe in-vi tro fert iIisa t i on of supernunerary hunsn oocytesin a gsmete intra-fallopian transfer progrsmne

35

31

J

IX

J

47 Stevenson, G T Edey, T N Hinch, G N11J.e influence of long te1TT1 weaning weight selection inmerino ewes on age. 1iveweight. fat and stage of maturityat pUberty

Session 4: FERI'ILI1Y .AND MANIPULATICN OF REPRCIX..CI'ICN

CRAL PRESFNrATIav8

Chainnan: Prof. T.J. Robinson

Time: 1600-1730

48 Fry, R C Clarke, I J Cunnins, J T Bindon, B MPiper, L Cahill, L P

Induction of ovulation in hypophysectanised booroola ewes

49 Nottle, MB Armstrong, D T Setchell, BP Seamark, R FLupin supplementation. FSH secretion and ovulation rate inthe ewe

50 Ryan, J P Maxwell, WM C Hunton, J R Wales, R GMetabolism of lactate by sheep ova fertil ised by fresh andfrozen-thawed semen

51 Ovens, J LWithin year repeatability of ovulation rate for booroo1amerino ewes at 1.5 years of age

52 Matthews, C D Lewy, A J Kennaway, D J Watson, RSeamark, R F

Serun melatonin (8M!') patterns in sheep under naturalphotoperiodic conditions and SUbjected to extendeddarkness. A circadian basis for seasonal breeding

53 Shaw, P Kennaway, D J Seamark, R FFailure of melatonin to synchronise a free-rzmning rhythm

INFCR\1AL S:X::IAL EVENIN3

Time: 1900h (Further details at conference>

Tuesday 2nd September

Sess i on 1: REPRCDU:::TlVE FNIXXR.INJLCX]YCRAL PRESFNrATIav8

Chairman:Dr B.M. Bindon

Time: 0830-1000

54 Fry, R C Cahi II, L P Ctmnins, J T Bindon, B MPiper, L P Clarke, I J

The half-1 ife of FSH in entire and chronicallyovariectanized Booroola and AB20 merino ewes

55 Jabbour, H N Evans, G Moore, N WSteroidogenic and OVUlatory response of merino ewes to FMSGand porcine FSH

56 Papachristoforou, C D'Occhio, M J Setchell, B PReproductive deve10pnent in male and female sheep followingsuppression of the hypothalamo-pituitary axis during theprepuberta I period

57 Sharpe, P H MacGregor, H E A..'"'mStrong, D TSuperovulation of ewes with constant, SUbcutaneous infusionof follicle stimulating hor.mone

58 Maddocks, S Zupp, J L Sowerbutts, S Setchell, B PTestosterone levels in rat testicular interstitial fluid

59 Risbridger, G P Leung, A Jenkin, G DeKretser, D MEvidence that ovarian factors influence testicularsteroidogenesis and vice versa

Sess i on 2: REPRCI:lO::TIVE FNIXXR.IJ.'iOI.(Xjy

Chairman: Prof. D. de Kretser

Time: 1030-1200

Lee, V WK Zhang, Z W Carson, R S Burger, H GInhibin secretion fran rat granulosa cells in vitro:effect of gonadotrophins and steroids

x

/ 61Ford, J R Nicholas, JC Boussioux, AM

A ne~ bioluninescence assay for oestrogens in sheep plasm8

XI

62 Cutler, S A Evans, G scarwnuzzi, R J Downing, JPhotoperiodic entrainment of seasonal patterns of Ufsecretion in crossbred ewes

Session 2: a:MPARATlVE REPRODUCI'ICN

a::N:::URR.ENI' POSTER PRESFNI'ATICNS

71 Gaston-Parry, 0 Robinson, T JPlasma. concentration of fluorogestone acetate (FGA;crono1one; Searle) in ewes treated with FGA impregnatedintravaginal sponges

63 Wide, LPituitary follicle-stimulating hor.mone in children andadults - different molecular forms in relation to sexand age

64

65

66

67

68

69

V70

Salamonsen, L A Cherny, R A Findlay, J Koestrogen and progesterone in vitro influence proteinsecretion by epithelial endometrial cells

Gaston-Parry, 0 Nemorin, J K E Robinson, T JA radioirrmunoassay for fluorogestone acetate (FGA;cronolone; Searle)

Willcox, D L Yovich, J L Bruce, N W McColm, S CEffect of medroxyprogesterone acetate on progesterone andoestradiol in plasma. of pregnant rats I

D'OCchio, M J Weatherly, T Sowerbutts, S Setchell, B PMaturation of the hypotha1smo-pituitary axis in 1118.1e andfemale sheep early in 1ife: Role of the gonads

Slni th, W Fairclough, R J Egan, A REffects of oxytocin a.nd nutrition on prostaglandin Fmetabolite levels in post-partum ewes

Handelsman, D J Spaliviero, J-A Scott, CD Baxter, R CHor.monal regulation of the peripubertal surge in circulating 1insul in-l ike growth fac tor-I (IGF-I) 'j,('/"~

Downing, J A Baird, D T Cwnpbell, B K Scarwnuzzi, R JSteroid secretion rates from the autotransp1anted ovary ofthe booroo1a merino

r/7273

74

76

77

78

79

80

Chairman: Dr. R. C. Jones

Time: 1030-1200

Stone, G M Jones, R C Hinds, L AOCcurrence of the initial segment (JS) in the epididymis ofthe tSITm8.r, Macropus eugeniJ

Clmnins, J M Robson, S KRouse, G W Graydon, MU1 tras truc ture of the i onophore-induced acrosome reac t i onin spermatozoa of the grey flying fox, Pteropus pol iocephalus

Dl:l.1rI, M Ullmann, S LOvarian developnent and polyovular follicles in the potoroo(Potoro~ tridacty1us, macropididae, marsupia1ia)

Clulow, J Jones, R CComposition of the luminal fluids in the 1118.1e genital ductsof the Japanese quail, Coturnix coturnix

GemneII,RTThe effect of melatonin on the initiation of the breedingseason of two I118.rsupia1s

Minj ie, L Thorne, M H Martin, I C A Sheldon, B LHistology of the gonads of triploid fowls

Fletcher, T P Renfree, M BOrigin of the transient peaks of progesterone and oestradiol17B occurring early in the oestrous cycle and pregnancy ofthe tSITm8.r wallaby, Macropus eugenii

Jolly, S Blackshaw, A WA novel reproductive strategy in the 1118.1e sheath-tail batTaphozous georgianus

Tyndale-Biscoe, D H Hinds, L ARapid response of the tSITm8.r to injection of melatonin

McGuckin, M A Little, L M Blackshaw, A WSeasonal changes in testis size, plasma. testosteroneconcentration and body weight in captive 1118.1e grey-headedfrui t bats, Pteropus pol iocephalus

XII XIII

KEITI1 HARRISCN MEM1UAL LOCIURE

Time: 1200-1300

Venue: Schone 11 TI1ea tre

Professor M.a. TI1orner: Advances in understanding ot themechanisms controlling growth hormone secretion

Session 3: CINADS AND GAMETES O. FEMALE)

a::N::URRENI' J?(E['ER PRESENI'ATICN

Chairman: Dr. I. Clarke

Time: 1400-1530

82 Fairclough, R J Baird, D T Downing, J A Scaramuzzi, R JOXytocin secretion by the ovary during the luteal phaseand following a prostaglandin stimulus

83 Peterson, A J Fairclough, R J Smith, J F Tervit, H RMoore, L G Watkins, W B

Administration of progesterone early in the oestrous cycleoauses premature release of PGFM and oxytooin-neurophysin

session 3: UI'ERUS, FDETUS, PLACENl'A AND LACfATICN (1)

a::N::URRENI' FOSTER PRESENI'ATICNS

Chairman: Dr. G. Stone

Time: 1400-1530

88 Bruce, N W Rahima, A Bluner, W F CSpacing of rat conceptuses in the uterine horn: Bnpiricalobservations and a "carputerus" estimate of the strengthof mechanisms involved

89 cerini, ME D O'Shea, J DIsolation of granulated cells from the metrial gland of thepregnan t ra t

90 Hall, S Pennefather, J NEffects of forskol in on guinea-pig mycmetriun: Theinfluence of ovarian steroids

91 Kleeman, D a Walker, S K Walkley, J R W ~ith, D MGrimson, R J Verhees, P J Ponzoni, R W Stone, B A

&; Seamark, R FUterine presensitization and embryo survival in booroolacross ewes

92 Lee, C S Wooding, F B P Brandon, MRUltrastructural localisation of a placental antigen with amonoolonal antibody in the placenta of the sheep

84l

v'

85

86

J87

Roche, P J Jenkin, G Gibson, WRCiroulating progestins in pregnant rats with denervatedovaries

Adams, N R Atkinson, SLow amounts of oestradiol-17B reduce the OVUlation rate ofmerino ewes

Meyer, G TThe influence of proximity to blood capi llaries onluteinization

swann, R T Bruce, N WSteroid seoretion from whole oorpora lutea in vitro,oOl1f>8.ri son of media and to in-vivo seore tion ra tes in ra tsat day 16 of gestation

XIV

93 McRae, A C Omrch, R BA topographical cOOJ)arison of inplanting and delayed­illplanting blastocysts fran naturally mated, randanly bredmice

94 Martin, L Crane, L HContinuous recording of mycmetrial activity in the rat byvideo-laps.ros'copy

95 Murray, C Stone, GDelayed'growth response of the mouse uterus to progesterone

96 Putra, D K H Bl ackshaw, A WThe effect of exogenous' ovarian steroids on early embryoniomortal ity and eleotron microscopy of the endcmetriun of thepig

97 Rogers, P Macpherson, A Beaton, LEmbryo-uterine interaction using the anterior eye chBlTlber inthe rat

XV

111----------------.--------------'i;:;p""

Sess i on 4: <nW)S AND GAMETES (2. MALE)

alAI. PRESFNI'ATIClVS

O1airman: Dr. B.P. Setchell

Wednesday 3rd september

Session 1: UI'ERUS, REIUS, PLACENTA AND LACI'ATICN (2)

alAI. PRESFNI'ATIClVS

Chairman: Dr. P.E. Hartmann

Time: 0830-1030

J

Time: 1600-1730~.-.,.,.,,,.._"'.

98 ,/;At~inson, S....)pOrd, J R Oldham, eM Pearce, D T\,,,,,,,,,~htfe increase in utero-ovari811 oestradiol fran the

ovar!....t?-!stined to ovulate after PO administration"""-04,,-

99 Cameron, H M) Bruce, N WEff~S.ts'·6f hunan chorionic gonadotrophin on peripheral

"""prdiestagen concentrations in conscious rats: A cCX1fJ8.risonwith luteal regression

100 ~be11, B K Scaramuzzi, R J Cognie, Y Downing, J AInhibition of cOl'lpensatory hypertrophy in ewes by bovinefollicular fluid

101 Djakiew, D Hadley, M A Byers, S W Dym, MIron is transcytosed across sertol i cells by two forms oftr811sferrin, whereupon the iron 1s secreted as testicularFe-tr811sferrin which subsequently binds to germ cells

102 DeGaris, R M Pel1.nefather, R MReirmervation of the rat vas deferens followingvasovasostomy after vasectomy: A long-term study

'~-"---.......103 {'s~arrow, R L~Kenzie, I F C Lutjen, P J

\,,_.Jl!feiJl,$~of a retroviral gp70 envelope-relatedglycoprotein on the irmer acrosomal membrane ofnonnal hunan spe:rmstozoa

ASRB .m.mAL GENERAL MEETINJ

Time: 1750h

Venue: Schone11 Theatre

105

106

107

108

109

Adamson, L M Stanger, J D smart, Y C Murdoch, R MRoberts; T K

Early pregn811cy associated thranbacytopenia (EPAT) - Anecessary pregn811cy phenanenon

crane, L H Martin, LPost-copulatory myanetrial activity in the rat as seen byvideo-laparoscopy

Kind, K OWens, J A Falconer, J Robinson, J S111e growth-retarded fetus suppl ies its placenta with 8I1lino.acids in late gestation

Shepherd, V Parkington , H Jenkin, G Ralph, MMThorburn, G D

Activity of ovine circular and longitudinal myometriunduring pregn811cy and parturit ion

Vini jsanun, A Martin, LProgesterone deprivation and embryo develO[m8nt inmice

Zhang, X Miller, B GInmunosuppressive factors during pregn811cy in the sheep

Sess i on 2: Ul'BRUS, IDE:IUS, PLACFNl'A AND LACI'ATICN (2)a:N:URRENT POS'I'FR PRESFNI'ATICl'YS

Chairman: Dr. C. D'Nei 11

Time: 1030-1200

ASRB-ESA Al'NJAL DI~

Time: 1930 for 2000h

Venue: Sheraton Hotel ($40 ea , busses free)

XVI

110 Arthur,'p GPotter, J Hartmann, P EPlasma lactose levels at the initiation of lactation inbreastfeedir.g mothers

111 Crowe 11, D H McGhee, R I Ogasawa, G Shanna, S Dseto, D Wi 11court , R J

Race/ethnic-specific patterns of fetal growth

112 Holmes, M Whitely, J Stokes, G Arthur,P Hartmann, PGlucose, insulin, progesterone 8lld lactose in blood plasmaduring lactogenesis in sows

XVII

a:N::URRENr RJS'1'ER PRESFNI'ATICNS

Session 2: CQW)S AND GtVdETES (2. MALE)

CR4L PRESFNI'ATICNS

Sess ion 3: aMPARATlVE REP.Ra::U:ITICN

SlID, Y-T Irby, D C Robertson, D M deKretser, D MEffect of chronic actninistration of testosterone on spermproduction and plaS1D8 testosterone, FSH, IE, andinterstitial fluid testosterone levels

Time: 1400-1530

Olainnan: Dr. L. Martin

129 Gordon. K Fletcher, T P Renfree, MBReactivation of the diapausing embryo after the temporaryretOOval of the sucking stimulus in the tamna.l' wallaby

130 Jones, R C Clulow, JRole of the initial segments (IS) of the epididymis of thetamnar, Macropus eugenii

128 Hancock, A D Johnson, J de Kretser, D MThe ultrastructure of spermatozoa fran men withasthenozoospermia

Gatie, S J Glover, T D Blackshaw, A WThe effects of ligation and cryptorchidism on the epididymisand epididymal sperm of the guinea-pig

127 Baker, H W G Cooper, E JProtein carboxyl methylase in Rsthenospermia

126

"1'/125/

Handelsman, D J Spaliviero, J A Scott, CD Baxter, R Ccellular origins of testicular insul in-l ike growth factor-I

Wide, MPrenatal exposure to lead (PbCI2) affects fertiI ity in thefemale mouse

Whi tely, J L Bryant-Greenwood, G Wi llcox. D L Hartmann. P EThe effect of MPA actninistration on the hormonal eventsassociated with parturition in sows

OYens J .A. Kind, K Falconer, J Robinson, J SRestriction Of placental growth alters utero-placentalmetabolism in vivo

Murdoch, R NGlycolysis in the post-il1fJlantation mouse uterus inresponse to acute doses of ethanol

M>ore, N W Xu, YiqlmThe effects of ovariectany and of exogenous progesteroneon pregnancy and on placental production of progesterone

Hoy, J Lutjen, PThe effect of clanJphene citrate on cervical mucussJalylation

Miller, B G Zhang, XEffects of cronolone on the uterus and on pregnancy

113

114

115

116

117

118

119

\/120

Olainnan: Dr. A.W. Blackshaw 131 Ullmann, S LThe origin of the granulosa cells in bandicoots

Time: 1030-1200

121 Keogh, E J Carati, C J creed, K E Earle, C MWisniewski, S Tulloch, A G S Lord, D J

P.harmacolqgical control of penile erection

crichton, E G Krutzsch. P HThe reproductive biology of the little mastiff bat,Mormopterus planiceps (Oliroptera: Molossidae) insouth-east South Austral ia

122 Liu, D Y Jennings, M G Baker, H WGReactivation of flagellar motion of detergent treated hll1l8nspermatozoa by adenosine triphosphate

123 O'Brien, G M Clulow, J Jones, R CPreparation of spermatozoa for analysis of inorganicelectrolytes

I

133

134

Bourne, A R HUf, P A Watson, T GBiosynthesis of androgens in the lizard Til iqua rugosa

Goodall, J A Blackshaw, A W Capra, M FReproduction in free and captive swmer whiting(Sillago ciliata) ,

124 Wang, Z X Risb;ridger, G P Wreford, N G M deKretser. D MA quantitative analysis of sertoli Cells in postnatal rats

XVIII XIX

AUTHOR INDEX AUTHOR INDEX

Adams, N R 85 deKretser, D M a2,59,124,125,128 Killeen, D 24 Pennefather, R M 102Adamson, L M 104 Djakiew, D1°1 Kind, K 106,117 Peterson, A ,] 8:3Amrni t , A J 1,2,10 Downing, J A 62,70,82,100 Kirby, C 30,35 Piper, L R 44,48,54Amos, E 11 Dym, M 101 Kleeman, D 0 81 Ponzoni, R W 91Armstrong, D T 14,49,57 Ear~e, C M 121 Krutzsch, p H 132 Pope, A K 29,33

Armstrong, J R 46 Edey, T N 47 Laws-King, A CJ 4 Potter, J 110Arthur, P 112 Egan, A R 68 Lee, C S 6,92 Putra, D K H 96Arthur, P G 110 EIlendorff, F 22 Lee, V W K 60 Quinn, P 9,J2,13Ashman, R J 9,13,14 Eppleston, J 19 Leung, A 59 Rahima, A 88Atkinson, S 45,85,98 Evans, G 18,55,62 Lewy, A J 52 Ralph, M M 107Ayton, B 26,27 Fairclough, R J 37,68,82,83 Little, L M 81 Reeve, J 23,25,26Baird, D T 70,82 Falconer, J 10G,117 Liu, D Y 122 Renfree, M B 78,129Baker, H W G 122,127 FarqUhar, P A 46 Lord, D J 121 Risbridger, G P 59,124Baldo, B A 2 Findlay, J K 64 Lutjen, P J 103,113 Roberts, T K 104Battye, K M 19 Fletcher, T P 78,129 MacGregor, H E 57 Robertson, D M 125Baxt.er, R C 69,120 Foote, M 23,24 MacplHH'son, A 97 Robinson, J S 106,117Beaton, L 87 Ford, J R 61,98 Maddocks, S 58 Robinson, T J 65,71Bindon, B M 39,44,48,54 Fry, R C 48,54 Mar tin, I C A 77 Rotlson, S }( 73Blackshaw, A W 79,81,96,126,134 Gardner, H 3 Mart in, L 94,105,108 Roche, P J 84Blumer, W F' C 88 Gaston-Parry, 0 65,71 Matson, p L 28 Roelofs, J H W 43Bootsma, B 28 Gatie, S J 126 Matthews, C D 52 Rogers, P 35,97

J Bourne, A R 133 Gemmell, R T 76 Maxwe I I , W M C 17,19,20,21,50 Rouse, G W 73Boussioux, A 1\1 61 Gibson, W R 84 'VleColm, 8 C 66 Ryan, S P 17,20,21,50Brandon, M R 6,92 Gidley-Baird, A 11 McConne I j , S ,J 22 Salamonsen, L A 64Breen, T M 29,33 Gifford, D R 38 McGhee, R I 111 Sathananthan, A H 4Brown, G A 40 Glover, T D 126 McGuckin, M A 81 ~aunders, D M 1,2,10Bruce, N W 66,87,88,99 Gogolin-Ewens, K 6 McKenzie, [ F C 103 Sawyer, -G J 45Bryant-Greenwood, J 119 Gooda II , J A 134 McPhee, S M 2~{,24,25,26,27 Scaramuzzi, It J 62.70,82,100Burger, H G 60 Gordon, K 129 McRae, A C 93 Scott, C D 69,120Byers, S W 101 Graytion, M 73 Mercer, W R 6 Seamark, R F 9,12,13,14,16Cahi 11, L P 36,48,54 Grimson, R J 91 Meyer, G T 86 52,53,81Cameron, HM 99 Hadley, M A 101 lVl i c Illl 1 S k a , A 12,13,14 Setchell, B P :18,49,56,58,67Campbe 11, B K 70,100 Hall, S 90 Mi I ~!s, M A 37 Seto, D 111Capra, M F 1 ~{4 Hancock, A 0 12 R Mi I Jer, B G 109,114 Sharma, S [) 111Carati I C J 121 Handelsman, D J t1 ,120 l\1i nj ie, L 77 Sharpe, P H 57Carson, R S 60 Harrison, K L B , ~~:"l Moorp, L G 83 Shaw, P 53Cer in i , M E D 89 Hart.mann, P E 110,11 , 11!J Mool'e, N W 18,5f),115 Sheldon, B L 77Cherny, R A 64 HaWkins, C D 45 Mote, t' 11 Shelton, .J N 15,34Chiappazzo, L 35 Hf!nnessey, J F 29, ~l:l Munro, R l{ 15 Shepherd, V 107Church, R R 93 Hi lIard, M A 44 M'!i'doch, R M 104,116 Smal, M A 2Clarl<e, I ,] 48,54 Hinch, G N 43,47 Murray, C 95 Smart, y C 104Clulow, J 75,123,1:30 Hinds. L A 72,80 Nemorin, ,} K E 65 Smi th, D H 6,19Cognie, Y 100 Holmes, M 112 Nethery, R D 44 Smi th, D M 91Collier, M 1 0 Hood, G 20 Nicholas, J C 61 8mi th, J F 83Cooper, E J 127 Hoy, J 113 Nottle, M B 49 Smi th, W 68Cox, R I 38,41,42 Buf, P A 133 0' Ne i 11 , C ).,2,10,11 Sowerbutts, S 58,67Crane, L H 94,105 Hunt.on, J R 17,20,21,50 O'Shea, J D 89 Spa I i vi ero, J A 69,120Creed, K E 121 Irby, D C 125 Cl' Shea, .T 39 Sparrow, R L 103Crichton, E G 132 Jabbour, H N 18,55 O'Brien, G M 123 Spinks, N R 1Croker, K P 40 Jenkin, G 59,84,107 Ogasawa, G 111 Spittle, J 28Crowell, D H 111 Jennings, M G 122 Oldham, C M 98 Stanger, J D 104Cummins, J M 29,3a,7~{ Johns, M A 40 Owens J .A. 106,117 Staples, L D 23,24,25,26,27Cummins, .J T 48,54 Johnson, J 128 Owens, J L 46,51 Stevenson, G T 47Cummins, L J :~ 9 JolI y, S 79 Papachristoforou, C 56 Stokes, G 112Cutler, S A 62 Jones, A 23 Parkington, H 107 Stone, B A 13,91D'Occhio, M 38,56,67 Jones, R C 72,75,123,130 Parr, R A 36,37 Stone, G M 72,95Dairia, M 74 Kaye, P L 3,8 Paull, D R 41 Sun, Y-T 125Davis, G H 46 Kennaway, D 5:J , 53 Peake, R 25 Swann, R T 87Davis, I 36,37 Keogh, E ,] 121 Pearce, D T 98 Szell, A 34DeGaris, R M 102 Khurana, N K 7 uennefather, J N 90 Tervi t, H R 83

(XX) (XXI)

:

AliTHOH INDEX

Thomas, C J ~~ 2 Wide, L 63Thompson, J G E 5 Wide, M 118Thorburn, G D 107 Winins, J 23,24Thorne, M H 77 Wi I Icourt, R J 111Tjondronegoro, S 45 Wi Ilcox, D L 66,119Trounson, A [) 4,aO,31,::l2,35 Wi II iams, A H 23,24,25,26.27TUlloch. A G S 121 Wi 11 iams, T J 15Ty.ndale-Biscoe, D 80 Williamson, P E 45Ullmann, S L 74,131 Wi Ison, L M 29,33Verhees, P J 91 Wi IRon, P A 41Vinijsanun, A 108 Wi I ton, L J 31Vize. P 12,13,14 Wisniewski, S 121Wales, R G 5,7,50 Wong, M S F 41,42Walker, S 1\ 8, .I 6,91 Wooding, F B P nzWalkley, ,J R W 91 Wreford, N G 1\1 124Wang, Z X 124 Xu, Yiqun 115Wa tkins" W B 83 Yates, C A 32Watson. R 52 Yovich, J L 28,66Watson, T G 133 Yovich, J M 28Waugh, E 5 Zhang, X 1Of!, 114Weatherly, T 38,67 Zhang, Z W 60We lis. J R E 12. 1a , 14 'l.upp, ,I L 58Whit.ely, J L 112,119

(XXII)

1

EMBRYO-DERIVED PLATELET ACTIVATING FACTOR (PAF): KINETICS OF ITSPRODUCTION IN VITRO.

N.R. SPINKS, A.J. AMMIT, D.M. SAUNDERS AND C. O'NEILL.

Human Reproduction Unit, Department Of Obstetrics & Gynaecology,Royal North Shore Hospital, St. Leonards, NSW. 2065.

The pre-implantation embryo of a number of species produces aplatelet activating factor in vitro and in vivo. This soluble factor,with similar biochemical and biological effects as 1-0-alkyl-2­acetyl-sn-glyceryl-3-phosphocholine, can be detected by a bioassayin medium in which embryos have been cultured. Preliminary evidencesuggests that production of this factor shows a correlation with theviability or pregnancy potential of human embryos produced by invitro fertilization (1). In this study we examine the time courseof production of embryo derived PAF by mouse embryos in vitro.

The bioassay for the detection of embryo derived PAF involvesmonitoring the change to the peripheral blood platelet count of splen­ectomized mice following intra-peritoneal injection of embryo culturemedia and is expressed as the percentage reduction in the singleplatelet counts. The details have been pubJished in (2).

Six week old· Quackenbush strain female mice were superovulatedusing 10 i,u. pregnant mares serum gonadotrophin and human chronionicgonadotrophin 48 hrs apart and mated with fertile males. Two-cellembryos were flushed from the oviduct 36 hrs after ovulation usingHepes buffered medium and washed twice before the transfer to culturemedium. Thirty embryos were cultured in Falcon-5ml culture tubescontaining 1ml of Quinns Human Tubal Fluid medium with 3mg/ml bovineserum albumin (CSL Melbourne). The culture tubes had all been equil­ibrated with 5% C02 in air at 370 C overnight. At various intervalsembryos were removed from the tube, their stage of development andmorphology assessed and transferred to fresh tubes. The culture mediawas immediately placed on ice and assayed for embryo derived PAFactivity within 4 hrs of collection.

One of three cultures of 2-cell embryos displayed activity follow-ing 1 hour of culture while all cultures displayed activity by 4 hours.Eight of ten cultures displayed activity after 24 hours of culture.However, only 5 of 10 cultures displayed PAF activity at the thirdsUbpassage i.e. after 72 hours in culture. There was no apparentcorrelation between the morphology of the embryos and PAF productionwith all cultures producing a high proportion of expanded blastocysts.

These results confirm that detectable levels of embryo derivedPAF are produced by ~ouse embryos in vitro within 1-4 hours, butdifferences in its production became apparent with increased time inculture.(1) O'Neill C. &Saunders D.M. Lancet, 2: 1034-1035 (1984).(2) O'Neill C. J. Reprod. Fert. 73: 559-566 (1985).

2 3

FURTHER CHARACTERISATION OF EMBRYO-DERIVED PLATELET ACTIVATING FACTOR(EDPAF) IN MICE.

THE EFFECT OF INSULIN TREATMENT DORING PREIMPLANTATION CULTURE ONFOETAL DEVELOIMENT IN THE MOUSE

B.A. BALDO*, M.A. SMAL*, A.J. AMMIT**, D.M. SAUNDERS** AND C. O'NEILL** Heather G. Gardner and Peter L. Kaye

*Kolling Institute and ** Human Reproduction Unit, Rcyal North ShoreHospital, St. Leonards, NSW.

Department of Physiology and Pharmacology, University of QueenslandSt. Lucia 4067, QLD

0~lture in medium containing insulin accelerated development of 2-cellembryos to early blastocysts but had no effect on the percentage ofembryos developing from transferred blastocysts, or the sex of thefoetuses. (Table 1).

Foetal weights of insulin treated embryos were greater than thoseof the l.+ntreated controls (Table 2). No differences in placentalweight or skeletal deformities were found.

Thus in culture insulin increased the rate of development ofpreimplantation embryos, and this effect was later manifested inincreased foetal weights. The role of hormones in the development ofpreimplantation embryos and subsequent· foetal development needsclarification which may contribute to improving I.V.F. foetalviability in animals and humans.

Although development of embryos cuItured during thepreimplantation period appears normal, viability of human IVF embryosis reduced (1 ). Addi tion of insulin as a growth factor topreimplantation culture medium accelerated development of 2-cell mouseembryos to blastocysts (2) • Effects on subsequent foetal developmentare described here.

Two-cell embryos were collected from superovulated mice in M2 ±BSA and cultured 48 h in BMOC-BSA ± 170 nM insulin. The most advancedblastocysts were transferred to uteri of 3 day pseudopregnantrecipients. On day 19 placental and foetal weights, and number andsex of viable foetuses were recorded. Foetuses stained wi th Alizarin0.1% were examined for skeletal deformities.

'l'ABLE 1: 'Ihe ~fects ofTreatment % Developing to

Blastocyst

0.149 ± 0.0190.151 ±0.033

Mean ± SEM; 3 Replicates)

Weights (grams) at Day 19Placental weight

82.75 ± 6.39 19 : 2181.79 ± 3.69 17 : 18

Mean ± SEM; 3 Replicates)

Insulin (170 nM.) on Developnent% Foetuses/Blastocysts Males/Females

Transferred (numbers)

British Medical Journal, 291, 1160-1163.P. L. (1 984) Proc. Aus""LSoc. Reprod.

1.162 ± 0.029**1.298 ± 0.026

paired t-test; n>35;

Placental and FoetalFoetal weight

16.29 ± 0.79* 25.42 ± 1.45

(*p<0.025, n>200 embryos;

Lancaster, P. A. (1985)Gardner, H. G. and Kaye,

BioI., ..l.§.., 107.

(** p<O. 001 ,

ControlInsulin

(1 )(2)

Table 2:Treatment

ControlInsulin

The pre-implantation embryo produces a soluble platelet activatinGfactor (1) which, in a bioassay, showed similar kinetics of action as1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether) (2).Embryo derived platelet activating factor (EDPAF) could be deactivatedby phospholipases A2, C and D and could be extracted into chloroformsuggesting a phospholipid structure (3).

In this study the chemical nature of EDPAF in mouse embryo culturemedia was fur-ther characterised using inhibitors of PAF acether andthe ability of an antisera against PAF-acether to neutralize itsbiological activity.

Mouse embryos were obtained by superovulating six week old Quacken­bush strain female mice with 10 i.u. of PMSG and HCG (Intervet) 48 hrapart followed by mating with fertile males. Two-cell embryos wereflushed from the oviduct 36 hr after ovulation using Hepes bufferedmedium and washed twice before transfer for culture ,to lml of Quinn'sHTF medium with 3mg bovine serum albuminlml (CSL). The culture tubeshad all been equilibrated with 5% C02 in air at 370 C overnight. After24 hr incubation the embryos were transferred to fresh culture tubesand the remaining media assayed for EDPAF.

The bioassay for EDPAF was ~arried out as previously described(2). Briefly, intraperitoneal (i.p.) injections of inhibitors orantisera were given to spler,ectomised mice, followed immediately bya second i.p. injection of either PAF-acether (Sigma) or mouse embryoculture media. Counts were performed immediately before and 30minutes after injection by removing 10ul of blood from the peri­orbital plexus and diluting in lml of 1% (w/v) ammonium oxolate.Platelets were enumerated using neubauer haemocytometers with phasecontrast microscopy.

Both PAF-acether and mouse EDPAF were inhibited by the drugsIloprost (Schering) and Alprazolam (Upjohn) showing parallel doseresponses and the capability of inhibiting O.lug of PAF-acether anda similarly active amount of EDPAF at concentrations as low as 4ugand 6ug respectively.

The antisera completely neutralized the effect of O.lug of PAF­acether and an equivalently active dose of EDPAF at a 1/10 - 1/100starting dilution, while pre-immune rabbit sera had no inhibitoryeffect.

The results are further evidence that EDPAF is homologous to PAF­acether and the cross-reactivity of the antisera provides promisefor the development of RIA.(1) O'Neill C. J. Reprod. Fert. 73 (2): 559-566 (1985).(2) O'Neill C. J. Reprod. Fert. 73 (2): 567-577 (1985).(3) O'Neill C. J. Reprod. Fert. 75: 375-380 (1985).

Supported by an NHMRC Grant to PLK, HGG is a NHMRC Biomedica~ Scholar.

45

FERTILIZATION OF HUMAN OOCYTES FOLLOWING MICROMANIPULATIONOF A SINGLE SPERM UNDER THE ZONA PELLUCIDA

DEVELOPMENT OF A SUPEROVULATORY REGIME FOR THE COLLECTION OFOWLATED SHEEP OOCYTES AND EARLY EMBRYOS.

School of Veterinary Studies, Murdoch University, WA, 6150.

* - Total stimulations = ovulations + large follicles (> 4mm)

J.G.E. Thompson, E. Waugh and R.G. Wales

Split PMSG/FSH-P (1) and GnRH (2) administration have both beenshown to aid the superovulatory response and ovulation rate in sheep.We have developed a superovulatory regime, based upon these twotechniques, especially for the collection of tubal oocytes immediatelyafter ovulation.

Merino and Merino x BL ewes were treated in the following manner10 days after sponge insertion: Day 10 - 500iu PMSG (Fo11igon,Intervet) + 5mg FSH-P (Burns-Biotec). Day 11 - 400iu PMSG + 4 mgFSH-P, Day 12 - 3mg FSH-P + sponge removed. Day 13 - 100~g GnRH(Fertagyl, Intervet). Oocyte collections were performed at 18-21(Group A), 21-25 (Group B) or 25-29 (Group C) hours after GnRHadministration. Oocytes were recovered by a retrograde flush of bothoviducts and the results are given in the following table:

21.450.056.3

%Recovery

0.6±0.66.0±1.87.0±1.5

Oocytesmean±s.e.m.

15.0±2.214.3±2.013.8±2.0

Total stim*mean±s.e.m.

2.8±1.512.0±1.912.4±1.8

Ovulationsmean±s.e.m.

579

n

A

B

C

Group

A.O. Laws-King, A.H. Sathananthari'and A.O. Trounson

Centre for Early Hu~an Development, Monash University,Queen Victoria Medical Centre, Melbourne, 3000 and

*Department of Biological Sciences, Lincoln Institute, Carlton, 3053.

Preovulatory human oocytes were collected at laparoscopy after ovarianstimulation and follicular maturation. Six hours post laparoscopy the cumulus cellswere removed with bovine hyaluronidase (150 units/mlj Type 1-5, Sigma) and theoocytes classified as mature if a polar body was visible. Semen samples wereobtained from healthy donors whose semen analysis characteristics were within theaccepted World Health Organization normal range. I Sperm were prepared asdescribed by Mortimer(3) using an overnight incubation in a modified Tyrode'smedium containing 2.!.J. mM SrC12. On 7 occasions individual sperm weremicroinjected under the zona pellucida of mature oocytes with the aid of 2 Leitzmicromanipulators to which glass holding and injection pipettes were attached.Twelve hours following microinjection the oocytes were assessed for the presenceof pronuclei and were fixed in cacodylate-buffered glutaraldehyde for electronmicroscopy (EM). Fifty-fou:' percent of the microinjected oocytes fertilized (Tableo.

The microinjection of intact sperm heads directly into the ooplasm of mouseoocytes results in the formation of pronuclei (1)(2). Nevertheless,· mostmicroinjected oocytes lyse following penetration of the injection pipette(l)(2)which is a severe difficulty with this technique. The aim of this study was toachieve fertilization by microinjecting a single sperm under the zona pellucida ofhuman oocytes in an attempt to establish a micromanipulative technique forfertilization with a single spermatozoon.

Table.!: Number of oocytes that fertilized following micromanipulation.

Experiment: 2 3 4 5 6 7

No.oocytes 3 2 2 2 -1-2-

inseminatedNo. fertilized 2 0

Electron microscopy showed the normal criteria for fertilization. In serialsections of one oocyte EM revealed 2 polar bodies, 2 normal pronuclei, the remnantof a sperm tail axoneme and release of cortical granules.

These results indicate that, in the future, this technique could be used to study thephysiology of fertilization and may eventually be used to aid severely infertile menachieve fertilization.

(1) Thadani, V.M. (1980). J.Exp.Zool. 212: 435-453.(2) Markert, C.L.(1983). J.Exp.Zool. 228: 195-201.(3) Mortimer, D. (1986). J. Exp.Zool."237: 21-24.

The level of cellular vestment surrounding collected oocytes wasnoted. Oocytes were grouped into four catagories: I - Surrounded byan expanded cumulus/corona complex. II - Surrounded by a corona only.III - Devoid of all cellular vestments. IV - Abnormal ordegenerating. In Group A, 100% (n=3) oocytes were Type I. In Group B(n=42), 16.7% were Type I, 54.8% Type II, 26.2% Type III and 2.3% TypeIV. In Group C (n=63), 6.3% were Type II and 93.4% were Type III.

These results indicate that ovulation takes place approximately18-21 hours after GnRH, and is mostly complete by 25 hours. Oocytesproduced by this regime are fertile in vivo (23 cleaving embryos w~re

collected from 3 mated-ewes 48 hrs post GnRH). The significance ofthese results will be discussed.

(1) Ryan, J.P., Bilton, R.J., Hunton, J.R. & Maxwell, W.M.C. (1984)Reproduction in Sheep (Eds D.R. Lindsay and D.T. Pearce) p 338­341. Australian Academy of Science.

(2) Nancarrow, C.D., Murray, J.D., Boland, M.P., Sutton, R. &Hazelton, I.G. (1984) Reproduction in Sheep (Eds D.R. Lindsayand D.T. Pearce) p 289-290. Australian Academy of Science.

6

A SHEEP PREGNANCY SPECIFIC PROTEIN

W.R. Mercer, C.S. Lee, K.J. Gogolin-Ewens, M.R. Brandon,

Department of veterinary prec~inical Sciences, university of Melbourne.

During early pregnancy the secretions of the pregnant uterus un­dergo specific alterations to facilitate the maintenance and develop­ment of the conceptus. The biochemical nature of these changes islargely unknown, however proteins secreted by the rabbit (1) and pig(2) endometrium have been described which appear to play an importantrole in implantation and maintenance o~ the conceptus. Also in vitrostudies of the ovine uterus suggest that distinct proteins are producedin response to the presence of the blastocyst. (3)

In pregnant sheep, the post-implantation trophoblast and uterineepithelium of the interplacentomal. areas are separated by a narrowirregular space containing a number of secretory products contributedby the trophoblast, the uterine epithelium and the endometrial glands.To study molecules expressed at this interface and their possible rolein implantation and pregnancy, monoclonal antibodies were produced tofoetal trophoblast membranes. One monoclonal antibody recognises anantigen designated SBU-4, present only in interplacentomal areas.

Histological sections of uteri from 25 she;ep with gestation per­iods ranging from 10-145 days were stained with the monoclonal antibodyusing the immunoperoxidase technique. Results indicate that the SBU-4antigen is present in sporadically localised sites distributed only inthe interplacentomal areas, with the openings of the endometrial glandsvery positive. The uterine epithelial cells at these sites showintense staining in the apical cytoplasmic region and brush border.Staining is also obser_ed in the glycoprotein layer and in vacuoleswithin the uninucleate cells of the trophoblast lying adjacent to thereactive uterine epithelium. This pattern of distribution is observedthroughout pregnancy with the amount of antigen increasing from lowlevels at approximately day 17 of pregnancy. Biochemical studiesusing material purified by monoclonal antibody affinity chromatographysuggest that the SBU-4 antigen is a high molecular weight complex oftwo disulphide bonded 100 KD proteins non-covalently associated with a50 KD protein.

It is concluded that the SBU-4 antigen is an ovine pregnancy spec­ific protein expressed throughout pregnancy being first found about thetime of implantation. Furthermore, the data suggests that it is secre­ted by the uterine epithelium and is subsequently taken up by the adja­cent uninucleate cells of the trophoblast. This glycoprotein may beassociated with functional changes in maternal uterine metabolisminduced by the presence of a viable conceptus.

(1) Beier H.M. In Proteins and Steroids in Early Pregnancy. Eds BeierH.M. Karlson (1982).

(2) Bazer F.W. J. An. Science ~ 1376 (1975).

(3) Godkin J.D. Bazer F.W. Roberts R.M. 114 120 (1984).

7

EFFECTS OF OXYGEN TENSION ON THE METABOLISM OF GLUCOSE BYMOUSE MORULAE-EARLY BLASTOCYSTS IN VITRO

N.K. Khurana and R.G. Wales

School of Veterinary Studies, Murdoch University, WA, 6150.

As changes in intrauterine oxygen tension alter the carbohydratemetabolism, including glycogen turnover, of endometrium (1),experiments were carried out to study the effects of changes in oxygentension on glucose metabolism, and in particular glycogen turnover, ofday 4 mouse embryos.

In first of two experiments, embryos were labelled by pulseculture for 2 h in [U_ 14C] glucose (0.28 mM, 30 ~ ci/~ mole) and thefate of carbon incorporated into glycogen was followed during 24 hchase culture under different oxygen tensions. In the secondexperiment, the rate of catabolic utilization of glucose underdifferent oxygen concentrations was estimated by measuring CO

2and

lactate production (p mole/embryo h- 1 ) during 4 h incubation inradiolabelled glucose. The results (mean ±sem) for the twoexperiments are presented in the following table.

Oxygen tension Glycogen turnover Carbon diOXide Lactate(% ) ( % degraded during produced accumulated

chase)

20 -2 ± 6 3.7 ± 0.1 3.4 ± 0.35 13 ± 4 4.5 0.3 4.9 ± 1.12.5 46 ± 9 4.4 ± 0.3 3.1 0.31.0 61 ± 4 4.4 ± 0.5 3.3 ± 0.2

The turnover of glycogen was significantly enhanced (P < 0.01) bylow oxygen concentrations and the lower the oxygen tension the higherwas the degradation of glycogen. However, the catabolic utilizationof glucose was not reduced by lowering the oxygen tension. Assumingintrauterine oxygen tension in the mouse is similar to that in otherspecies at approximately 5% (1), the results of this study indicatethat the increased degradation of embryonic glycogen in vivo reportedin an earlier study (2) results from the low oxygen tension in theuterus. This could provide an explanation for the difference inglycogen content of cultured-and freshly collected blastocysts (3).

(1) Yochim, J.M. (1971) The Biology of Blastocyst. (Ed. R.J.Blandau). p 363. University of Chicago Press.

(2) Edirisinghe, W.R. and Wales, R.G. (1984) J. Reprod. Fert. 7267-73.

(3) Ozia~, C. and Stern, S. (1973) BioI. Reprod. ! : 467-472.

•

8

AMINO ACID TRANSPORT IN '!'BE MOUSE BLASTOCYST

Peter L. Kaye

Department of Physiology and Pharmacology, University of Queensland,St. Lucia, Queensland, 4067.

9

PROTEIN SUPPLEMENTATION FOR THE CULTURE OF ONE-CELLEMBRYOS OF SHEEP

~J!.alkeE' P. Quinn, R.J. Ashman. D.H. Smith* and R.F. Seamark

Blastocysts were collected from superovulated mice in M2 medium96 h post hCG and uptake of 3H- amino-acitds was measured over 10 min at37°C in M2 medium (2) • The kinetics for uptake of glycine in thepre7ence of alanine and vice versa, for glycine in the presence oflys~ne, and the effects of varying Na+ on these kinetics weredetermined using a non-linear curve fitting procedure. '

Alanine and glycine were competitive inhibitors of each other'suptake wi th sim~lar Vmaxi IS. for glycine inhibition of alanine wasclose to :he Km for g~ycine and vice versa. These results suggestth~t glyc~ne and 0 a;an~ne are carried ~ the same transport systemwh~ch resembles B '. Increasing the Na concentration decreased the~ for+ glycine. a~d s~ightlY increased Vmax ' with a Hill coefficientfor ~a of 2 ~nd~cat~ng co-transport of 2 Na+ with each glycine (oralan~ne). The strong dependence of the affinity of the transporter onth~ Na+ .concentratio~ suggests an important part for this system inam~no-ac~d uptake, Na -transport and blastocyst expansion.

The external cell membranes of the mouse blastocyst containsystems for simple facilitated diffusion and for active uptake ofamino-acids from their environment. The uterine fluids contain highlevels of amino acids, thus these transport systems represent asignificant metabolic interaction between the embryo and the maternalenvironment. Recently anew, wide specificity, Na+-dependent,transport system was described in blastocysts activated from diapause(1). This system: BO,+ shared many features of the systemtransporting glycine (2). We have attempted to assess the identity ofthese two systems using competitive kinetics.

(1) Van Winkle, L.J. et al.. (1985) J. Bioz.. Chern.12123.

(2) Hobbs, J.G. and Kaye, P.L. (1985) J. Reprood. Perot.

This work was supported by NHMRC.

260, 12118-

74, 77-86.

Department of Obstetrics and Gynaecology, Uniyersity of Adelaide, The QueenElizabeth Hospital. Woodville, S.A. 50ll and °Turretfield Research Centre.Department of Agriculture. Rosedale, SA 5350.

The successful culture of one-cell embryos is limited despite its potentialuse in gene transfer studies. One-cell embryos of sheep can be cultured inmedium supplemented with glucose (1) but the protein requirements for suchdevelopment have not been examined. In this study we assessed the suitabilityof sheep serum (SS). human serum (HS), ovine lymph (OU. bovine lymph (BL).BSA D2mg/ml) and NU"Serum (Collaborative Research Inc.) as proteinsupple ments in the culture of one-cell embryos of sheep.

Embryos were collected approx. 13 ·19h after ovulation and randomlyallocated to treatment groups. The medium used was synthetic oviduct fluidbuffered wit!! 12.5m M sodiu1)l bicarbonate and 12.5m M Hepes. Cultureoccurred in SOul droplets at 37 C in an atmosphere of 5% CO 2, 5% O2 and 90%N2' Embryos were stained after culture and the nu mber of nuclei counted. Theresults of two experiments are presented below.

Protein Fertilized Final develop mental Stage (%) Days (ra~ge) for

Source ova(n) l-6cells 7-16cells B HB B formation

EXp.l--_._------

S8 27 33.3 14.8 40.7 11.1 57

HS 28 46.4 10.7 32.1 10.7 6-7

OL 19 78.9 10.5 10.5 0.0 8

BL 31 67.7 16.1 16.1 0.0 7-8

BSA 28 25.0 39.3 35.7 0.0 7·10

NS 28 53 6. 14.3 25.0 7.1 6· 8

Exp.2HSOO%) 35 57.1 22.8 17.1 2.9 6 8

H8(20 %) 15 46.7 20.0 26.7 6.7 5 6

HsOO %)+NS(5%) 36 44.4 22.2 30.6 2.8 5 8

HSOO %)+NS(lO%) 30 53.3 13.3 23.3 10.0 5·8

Sera/ly mph heat inactivated and 10 % cone. used. -'unfess otherwise shown;B=blastocyst, HB=hatched/hatching blastocyst

SS and HS proved superior in the pro motion of blastocyst/hatched blastocystform ation. The nu mber of culture days required for blastocyst form ation was alsogenerally less with these two sera. The results of Exp. 2 indicate that embryodevelopment with HS supplementation can be improved by either increasing theconcentration of serum or by the addition of NS. The reason for the lowerpercentage of blastocyst formation in Exp. 2 compared with Exp. 1 is not known.

(1) Betterbed, B. & Wright. R. w, (1985) Theriogenology .2;1:547553,

10

DEVELOPMENT OF A QUANTITATIVE BIOASSAY FOR EMBRYO DERIVED PLATELETACTIVATING FACTOR (EDPAF).

A.J. AMMIT, M. COLLIER, D.M. SAUNDERS & C. O'NEILL

Human Reproduction Unit, Department Of Obstetrics & Gynaecology,University of Sydney, Royal North Shore Hospital, St. Leonards, NSW.

The human and mouse pre-implantation embryo releases EDPAF (1 l.It was suggested that the production of this factor may serve as amarker of embryo viability (2). Its presence in human or mouse culturemedia was detected using a bioassay in which the fall in single peri­pheral platelets subsequent to an intra-peritoneal injection of cult­ure medium in splenectomised mice was measured (3). This assay howeversuffers the known limitations of bioassays in that it is time consum­ing, technically demanding as well as being non-quantifiable. Inorder to assess the use of EDPAF as a marker for embryo viability arapid, sensitive and quantitative assay for platelet activation isreqUired, characteristics which are lacking in the current methodsused to assess platelet activation in vitro e.g. aggregometers andmeasurement of the release of factors from platelets.

We therefore developed a bioassay in which the reduction in single,non-aggregated platelets in rabbit whole blood in vitro after incub­ation with standard concentrations of synthetic PAF-acether or EDPAFwas measured.

Culture medium contains serum proteins which can themselves causeplatelet activation, therefore culture medium was deproteinisedby the addition of methanol, 4oC,and the protein removed by centri­fugation. The methanol-culture medium mixture was removed and theEDPAF extracted from it into chloroform. The chloroform fraction wasdried down under nitrogen and the EDPAF taken up in Dulbecco phosphatebuffer (calcium and magnesium free) containing 0.35% bovine serumalbumin (PBS-BSA1.

Blood from male New Zealand white rabbits was collected from themarginal ear vein into citrate using a 20G telfon catheter and kepton ice. A 50 ul sample of rabbit blood was placed in each of thewells of a temperature controlled (370 C) multiwell teflon micromixingdevice. After a 2 minute eqUilibration period a 50 ul sample ofextracted EDPAF or synthetic PAF-acetller (Sigma, USA) in PBS-BSA wasadded and 10 ul samples taken immediately and after a 15 minuteincubation period. The single, non-aggregated platelets were countedon a Baker 810 platelet analyser set to the regions of 2-24 um3 .The single platelets remaining after a 15 minute incubation periodwere expressed as a percentage of the initial count. A control sampleof PBS-BSA was run in every series, and the multiwell device allowedconstruction of a standard.curve or the simultaneous reading of manysamples.

The new assay for EDPAF requires only small volumes of wholerabbit blood, a 15 minute incubation period, it is sensitive and itallows the EDPAF to be quantified.(1) 0' Neill C., et. a1. Annals N.Y. Acad. Sci. 442: 429-431 (1985)(2) 0' Neill C. & Saunders D.M. Lancet 2: 1034-1035 (1984)(3) 0' Neill C. J. Reprod.Fert. 73: 559-566 (1985).

11

HISTOLOGICAL AND ULTRASTRUCTURAL EVIDENCE FOR PLATELET ACTIVATION INTHE REPRODUCTIVE TRACT DURING EARLY PREGNANCY.

C. O'NEILL, E. AMOS, *P. MOTE AND *A. GIDLEY-BAIRD

Human Reproduction Unit, Royal North Shore Hospital, St. Leonards,N.S.W. and * Dept. Vet. Physiology, University of Sydney. NSW. 2006.

The preimplantation embryo produces a soluble platelet activatingfactor with a structure similar to 1-0-alkyl-2-acetyl-sn-glyceryl­3-phosphocholine. In Vitro, this factor has been shown to causeplatelet activation and aggregation which in vivo causes a mildthrombocytopenia. Platelet activation is known to be a potentmediator of inflammation and vascular changes. The production of theembryo derived PAF would therefore be expected to cause considerablelocalized platelet activation and inflammation.

Ten week old Quackenbush female mice were induced to ovulate with5iu PMSG and HCG and either not mated or mated with fertile or vas­ectomized males. The resulting non pregnant, pregnant and pseudO­pregnant females were autopsied 36 hr after the expected time ofovulation. The reproductive tract was examined for gross signs ofinflammation. The reproductive tract on the right hand side wasdissected out and fixed for either paraffin sectioning and stainingwith haematoxylin and Eosin or sectioning and staining for trans­mission electron microscopy. The left hand side of the reproductivetract was flushed with saline and the flushings examined for unfert­ilized oocytes or 2-cell embryos to confirm the state of pregnancy.

Gross examination showed that the ovarian bursa of pregnant micetended to be inflammed compared with that of non pregnant or pseudo­pregnant mice. Fine needle aspiration of the contents of the ovarianbursa and examination showed that the fluid from the pregnant micehad a rich cellular content with numerous red blood cells and manynucleated and multi-nucleated cells. This observation suggestsdiapesis from the blood vessels surrounding the ovarian bursa, acommon feature of inflammatory reactions. The nucleated cellsappeared to be predominately polymorphonuclear leucocytes while themultinucleated cells had many of the features of megakaryocytes, theplatelet precursor cells. .

Sections of the oviduct of pregnant and pseUdopregnant mice showedthat the blood vessels surrounding the section of oviduct containingthe embryo had a considerably larger diameter than the vessels ofpseUdopregnant mice next to the unfertilized oocytes. Transmissionelectron microscopic views of these blood vessels show that the bloodplatelets are clearly activated, having undergone shape change anddegranulation and displaying pseudopods. The endothelium of the vesselsshows the characteristic features of an inflammatory site with consid­erable vesicularization and a loss of integrity. The blood vesselsof pseUdopregnant animals on the other hand displayed normal endotheliawith discoid platelets still containing~and dense granules.

This histological and ultrastructural examination of the reprOduct­ive tract in early pregnancy provides evidence for a mild inflammatoryreaction as a consequence of early pregnancy and is consistent with,and provides indirect support for, previous observations of the prod­uction of an embryo derived platelet activating factor.

12

TRANSGENIC MICE-GROWTH REGULATION BY TWO PORCINE GROWTHHORMONE GENE CONSTRUCTS

A. Michalska l , P. Vize 2, P. Quinn l , J.R.E. Wells 2 and R.F_ Seamark l •

Departments of lObstetrics and Gynaecology and 2Biochemistry, University ofAdelaide, Adelaide, South Australia, 5000.

Recombinant DNA technology provides the prospect of adding specificgenes to existing animal breeds to enhance selected productioncharacteristics. This offers a new dimension to animal breeding practices. Inour studies we use the mouse as a model for incorporation and expression ofporcine growth hormone gene constructs, that we are currently injecting into

- fertilized one-cell pig embryos.Porcine growth hormone gene (pH MPG.4) was constructed using the human

metallothionein IIA promoter, the pig growth hormone coding sequence fromthe cD NA and the 3' processing signals of the genomic clone. The expression ofsuch a construct can be induced by heavy metals One--cell in vivo fertilizedzygotes from superovulated (C57xCBA) female mice were collected 21-23 hrafter hCG injection. Approximately 600 copies of the pHMPG.4 were injectedinto the male pronuclei of one-cell embryos. After injection the zygotes weretransferred to the oviducts of pseudopregnant (C57xCBA) Fl mice.

Of 66 offspring analysed, 19 were found to have incorporated between 1 and30 copies of pH MPG.4 and 12 showed increased growth rates. However, therewas no correlation between the growth rate and the nu mber of gene copiesincorporated. To determine whether the pH MPG.4 gene and the large size areheritable, mice were bred with control animals. Seven out of eight males siredlitters_ The gene construct was inherited by 50 % of the offspring; the largephenotype was observed in 100 %. 50 % and 0 % of the transgenic progeny of 2,3and 2 sires respectively. Five. out of ten fe males were fertile and producedlitters. The injected gene was passed to the second generation by two females(5/12 and 1/18 transgenic offspring)_ Only one of the fertile fe males had anincreased growth rate. but none of her offspring carried the pH MP G.4 gene.

To obtain better control of the growth rate of transgenic anim als, theoriginal pH MP G.4 gene construct was modified by deleting the 200 bp basalsequence (which probably was responsible for the gene expression without heavymetals induction) from the human metallothionein promoter. The newconstruct (pH MAPG.U was injected into mouse eggs as described above. Thirtynine mice have been born and 11 were found to carry the pH MAPG.l gene. Onlytwo of the transgenic males showed a slightly elevated growth rate. All micewere bred with control animals and the offspring from these matings arecurrently being analysed for the presence of the pH MllPG.1 gene.

To determine whether expression of the pH MAP G.l gene can be regulated byheavy metals so me of the transgenic offspring will be put on a diet containingincreased levels of zinc.

13

EXPRESSION OF PORCINE GROWTH HORMONE cDNA IN TRANSGENICPIGS

A. Michalska l , P'l

Vize 2, R.J. Ashman l , B.A. Stone l P. Quinn l , J.R.E. Wells2

and R.F. Seamark

Departments of IObstetrics and Gynaecology and 2Biochemistry, University ofAdelaide, Adelaide, South Australia, 5000.

The dramatic advances in molecular biology which have occurred inrecent years, and the development of powerful new technologie~ ofreco mbinant DNA and gene cloning have provided animal breeders w~th apotential ne w tool for genetic i ~ prove me~t by direct gen.e trans~er, as asupple ment to conventional select~ve breed~~g meth~ds•. ThlS techmque ~ aylead to production of transgenic farm ammals w~th lmproved productlOncharacteristics, such as superior growth rates and food utilizationefficiencies. In this paper we report on the introduction of porcine gr?wthhorm one cD NA into pigs by pronuclear injection and its subsequent expresslOn.

One -cell pig zygotes fertilized in vivo were collected from. sup:rov:-rlatedLarO'e-White sows 5 hr after mating. To enable adequate vlsualizatlOn ofpro~uclei, ova were centrifuged at 7000 x g for 3 min. Approximately 600copies of a human metallothionein porcine growth hormone gene. construct we~e

injected into one pronucleus of the embryos. After overnlght cultu~e 10

l1inimal Essential Medium (U, microinjected embryos were surglcallytransferred to the oviducts of synchronized recipient sows.

A total of 327 embryos were transferred to 11 recipients (29.7 1: 5.6 [SE]embryos transferred per recipiend, four of which ret~rned to o~strus durin~ ~he

fourth week following transfer. Two sows farrowed litters and 10 the remalmngfive recipients, pregnancy was detected by elevated seru m oestrone sulphatelevels.

The DNA was isolated fro m tissue sa mples taken fro m 8 born piglets and theincorporation of injected cD NA was analysed by dilution dot. hybridization.Three piglets, 2 females and 1 male, were found to be transgemc. They werealso found to be positive by Southern blotting. One of the females ha~ a ?rowthrate at three months of age which is 25 % bigger than her nontransgemc slsters.

1. Michalska, A., Stone, B.A., Quinn, P. and Ashman, R.J. (985) Proc. Aust.Soc. Reprod. Biol l.2.:88.

14

GENE T RAN SF ERIN GOA TS

D.T. Armstrong l ,3, A. MichaL'3ka l , R.J. Ashman l P. Vize 2, J.R.E. Wells 2 andR.F. Seamark l .

Departments of 1Obstetrics and Gynaecology and 2Biochemistry. University ofAdelaide, Adelaide, Australia, 3The MReGroup in Reproductive Biology,University of Western Ontario, London, Canada.

A protocol has been developed to allow collection of single cell goatembryos at a stage which allows microinjection of genetic material as an i::titialstep in a program aimed at the creation of new goat breeds with enhancedproduc tion charac teristics.- Progestagen treated does were superovulated using FSH··P (total dosage

l6mg) either alone or in combination with PMSG. Synchrony of ovulation wasachieved by treating does with GnRH (50ug) 25 to 26 hr after sponge removal.Does were inseminated 14, 18 and 22 hr after GnRH injection and embryosrecovered 14 to 22 hr later (ie. 62,-70 hours after sponge withdrawal>. Of the 24does treated, 16 yielded 72 pronuclear embryos, 53 at a stage suitable for geneinjection. Centrifugation at 7000 x g for 2 minutes was routinely perform ed tofacilitate microinjection. I

A pronucleus of each of the 20 embryos was injected with 600 copies of aproven porcine growth hormone (pG H)- metallothionein gene construct and 35embryos were given a control injection of phosphate buffered saline (PBS). Theremaining 17 pronuclear ova were not injected, and were placed in culture inmodified synthetic oviduct fluid medium (1) containing 10% heat inactivatedgoat serum to derermine their ability to develop in vitro. All cultured ovadeveloped to the morula-blastocyst stage and 7 blastocysts hatched.

The injected ova were transferred to the oviducts of two synchronizedrecipients; the reproductive tracts of these recipients were flushed three dayslater to assess stage of developm ent in vivo. All the transferred ova wererecovered. Fifteen of the 26 PBS-injected and 14 of the 20 gene-injected ovahad cleaved to the 8-cell stage or beyond, at the time of recovery. They weretransferred to recipients to determine capacity for further development invivo. ---Ultrasound scanning was performed 50 days post-fertilization. Three out offour and two out of five recipients which received control and injected embryosrespectively were found to be pregnant. The microinjected em bryos will beharvested by hysterectomy at 70 days gestation for assessment of DNAincorporation using dot blot hybridization techniques.

1. Walker, S.K., Quinn, P., Ashman R.J.) Smith, D.H. and Seamark, R.F.(1986) Proc. Aust. Reprod. BioI. ..!~.

15

PRODUCTION OF OVERT INTER-SPECIES BOVINE CHIMAERASBY AGGREGATION OF DEMI-EMBRYOS

R.K. Munro, T.J. Witliams and J.N. Shelton

Division of Tropical Animal Science, C.S.I.R.O., Rockhampton andDepartment of Immunology, Australian National University, Canberra.

The aim of the work reported here was to produce primary(whole-body) chimaeras between Bos taurus and Bos indicus whichmight prove useful in stUdying bovi~lopmen:tanddIff'erences

between species in physiological and productive traits. Bos taurusand~ indicus donors were stimulated to superovula.te by~e~with FSH or PMSG towards the end of treatment with PRIDS. Oestrousdonors were inseminated with semen of the same breed and embryoswere recovered nonsurgically 5.5 to 6.5 days after the onset ofoestrus. Embryos were bisected with a microscalpel and half of eachembryo was removed from the zona pellucida using a micropipette (1)

and aggregated with a demi-embryo of the other species to produceinter-species chimaeric embryos which were transferred nonsurgicallyto synchronized recipients 1 to 6 hr after microsurgery. Inexperiment 1, 30 chimaeric Friesian-Brahman or Charolais-Brahmanembryos were' transferred to 30 recipients of which 9 were pregnantat 8 weeks and 7 at 6 months. These produced 2 sets of mixedspecies (Friesian and Brahman) twins and 5 single calves. One ofthe single calves was an overt male chimaera and had been derivedfrom an embryo made up of half a Friesian morula and half a Brahmanmorula. The calf was of Friesian conformation; at birth thepredominant coat colour was light brown grading to light gray on theunderline and inside the legs. There were light gray hairs in theears and in a symmetric rim around the mouth and nostrils. At 4months of age the calf was almost completely black with a reducedarea of gray hairs around the mouth and a gray stripe (approx 25 cmx 3 em) in the mid dorsal lumbar region. The erythrocytes werecompletely Friesian in type while 31% of the lymphocytes were of Bosindicus (Brahman) karyotype. In Experiment 2, 15 chimaeric embryos(Hereford x Shorthorn-Brahman) were transferred to 15 recipients ofwhich 3 were pregnant at 8 weeks and produced 3 single calves. Onewas an overt male chimaera. This calf had a Brahman hump and earswith Hereford x Shorth,orn (HS) conformation of the body. At birthit had a red-brown body with roan face and brisket and blacksocks. At 6 months the body colour was gray with a lighter roanface. The erythrocytes were of mixed type wi th no chimaerism and44% of the lymphocytes' were of taurus (HS) karyotype. The other twocalves were phenotypically taurus and one was an erythrocytechimaera. It is not possible to discern whether this erythrocytechimaerism was of primary or secondary origin.

This is the first report of the synthesis of overt inter­species bovine chimaeras.

(1) Williams, T.J., Elsden, R.P. & Seidel, G.E. (Jr). Proc. Ann.Conf. A.I. and embryo tranfer in beef cattle Denver, Co. Jan. 1983pp. 45-51.

.,-16 17

FERTILISATION OF OVA IN SUPEROVULATED MERINO EWES FOLLOWING AIWITH FRESH AND FROZEN - THAWED SEMEN

SYNCHRONIZATION OF TIMING OF OVULATION IN THE ARTIFICIALINSEMIN ATIO N OF SHEEP

D.H. Smith, S.K. Walker and R.F. Seamark

J.R. Hunton, W.M.C. Maxwell and J.P. Ryan

Animal Breeding and Research Institute,Katanning, Western Australia

(1) Walker, S.K., Smith, D.H. and Seamark. R.F. (1986). J. Reprod. Fert.77:135-142.

Turretfield Research Centre, Department of Agriculture, Rosedale, SA 5350and Department of Obstetrics and Gynaecology, University of Adelaide, TheQueen Elizabeth Hospital, Woodville SA 5011,

GnRH treatment failed to improve pregnancy rates irrespective of thenumber of motile sperm inseminated. In a second study. ewes were inse minatedbetween 0 4h before the median ovulation. Pregnancy rates were depressed(range 31.3-52.9 %) presumably due to a superovulatory response recorded insome ewes. However, when GnRH was administered at 24h, 18/34 ewesinseminated with the low number of sperm became pregnant; this compareswith 9/47 when insemination occurred earlier (6-Uh). These results indicatethat GnRH treatment may facilitate the use of low numbers of sperm subjectto appropriate timing of insemination.

There is little information on the use of fresh and frozen­thawed semen in sheep embryo transfer programmes. This experimentexamined the rates of fertilisation of ova in superovulated ewesfollowing surgical insemination with fresh and frozen semen 24 or 48hr after progestagen sponge removal (SR). .

In October 1985, 33 mature Merino ewes were superovulated w1tha combination of 400 i.u. PMSG (Batch 0971 Folligon, Intervet) + 8mg FSH-P (Batch 526K85, Burns-Biotec) as previously described (1).Semen was collected from two rams (A and B) by artificial vagina foruse either fresh or after freezing in pellet form and thawing (2).For surgical insemination, the uterus was exposed after mid-vent~al

laparotomy (general anaesthesia) 24 or 48.hr afte~ SR. ~oth uter1nehorns were ligated midway between the t1P and b1furcat1on. Semenwas then deposited in the lumen of the uterus above the ligatio~:

frozen semen 'in the left horn and fresh semen from the same ram 1nthe right horn. Forty eight to 52 hr after insemination, uterineligatures were removed, ovaries examined for corpora lutea and cellstage of recovered ova recorded. .

Semen from ram B fertilised a higher proport1on of ova thansemen from ram A (65/101 v. 53/158, p<O.OOl). The remaining data(pooled for rams) are presented in table 1.

'columns with unlike superscripts differ (p<0.05)Recovery rate~ for insemination 24 hr after SR were higher than

for insemination at 48 hr (109/143 v. 150/266, p<O.OOl). However,fertilisation rates were higher for insemination at 48 hr than at 24hr (92/150 v. 26/109, p<O.OOl). Fertilisation rates were betterafter using fresh than frozen semen (77/143 v. 41/116, p<0.005).

(1) Ryan, J.P., Bilton, R.J., Hunton, J.R. and Maxwell, W.M.C.(1984). In: Reproduction in Sheep. Eds D.R. Lindsay and D.T.Pearce. Australian Academy of Science, Canberra p. 338 - 341.(2) Visser. D. and Salamon, S. (1973). Aust. J. bioI. Sci. 12.: 513516.Supported by Aust. Meat &Livestock Research &Deve~opment Corp.

39.609/48)

GnRH treated ewesm------36-h-----

24.5 (12/49) 58.7 (27/46)--

19.1 (9/47)

Observations in superovulated sheep indicate that GnRH treatment producesa synchrony in the timing of ovulation (1). Approximately 95 % of oocytes arefertilized when intra-uterine insemination with low doses of thawed-frozensperm occurs within 6h of ovulation (S.K. Walker and D.H. Smith.un"published). The aim of this study was to determine if pregnancy ratesfollowing artificial insemination could be improved in the nonsuperovulated ewetreated with GnRH.

Progestagen/PMSG (400IU.) -treated ewes commenced ovulation between5l-72h after treatment (median 63h). When GnRH was administered 24h or 36hafter treatment, figures were 45-51h (median 48h) and 51-63h (median 60h)respectively. Month of breeding season (Nov. v. Feb.) was unimportant. Ewesin these treat ment groups w~re inse minated (:U1tra-uterus) with pooled thawed­frozen semen (either 10xlO or lxl06 motile sperm/uterine horn) between 6­Uh before the median ovulation. The number of pregnant ewes (day 60 scan) asa percentage of ewes inseminated is presented below

37.0 (17/46)

-M oTlle- sperm- - - Controlper horn eweslOXTo-o---57.4C58/10l)

18

TIME OF INTRAUTERINE INSEMINATION OF SUPEROVULATED EWESUSING FRESH AND FROZEN SEMEN

G. Evans, H.N. Jabbour & N.W. Moore

Department of Animal Husbandry, University of Sydney, NSW 2006.

19

EFFECT OF THAWED MOTILITY AND INTRA-UTERINE DOSE OFMOTILE SPERM ON FERTILITY IN EWES

J. Eppleston, W.M.C. Maxwell*, K.M. Battye and E.M. Roberts.

D partment Wool Science, University of New South Wales, N.S.W.;*~imal Breeding and Research Institute, Katanning, W.A.

There was no significant effect on fertility of either grade ofpost thawed semen, or of number of motile sperm inseminated; nor wasthere any significant interaction between these variables. We concludethat semen in which only a low proportion of sperm retain motilityafter freezing can be used successfully for intra-uterine inseminationby adjusting inseminates to the required number of motile sperm. Inaddition these results suggest that acceptable fertility can be obtainedwith frozen thawed semen using an intra-uterine inseminating dose aslow as 5 x 106 motile sperm per ewe.

(1) Killeen, I.D., and Caffery, G.J. Aust. Vet. J. 59:95 (1982)

(2) Salamon, S. Artificial Insemination of Sheep, Sydney university.

Wide variation exists in the post thawing motility of semen, bothbetween rams and between ejaculates within rams. This variatio~ can beadjusted for by diluting thawed samp17s to a co~n dose of mot~17 .sperm but there is no information ava~lable on ~ts effe7t on f7~~l~ty.

This trial reports the effect on fertility of post ~haw~~g mot~~~ty

of frozen ram semen at three doses of motile sperm ~nsem~nated ~nto

the uterus with the aid of a laparoscope. (1)

Semen was collected from 6 Merino rams and frozen in pellet form(2). Sample pellets from each ejaculate were thawed and.inc:mated for6 hours at 37°C. The percentage of motile sperm was subJect~vely

assessed at two hourly intervals and the mean was used to class.e~ch

ejaculate as Grade 1 (mean motility> 30%) or grade 2 (mean mot~l~ty <20%) so that each ram was represented by semen of both grades. Within

15 minutes of insemination, semen was thawed and diluted with Trisbuffer so that the inseminating doses of 5, 10 or 20 x 106 motile spermwere contained in O.lml (0.05m1 per uterine horn). Mature Merino eweswere treated with intravaginal sponges containing 60mg MAP (Repromap,Upjohn) for 13 days, and 400iu PMSG (Pregnacol, Herriot Agencies) atsponge withdrawal. Laparoscopic intra-uterine insemination wascarried out 60-66 hours after sponge withdrawal. Pregnancy was deter­mined at slaughter 50 days after insemination. Results were analysedby least squares analysis of variance (Table 1). Ram had no effect onfertility and was omitted from the table.

TABLE 1 Effect of Grade and Dose of thawed semen on fertilityfollowing intra-uterine insemination

Intrauterine insemination is necessary to achieve satisfactory fertilityin superovulated ewes. This experiment was designed to investigatethe optimum time for intrauterine insemination of superovulated eweswith fresh or frozen-thawed semen.

Semen was collected from 3 rams, pooled and frozen in glycerol-eggyolk-citrate diluent. Fresh semen from the same rams was used afterdilution with PBS. Forty-six mature Merino ewes Were treated withintravaginal progestagen pessaries for 12 days and with one of threegonadotrophin regimes previously described (1) to induce asuperovulatory response. Intrauterine insemination was performed bylaparoscopy under local anaesthesia at 24, 44 or 64 h after removal ofpessaries. 50 x lOs motile spermatozoa were deposited in a volume of0.1 ml in each uterine horn (total 100 x I06 / ewe). Ova were recoveredand ovaries inspected by laparotomy under general anaesthesia 88 hafter removal of pessaries; ovulation points were recorded. Onexamination, embryos, or single cell ova 'with pronuclei visible afterstaining, were recorded as fertilised ova.

The mean ovulation rate was IO.8±0.9. Mean recovery of ova (no.recovered/no. of ovulation points) was 61%. Multiple X 2 analysis wasperformed. There was no difference in ovum fertilisation rates betweengonadotrophin treatments; the data were therefore pooled and arepresented in Table 1.

Table 1. Fertilisation of ova recovered (No. fertilised/ No. recovered)from superovulated ewes inseminated with fresh or frozen semen atdifferent times in relation to withdrawal of progestagen pessaries.

Time of Fresh semen Frozen semeninsemination: 24h 44h 64h 24h 44h 64h

39/65 75/80 36/41 18/39 55/56 18/69(4/7) (8/8) (6/6) (3/6) (9/9) (10/10)

*Figures in brackets = No. animals with fertilised ova/No. inseminated.

Fertilisation rates were higher for fresh than frozen semen (P<O.OI)and there was a significant interaction with time of insemination(P<O.OI). There was no significant difference in fertilisation ratesbetween times of insemination in the fresh semen group; however, in~he ~roz~n semen group the fertilisation rate was higher aftermsemmatiOn at 44 h than at 24 h or 64 h (P<O.OI).

These data indicate that fertilisation rates in superovulated ewes are~ighe~ ~sing fresh than frozen semen. The optimum time of~nsem~nat:on was 44 h after pessary withdrawal, but the time ofmsemmatiOn was less critical using fresh than frozen semen.

1) Jabbour et a1 (1986). Proc. Aust. Soc. Reprod. BioI., 18.

This work was supported ,by the Australian Wool Corporation.

MAIN EFFECT

Grade

DoseMotile sperm(x 106 )TOTAL

125

1020

NUMBER EWESINSEMINATED

184188138123111372

LEAST SQUARES MEAN ± S.E.EWES PREGNANT/EWE INSEMINATED %

53± 3.744± 3.750± 4.346± 4.549± 4.749± 2.6

20 21

EFFECT OF GnRH ON TIME OF OVULATION IN SUPEROVULATED MERINO EWES EFFECT OF OVARIAN RESPONSE ON DISTRIBUTION OF OVULATIONS INSUPEROVULATED MERINO EWES

-------------------------------------------------------------------6a 15a l6a 27a 73aOb 24a 31b 33a 61a

·Oab 4b Oc 3b 37b2ab 4b Oc Ib 35b

Animal Breeding and Research Institute,Katanning, Western Australia

(1) Naxwell, W.M.C., Hunton, J.R. and Ryan, J.P. (1986). Theseproceedings.

W.M.C. Maxwell, J.P. Ryan and J.R. Hunton

Spring ~12

>12Autumn ~12

>12

;~~~~-n~-:;~~-~-i-t-h-diff~~~-n-;~~~;~~-s-;;:;~~-~-:;hi~-~-o-l-:~~~--;ifi~ rsignificantly (p<O.OS). , , .

Irrespective of ovulation rate, the maJor1tyof ovulat10ns forewes treated in autumn occurred between 48 and,54 hr after SR,whereas in spring a greater distribution of ovulat10ns was ~bserved

with some 70% occuring prior to 48 hr after SR. In spr1ng~ nodifference was 'observed between the percentage of ovulat10nsoccuring at 30, 36 and 42 hr after SR, yet all were greater thanthat observed for ewes at 24 hr. This suggests that there may betwo peaks of ovulations for ewes treated in spring, one between 24and 30 hr and another after 42 hr. This effect was not apparent forewes in autumn. Further, for ewes with >12 CL in spring, a greaterproportion of ovulations occured between 30 and 42 hr than for eweswith <12 CLs (154/S24 - 29% v. 58/302 - 19% p<O.OOS) indicating thatOVUlation may occur earlier in ewes, with a high ovarian response.

Ovarian response in sheep follOWing sup~rovulati~n with exogenousonadotrophins is highly variable. The t1me at,wh1ch superov~lated

:wes start to ovulate is an important variable 1n the product1~n ofembryos, especially when pronulc~ar s:age embryos are requ1red.However, the distribution of ovulat10ns 1S thought to have a,greater, fluence on yield of viable embryos. The present study exam1ned.the~~lationshiP between ovulation rate and distribution of ovulat10nsin ewes superovulated in spring and autumn. .

The experimental animals were 200 mature Merino ewes treate~ w1th400 i.u. PMSG + 12 mg FSH-P in spring or autumn as pre:10uslydescribed (1). The percentage of ovulations observed at d1fferentlaparoscopic ovarian examinations are presented (Table 1) for eweswith ~ 12 (low response) and >12 (high response) corpora lutea (CL)8 days after progestagen sponge removal (SR).

Table 1. The percentage of the final ovulation r~te observed atdifferent times of ovarian examination for ewes w1th ~ 12 and >12CL in spring and autumn.

S~~~~~-N~~---------T~~~-~f-~~~;~~~-~~~~i~~~i~~-(h;-~f~~;-SR)-------CL 24 30 36 42 48 54

Animal Breeding and Research Institute,Katanning, Western Australia

Table 1. Proportion of ewes wi th recent ovulations folloWingGnRH treatment in spring and autumn.

W.U.C. lfuxwell, J.R. Hunton, J.P. Ryan and G. Hood

--------------------------------------------------------------------

GnRH has been used to synchronise the time of ovulation in ewessuperovulated with PMSG and FSH alone (1). Our previous reportindicated the advantages of a combination of PMSG and FSH-P forsuperovulation of ewes (2). This study examined the effect of GnRHon time of ovulation in ewes superovulated with PMS + FSH-P inspring and autumn.

A flock of 200 mature Merino ewes was randomly allocated togroups for treatment in September 1985 (spring) and March 1986(autumn). In each season 100 ewes were superovulated with 400 iou.PMSG (Batch 0971 Folligon, Intervet) + 12 mg FSH-P (Batch 526K85,Burns-Biotec) as previously described (2). At progestagen sponge(Repromap, Upjohn) removal (SR) the ewes were randomly allocated to5 groups of approximately 20 for ovarian examination by laparoscopy(local anaesthesia) 24, 30, 36, 42 and 48 hr after SR when thenumber of large follicles and recent ovulations were recorded. Inautumn, a random sample of 19 ewes was re-examined 54 hr after SR.Twenty four hrafter SR half the ewes in each group received an i.v.injection of GnRH [100 pg Fertagyl, Intervet](+GnRH) while the otherhalf remained untreated (-GnRH). The final ovulation rate wasdetermined at laparoscopy 8 days after SR.

The final ovulation rates for + GnRH were 15.8 + 1.10 and 12.0+ 0.68 (p<O.OOl) in autumn, and 11:-6 + 0.87 and 13.1 + 0.95 (n.s.)in spring. The time of ovulation data-are presented in-table 1.

Season GnRH Time of ovarian examination .iliE. after mQ~- -3-0-- 36 42 48 54

-----------------------------------------------------~------------~--spring + 0/12 4/12 5/10 7/11 12/122/09 6/12 3/10 7/12 08/09autumn + 2/10 1/11 0/09 1/09 07/09 9/091/09 2/08 0/10 1/09 01/10 7/10

--------------------------------------------------------------------Treatment of ewes with GnRH had no effect on ovulation time in

spring. .In autumn, the majority of +GnRH ewes ovulated between 42and 48 hr (p<0.05), whereas the majority of -GnRH ewes ovulatedbetween 48 and 54 hr after SR (p<0.05). Ewes appeared to ovulateearlier in spring than autumn.

(1) Walker, S.K., Quinn, P., Ashman, R.J. and Smith, D.H. Proc.Aust. Soc. Reprod. BioI. (1985). p. 32(2) Ryan, J.P., Bilton, R.J., Hunton, J.R. and Maxwell, W.M.C.(1984). In: Reproduction in sheep. Eds D.R. Lindsay and D.T.Pearce. Australian Academy of Science, Canberra. p. 338 _ 341.Supported by Aust. Meat &Livestock Research & Development Corp.

22 23

ATT.E:MPTS TO IMPHOVE REPRODUCTIVE EFFICIENCY OF EARLY JOINEDMERINO, BLM AND CORRIEDALE EWES WITH VARIOUS MELATONIN TREATMENTS

L. Staples, M. Foote~, A. Jones.*, S. McPhee, J. Reeve.J. Wilkins, I and A. Williams.

Dept. Agric. & Rural Affairs, ARI, Werribee, 3030. Vic. #Tas. Dept. Agric. *, N.S.W. Dept. Agric. + & Private Consultant

Seasonal limitations to reliable early joining and highlambing percentage can be overcome by artificiallY shor~ening

daylength (1) or by administration.of melatonin to ew~s pr10r tojoining (2). This series of exper1ments, conducted 1n 1984/85, ~

assessed two methods of melatonin treat~ent under. fieldconditions for early joined Merino, Border.Le1cester x Mer1no andCorriedale ewes in Vic., N.S.W. and Tasman1a,

Experimental groups of 100-110 mature ewes were treated ~or

three weeks prior to joining. Ewes were observed for.mat1ng·marks for a 6-8 week joining period and pregnancy was d1agnosedby ultrasound. Control (C) groups were fed a supplement of sheeppellets at 100g/ewe/day. Melat.onin fed ~F) group~ received thesame pellets containing 2 mg/100g melaton1n fed da1ly at 1600hand implanted ewes (1-3) received control p~llets and pr~totype

subcutaneous melatonin implants. These 1mplants pro¥1ded a·transient elevation in blood melatonin levels lasting 7 0: .10days. Implants were inserted at 7 or 10 day intervals P~0~1~1nga "sawtooth" melatonin profile during the. 3 w~eks before J~lI;ung.

Table 1. Mating times and potential lamb1ng 1n ewes rece1v1ng-_._--melatonin treatment commencinE!' 3 weeks before ·oininE!'.Trial Breed Location Joining Days to 1st mating Lambing %

(day 0) C F I~3 C F 1-31. Mer. Cowra 7 Jan 22 22 21+ 99 92 892. BLxM Ruther-a) 27 Oct 27 21 23+ 99 118 1063. glen b) 30 Oct 19 22 22 106 120 1114. " Tamworth 20 Dec 17 17 18 151 143 l~g

5. " Macedon 19 Dec 24 23 22 124 133 143p. Corr. Werribee 19 Dec 24 22 22 110 101 1037. " Launceston 14 Feb 24 - 25 111 - 121Values under11ned are significantly (P<0.05) different to contrnJ

Ewes 1n all experiments showed character1st1c.of ram-1ndu?edmatings, the onset of which was not enhanced by e1ther the da1lyfeeding or the :3 week "sawtooth" ~lelatonin treat~ent (Table 1).Neither treatment consistently 1ncreased .lamb1ng percentage.Differences between trials were not cons1stently related tobreed time of joining or latitude. Responses to theseconti~uous and discontinuous melatonin treatments for 3 weeksbefore joining are ineffective in breeds which are responsive toram-effect. These results contrast with those obtained fromsimilar treatments in British breeds (3). They also contrastwith those achieved with improved continuous delivery implants ina subsequent study (4). .. ..

This work was supported by Gene L1nk Austra11a L1m1ted.(1) Dunstan, E.A. (1977). Aust.J.Exp.Ag.Anim.Husb. 17:741-745(2) Kennaway, D.J., Gilmore, T.A. and Seamark, R.F. (1982).

Endocr. 110:1766-1772(3) Staples, -L~, Foote, M., Killeen, D., McPhee, S., Wilkins, J.

and Williams, A. (1986). Proc A.S.R.B. 1~

(4) Staples, L.D., McPhee, S.R., Ayton, B.M., Reeve, J. andWilliams, A.H. (1986) Proc. A. S.R.B. 1?

ABSENCE OF NOCTURNAL PLASMA MELATONIN SURGE UNDER LONG ANDSHORT PHOTOPERIODS IN THE DOMESTIC SOW

S.J. McConnell l and F. Ellendorff2

lCS~RO, Divisi?n of Animal Production, PO Box 239, Blacktown, NSW 2148Institut fur Tierzucht und Tierverhalten (FAL) , Mariensee 3057,

Neustadt 1, Federal Republic of Germany.

Reproductive performance of the domestic pig fluctuates throughoutthe year. Reports of summer infertility are common and a decline inperformance during winter is also apparent. The responsible factor(s)are ill-defined, but recent evidence suggests that changes inday length are involved (1). Changes in the secretory pattern ofmelatonin by the pineal mediate the effects of day length onreproduction in many mammals. This stUdy describes the changes inconcentrations of melatonin in plasma samples collected from GermanLandrace sows at hourly intervals for 31 h after exposure to each ofthree different photoperiods. Blood was obtained from jugularcatheters and melatonin concentrations were measured byradioimmunoassay. Lights were turned on at 0730 h throughout thestudy. The intensity of illumination during the light phases (L) was320-550 lux and, during the dark phases (D), total dark was used.

Under 12L:12D melatonin concentrations during the light phaseranged from 22 ± 5.9 to 96 ± 25.1 pg/ml (mean ± S.E.M., n = 4). Duringthe dark phase the individual concentrations increased 2 to 5-fold overthe peak individual light phase values in 3 out of 4 sows. '!'hisnocturnal surge was of 3.8 h duration and peaked (190, 294 and546 pg/ml) at 0100 h which was 5.5 h after the onset of dark. '!hesurge did not occur in these animals after exposure to 16L:8D and couldnot be reinstated by the subsequent exposure to 8L: 1 6D. During thelatter two photoperiods .the mean concentrations were consistently<30.8 ± 10 pg/ml during the dark phase and <55 ± 21.9 pg/ml during thelight phase. All the sows displayed regular oestrous cycles during thestudy and the day of the oestrous cycle on which the samples wereobtained under each photoperiod was not significantly correlated withthe presence or absence of the melatonin surge. A separate experimentusing different animals confirmed the surge under 1 2L: 1 2D. In two offour cycling sows the peak concentrations during the dark phase(122 and 110 pg/ml at 0400 hand 0500 h) were 2 to 6-fold higher thanthose of the light phase. Absence of the nocturnal melatonin surgeunder long and short daylengths may be a factor contributing to thedecline in reproductive prolifacy during the summer and winter months.

(1) Claus, R., and Weiler, U. J. (1985) J. Reprod. Fert., suppl. 33:185-197.

24 25

~mLNroNIN 'l'HEi'ITHF:NTi3 TO IMP]{OVE ElIli:LY ,.lOWING OF BOHDEH LEIC);;S'l'Eh'AND HOHNEY EI'i'ES

THE EFFECT OF SEASON ON THE RESPONSE OF BORDER LEICESTER XMERINO (BLxM) EWES TO SUBCUTANEOUS IMPLANTS OF MELATONIN.

(1)

(2)

TABLE 1. Mating and fecundity in BLxM ewes implanted withI II dOff t' f S 1985 d S 1986I Requlin at ~ eren t~mes 0 ll::r~nq an ummer

First implant 19 Aug 30 Sep 11 Nov 23 Dec 3 FebJoininq date 30 SeD 11 Nov 23 Dec 3 Feb 10 Mar

C T C T C T C T C TNumber of 0 18 17 6- 5 17 12 18 13 8 12ewes 1 70 52 84 59 66 53 46 39 42 45bearinq >1 14 33 12 39 18 38 38 50 52 46oetus % 96 117 106 133 101 127 122 138 143 135~A from C fr21.4%** +25.6%*** +25.9*** +13.7NS -5.7NS** P<O .01, ***P<0.• 001 , NS = P>0.05 ~Chi square

J. Reeve, A.H. Williams+, S.R. McPhee+, R. Peake & L.D.Staples,+

Department of Agriculture and Rural Affairs, Rutherglen ResearchInstitute and + Animal Research Institute, Werribee, Vic.3030.

Exposure of anoestrous Corriedale ewes to a simulatedreduction in photoperiod by administration of a controlleddelivery form of melatonin causes an early onset to the seasonalpattern in oestrus and ovulation rates (1). When fertile ramsare introduced to the flock at 3 to 6 weeks after implants areinserted, matings occur in response to "ram effect" or duringnatural cycles at the time of optimum ovarian response totreatment and so foetal percentages are increased (2).Although the "Re~ulin" implants were effective in Merino, Romneyand 8lxM ewes (2), no systematic study has been made of the timeof year which implants can be used effectively in any breed.

In this study 1021 mature BLxM ewes were maintained inisolation from rams and bucks. At intervals of six weekscommencing 19/8/85 groups of 100-102 ewes (T group) received"Regulin" implants (s.c.). A similar number of ewes were selectedas untreated controls (C). The implants maintained daytime plasmamelatonin at 2597±279 p molar for 37 days. A second implant,given 4 weeks after the first, provided a continuous treatment forat least 9 weeks. T and C groups were grazed together but inisolation from the residual flock and harnessed sexually activerams (3%) were introduced 6 weeks after the start of treatment.Matings were recorded over a 6 week joining period and pregnancywas diagnosed by ultrasound.

All matings followed typical patterns for "ram. induced"oestrus. Regulin stimulated twinning in ewes joined from Springto early Summer but did not enhance peak seasonal reproductiveperformance for ewes joined in early Autumn (Table 1). Regulinthus provides an effective way to increase fecundity of BLxM ewesjoined before and around the summer solstice.This study was sponsored by Gene Link Aust.Ltd.(1) Williams, A.H., McPhee, S.R., Ayton, B.M. and Staples, L.D.

(1986). Proc. A.S.R.B.(2) Staples, L.D., McPhee, S.R., Ayton, B.M., Reeve, J.L. and

Williams, A.H. (1986). Proc.A.S.R.B.

G. HePbee, L. Stq.ples, M. Foot~E:'I, D, KiIJeen-l, ,J. Nilkins*and A. Nill i am~; .

Dept. \if Ag~ic. & I,ural Af~airs, :}..l': I. ~'iJerribee, 312'30, Vic.N.S.h. Dept. of Agrlc. & PrIvate Consultant.

The . u~e of rnelat.onin tlJerapy to induce responsescbarac~e~'li~tlc of. short days offers particular advantages forearly JOInIng and Increased lambing percentage in British Breeds.These.trials, conducted in 1984/85, compared the effectiveness ofa dal ~y feed ,trea"trnent wi tb 3 different continuous de] i verytherapIes to stImulate breeding in early joined Border Leicester'(BL) and Romney Marsh ewes (Rom) in N.S.N.

Control (C) groups received 100 grn/day supplement ofuntreat;E:d sheep pelle·ts; melatonin fed ewes (F) received similarpellets containing 2mg/100g melatonin group fed at 1600 h . dailyfor 3 weeks before joining; implanted animals received control1?ellets pll~s r:~elatonin via. protot,ype s. c. implants. TheseJlriplants malntaln:~d plasma melat.onin levels above 1000 pmolar for~0 days." New Implant.s were insert.ed every 10 days to give asawtooth treatment profi~e: . Treatments (n=50-100 ewes/group)

wer~ for 3 weeks before JOInIng (1-3), 3 weeks after joining(1+0) or both (16). Harnessed rams'(2-3%) were introduced witball groups run as one mob. Pregnanc~'l was diagnosed by ultrasound.

Trial/ Location Joining ,Joining t.o 1st oestr;!" % mated ea~lBreed r-:(~la':"":\~'·::-"Cc;;."j),--+.-,C",,-..~.:..LJ.Q __L!:.?_C F 1--3 16 1+3

T1-"BL"-T~~h 24 Dec --- No ewes matedT2 HI. Tamv?Qrtb 28 Jan 33 36 32 - - J56 73 74'1'3 BL Finley 29 Jan 52 43 44 44 44 8 42 38' ~1 29.1'4 Rom. Cowra 8 Jan 3~. 3~_~~._- -_ ~2 ~.~ 1~

If!!21~ 1.!- Effe~t of melaton~n t~rea~ment~ c:n. ITIl;-,m days ~rom joiningto 1st oestrus and proportIon of group JOInIng early .

+ Mean time to 1st oestrus for only those ewes mating.* % of flock ~ated ~y day 42 (1'2 & 1'3) & day 28 (T4).Values underlIned dlffer significantly (P<0.025) from control.

. In contrast to less seasonal breeds (1), BL and Romney ewosdId. not show an oestrous pattern characteristic of ram inducedr!a.ttln~s. Feed and implant t.reat,mont." enhanced onset. of oes'LrusIn trIals 2, 3 & 4 but were not effective for BL ewes joined inDe~em~~~ (~1). . Potential lambing percentage was increased(~,0:~~a) from.102 (C) to 124 (F) and 119 (1-3) in T2 but was notsl~rJlflcant.ly ~mprove.d in other tTials. The results suggest thatbot.h. feed a~Jd Implant therap~es oowmeneln[:: 3 weeks before 50iningr~~:glna.llY lmprove reproduct~lve :perfol"IlliWOC' in f.:C1.rly joined ewesv,lnch do not show ~ ram induced mating, Subsequent irnpJ:::ntdevelopments have aclneved improved l-e;.;ponf~es (2).

This work was support.ed by Gene Link P.II,~:t.ri'.l1ia Limi ted.s:.~p~:s;" I.., _Foot.~, . H. , Jonps, A .• j'1r.·plwe. S., Reeve, ~J.,~LIJ.'.ln." ,J., ,:md IhlllElifJ:3, 11., (l!l8i.i hr:H~. !I.S.R.B,

St;aples, L., MoPhee, S., IIvt.cm 1'1 J 8· Vi IIi· A(1986) Proe. A.S.R.B. ' ,.. ~. " I. alll~.>, .

,-26 27

1234567-9 -9 -6 -6 -359595

<troup (i) and per ewe ovulatin<t (ii).

Day 28 53 74 96 124 162 203 Mean date

Date 21/11 16/12 60 28/1 2fl/2 3/4b~b 1st Oestrus

(i) 1f 6 74a-m a 133 128 109 128 9/1/86

L 8 38b 8Bb 138 138 153a NE 17/1/86

C 12 24b 55 c 137 142 16Zb lS5a 26/1/8nx---x 1. 39 1. 29 1. 21 X 1. 30

X

(ii)H 1. 34 1. 70L 1. 23XY 1. 15: 1. 43 1. 38 1.55Y NE

C 1. 04Y 1. 08Y 1. 39 1. 44 1. 67 z 1. 57Y~ '"For each date numbers w~th dIfferent super~crlpt~ dIffer (1)

a,b,c P<0.01-0.001 and (ii) x,y,Z P<0.05-0.001. NE=not examin~d.Implant treatments resulted in an earlier onseL lo breedIng

and increased both the proportion ovulating and the number ofovulations per ewe ovulating (OR) at 53 and 74 days aft.ertreatment. Thereafter responses to treatment declined so that byday 162 OR's, but not proportion ovulating, were lower ~n group H(P<0.001) and group L (P<0.05) than Control. Chang~s In OR ~eredue almost entirely to an increased occurrencp. of tWIn ovulatIons.

The Regulin implants induced an earlier onset but shorterduration of the seasonal pattern in ovarian activity in 'a dose­dependent. manner. Peak seasonal OR was observed 12 weeks earlierin the H group than in controls with increases of up to 300% ofControl early in the season. Regulin thus enables peak seasonalreproductive activity to be attained at a Spring joining.

This study was support.ed by Gene Link Australia Limited(1) Arendt., J., Symons, A., Land, C. and Pryde, S. (1983) J.Endecr. 97: 395--400(2) Kennaway, D. , Gi lmore, T. and Seamark, R. (1982)

Endocrinology J.jQ@.: 1766-1772(3) Nowak,R. and Rodway, R.G. (1985) J.R~prod. Fert. 74:287-293(4) Linooln, G.A. and Elbing, F ..J.P. (1985) J.Reprod. Fert.:n,: 241-253

A. H. I'lil Iiams" S. R. McPhee, Ayton, B. M. and L. D. 5t.aples

Dept. Agric. & Rural Affairs, A.R.I., Werribee, Vic., 3030

The stimulatory effects of short days on the reproductiveactivity of sheep can be mimicked by the administration ofmelatonin daily as a feed supplement (1,2) or continuously viaintravaginal sachets (3,4) .. Apart fr~m reports of a sli~htlYincreased lambing percentage 1n melatonIn treated flocks, llt~leis known of the dynamics and dose-depend~nce of the ovar1anresponse to continuous melatonin treatment In ewes. .

Anoestrous Corriedale ewes (n=100/group) rece1ved (s.c.)propriety melatonin ( "Regul ~n") implants which maintainedmel at.on in plasma levels contInuously at 2597 279 pmolar (IIgroup) or 1095 128 pmolar (L group) fro~ days 1-37 afterimplantation. Control ewes (C group) receIved no treatment.Treated ewes were implanted on 24 Oct (day 0) and 21 Nov '85 (day28) to give a continuous treatment duratio~ of at least 9 weeks.Ewes were maintained on irrigated pasture In seperate paddockswith vasectomised rams throughout the experiment. Oestrousobservations were made daily and ovaries were examined at 3-4week intervals by laparoscopy under local anaesthesia.Table 1. Ovulation rate as corpora lutea (CL) per 100 ewes in

DOSE-DEPErlDENT EfI'FEC'I';"; OF CONTINUOUS MELATONIN ADMINISTRATION ONTHE SEASONAl. PATTEI?N OF' OES'I'ROUS CYCLICITY AND 8VULP.TION RATE IN

CORRIEDALE ElvES

ewes were grazed with treated ewes (Gp 6) orisolation (separate ewe effect control, Gp 7).Mean time from joining to conception (CD, days), midfoetal % (foetuses/100 ewes in group) and minimum

OPTIMUM MELATONIN TREATMENTS TO IMPROVE REPRODUCTIVE PERFORMANCEOF MERINO, BORDER LEICESTER x MERINO AND ROMNEY ElvES JOINED IN

SPRI~G AND EARLY SUMMER

L. Staples, S. McPhee, B. Ayton, J. Reeve & A. Nilliams.

Dept. Agric. & Rural Affairs, A.R.I., Werribee, Vic. 3030

St~dies have shown that parenteral (s.c.) administration ofmelat?nln to anoestrous ewes causes an early onset to thebreedIng season (1) and a precocious seasonal peak in ovulation:ate (2). To exploit the potential benefits of melatonin~m~l~nts, the timing and duration of treatment relative toJOln:ng must be such that conception occurs at a time of maximumova:lan response. It is also necessary to determine whetherop~lmum ~:eatments vary between breeds having different seasonalpatterns In reproductive activity.. Under farm conditions we tested the effects of 5 differentImplan~ treatments on mating, conception patterns and fecundity(by mld-?regnancy ultrasound) in maiden Merino (n=819), parousBorder Lelce~ter x Mer~no ~n=944) and Romney ewes (n=694) joined~or Autumn-~lnter.l~mbln~In S?uth~rn Victoria. Proprietry s.c.Implants (Regulln) whIch maIntaIned daylight plasma levels at2597+/- 279 pmolar from 1-37 days were used. Treatmentscommenced 9, 6 or 3 weeks prior to joining with durations of 9 or

period over which 75% of ewes conceived (CMF, days).

U"oinedMaiden Merino Parous BLxM Parous Romney

8/11/85 (day 0) 18/11/85 (day 0) 20/1/86 (day 0)Mean I Lamb 1 75% Mean -, Lamb I 75% Mean I Lamb 175%

Gp CD % CMP CD % CMP CD % CMP1 15 99 c · 16 30 94 c 17 24 100E' 152 13 101 c 14 26 106bc 20 22 126ab 133 13 112ab 14 27 110b 10 26 118bcd 94 13 117 a 14 27 129a 10 28 138 a 85 15 118a 14 27 127 a 7 30 130abc 86 17 104bc 16 30 100bc 14 33 114bcd 117 18 98 c 18 34 101 bc 15 37 114bcd 20

Means wIth dIfferent superscrIpts dIffer P<.05-0.001.Treatments commencing 3-6 weeks prior to joining (Gps 3, 4 &

5) Ind~ced an earlier conception, rendered treated animalsrespons~ve to. "ram effect", and defined and shortened thecOnCe?tlon perIod, especially for BLxM and Romney ewes. Regulinalso Increased expected lambing percentages for all three breeds." W~" ~onclude tha~ melatonin treatment in the form ofRegulln .l~p~ants prOVIdes a practical solution to the proble

of early JOInIng. msThis study was s~pported by Gene Link Australia Limited

(1) Stap~es~ L., Foote, M., Killeen, D., McPhee, S., Wilkins J~ W:lllams, A. (1986) Proc. A.S.R.B. ' .

(2) Wllhams, A., McPhee, S, Ayton, B. & Staples, L. (1986) Proc.A.S.R.B. .

28. 29

THE IN-VITRO FERTILISATION OF SUPERNUMERARY HUMAN OOCYTES IN AGAMETE INTRA-FALLOPIAN TRANSFER PRqGRAMME

J.L. Yovich~ P.L. Matson, J.M. Yovich, B. Bootsma, J. Spittle.

PIVET Medical Centre, 166-168 Cambridge Street, Leederville,Western Australia 6007.*S:nior.Lecturer, Department of Obstetrics & Gynaecology,Unlverslty of Western Australia.

5 pregnancies were achieved from 12 (42%)h d • normospermic couples who

a no oocytes fertilised in-vitro.It is concluded that the failure of small numbers of supernumeraryoocytes to fertilise in-vitro is of no diagnostic value and thata lack of fertilisation as such should not preclude a f~rtherGIFT attempt.

SEMEN QUALITY TOTAL PTS. FERTILISATION NO FERTILISATION

PTS. PREGS. PTS. PREGS.

Oligospermia 5 1 0 4 0Normospermia 29 17 6 12 5

Delay Between Oocyte Aspiration and Insemination (hours)

~-1~ 2-2~ 3-3~ 4-4~ 5-5~ 6-6~ 7-7~ 8-8~

Number19 85 74 208 303 212 61 30of Oocytes

Fertilization53% 60% 68% ' 61% 67% 59% 61% 57%Rate

It is apparent from the results that there is no significant effecton fertilization by inseminating ocicytes in the range 2-8 hours afteraspiration. Whether insemination immediately after aspiration isacceptable awaits greater numbers. It is however logisticallydifficult to do this unless the spermatozoa have been preparedbeforehand. A major advantage of earlier insemination of oocytes isthat it allows a fertilization check and possible reinseminationbefore the maximum time for successful fertilization is reached.

FERTILIZATION OF HUMAN OOCYTES IN RELATION TO DELAY BEFORE INSEMINATION

*K.L.Harrison, L.M.Wilson, T.M.Breen, A.K.Pope, J.M.Cummins and J.F.Hennessey. Queensland Fertility Group, 225 Wickham Terrace, Brisbane4000, and *Depattment of Animal Sciences and Production, University ofQueensland, St.Lucia 4067.

As part of the group's in vitro fertilization (IVF) programme, it hasbeen our policy to inseminate the oocytes approximately 5 hours afteraspiration. This protocol was influenced largely by the work ofTrounson et a1.(1) who found that a short period of culture in vitroallowed completion of oocyte maturation. They found poor fertilizationrates at O-~ hour and optimal fertilization at 5-5~ hours but withlittle information on the period 1-4 hours.

For various logistical reasons we had occasionally inseminatedoocytes in the range 2-4 hours after aspiration and had achievedacceptable fertilization. This was supported by reports such as thatof Fishel et al.(2) which achieved fertilization in the same timeinterval. The introduction of the successful gamete intra-fallopiantransfer (GIFT) technique by Asch et al.(3) which involves effectiveinsemination at less than 1 hour caused us to further question our5 hour insemination policy.

This paper presents preliminary results of a study to determinefertilization rates for post-aspiration insemination delays of 1-8hours. The study began at 5 hours, verified success at 4 and 6 hours,and gradually widened to cover the 1-8 hour range. Fertilization ratesare shown in Table 1.

Table 1. Fertilization Rates for Varying Oocyte Post-AspirationIncubation Times.

1. Trounson,A.O.,Mohr,L.R.,Wood,C. &Leeton,J.F. (1982) J.Reprod.Fert. 64: 285-294.

2. Fishe1-:-S.B. ,Edwards,R.G., & Purdy,J .M. (1984) Fertil.Steril.42: 191-197.

3. Asch,R.H.,Balmaceda,J.P.,Ellsworth,L.R. & Wong,P.C. (1985) Int.J.Ferti1. 30: 41-45.

Asch, R.H., Balmaceda, J.P., Ellsworth, L.R., & Wong, P.C.(1986) Fertil.Steril. 45: 366-371.

Yovich, J.L., Stanger, J.D., Tuvik, A.I., & Yovich, J.M.(1984) Med.J.Aust. 40: 645-649.

Gamete intrafallopian transfer (GIFT) is a technique which isbecoming widely used in the treatment of infertility (1). Folli­cular growth is stimulated using clomiphene citrate (CC; Clomid,Merrell-Dow), human menopausal gonadotrophin (hMG; PergonalSerono) or a.combination of the two drugs. Preovulatory oo~ytesar: then aspnated from Graafian follicles 35h after an ovulatorytrlgger of 10,?00 IU hCG with a double-lumen needle, and a maximumof 4 replaced lnto the fallopian tubes together with approximately100,000 motile sperm, using a 16-gauge Teflon catheter.

I~ an attempt to determine whether fertilisation would occur in­V1VO, supernumerary oocytes remaining after the GIFT procedure(me~n 2.8 oocytes per patient) were each inseminated with 100,000motlle spermatozoa, and fertilisation in-vitro confirmed 18h laterby the presence of 2 pronuclei (2).

(1)

(2)

30 31

THE VIABILITY OF MOUSE EMBRYOS CULTURED IN HUMAN AMNIOTIC FLUID

Carol Kirby and A. Trounson.

Centre for Early Human Development, Monash University,Queen Victoria Medical Centre, Melbourne.

It has been shown that mouse embryos develop from 2-cells to blastocysts in humanamniotic fluid (HAF) at rates comparable to culture in medium specificallydesigned for preimplantation embryo development (0. It was noted thatsignificantly more blastocysts hatched in HAF (0. In the present experiments wehave examined the viability of mouse embryos grown in HFA by transfer topseudopregnant recipients prior to study of the use of HFA for culture in human invitro fertilization.

* xI = 4.97, P <.05

!here was a signific~ntly higher proportion of normal fetuses at 16 days gestation~n embryos cultured 111 t:IAF (Tabl.e 0 although there was no significant difference111 the total number of ImplantatiOns (normal fetuses and implantation sites). Theresults have encouraged us to undertake a controlled trial using HAF for culture inhuman IVF.

Amniotic fluid was obtained from patients undergoing amniocentesis at 16-21weeks gestation. After removal of fetal cells the supernatant was heat inactivatedat 56°C for 45 mins. antibiotic added (penicillin - 60 mg/l; streptomycin - 50 mg/l)filtered (0.221-1 pore size) and stored at 4°C for up to two weeks before use. Two~cell mouse embryos obtained from superovulated donors were cultured in eitherHAF or Whittingham's T6 supplemented with 1096 fetal calf serum (FCS) in Falcontissue culture tubes at 37°C under 596 CO2 in air for 48 hrs. A random selection ofembryos from the two culture media were transferred to the uteri ofpseudopregnant recipients. I

8C85.2 (61)90.5 (42)

4C39.0 (77).90.5 (63)

BIOPSIEDCONTROL

Leeanda J. Wilton and Alan O. Trounson.

Table 1 shows. pooled results from several experiments. anddemonstrates that embryo' viability is greater when biopsies areperformed at the 8C stage. However proliferation of biopsied cells isgreater when blastomeres are taken from 4C embryos. 58.6% and 63.8% ofsingle blastomeres taken from 4C and 8C embryos respectively divided to2 cells. Only 6.4% of blastomeres from 8C embryos divided further (to 3cells) whereas 29.3% of blastomeres from 4C embryos continued to divide,some going to 6 cells.

We are currently attempting to karyotype biopsied cells and modifythe biopsy technique and post biopsy culture to improve embryo viabilityand proliferation of isolated blastomeres.

VIABILITY OF MOUSE EMBRYOS AND BLASTOMERES FOLLOWING BIOPSY OF A SINGLECELL.

TABLE 1 %EMBRYOS FORMING BLASTOCYSTS eN)

Centre for Early Human Development, Monash University, Queen VictoriaMedical Centre, Melbourne, 3000.

Many genetic diseases are diagnosed prenatally using DNA probes andgenetic markers on fetal tissue collected at 6-14 weeks gestation. Alsogross chromosome anomalies are detected in karyotypes of such fetalcells. First trimester terminations of fetuses positive for thesediseases could be avoided if pre-implantation embryos obtained throughin vitro fertilization could be screened for genetic abnormalities.Th~would involve removal of a single blastomere from the embryo andproliferation of that cell for genetic testing. We present results ofpreliminary studies to determine the optimal cell stage for embryonicbiopsy where viability of the embryo and proliferation of biopsied cellsare maximum.

Four cell (4C) or eight cell (8C) embryos were collected fromsuperovulated Fl mice and cultured for 0.5-2 hours in Ca++/Mg++-freemedium M2 to decrease cell-cell contacts. For biopsy, embryos were heldin place by suction through a glass holding pipette attached to a Leitzmicromanipulator. The zona pellucida was punctured with a glassaspiration pipette (AP) attached to a Zeiss motorized micromanipulatorand a single blastomere was sucked gently into the AP. The AP wasremoved from the embryo and the blastomere expelled into the medium.Embryos and matching blastomeres were cultured in microdrops of T6+10%fetal calf serum and were checked at 24 (8C) or 48 (4C) hourspost-biopsy for growth to blastocyst (embryos) or cell number(blastomeres) by counting stained nuclei. Control embryos were treatedidentically to experimental embryos but were not biopsied.

96Totalimplantations

5954

46*30

101102

9882

96 morulae No. Morulae 96 normaltransferred fetuses

210208

Viability of embryos grown in HFA and T6 + FCS for 48 hrs.

Gianaro~i, L., Serachioli, R., Ferraretti A.R., Trounson, A., Flamigni, C.and Bovicelli, L. Fertil. Steril. (in press).

HAFT6 & FCS

TABLE 1.

(1)

Culture medium No.2-cellscultured

32

MALE FACTOR PATIENTS WITH ANTISPERM ANTIBODIES IN IVF

C.A. Yates, C.]. Thomas*, A.O. Trounson+, and D.M. de Kretser

Department of Anatomv, *Inferti litv Hedical Centre, Epworth Hospitaland +Centre of Early Human Developmenmt, Oueen Victoria MedicalCentre, Monash Universitv, Melhourne, Victoria.

33

ULTROSER G AS A SERUM SUBSTITUTE IN EMBRYO CULTURE MEDIUM

*A.K.Pope, K.L.Harrison, T.M.Breen, L.M.Wilson, J.M.Cummins and J.F.Hennessey. Queensland Fertility Group, 225 Wickham Terrace, Brisbane4000, and *Department of Animal Sciences and Production, Universityof Queensland, St.Lucia 4067.

The lack of uniformity between serum samples and the problemsassociated with their preparation and storage have led to theproduction of synthetic serum substitutes for cell culture. In thisreport we describe the effects of substituting a product, UltroSer G(L.K.B. Reactifs, lBF, France) for human serum in growing two cellmouse embryos. This was carried out to evaluate it as a possiblealternative to serum in human in vitro fertilization (IVF) culture.

Two cell mouse embryos from Fl hybrid CBA/C57 female mice bred toSwiss Studs were cultured in HTF medium (1) supplemented with 1,2,3,5and 10% (v/v) of UltroSer G. HTF medium supplemented with 10% heatinactivated pooled human serum was used as a control. Hatchingblastocysts were observed after 72 hours in all concentrations ofUltroSer G, but in most cases lower percentages were observed whencompared with controls. Data are presented in Table 1, which isbased on 8 replicate experiments.

Table 1. Percentages of Two Cell Mouse Embryos forming HatchedBlastocysts in HTF Medium supplemented with UltroSer G.

79 78 78 74 52 79(87/110) (76/97) (82/104) (70/94) (74/142) (85/107)

The use of in vitpo fertilization (IVF) techniques for thetreatment of male factor infertility has heen nescri hen previouslv(Yates et al, 19R5) for various groups. The aim of this study was toobserve how effective the use of IVF techniques was for patients withboth IgG and IgA classes of antispermatozoal antibodies coating thesperm.

The patients were classified as having antisperm antibodies in atleast two semen analvses prior to the treatment cycle, with theimmunobead test (Clark et al., 19R4) being usen to netect the presenceof IgG and IgA antibodies. Only patients with greater than 60~ ofspermatozoa coated with hoth' classes of anti hocHes were used in thestudy.

Once eggs had been obtained, the male was asked to pronuce asplit ejaculate. Only the initial part of the ejaculate was used anoculture media was layered directly onto the semen sample. The nor~alwashipg and centrifuging of the entire sample before lavering was notcarried out due to the large amount of agglutination of the male spermthat occurs. After layering for 1-2 hours, the top layer was removedand washed. The· eggs were then inseminated with 250,000 motilespermatozoa and checked for fertilization 14-20 hours afterinsemination. The results are summarized in Table 1:

Concentration ofUltroSer G

% of HatchingBlastocysts

1% 2% 3% 5% 10% Control

The results show that there is a reduced fertilization rate inthese patients when compared to the normal fertilization rate (63%).However, there is a much higher pregnancy rate per treatment cyclecompared to the normal (16.2%) and the abortion rate is lower to thatof the normal (33%).

The use of IVF for the treatment of male factor patients withboth IgG and IgA antisperm antibodies is encouraging especially withthe good pregnancy rate observed. Thus, IVF offers good potentialtreatment for this group of male factor infertility.

1. Yates, C.A., Trounson, A.D. and de Kretser, n.M. (I9R5)Proc.Seventeenth Amm. Aust.J.Reprod.Biol. p.8 •.2. Clarke, C.N., Sto.ianoff, A., Candin, M.N., McRain, .r.C., Spirs,A.L. and Johnston, W.I.H. (1984) Amm.J.Reprod.Immunol. 5:61.

~:

No. oftreatment cycles

14

No. of eggsinseminated

63

% eggsfertilized49.2(31/63)

%preg./Treat:cyc.

30.R

% abort./preg.25%

Concentrations in the range 1-5% of UltroSer G gave virtuallyidentical results with the control. However concentrations of 10%~ltroSer G showed significantly less growth (P,.OOI: Chi-squaredtest) to hatching blastocyst. This supports the manufacturer'srecommendation of 1-3% of this product for use with cell lines.Similar results have been obtained by a group using Ham's FlO as thebasic medium (2).

While these results indicate that UltroSer G can De usedsuccessfully as an embryo growth medium supplement, test with humancumulus and spermatozoa revealed drawbacks. The cumulus matrixdegenerated into a dense gelatinous mass after 24 hours in thepresence of 2% UltroSer G, and spermatozoa showed impaired motilitycompared with those cultured in control media for the same period.Thus UltroSer G would appear to have only limited application in IVF.

1. Quinn,P., Kerin,J.F. & Warnes,G.M. (1985) Fertil.Steril. 44:493-489.

2. Bernart,W., Rabe,T., Kiesel,L. & Runnebaum,B. (1986) Hum.Reprod.Supp!. 1:.: A26.

34

SURVIVAL OF ULTRA-RAPIDLY FROZEN MOUSE EMBRYOS AS A FUNCTION OFGLYCEROL PERMEATION

A. Szell and J. N. Shelton

Department of Immunology, The John Curtin School of Medical Research,Australian National University, Canberra, A.C.T.

The role of glycerol permeation in the cryoprotection of ultra-rapidlyfrozen Day-3 mouse embryos was examined by studying the effect of theduration and temperature of equilibration. In Exp. 1 the volume ofembryos was determined after I-step additon of 2.0, 3.0 or 4.0 M­glycerol + 0.25 M-sucrose at 20°C. In Exps 2 and 3 the embryos wereequilibrated in 2.0, 3.0 or 4.0 M-glycerol + 0.25 M-sucrose for 2.5,5, 10, 20 or 40 min at 4 (Exp. 2) or 20°C (Exp. 3) before freezing. InIn Exp. 4 the embryos were either dehydrated by 0.25 M-sucrose (10min) or permeated by 4.0 M-glycerol (10 min) or left in PBS + 5% FCSbefore being equilibrated in 4.0 M-glycerol + 0.25 M-sucrose for 1 24, 8 or 16 min at 20°C and then frozen. ' ,

The embryos were photographed at preset times after placing theminto experimental solutions and their volume was estimated from thecross-sectional area assuming a spherical shape. In Exps 2, 3 and 4the embryos were frozen in 0.5 ml straws by direct transfer intoliquid nitrogen vapour (about -180°C) as described in detailpreviously (1). The number of frozen-thawed embryos developing toblastocysts in culture was expressed as a proportion of those frozen.

The mean volume of embryos 2.5, 5, 10, 20 and 40 min after placingthem into 2.0, 3.0 or 4.0 M-glycerol + 0.25 M-sucrose was 51 59 6467 and 68% respectively (P(O.OOI) (Exp. 1). ' , ,

In Exps 2 and 3, the overall survival was lower when theequilibration was done at 4°c (Exp. 1, 35%) compared to 20°C (Exp. 2,49%; P(O.OOI). The viabilty of embryos after 2.5, 5, 10, 20 and 40 minof equilibration at 4°C (Exp. 2) was 5, 23, 36, 54 and 57%respectively (P(O.OOI). The regression of the survival rate on theduration of equilibration was linear (P(O.OOI). At 20°C (Exp. 3) theduration of equilibration period did not affect the response (P>O.l).In both experiments the viabilty was higher for embryos frozen in 3.0or 4.0 M-glycerol compared to 2.0 M (P(O.OOI).

The mean survival rate for embryos equilibrated for 1 to 16 min at20°C after previous dehydration, permeation or without pretreatmentwas 40, 78 and 55% respectively (Exp. 4). The differences between anytwo pretreatments were signifcant (P(O.OOI). The duration ofequilibration period did not affect the viability of previouslypermeated embryos (P>O.I), but had a significant effect (P(O.OOI) onthe other two groups.

These experiments demonstrated that the viabilty of ultra-rapidlyfrozen mouse embryos is ·dependent on the temperature and duration ofequilibration. It is concluded that increases in the intracellularglycerol level are beneficial for the survival of ultra-rapidly frozenmouse embryos and previous or concomittant exposure to sucroseunfavourably affects glycerol permeation.

(1) Szell, A. & Shelton, J. N. (1986) J. Reprod. Fert. 76, 401-408.

35

THE USE OF HUMAN AMNIOrrC FLUID AS AN EMBRYO aJLTURE MEDIUMFOR HUMAN IVF

L. Chiappazzo, P. Rogers, C. Kirby and A. Trounson*

D partment of Obstetrics and Gynaecology and *Centre for Early HumanD:velopnent, Monash University, 172 LOnsdale Street, Melbourne, 3000.

It has been reported that human amniotic fluid (HAP) may be used tosuccessfully culture mouse emb:YOs (1). The v~~ility of mouse embryos

• U7\'C' for 48 hours is hlgh (2) and prellmmary results of the usegrown In no=: , ", h h 1f HAF for human in vltro fertlllzatlOn (IVF) ave s own norma~ertilization rates in vitro and high pregnancy rates when,embryosfertilized and cultured in HAP were transferred to IVF patlents (1).

The studies described in the present paper confirm that.~ can JJ: usedfor fertilization and culture of human embryos. A prellmlnary t:lal

amined fertilization and cleavage of one or two oocytes from elght~tients in whom six or more ooeytes were obtaine~ by ultraso~d guidedaspiration. The results using HAP are compared wlth those usmgWhittingham's T6 supplemented with 10% human serum, (T6+10% HS) for the

remaining oocytes.

TABLE 1. Fertilization and cleavage of human embryos in HAP and T6+l0% HS

Medium No. of No. Embryos Suitable for

Oocytes Fertilized Transfer or Freezing(%) (%)

EAF 19 15 (79%) 9 (60%)

T6+10% HS 48 34 (71%) 26 (76%)

A large scale clinical control trial has been initiat~ to assess the .reported improvement of mouse and human embryos grown m HAP compared WlthT6+l0% HS (1,2). Alternate patients were allocated to HAP or T6+10% HS.Of 14 patients allocated to HAP, 11 (85%) were transferred 24 embryos. Of15 patients allocated to T6+10% HS, 15 (100%) were transferred 42 embryos.In both groups 56% of the embryos reached at least the 4-ce11 stage, HAP14/25, T6 +10% HS' 30/54. These results confirm that HAP may be used for

IVF.

1. Gianaroli L Serachioli, R., Ferraretti, A.R., Trounson, A.,Flamighi,'C.' and Bovicelli, L. Fertil. Steril. (in press) (1986).

2.' Kirby, C. and Trounson, A. Aust. Soc. Reprod. BioI. Abstracts (1986).

36 37

MAINTENANCE OF PREGNANCY IN OVARIECTOMISED EWESWITH SYNTHETIC PROGESTAGENS AN INTERACTION BETWEEN NUTRITION AND PROGESTERONE

REDUCES EMBRYONIC SURVIVAL IN SHEEP.

I.F. Davis, R.A. Parr and L.P. Cahill

Department of Agriculture and Rural Affairs, Vic.Animal Research Institute, Werribee, 3030.

R.A. Parr, I.F. Davis, R.J. Fairclough and M.A. Miles.

Animal Research Institute, Department of Agricultureand Rural Affairs, Werribee, Victoria, 3030.

Low+

Day 12 Prog. Ewes Preg. Foetuses/Ewe(ng(ml) % Preg. %

Nutrition Prog.Suppl.Group (CIDR 8-14)

Although 20 to 30% of fertilized sheep ova die in the.first weeks of pregnancy (1) little progress has been made 1nunderstanding the role nutrition might play in this large 107s.Extremes of nutrition have been linked with embryonic mortallty(1) and nutrition is inversely related ~ith progesteron: ~2).Our aim was to investigate a possible Ilnk between nutrlt1on,progesterone and embryonic mortality. . .

Mature Merino ewes (n=330) were synchronlzed uSlng prog.pessaries (CIOR Hoechst Aust.) and then mated with entire rams.From day 2-14 after mating, the ewes were placed in a feedlotand fed daily a low, medium or high ration (25, 100 or 200% ofmaintenance respectively). Prog. supplement was delivered ondays 8-14 using a second CIDR in treatment ewes. Blood sampleswere taken on day 12 to measure plasma progesteroneconcentration. On day 14 after mating all ewes were returned topasture. Pregnancy rate was determined by returns to oestrusand was later confirmed using ultrasound.TABLE 1. The influence of nutrition and exogenous prog. onDay 12 peripheral prog. levels, pregnancy and foetuses perewe pregnant.

Exogenous administration of fluorogestone acetate (FGA),medroxyprogesterone acetate (MPA) in sponges and progesterone incontrolled internal drug release (CIDR) devices are commercialpreparations used to regulate oestrus in ewes. Furthermoresupplementation of ewes on a high plane of nutrition withprogesterone during days 8-14 after mating has been shown toreduce embryo mortality (1). The ability of intravaginaldevices containing FGA or MPA to maintain pregnancy inovariectomised (ovex) ewes may indicate their suitability forpost-mating use to reduce embryo mortality. However FGAinjection has been shown not to maintain pregnancy in ovex miceand rabbits (2) although MPA injection does maintain pregnancyin ovex ewes (3).

Ewes were allotted to five groups each of 14 ewes on Day 3after mating and received either FGA, MAP or CIDR devices.Positive and negative control groups were either ovex or entire.On Day 4 all ewes underwent laparotomy under generalanaesthesia, Thiopentone Sodium (Intraval Sodium, May and Baker)and all except the entire control animals were ovex. Theintravaginal devices were renewed every 6 days until Day 21 whenembryos were recovered at laparotomy. Data were examined byanalysis of Chi-square~

Table 1 • Percentage of ewes pregnant at day 21 Med+

The work was supported by WRTF of the Australian WoolCorporation.

The results show that MPA, FGA and ClDR intravaginaldevices can maintain pregnancy in ovex ewes. Furthermore MPAand FGA sponges administered Days 8-14 after mating could reduceearly embryo loss depending on nutritional status of ewes.

TreatmentEwes pregnant %

a,b;

MPA FGA CIDR Entire71 a 50a 50a 75a

data with unlike superscripts differ.

OvexDb

High

Unlike superscripts differ significantly; (80 P<0.05, xy P<O.01)There was a decline in plasma progesterone level with

increasing rati08. The pregnancy rate in high ration ewes wassignificantly reduced but this did not occur when ewes ~ere

given exogenous progesterone (CIOR) over days 8-14. ThlSphenomenon was not seen in ewes fed low or medium rations. Thenumber of foetuses per ewe pregnant was not influenced by levelof nutrition or progesterone treatment.

These results demonstrate a direct link between nutrition,progesterone and embryonic mortality.

Parr, R.A., Davis, I.F. and Fairclough, R.J. Proc.Aust. Soc. Anim. Prod. 16: 425 (1986).Zhang, X., Stone, G.M. and Miller, B.G. (1985) Proc. Aust.Soc. Reprod. BioI. 17: 90.Ricketts, A.P. and Flint, A.P.F. (1980) J. Endocr. ~:337-347.

This work is supported by the WRTF of the Aust.Wool Corp., (1) Edey, T.N. Anim.Breed.Abstr. ]1, 173 (1969).

(2) Williams, A.H. and Cumming, I.A. J.Agric.Sci.Camb. ~517 (1982).

38 39

OVARIAN RESPONSES OF BEEF HEIFERS TO A BOVINE SERUMALBUMIN-TESTOSTERONE BASED IMMUNOGEN

Body Weight Ovarian Weight (g) Corpus luteum Plasma(kg) luteal non-luteal weight (g) P4Control 448 ± 19 7.2 ± 0.5 3.7 ± 0.3 2.6 ± 0.2 2.9 ± 0.2Anti-T 411 ± 14 7.8 ± 0.3 4.5 ± 0.4 3.4 0.2± 4.5 ± 0.3

Anti-testosterone titres ranged from 1:50 to 1:2,000 whichcould be co~sidered moderate even for cattle. The increased(p,< 0:05) we~ght of the non-luteal ovary in Anti-T heifers resultedpr~mar~ly from greater numbers of follicles in the 2-4 mm .-these animals (5 4 0 8 range ~n

. .• ± • ~ 3.9 ± 0.6). The corpus luteum was also~eaner (p < 0.05) in Anti-T heifers and this was reflected in~ncreas,ed (p < 0:05) pl~sma P4 concentrations. '!Wo Anti-T heifershad tmn ovulat~ons wh~ch in both animals occurred on theovary. These r~sults indicate that immunization against a ~:;:~te~tosterone conJugate can increase ovarian activity in beefhe~fe~s. In addition to the prospect of increased incidence oft~nn~ng,. the ~ene~al i~crease in mass and steroidogenic activity oflut~al t~ssue u~ ~mmur:uzed heifers may confer some advantages in Pmed~ated responses of early pregnancy in cattle. '4

study supported in part by a grant from the J.B. Davies Bequest.

Department of Animal Sciences, Waite AgricUlt 1 ResearchInsti tute 1 t ura. ' G en Osmond, S.A., 5064; S.A. Department ofAgr~culture, Turretfield Research Centre, Rosedale S.A. 5350 and'!' C.S.I.R.O., Division of Animal Production, pros~ct, N:S.W., 2149.

The overall objectives of the present work are to determine the~munologic and reproductive responses of heifers to steroid~mmm:i~ation us.ing homologous proteins as carriers. In aprel~m~nary tr~al, heifers were immunized against eithertestosteron~, androsten~ione or oestradiol-17S conjugated to bovineserum album~n (BSA). He~fers showed significant immune responses toall three ~SA-ster~id conj uga tes and antibody ti tres were maintainedover a per~od of.e~ght months by periodic booster injections. In asub~equen~ exper~ment, 14 pubertal Hereford heifers were immunizedaga~nst e~ther. BSA alone (Controls; n = 7) or a BSA-testosterone~onJugate (Ant~-T; n = 7). Heifrrs received the same amounts of~mmunogen (,equivalent to 8 mg of protein emulsified in Freund'scom~l~teadJuvant) for primary immunization and 6 booster injectionsadm~n~stered at approximately monthly intervals. At the time flast boo~ter inj ection ovarian cyclicity was synchronized uSingpG~2a andhe~fers were then slaughtered on day 8 of the ensuing oestrouscycle. , Data were .analyzed by ANOVA and results (mean ± SEM) forbody we~ghts, ovar~an characteristics and plasma concentrations f~rogesterone (P4 , ng/ml) on the day before slaughter are present~d~n the Table.

L.J. Cummins,* T. O'Sheat and B.M. Bindon#

*Pastoral Research Institute, Box 180, Hamilton, Victoriatuniversity of New England, Armidale, N.S.W.iCSIRO Division of Animal Production, Armidale, N.S.W.

"INCREASED OVULATION RATES IN CATTLE VACCINATED WITH A PARTIALLYPURIFIED FRACTION OF OVINE FOLLICULAR FLUID"

Increased ovulation rates have been recorded following vaccination ofewes with a partially purified extract of bovine follicular fluid (1,2).Here we report on the active vaccination of heifers with a similarextract of ovine follicular fluid.Follicular fluid was collected from the ovaries of sheep slaughtered ata commercial abattoir. The fluid was chromatographed on an affinitymatrix (Reactive Red 120. Sigma) and the steroid free second peak (3)was stored frozen. As required this was thawed, and after mixing withequal quantities of Freund's complete adjuvant and water and a fewdrops of Tween detergent it was emulsified. At each vaccination timeeach cow was injected with 3mls of this emulsion. Based on previouswork this dose of vaccine may have contained approximately 2mg offollicular fluid protein. The vaccine was given as a divided dose atintramuscular and subcutaneous sites.Twenty four Hereford heifers in store condition and approximately12 months of age, were allocated to the experiment in June 1984. Therewere 10 untreated controls and 14 in the vaccinated group. The heifersremained in store condition throughout the experimental period. In thefirst half of 1985 repeated mid-ventral endoscopy under generalanaesthetic (4) was carried out during the mid-luteal phases of oestruscycles.Vaccinations were carried out on 15.6.1984; 22.8.1984; 13.2.1985;18.3.1985; 22.4.1985 and 19.6.1985. In the vaccinated group at thefirst endoscopy (February/March) 1/11 heifers had a mUltiple OVUlation;at the second endoscopy (March/April) 2/13 heifers had mUltiple OVUlations(1 with 4 corpora luteal i at the third endoscopy (May) 6/12 heifers hadmultiple OVUlations (4 with 2 corpora lutea, 1 with 5 corpora 1utea and1 with 6 corpora luteal and at the final endoscopy (July) 2/9 heifers hadmultiple ovulations (1 with 4 corpora lutea). Nutritional and seasonalconditions caused some heifers to become anoestrus during the experimentbut both vaccinated and control animals appeared to be affected insimilar proportion~. No mUltiple ovulations were observed in the controlgroup.This experim~nt demonstrates the potential for increasing ovulation rateby vaccination with proteins of follicular fluid origin.(1) O'Shea, T., Cummins, L.J., Bindon, B.M. and Findlay, J .K. (1982)

Proc. Aust. Soc. Reprod. Biol. 11:85.(2) O'Shea, T., AI-Obaidi, S.A.R., Hillard, M.A., Bindon, B.M.,

Cummins, L.J. and Findlay, J.K. (1984) Reproduction in Sheep.Editors Lindsay, D.R. and Pearce, D.T. p.335-337. Published byAustralian Academy of Science and the Australian Wool Corporation.

(3) Jansen, E.H.J.M., Steenbargen, J., de Jong, F.H. and van der Molen,H.J. (1981) Molec and Cell Endocrinol. £1:109-117.

(4) Holland, E.J., Bindon, B.M., Piper, L.R., Thimonier, J.,Cornish, K.A. and Radford, H.M. (1981) An. Reprod. Sci. !:127-135.

T. WeatherlyM.J. D'-Occhio, D.R. Gifford t , R.I. COX'!'and B.P. Setchell

The poor lambing response in the A immune ewes reflects thatsometimes seen under field conditions with Merino ewes. Bothprocedures increase ovulation but the use of several steroids in activeimmunization may offer advantages with improved lambing performance.

TABLE 1. Reproductive performance of immunized ewes.GROUP NO. RECIPROCAL NO. EWES WITH OR LAMBS

EWES TITRE 2 3 0 1 2 PERMEAN ± SEM EWE

A, , T, E OVULATIONS LAMBS JOINED

Control 50 48 2 0 4 43 3 1.04 0.98A immune 50 2010 35 14 1 5 39 6 1.32 1.02

±580ATE immune 50 820, 500, 1060 31 19 0 6 29 15 1.38 1.18

±300 ±170 ±240

Active immunization against either steroid androgens or oestrogenscan increase ovulation rate (OR); however the gain in lambing percen­tage, particularly with Merino ewes ~ is n~t always realized due. tovarious reproductive losses (1). It ~s posslble that controlled actlveimmunization against several steroids simultaneously may cause lessdisturbance to the endocrine balance and result in increased ovulationwith less embryo wastage.

The effect of multiple immunization was examined in a flock ofmedium-wool Merino ewes (n=150) aged 3 years which were randomlyallocated to a control group, an androstenedione (A) immunized groupand a gro~p immunized against (A), testosterone (T) and oestrone (E).Immunogens were inj ected with DEAE-dextran adj uvant in March 1985; abooster immunization was given 21 days later. After a further 3 weeksthe ewes were joined with entire rams for 5 weeks. Laparoscopy wascarried out folliwng oestrus markings. Steroid antibody titres weremeasured in plasma samples (1 O/group) one week post boosterimmunization.

All ewes showed normal oestrus behaviour. Both immunizationprocedures produced significant increases in O.R. (P<0.01, Fisher'sExact Test) (Table 1). However, the number of lambs born wasconsiderably less than expected in the A immune group but not in theATE immune group. Only multiple immunization resulted in a significantincrease in lambs born (P<0.01).

R.J. , Sutton, R. ,C.D. and Moran, C.

Canberra, Australian

41

IMPROVED PROLIFICACY IN. MERINO EWES IMMUNIZED AGAINST ACOMBINATION OF ANDROGENS AND OESTROGENS

CSIRO, Division of Animal Production, prospect, NSW andtCSIRO, Division of Animal production, Armidale, NSW

Patricia A. Wilson, R.I. Cox, M.S.F. Wong and D.R. Paullt

Boland, M.P., Murray, J.D., Scaramuzzi,Hoskinson, R.M., Hazelton, I.G., Nancarrow,(1984). Reproduction in Sheep, p.137-139.Academy of Science.

(1)

Table 1. Performance of crossbred ewes immunized with Fecundin~

Treatment Age Weighta No. O.R. b Barren Multiple(y,:s) (kg) ewes ewesc (%) pregnanciesd (%)

Untreated 1.5 50.2 58 1.10 12.1 0> 1.5 54.6 91 1.28 3.3 19.3

Immunized- 2 ml 1.5 48.8 57 1. 65 14.0 32.7

Boosted- 2 ml > 1.5 57.4 71 1.80 8.5 47.71 ml > 1.5 56.0 72 1. 79 0 38.9

PERFORMANCE OF CROSSBRED EWES RECEIVING DIFFERENTFECUNDI~ BOOSTER DOSES

a Weight when rams joined. b 0 1 t'b d 17 f . " c vu a ~on rate of ewes Ovulating

y ay 0 Jo~n~ng. % of ewes scanned with RTUS. d % ofpregnant ewes.

~vulation rates (O.R.) and lambing performances of ewes can be~ncreased following treatment with Fecundin~ in a range of breeds~nd env~ronments (1). However, in Western Australia the increases~n mark~ng percentages following treatment are variable and only ona few ~roperties have they been large (2). The recommended dose ofFecund~n· for both the initial injections and later boosterinjections is 2 ml, but if the volume injected could be lowered thecost of treatment would decrease.

~~~ Smith, J.F. (1985) Proc. N.Z. Soc. Anim. Prod. 45: 171.Croker, K.P. (1986) J. Agric. West. Aust. 27: 27:

K:P. Croker, M.A. Johns and G.A. Brown*Western Australian Department of Agriculture, South Perth and Moora*

In the third year of a study of Dorset x Merino ewes, differentvolumes of booster injections were examined. Half of the maiden 1.5year-old ewes received 2 ml of Fecundin·, injected 8 and 4 weeksbefore t?e introduction of entire Poll Dorset rams on 20.12.1985.T~e.prev~ously treated ewes (2.5 year-old and older) were randomlyd~v~ded ~nto 2 groups, one received 2 ml and the other 1 ml of~e:U?din· 4 weeks before the rams wer~ joined. The length ofJo~n~ng was 8 weeks.

40

~or.both the young and older ewes there were significantly higher1nc~dences of multiple ovulations (P < 0.001) and pregnancies(young ewes and 2 ml boost P < 0.001· 1 ml boost P <0 OS) .th . • ' • ~n. e Fecu~d~~ .treated ewes than in untreated ewes. Of importance~s the s~gn~f1c~nt response with the low dose of Fecundin~. There1S need to conf~rm the widespread response to the use of a 1 mlbooste: injection which would substantially reduce the cost forproduc~ng extra lambs from treated ewes.

42

LOW PREGNANCY RATES IN MERINO EWES BOOSTEDWITH TESTOSTERONE:HSA

POST MATING'

43

LUPIN FEEDING AND INSULIN INFUSION DURING THE LATE LUTEAL PHASECAN INCREASE OVULATION RATE IN SHEEP

M.S.F. Wong and R.I. Cox

CSIRO, Division of Animal Production, Prospect, NSW

G.N. HINCH apd J.H.W. ROELOFSDepartment of Animal Science, University of New England, Armidale, NSW

rate (OR), follicle number (FN) andof Merino ewes from the three flocks.~ ~ Liveweight

1.15 1.68 37.8 ~ 0.281.52 1.49 38.6 ~ 0.311.33 2.11 38.5 ~ 0.29

of 4IU insulin0.08 0.47

TABLE 1: Mean ovulationliveweight (kg)

Treatment/head/dayA Control: 600g sorghumB Lupins: 600g lupinsC InSUlin: 600g sorghum

+ infusion of a totalSE of Diff.

Ovulation rate was higher (P<O.Ol) for both treatments Band Ccompared With A. Liveweight effects were also significant (P<0.05)with an OR increase of 1.7% per kg across treatments. There was noeffect of flock on OR but FN did differ between flocks (P<0.05) aswell as between treatments (P<0.05).

These data support the concept of a -sensitive- period for the'flushing' action of 'lupins dur ing the late luteal phase (1) • Inthis experiment a consistent increase of around 30% was obtained inthe OR of three different flocks. Secondly the data support thehypothesis that insulin infusion during the latter part of the lutealphase can increase OR in sheep to a level similar to that obtained bylupin feeding. Increases in OR via insulin infusion, at similarlevels (0.1 IU/kg) have also been obtained in pigs and cattle (2,3).Whether the mechanism of action of lupins is mediated via insulin hasyet to be tested.

Long study of mechanisms and timing of nutritional effects onovulation rate in sheep have recently led to the identification of aspecific 'sensitive' period during the late luteal phase (1). Thisexperiment was designed to examine the hypothesis that an increase inOVUlation rate, similar to that obtained by nutritional flushing withlupins, could also be obtained by insulin infusions during the'sensitive' part of the late luteal phase.

In March/April 1986 three flocks of Merino ewes (approximately 60per flock) had progestogen sponges inserted. On day 10 after spongeinsertion each flock was divided into three treatment groups (A, B,C; Table 1). These treatments continued to day 14 at which timesponges were removed. Ovulation rate (OR) and the number of large()3rnm) follicles (FN) were recorded by endoscopy 6 days after spongewithdrawal.

Analysis of the data was by least squares techniques, the modelincluding flock, treatment, their interaction and a liveweightcovariate. There was no liveweight change over the period of thetreatments and initial liveweight was used as the covariate.

(1) Stewart, R. and Oldham, C.M. (1986). Proc. Aust. Soc. Anim.Prod. ~: 367-370.

(2) Cox, N.M., Althen, T.G., Bennett, W.A. and Rather, H.M. (1984).lOth Int. Congr. Anim. Reprod. 2: 159-161.

(3) Randel, R.D. (1985). Personal communication.

1.145.71±0.99

LOG TITRE*t OR % % EWES~M_E_AN__±_S_E_M_:) MA,_T_ED__L_AM_BING

FCAT.HSA

T.HSA

485044131413

ControlImmuneControl

Immune

Immunization of ewes against testosterone () can 'increaseovulation rate (O.R.) a d 1 b' T.immunization against T . n ,am ~ng percentages unless a booster

Pre ~s ,g~ven shortly after mating when a lowgnancy rate resulted. 'lh~s effect was examined

Merino ewes (n=100, 2-3 years old f,:rther.to a control or treatment group Th) w~re randomsed and allocatedEstrumate® on the 1st and 13t'h e, atter was synchronized witht t Apr~l, 1981 and ~ 'des osterone-3-carboxymethyloxime' HSA (T .mmun~ze with

emulsion with Freunds complete d" (.HSA) (1.2 mg/sheep) in an, a Juvant FCA) on 23rd A 'l .....

w:re Joined with entire rams (10%) on 4th pr~ • ,.1.11e ~westlon was given to all ewes at ab t 17 d May and a booster ~mmun~za-were joined with entire rams (2~~ 'lh ay,s of ,pregnancy. Control ewes1982) were randomly assigned t ~h e ~mmunlzed ewes (re-boosted ingiven Estrumate® On 3rd and 13t~ A r~~ gr~u~s (n=13, 14,13) in 1984,24th April. The first sub prl an Joined with entire rams onwith FCA at 17 days of p;~;~:~ was ~oo~ted w~th T:HSA/FCA, the secondoriginal control group no 44 cyan, t e thlrd left unboosted. '!he

h ' w ewes, was untreated b t ' ,t e same time as a single flock. u was Jo~ned at

Oestrous activity in 1981 aEter the rimarbefore the secondary was similar to t p y immunization butproportion of ewes lambing was sign' f' hO~~ of untreated ewes, but the

~ lcan y less (P<0.05) (Table 1).

TABLE 1. Reproductive performance'1n immunized Merino ewes

'YEli:R--GR~-~IMMUN- ADJU-

OGEN VANT1981

1984100 83100 42*

FeA 5 + 1.32 100 89.61-0.06 100 38*

FCA 4.14±0.10 1.90 100 64--_______ 3.72±0.10 100

*P<0.05, Fisher's EXact Test. ---------------_.l..!.-_t 7 days post-boost.

A significant decrease in the 1 mb'the T:HSA/FCA (Table 1) (P<O 05) - b a lng ~ercentage was observed forconfirmed the 1981 results and' ind' u~ ;0: ~n the ?ther groups. Thishigher concentration resul tin f lca eh hat sterold antibodies at theeffect. ,g rom t e boost were required for the

The timing of the b t . , ,could be related to impair::

ster

Ell~munllzatlon suggests that the effectEf . n 0 lmp antation but wh th '

e ect through the neutralization of testo e er Vla a directThe data show that a booster' 't' sterone cannot be concluded.

h 1 lmmun~za ~on of Meri 'Sort y post-mating, results in a s' " no e,wes against T,percentage and is relevant t ' ~~nlf~cant reductlon in lambingreproductive perfor~ance. 0 lmmunlzatlon strategies to improve

44 45

OVULATORY RESPONSE TO EXOGENOUS FSH BY CATTLE SELECTED FOR TWIN BIRTHS

B.M.Bindon, L.R. Piper,M.A.Hillard and R.D. Nethery

TABLE 1. Ovulation rates following FSH trea tments •

FSH Dose Control(Test) (mg) Ovula tion Ra te

Twin HerdOvula tion Ra ten Mean S.E. (Range) n Mean S. E. (Range)

0 6 1.0 0 (1-1) 5 1.8 0.4 (1-3)FSH-P-BB 10 6 1.2 0.2 (1-2) 6 6.6 3.8 (1-21)(1) 30 7 7.9 3.2 (1-19) 6 4.3 2.0 (1-13)FSH-O-HH 0 6 1.0 0 (1-1) 6 1.0 0 (1-1)( 2) 10 6 2.3 0.5 0-4) 7 7.2 3.5 (1-23)30 7 13.7 1.3 00 19) 6 7.8 3.9 (1-23)FSH-O-HH 8 19 2.4 0.5 (1-9) 19 8.5 1.6 ( 1-21)(3)

Table 1 shows that in all 3 tests cattle selected for tWinningmore sensitive to exogenous FSH than control animals. wereevident from h f This issuperovulation ~e~g. a~~o that some twin herd cattle recordedcontr 1 i 11 3 ovulations) at lower FSH doses than

i 0 s n a tests. In test 3, involving meaningfUl numbers ofan mals, the difference between genotypes was significant (P<O 01)

fIn tests 1 and 2, however twin herd ovulation rates did not inc;eas~urther with the high dose of FSH.

The study also demonstrates the excellent superovulatory response bycon trol cattle to FSH-O-HH. A dose of 30 mg of thiproduced s prepara tion

a very uniform ovulatory response (13.7 + 1.3) withovarian over-stimulation. A dose of 8-10 mg produced a mOde~~increase in ovulation rate of potential value for induced tWinningin cattle.

1. Bindon, B.M., Ch'ang, T.S., and Turner, H.N. Aust. J. Agric.Res. 22: 809-820 (1971).

Sheep selected for prolificacy are known to be more sensi tive toexogenous gonadotrophin (1). The present study compared the ovariansensitivity to FSH of nulliparous Hereford heifers aged 3 years withanimals of mixed age from the CSIRO herd selected for twinning Isepara~e tests in July and October 1985 and February 1986 th~animals oestrous cycles were synchronized by two injections (500i/m) of prostaglandin analogue (PG) (Estrumate; ICI) at 11 d;;intervals. On day 8-10 of the sUbsequent cycle the animals commenceda 4 day FSH treatment (2 subcut. injections per day at 0900 and1600hr) with the total FSH dose being given in a decreaSing ratio of4: 3: 2: 1 over the 4 days. PG analogue was again inj ec ted on themorning of the third day. Ovulation rate was assessed by laparoscopyunder barbi tura te anaes thes ia 4-6 days af ter oes trus. Tes t 1evaluated FSH-P (Burns- Biotec USA) with bi, ••• a opo tency ofapproximately 2 x NIH-FSH-Sl. Tests 2 and 3 utilized 2 batches ofovine FSH prepared in Aus tralia for Herio t Agve t wi th a bio tof 4.5 x NIH-FSH-Sl. 'po ency

EFFECTS OF PROGESTERONE INTRAVAGINAL DEVICES ON OESTRUSSYNCHRONISATION IN POSTPARTUM DAIRY COWS

41-49.

School of Veterinary Studies, Murdoch University, Murdoch, W.A. 6150,*Department of Agriculture, Bunbury, W.A. 6230, +CSIRO, Division ofAnimal Production, Private Bag, Wembley, W.A. 6014.

Prolonged postpartum anoestrus, a common cause of poor reproductiveperformance in dairy cows, is due to a combination of a delayedinterval to first oestrus, silent oestrus after parturition and pooroestrus detection. Synchronisation of oestrus may provide a means ofincreasing the proportion of cows showing oestrus in a specifiedperiod, enabling more accurate oestrus detection. In this trial theeffects of two different intravaginal treatments of progesterone onthe synchronisation of behavioural oestrus were compared in 3 groupsof lactating dairy cows at 7-8 weeks postpartum. Group 1 (47 cows)had Progesterone Releasing Intravaginal Devices (PRIDS; BomacLaboratories, Australia) with an attached oestradiol benzoate capsuleinserted for 12 days. Group 2 and 3 (38 and 44 cows) had ControlledInternal Drug Release (CIDR, AHI, New Zealand) devices inserted for 9or 12 days respectively. Milk samples (5 ml) were taken 3 timesweekly for 3 weeks then twice daily between 3 days before and 6 daysafter removal of the devices. Milk progesterone analysis was carriedout using an enzyme immunoassay (1). Oestrus detection was conductedby observing the cows at 4 hourly intervals, commencing 24 h afterremoval of the device.

Behavioural oestrus was detected as early as 24 h after PRID removal(Group 1), whereas oestrus was not observed earlier than 32 hand 36 hfor Groups 2 and 3, respectively. The proportions of cows showingoestrus within 5 days after removal of the device were 80.9%, 57.9%and 63.6% for Groups 1, 2 and 3, respectively. After removal of thedevices, the mean milk progesterone level of cows which did not showoestrus (all groups) was significantly greater than that of cows whichshowed oestrus (p < 0.05). In Groups 1, 2 and 3 respectively, 1/7,7/12 and 4/12 cows which failed to display oestrus (no visibleoestrus, NVO) had high milk progesterone levels after removal of theintravaginal device, indicating persistent corpora lutea (eL). Thiswas confirmed by rectal palpation. The remaining NVO cows haddecreased milk progesterone concentrations after removal· of thedevices, which indicates that these cows were cyclic, thoughbehavioural oestrus did not occur.

This study has shown that oestrus was best synchronised in Group 1when PRIDs with oestradiol benzoate were inserted for 12 days, withluteolysis precipitated by oestradiol benzoate. leading to enhancedsynchronisation. The use of a luteolytic agent with CIDRs isindicated to improve synchronisation.

(1) Munro, C. and Stabenfeldt, G. (1984). J. Endocr. 101

S. Tiondronegoro, P. Williamson, G.J. Sawyer*, C.D. Hawkins andS. Atkinson+

2350NSWCSIRO Divsion of Animal Production, Armidale

46

EFFECT OF PREGNANCY NUTRITION AND PRE-LAMB SHEARINGON LAMB BIRTH WEIGHTS IN PROLIFIC SHEEP

J.R.Armstrong, G.H.Davis, P.A.Farquhar and J.L.Owens

Invermay Agricultural Centre, Mosgiel, New Zealand.

Birth weight is an important factor influencing lamb survival.As litter size increases birth weights decrease and this contributesto the lower survival among mUltiple-born lambs (1). The effectsof mid-pregnancy nutrition and pre-lamb shearing on lamb birth weightsin prolific sheep were measured.

109 1/2 Merino 1/2 Romney ewes, 52 1/4 Merino 3/4 Romneys and60 Romneys each had an ovulation rate ~3 and conceived within 17 daysof joining. These ewes were allocated to one of four nutrition groupsand fed to either gain 21% (+21%), gain 5% (+5%), lose 6% (-6%) orlose 8% (-8%) of their liveweight from days 30-90 of pregnancy. Themean fasted weight, unadjusted for weight of conceptus, of all eweson day 30 of pregnancy was 48.3 kg. A random half from each nutritiongroup was shorn 5 weeks pre-lambing.

There was no significant effect of ewe mid-pregnancy feedinglevel on lamb birth weights. However within ewes not shorn pre-lambinglambs from +5% ewes tended to have the highest birth weights (Table 1).

47

THE INFLUENCE OF LONG TERM WEANING WEIGHT SELECTION IN MERINO EWES ONAGE, LIVEWEIGHT, FAT AND STAGE OF MATURITY AT PUBERTY

G.T. STEVENSON, T.N. EDEY and G.N. HINCHDepartment of Animal Science, University of New England, Armidale, NSW

Selection for and against weaning weight may result in correlatedresponses in factors associated with the onset of puberty, while aossible link between body fat and liveweight (LW) at puberty has

~ot been fUlly examined. These relationships were investigated inthe 1984 ewe lamb drop of the Trangie weaning weight selection flock.

Seventy five September-born ewe lambs of 3 genotypes (weight plus(W+), weight minus (W-) and control (C» were either (i) housed andaised artificially from birth with high levels of concentrate

;eeding post weaning, or (ii) raised at pasture with grainsupplementation. Oestrus was detected by use of vasectomised rams,concurrent ovulation checked by laparoscopy and verified bysubsequent plasma progesterone concentration. At pUberty, body fatwas determined by tritiated water measurement of total body water.

TABLE 1: Mean age, LW, percent fat (PF), percent mature liveweight(PMLW) at puberty, and rate of LW gain (RLWG) post weaningto 7 months, as affected by weaning weight selection

Genotype/ Lambs attaining puberty Age LW PF PMLW RLWGrearing number & proportion (dl ( kgl (%)a (%)a (g~

W+/housed 8 0.67 Ina 32.7a

27.3a

54.1b

119,Sb

W+/pasture 13 0.30 22la

31.8a

25.9b

67.5a

58.2a

C/housed 10 0.83 202 31.8 34.5b

57.8b

l17.7a

W-/housed 5 0.71 2l3a~a~--1..Q..J;.~(Different superscripts indicate significant differences (P<O.OS).)

At pUberty, differences between rearing method in W+ lambs werenot significant for age, LW and PF, but pasture reared lambs weresignificantly more mature and had a reduced RLWG. In the 3 housedgenotypes there were no significant differences between puberal ageand LW. However, clear trends exist with the heavier genotypesreaching puberty younger and at higher LW's. The W+ genotype wassignificantly leaner, and together with the control genotype was lessmature than the lighter genotype at pUberty.

Selection for and against weaning weight, while leading to fasterrates of LW gain, has not led to significant differences in pUberalage and LW as might be expected (1) yet it has led to the W- genoty~e

requiring a higher stage of maturity to reach puberty. As there ~s

no consistent level of PF between genotypes at pUberty, this is seenas evidence against the theory for a critical puberal fat level (2).

Dyrmundsson, O.R. (1981).Frisch, R.E. et ale (1977).

TABLE l. Lamb birth weights (kg) number of lambs in parenthesesNutritiorl Shearing Birth Ranktreatment treatment 1 2 3 4+21% unshorn 4.80 (2) 3.83 (24) 3.37 (33) 2.48 (4)shorn 5.45 (2) 4.62 (22 ) 3.33 (30) 2.95 (8)+5% unshorn 5.20 (7) 4.11 (16) 3.32 (27) 2.69 (16)shorn 5.25 (2) 4.08 (18) 3.49 (30) 2.91 (12)-6% unshorn 6.10 (2 ) 4.04 (20) 3.02 (27) 2.59 (20)shorn 5.00 (4) 4.33 (10) 3.44 (42) 2.63 (4 )-8% unshorn 4.97 (4) 3.87 (24) 3.02 (27) 2.25 (8)shorn 5.70 (4) 4.18 (21) 3.12 (18) 2.46 (8 )

Shearing ewes 5 weeks pre-lambing resulted in an 0.24 kg increase~P<O.~l) in b~rth weights of lambs. For the range of birth weights~n th~s exper~ment an 0.24 kg increase in birth weight should resultin the survival of an extra 1% of twins, 4% of triplets and 10% ofquadruplets, with no effect on the survival of singles (1). Resultsshow tha~ lamb b~rth weights were increased by pre-lamb shearing butnot by h1gh feed1ng levels during mid-pregnancy. This response topre-lamb shearing was previously observed in pregnant ewes artificiallyexposed to cold when the partitioning of nutrients between mother~nd f~et~s was altered, increasing the concentrations of glucose and1nsul~n ~n foetal plasma (2). As shearing ewes in late winter 5 weeks~re~lambing exposes ewes to greater risk from adverse weather, it~s ~mportant that they have access to effective shelter.

(1) Hinch,G.N., Crosbie,S.F., Kelly,R.W., Owens,J.L. and Davis,G.H.(1985) N.Z. J. Agric. Res. 28 : 31-38.

(2) Thompson,G.E., Bassett,J.M.,:Samson,D.E. and Slee,J. (1982)Br. J. Nutr. 48 : 59-64.

(1)(2)

Liv. Prod. Sci. .§..: 55.Proc. Natl Acad. Sci. USA Ii: 379.

48 49

INDUCTION OF OVULATION IN HYPOPHYSECTOMISED BOOROOLA EWES.

R.C. Frya, I.J. Clarkeb , J.T. Cumminsc , B.M. Bindond,L.R. Piperd and L.P. Cahilla

Ewes not Ewes ovulating Ovulation rateTreatment n ovulating 1 2 3 (mean + sem)Control 40 1 27 12 0 1.28 + O.08a

2 mg FSH 40 2 22 16 0 1.35 ±O.09~4 mg FSH 40 0 14 26 0 1.65 .±. 0.08

bLupin 39 2 8 27 2 1.74±.0.10

LUPIN SUPPLEMENTATION, FSH SECRETION AND OVULATION RATE IN THE EWE

M.B. Nottle, D.T. Armstrong, B.P. Setchell and *R.F. Seamark

Means with different superscripts are significantly different (P<0.05,Mann Whitney U-Test).

Supplement~tionwith lupin grain for the last ten days of the cyclesignificantly increased OR (P<0.05). 4 mg of FSH-P injected on days12-14 similarly increased the proportion of ewes with twin ovulations.

Together the results suggest that increases in OR in response toshort-term lupin supplementation are mediated by increases in FSHsecretion late in the luteal phase.1) Nottle,M.B., Hynd,P.I., Setchell, B.P. and Seamark, R.F. (1985)

Proc. Aust. Soc. Reprod. BioI. 12: 23 (abst).2) Brien, F.D., Baxter, R.G., Findlay, J.K. and Cumming, I.A. (1976)

Aust. Soc. Anim. Prod. 11: 237-240.M.B.N. is supported by the J.S. Davies Bequest.

Department of Animal Sciences, Waite Agricultural Research Institute,The University of Adelaide, Glen Osmond, SA 5064; *Department ofObstetrics & Gynaecology, The University of Adelaide, Queen Elizabeth

Hospital, Woodville, SA 5011.

We have previously reported that increases in ovulation rate (OR)in ewes supplemented with lupin grain can be explained by increases inpost-ruminal protein supply (1). However, the importance of associatedincreases in the concentration of FSH on days 12-14 of the cycle (2)has not been determined. The present study was undertaken to determinewhether changes to circulating levels of FSH on these days couldincrease OR.

160 six year old Merino ewes (mean liveweight 51.1 kg) wereassigned at random to four groups after being stratified according toliveweight. Animals were housed indoors in individual pens and fed amaintenance ration of wheaten hay. Oestrus was synchronised by meansof intravaginal sponges inserted for twelve days. Two groups wereinjected subcutaneously with either 0.5 mg or 1.0 mg FSH-P(Burns-Biotec, Nebraska, USA) on days 12 (1700 h), 13 (0800 and 1700 h)and 14 (0800 h) of the cycle. 500 g/ewe/day of lupins were fed to afourth group for 10 days beginning on day 7. OR was determined byendoscopy on days 5-6 of the subsequent cycle.Table 1. Mean ovulation rate of control, FSH injected and lupinsupplemented groups.Booroola

F-/-

22S0750o

Animal Research Institute, Werribee Victoria. aMedical Research Centre, Prince Henry'~ Hospitalb

St.Vincents Hospital, Melbournec &C.S.I.R.O., Armidale, NSWd

The high ovulation rate (O.R.) of Booroola ewes is welldocumented ~fter either spontaneous ovulation or inducedsuperovulat~on (1).

Th~s study ~nvestigated the contribution of the ovary indeterm~n7ng the h~gh O.R. characteristic of the Booroola ewe,by treat~ng hypophysectomised (Hypox) Booroola ewes withgonadotrophin and observing the O.R ••

/) Ewes diagn~sed, after repeated endoscopy, as carriers (F+ - or non-carr~ers (F -/-) of the Booroola Fgene underwent~ypox (2) and after one month an intravaginal progestagen­~mpregnated sponge was inserted in all ewes for 12d~ys. At sponge removal and 24 h later ewes were injectedw~th one of the following doses of PMSG· SOD + 2S0 i1000 + 500 i.u. and 1500 + 750,i.u. At'S6 h after s;~~geremoval all ewes received 1000 i.u. HCG and 3-4 days laterthe O.R. was determined by endosco~t. Results were analysedby ANOVA. 10 (62J,.." --___ (7q, ---

"O.R. ~,," (4) B(per ,,' ooroolaewe ,," F+/-ovul. ) ,,"

(4)f'(3)

i1S00PMSG(iu)

~ The mean (± s.e.m.) number of ovulations per ewe OVUlating(No. ewes) for F+/- and F-/- Booroola ewes treated with PMSG.

The induced O.R. were significantly higher (P<O 05) in~ooroola F+/-ewes than i~ control F-/- ewes (Fig.1) a~d appeared

a reac~ a peak at 1S00 ~.U. PMSG in both groups. Theproport~on of ewes OVUlating did not differ between groups andonly 53% ovulated after the first gonadotrophin treatment

The results ~how for a given level of gonadotrophin·Booroola F+/-ewes have double the 0 R of F / Th'It • • - - ewes. esere:u 7 suggest t~at the higher O.R. of Boorbola ewes is duepr~mar~ly to ovar~an rather than pitUitary factors.Th~s ~ork was supported by WRTF of the Aust. Wool Corp.(1) B~ndon, B.M., Piper, L.R. and Evans R (1980) pBooroola. Workship, Armidale CSIRO, Melb: P21-33 • roc.(2) Clarke,.I;J., Cummins, J.T. and de Kretser 6.M. (1983)Neuroendocr~nology, 36, 376-384. ' •

50 51

METABOLISM OF LACTATE BY SHEEP OVA FERTILISED BY FRESH ANDFROZEN-THAWED SEMEN

WITHIN YEAR REPEATABILITY OF OVULATION RATE FORBOOROOLA MERINO EWES AT 1.5 YEARS OF AGE

J. L. Owens

Year of Record1983 1984 Both Years

0.39 (42) + 0.07 0.15 (77) + 0.04 0.25(119) + 0.040.34 (70) +' 0.05 0.21 (70) +' 0.05 0.28(140) +' 0.030.12 (23) +' 0.07 0.33 (17) +' 0.10 0.23 (40) +' 0.060.31 (38) +' 0.07 0.45 (37) +' 0.07 0.38 (75) +' 0.050.66 135) +' 0.03 0.51(164) +' 0.04 0.59(299) +' 0.03

FFF+++M

BM were no cons1stent d1 erences 1n t between thegenotypes within years. Within year t estimates were different(P<0.05) between years for FF. For both years the 3 genotypes (FF,F+, ++) each had a low t estimate (0.23 t~ 0.28): In the totalpopulation comprised' of the 3 genotypes (le comb1ned BM) the la~gedifferences in ovulation rate between the 3 genotypes resulted 1n ahigh t estimate (0.59). This is.similar to ~ne~rl~er.stu~y (1) andsuggests that a high t estimate 1n a populat10~ 1S 1nd1catlve of asegregating gene with a major effect on ovulatlon rate.(1) Davis, G.H., Montgomery, G.W., and Kelly, R.W. (1982). In WorldCongress on genetics applied to Livestock production Vol VI1I pp674-679.(2) Davis, G.H., Montgomery, G.W., Allison, A.J., Kelly, R.W., andBray, A.R. (1982). N.Z. J. Agric. Res.~: 525-529.

EweGenotype

Invermay Agriculture Centre, Prlvate Bag, Mosgiel, New Zealand

Between year repeatability estimates (t) of ovulation rates forBooroola Merino (BM) F+ carriers (0.31) are similar to BM non-carriers(0.38) and Merino controls (0.32) (1). However, the combined BMestimate is high (0.61) indicating that the presence of differentBoorool a genotypes influence t estimates. This study reports on thewithin year t estimates for known BM genotypes (FF, F+, ++) and Merinocontrols (M) of one age group (1.5 years of age).

In January 1983, 135 BM and 38 M15 month old ewes were joinedwith a harnessed vasectomised ram. Mating marks were recorded every10 days and ewes marked were laparoscoped 2 days later. Thiscontinued until mid-May. In January 1984, a further 164 BM and 37 M15 month old ewes underwent a similar laparoscopy schedule. 8Mgenotypes were assigned by using ovulation rate classificationcriteria (2). Mean ovulation rates for both years were: FF! 4.0 ~0.07. F+, 2.7 + 0.04; ++, 1.4 + 0.04 and M, 1.2 + 0~03. Est1mates oft we~e calc~lated by intraclass correlation using 8 complete ovulationrate records per ewe for the combined BM genotypes then the FF, F+, ++and Mgenotypes respectively (table 1).

Table 1: Within year eitimates of repeatability (+ S.E.) forovulation rate of BM and Mewes age 1.5 years. (number of ewes).

J.P. Ryan, W.M.C. Maxwell, J.R. Hunton and R.G. Wales*

Animal Breedi¥g and Research Institute, Katanning, WesternAustralia, and School of Veterinary Studies, Murdnch University,

Western Australia.

Techniques that allow measurement of metabolic parameters ofsingle embryos have recently been developed (1). The present studyexamined the utilisation of an energy substrate by embryos derivedfrom ~rozen-thawed ~nd fresh semen. This was accomplished bymeasur1ng the product10n of CO2 from carbon-l position of lactate toassess the rate of entry of this substrate into the citric acidcycle during a short period of in vitro culture.- Forty eight hrs after progestagen sponge removal the uterinehorns of superovulated Merino ewes were ligated and the ewessurgi~ally in~eminated. Freshly collected semen was injected abovethe 11gature 1nto one uterine horn, and frozen-thawed semen from the~ame ~am ~n the other horn. Embryos were recovered 48 hr after1nsem1nat10n and rinsed twice in substrate-free medium prior totransferl~o drops of DUlbe:cos phosphate buffer + 1 mg/ml BSA + 1.12mM (1 - C) lactate. S1ngle embryos in 1.0 pI of medium weretransferred to 10 pg glass tubes which were attached to the innerwall of 1.0 ml micro-centrifuge tubes, previously loaded with 30plof 5.0 M NaOH to trap radio-labelled CO2, Tubes were capped and~laced .in an incubator (370C) for 2.0-2.3 hr. At the end of1ncubat10n the NaOH was quantitatively removed and counted.

.T~ble 1. Production of CO 2 (pmoles/embryo/hr) from carbon-1pos1t10n of lactate for embryos derived from fresh and frozen-thawedsemen.

---------E:~---------------------F~~;;~=~h~:;d----------------------

Ram No. Fresh semen semen Mean

~--------1----1~70-:-0~11-(-8)---1~39-:-0~15-(-4)----1~59-~-O~09-(12)2 0.31 +0.21 (2) 0.12 + 0.04 (5) 0.17 ± 0.06 ( 7)

B 3 2.19 +0.17 (3) 2.03 + 0.07 (4) 2.10 ± 0.08 ( 7)4 1.37 +0.09 (10) 1.24 + 0.11 (8) 1.31 ± 0.07 (18)5 1.40 +0.16 (5) 1.01 +0.29 (4) 1.23 ± 0.16 ( 9)6 0.22 + 0.09 (5) 0.08 +0.02 (2) 0.18 ± 0.07 ( 7)

Mean 1.22 ±0.11 (33) 1.05 ±0.13 (27) 1.18 + 0.11 (60)

(--)--~~~b~~-~f-;~b;;~~-----------------------------------~---------.Embryos derived from fresh semen produced more CO2 than embryos

der1ved f~om frozen-thawed semen (p<O.OI), suggesting that they maybe more v1able. Further, ~tilisation of lactate differed greatlybetween ewes (p~O.OOI). Th1S may be due to differences intrinsic toova from a part1cular ewe and/or to the oviductal environment of theewe.(1) Wales, R.G. (1986). J. Reprod. Fert. 76: 717 - 725.Supported by Aust. Meat & Livestock Research & Development Corp.

5.

52

SERLM MEI.MaUN(aMr) P.AT'.l'ERNS IN SHEEP UNDER NATURAL PfIJIOPERIOOICcnIDITICNS AND su:aJOCTED 'ill .&lClHIDED DARI<NESS. A CIRCADll'N MSIS EORSJ?ASCtW:, BREEDJ:N3.

C D Matthews, A J 1eI1y,* D J Kennaway, R watson and R F 8ealm:rk

IJepartDent of Obstetrics and Gynaecology, The Queen El.izabethl:bspJ.tal, UIuversity of J\delaide, S.A. 50fOl1. * 0r:e9XJ. SCiencesUniversity, USA.

The changll1g length of day throughout the year ~s known toprov~de the llIlfOrtant ze~tgeoer for the entraJ..nIlleI1t of an endogenousannual reproduct~ve cycle ~n Photoper~odJ.c spec~es. The serum aMI's~gnal, known to be dr~ven endogenously and be ooth entramed andsuppressed by 119ht (I), prondes cruc~al information to time theonset/offset of reproductive OOl'l'petence ill the sheep (2). ~s studyaddresses the mecharusm underlyillg fihotoper~octi.c breedIDg.

'ren Rotmey sheep kept ill natural photovenodic confutwns atlaUtude 35S (Ranye daylength ~.S-14.5 hr) v.ere mnded ~nto twogroups for Salilflhng throughout a 13 rronth per~od. Group 1 sheep weresampled wtule eXlJOsed to natural photoper~odic conditions. Group 2sheep were subJected to an extended. dark penod conmencing 5 hr pnorto dusk and conUnued 5 hr after natural dawn. Jugular venous bloodsamples \Yere drawn at 1~-3k.J mll1 illtervals on 7 occasions 111 U=!c, Feb,Har, May, Jul, Sept and O:::t, for a penoo of 25 hr throughout 1~l:M/85.

aMI' was assayed by the rrethod of Kennaway (3). The onset (offset) ofthe aMr rhythm was determmed by a rise (fall) of the aMI' valuesgreater (lesser) than 2XSD of assay sens~uv~ty. Mann-Hlutney rrethodswas used for stat~st~cal purposes.

Group 1 arurnals denonstrated a delay between the onset of the aMI'rhythm and the tune of natural dusk at the ~nter solsUce(l::::0 ± 6~ ICllll) (mean ±S1) (p=tJ. k.J3) • At the surrmer solstice the offsetof the aMI' rythm was 67 ± 21 nun (p=<0.kJ2) earl~er than dawn. Group 2arurrals showed an earl~er onset of aMI' than natural dusk at the surrrrersolsUce (ll:J4 ± 66 rru.n) (p=<kJ.kJ3). The offset of eMf was later thannatural dawn m lJecernber (5b ± 21::l~) (p=<kJ.2:'), and earl~er thannatural dawn m May (127 ± 61 ICllll). D~fferences between the twoyroups occurred at the onset of aMI' at roth the w~nter (p=<kJ. kJkJ3) andsunner tp=<kJ.kJI(32) solsUces, Group 2 arllma.ls derronstrating an earliernse at roth tunes. The li-earl durat~on of aMI' rhythms throughout theyear was 11.10 ± 1.4 hr (Range 9.k:l-1..:i.:' hr) m Group 1 and 12.3 ± kJ.bhr (Range 11. ~-lj .:; hr) 111 Group 2 aruma.ls.

The fID~9S suggest a stable circaman phase and duration to theaet~v~ty of the Melatorun generating system (the secret~on of aMI' fromGroup 2 aruma.ls) through the year but dayhyht ~llunu.nates a differentphase of the c~rcaman system depen~g on the tJ.ri'e of year. Theresults prov~de for an explanat~on of seasonal breedIDg qn classicalc~rcadJ.an lmes.1. J..e..Ny A.J. ~~ehr T.A. GoodwJ..n F.K. Newsome D.A. Markey S.P. (1~l::skJ)

SC~ence 21kJ:l~67-l~69.

~. Bntman B.L. Dempsey R.J. l\arsch F.J. (19tl3) Endocrillology113:227b-221::l3.

3. Kennaway JJ.J. Fr~th' R.G. Pml11pou G. Matthews C.D. oeamark R.F.(1977) Endocnnology 1101: 119-127.

53

FAIUJRE OF ME[A'I.UITN 'ID~ A FREE-RlJ.iINIt.'G BElY'l'fM

p.P. ShaW, D.J· :KeDnaWaY, R.F. seauark

:t of (j:)stet:rics and GyDaeCX>logy, '!be <)leen El.izabef:h~, university of Adelaide. S.A. 5I1J00

1 tonin (aMI') have derronstratedprevious studies of ovi,n~ p~asrra x:e a, hytl1m in continuous

that the pineal maintains ';l. di~tinct c:Lrca~ ~ve derronstrated. thatdarkness (00) (1,2). studies ~~: ~ither continuous light (LL)

free-running rh~~~) These rhythms are thought to beor OJ (:dg .,=v~ ~upra chias~tic nucleus SCN (4). It has ,generat WJ. . dail injection of aMI' can synchroruse apreviously been ~~ that a Yfree-running activ~ty rh~t(~.determine whether the neural centres

The present study soug . aMI' hythm after 'placement in a

(seN?) pe~SiS~~i:~a~~~~~(LL(and al~ to det~efree-runrung. .. . f aMI' ~uld influence this free-runru.ngwhether a daily :InJect:J..Ol1 0

rhythI;;e study involved two eX~i..r'rentsduSf~g~;~i,nr1=~:es,F' ewes were housed :In 00 an :Lve

Exp. 1. ~s were then bled hourly for 24 hours under 00.days, the . housed under LL for 32 days. From raysExp. 2. suteen ewe~v=~l 9.00 a.m. SC injections of either aMI' or4-32 the ~s were g .dni.' '"-t;f ray 32 the ewes were placed in DO andsaline vehicle. At rru. gubled hOurl~ for ~ hours. di 1" ed aMI' rhythms after four days, phase

DD an:unals m Exp. 1 ~p ay . tent with previous findings (1,2).delayed by 2-~ hour~. This1:L:l:n~~nstrated aMI' rhythms which \YereIL treated aJ.'l1.I'(\3..ls ill EXp. . that the neural centres (SeN?)phase delayed by 2-6 hours~ su:;s~;bwper.sist under condiHons (LL)which generate a free-runn.:-ny r . '::hJ.~ Both aMI' treated andwhich suppress tJ1e express~on of t-...his rhyt •d ' the 48 rotirs of DD.

, . """ 2 sh~d aMI' rhyt.l1rns urmgcontrol arurnals :m =-p. . 1 :in that LL ooes not'!he results of Exp. 2 ~p~rt those .~~ ~.ession in the form of an aMI'suppress the SL'N rhythmiC:L -y (only :L xpr . the time of the riserhythm) • Addi-t:.ionally, tl:ere ~s.noe~~:r:~:r treated or controlor the fall of aMT secr;at.1.on~~ train the free-running neuralgroup. Thus, ~ a.ppears unabt-~ ~rhythm. This is in disagreementcentre (SCN?) which generates e t the neural centres whiChwith 'previous wor~ (5) and. ~uygests ~ which generate aMI'generate acHvity rhythms d1.ffer fran lOse

rhyt-.luns .

1. ROllag M.D., bliswender G.D. (1976) Endocrin. V 98, P 482.

S.A. De,,:I1+..ment of Agriculture. Unpub. observations.2. Earl C.R. ~.-

3. Turek F.W. (1985) Ann. Rev. Physiol. V 47, P 49.

4. Nishino H., Koiyumi. 1-\.., Brooks C.M. (1976) Brain Res. V 112, P 45.

Redman J., .ArrnStron':j S., Ng K.'I'· (1983) SCi. V 219, P 1089.

54

STEROIDOGENIC AND OVULATORY RESPONSE OF MERINO EWESTO PMSG AND PORCINE FSH

55

GROUP 1 (PMSG) 2 (FSH) 3 (PMSG+FSi-1T

H.N. Jabbour, G. Evans and N.W. Moore

Total folliculardevelopment (CL + F) 17.1 ± 1.9a 8.3 ± 2.5b 12.7 ± 2.1

Ovulations (CL)' , 11.4 ± 2.5 7.2 ± 2.5 11.1 ± 2.1E per follicle (pg/ml) 23.2 ± 0.8a 10.8 ± 0.6 b 13.3 ± 0.4bP er CL n Iml 4.3 ± 1.4 1.6 ± 0.5 2.2 ± 0.6Values with different superscripts within each row differ P<O.Ol

These data demonstrate that PMSG is highly steroidogenic incompari son with FSH. Prob1ems of sperm transport, fertil i sa ti on andembryonic development previously observed after PMSG treatment may berelated to excessive oestrogen production. A combination of PMSG andFSH induces a hi gh superovul atory response without excessi ve steroi dproduction.(1) Ryan, et al. (1984). Superovulation of ewes with a combination of

PMSG and FSH-P. In Reproduction in Sheep, ed. Lindsay &Pearce.Aust. Acad. Sci., pp.338-341.

This work was supported by the Australian Wool Corporation.

Department of Animal Husbandry, University of Sydney, N.S.W. 2006

PMSG and pituitary extracts are both used to stimul ate super­ovulation in sheep. The use of PMSG may result in adverse effects onfertility, possibly associated with excessive ovarian stimulation. Acombination of PMSG and porcine pituitary FSH has also been used tosuperovulate sheep(I). This experiment investigated the effect ofdifferent superovulatory regimes on the ovarian response of Merino ewes.

Ewes were treated in the breeding season with intravaginal progest­agen pessaries (Repromap, Upjohn) for 12 days. Group 1 received 1200 lUof PMSG (Folligon, single injection 48 h before pessary withdrawal (PW),n::10); Group 2, 15 mg porcine FSH (Burns-Biotec, 6 injections at 12 hintervals ~qmmencing48 h before PW, n=10); Group 3, 700 lU PMSG + 11mg FSH (n=10). Oestradiol-17B (E), progesterone (P) and LH levels weremeasured in jugular venous plasma collected at 4-hourly intervals for 56h from PW and daily thereafter. Onset of oestrus was determined usingraddl ed teaser rams. Ovari es were observed vi a 1aparoscopy under localanaesthesia on Day 6 after PW and the number of corpora lutea (CL) andlarge follicles (F) recorded.

Time of onset of oestrus did not differ between treatment groups(24.7±2.3 after PW). The proportion of ewes ovulating was Group 1,10/10; Group 2, 7/10; Group 3, 10/10. The mean time of preovul atoryLH peak (30.6±2.0 after PW) did not differ between groups.

The ovarian follicular, ovulatory and steroidogenic responses areshown in Table 1. Peak preovulatory E levels adjusted for number offollicles and peak luteal phase P levels adjusted for number of corporalutea are shown.TABLE 1. The ovarian response of Merino ewes to different gonadotrophinregimes (mean ± s.e.m., n=10).

MEAN

110±81101 ±49

AB20

116±8a1107±96b

103±14a1096±53b

work was supported by WRTFBindon, 8.M. (1984). Aust~f the.Austra~ian Wool Corp.Montgomery G W C b' J. Blo1. SC1. 37 163-189P 11 , •• , ros le S F M t· -- •e etier, J. (1984) In 'R •• , ~r l~, G.8. andAkbar, A.M., Nelt, TM epr?ductlon ln Sheep'. Awe P23-25.Endocr. ~ No. 5; 1318~1~~~.NBwender, G.D. (1973)

THE HALF-LIFE OF FSH IN ENTIRE AND CHRONICALLYOVARIECTOMIZED BOOROOLA AND AB20 MERINO EWES

R.C. Frya, L~P.Cahilla, J.T. Cumminsc B .L.P. Piperd and I J Cl k' b· M• Blndond,•• ar e •

Animal Research Institute W .Research Centre Prince H' e~rlbee,.Victoriaa, MedicalH 't 1 ' enry s HOspltal b St V·OSPl a , Melbourne and eSIRO A' '. lncents., rmldale,N.s.Wd.

. A possible action of the F .hlgh ovulation rate of th B gene, responslble for themetabolism (1). This e ooroola ewe, is to alter FSH(FSH-t~) in Booroola a~~P:~2~ompar~s the. half-life of FSHhypophysectomy (Hypox). As cha~t~:l~ Merlno ewes followingfollowlng ovariectomy (0 ) g ln the clearance rate of LHals" vex occur in He d F

. 0 lnvestlgated the effect fIe rance ewes (2) weThe experiment was a 2 0 ong:term ovex on FSH-t~. '

Booroola (n=6), Entire AB20 (~_~)deslgn comparing EntireAB20 (n=3) ewes. Ovexwas carrl ' Ovex Booroola (n=3) and Ovexvenous cannulae wer'e' inserted ed out 6 months before jugularat 15 min. interVa}5 immediat~l Thr~e blood samples were takenperformed under R~lothane ana y pr:or to Hypox which wasr7moval of the pituitary lan~sthesla. Immediately after themln, for 2h, ~O min for 2~ a d't~loOd samples were taken each 15concentrations were measurednb Ren 3 h for 18h. Plasma FSHwas calculated assuming expone~ti;i·~~ca;~d the apparent FSH-t~

TABLE 1 FSH-t~ (min±s.e) .8ooroola and AE 20 merino ~:e:~tire and chronically ovex

Booroola

EntireOvex

56

study SUpported in part by a grant from the J.S. Davies Bequest.

REPRODUCTIVE DEVELOPMENT IN MALE AND FEMALE SHEEP FOLLOOINGSUPPRESSION OF THE HYPOTHALAMO-PITUITARY AXIS DURING THE

PREPUBERTAL PERIOD

57

P.H. Sharpe, H.E. MacGregor and D.T. Armstrong

Waite Agricultural Research Institute,Department of Animal Sciences,Glen Osmond, S.A. 5064.

'cle Stimulating Hormone (FSH) may be superior to PregnantFoll1 h' (PMSG) for superovulation and embryo

Mares' Se:um Gonado~:~pt~nimproved sperr,1 transport (1) and embryotransfer 1n sheep" of 7 or 8 FSH injections is required for ,recovery (2). A ser1es 'l in 'ection of PMSG. The aim of th1Ssuperovulation vs o~lY a S1tg

~'b,i,ty of developing a single-treatmentstudy was to determ1ne the eaS1 1 1

delivery syst~m for FS~o ture merino ewes were fitted with RepromapIn Exper1ment I, ~a R dalmere, N.S.W.) for 12 days. FSH

intra-vaginal sponges (Ui50hnTe~ty ewes were injected s.c. in thetreatments began on day • wOf FSH-P (Burns-Biotec, Omaha, Nebraska)axilla with 1x3, then 6x1.5 mg _ 12 mg) Another twenty ewes wereat 12 hour intervals (total dose ~ , • , um s (Alza, Palo Alto,treated with two modified A:zethm1n1,~~~ot1~a~h~ump was filled withCalifornia) implanted s.c. 1n t 7lax~ p~iyethYlene tubing to deliversaline and was connected to a ~~1h °rs Infused ewes also received a4.5 mg of FSH-~ in,74 ul over SH-~Uat'pump i~sertion (total dose 12single s.c. ~nJect10n of 3t~ ~YlaparOScopy, seven days after spongellI6). Ovulat10ns were coun e

removal. 't II the oestrous cycles of twelve 3-year old ewesIn Exper1men ~ , I Groups of four ewes were

were synchronized as ~n)Expe~~~en~b)'injections of 12 rug of FSH-P as inassigned to receive:, a ~o Of' 9 of FSH-P in 11.3 ml of saline,Experiment I or (c) 1nfus10n 72 ~ s using a Gilson Minipuls 2s.c. behind the sh~u~der~ overf FSH_~u:t' the start of infusion. In a

Plus a 3 mg ~nJect1on 0 h d f onepump, 'd d 'n 'ected ewes were switc e romsecond replicate, 1nfuse oanl 1t ,J were counted as in Experiment I.

.. t to the other vu a 10ns ttreavllien, • (+ SEM) ovulation rates were no

In Exper1ment I, mearl -, d (6 5 + 1 2) and injected ewes. 1 d'fferent between 1nfuse • _ .signif1cant y 1 "t II an ovulation rates over two(9.9 + 1.2). In Exper1men ,me d d 'nJ'ected ewes were 1.0 + 0.3,, ,- f t 01 pump-infuse an 1 - drep11cates or con r , 'l Both FSH-treated groups ha4.0 + 1.2 and 6.8 ± ~.2, respect1ve y. t ols (ANOVA P<0.05) butsignificantly higher ovulation rate~ ~han con ~u s was notthe difference between infused and 1nJected gr p

significant. 'f 'on of 12 mg of FSH-P overTwo different methods of s.c. :n US1

tsimilar to those obtained

72 h aused ovulat10n ra es ,approximately ours c , t. Therefore it is reasonablefrom 7 s.c. injections over the same 1me. administering ato expect that development of a vehi~~ef~~jection or implant is asuperovu.latory dose of FSH-P as a S

possibility.

D T (1984) J Reprod. Fert. 70:47-53.(1) EVffi1S, G. and Armstrong, •. . Sh e P.H. and Armstrong,(2) Evans, G., Holland, M.K., Nottle, M.B., L~:~S~ and D.T. Pearce,D T· (1984) In: Reproduction In Sheep. D.R. yads: Austraiian Academy of Science. pp 313-316.

OF EWES WITH CONSTANT, SUBCUTANEOUS INFUSION OF FOLLICLESUPEROVULATION STI~IDLATING HORMONE

IE

0.1 - 0.23.8 - 7.9

0.13

CE

0.2 - 4.110.2 - 20.7

12.0

IR

0.1 - 0.44.1 - 9.3

0.'07

LH (ng/ml)FSH (ng/ml)LlLH (ng/ml)

This study compared the relative rates of reproductivedevelopment in rams and ewes following long-term suppression ofnormal sexual, maturation during the prepubertal and pubertalperiods. A proven commercial implant (Revalo;r$) containing both anandrQgen (trenbolone acetate) and oestradiol-178 was used to suppressactivity of the hypothalamo-pituitary (HP) axis. Autumn-born lambsreceived repeated ear implants at 1, 3, 7 and 9 months of age withtreatment groups inclUding control rams (CR, n = 9), implanted rams(IR, n = 6), control ewes (CE, n = 10) and implanted ewes (IE,n = 9). Implanted rams and ewes had reduced (p < 0.05) serumluteiniZing hormone (LH) and follicle stimulating hormone (FSH)levels from 2 to 14 months of age (range of mean values during thisperiod are shown in the Table) and testosterone (T) levels in IR werelower (p < 0.05) than in CR Over the same period. Suppressedactivity of the HP axis in implanted animals was confirmed by veryPoor LH responses to exogenous LH-releasing hormone (5 ng/kg BW) at10 months of age (Table, ~H).

C. Papachristoforou, M.J. D'occhio t and B.P. Setchell

Department of Animal Sciences, Waite Agricultural Research Institute,Glen Osmond, S.A., 5064 and t C.S.I.R.O., Division of Tropical AnimalScience, Tropical Cattle Research Centre, Rockhampton, Qld., 4702.

In IR and IE, LH began to rise sharply and FSH more steadily between4 and 5 months after the last implantation, and in both groupsgonadotrophins reached respective control values 1 to 2 monthslater. Testosterone in IR rose sharply at the same time as LH beganto increase and within 2 weeks 'T levels were similar in IR and CR.In IR, the unchanged testis size from 2 to 12 months was followed byrapid and sustained growth so that within 7 months of receiving thelast implant (which coincided with the latter half of winter), testissize was the same for IR and CR. Semen characteristics at 18 monthswere also similar for IR and CR. Seven of the CE started cycling ataround 12 months of age, but none of the IE had shown any signs ofovarian activity by 18 months, 9 months after receiving the lastimplant. The differential response in reproductive developmentbetween IR and IE, when body weight and chronological age were notlimiting factors, suggests that in sheep, rams more readily overcomethe effects of long-term suppression of the reproductive axis. Thepresent findings also Substantiate the concept that the period ofsexual development is more protracted in ewes than in rams and, itwould also seem, more sensitive to seasonal influences.

s. r··:addoc~\s is supporteC: by· t' ,. .,ne .ioe I Research li"LSi: ;ur,cJ.

Hours after hCG 0 2 4 8 12 18 24 36 72 96

lestosterone I. F. 23 66 29 190 III(ng/lftl) T.V. 38 423

28 77 13 20 11359 S8S 470 237 2150 289 212' 96

(mean values frolT, 6-8 testes)

Risbridger et al (1986) J.Reprod.Fert. (in press)Erickson &Elseuh (1978) Endocrinology 102:1275.

1.2.

Department of Anatomy, Monash University, Clayton, Victoria 3168.

59

EVIDENCE THAT OVARIAN FACTORS INFLUENCE TESTICULAR STEROIDOGENESISAND VICE VERSA

G.P. Risbridger, A. Leung, G. Jenkin, D.M. de Kretser

We have previously reported the presence of a non-gonadotrophicfactor in testicular interstitial fluid (IF) that stimulates testos­terone production by purified Leydig cells. Based on observationsthat the ovary and testis often produce the same peptides (e.g. inhibin)we have examined bovine follicular fluid (bFF) to determine whether ornot it contains testosterone stimUlating activity. Conversely, weexamined the effect of testicular IF on estradiol production by granu-

losa cellS.Testicular IF was collected and charcoal treated as described byRisbridger et al (1). bFF was aspirated from ovaries and charcoaltreated twi~ prior to storage at _20oC. Testosterone (T) stimulatingactivity was determined by the addition of IF or bFF to Percollpurified Leydig cells. The Leydis cells were aliquoted in 48 wellplates at a concentration of 5xlO cells/ml and incubated in a volume of250ul for 20h at 340 C. Test fluids were added at increasing volumes upto a final concentration of 20%. Testosterone production was measuredby RIA and the results analysed using parallel line bioassay statistics.Granulosa cells were prepared from DES treated immature rats as descri­bed by Erickson & Hsueh (2). The effect of testicular IF on both preg­nenolene and estradiol production was determined after two days of

incubation.IF and bFF caUse linear dose-dependent stimulation of T productionby Leydig cells in vitro. The dose response lines were linear andparallel (p<0.05~~indexof precision (A) of the assay was 0.07.The estimated potency of bFF relative to IF was 0.83 (0.71-0.98 CL).Similarly, the effect of IF and bFF required protein synthesis whichcould be blocked by addition of cycloheximide. under conditions ofmaximal output of testosterone, pregnenolene production increased in alinear manner, indicating an effect of bFF on steroidogenesis prior to

the conversion of pregnenolene.The additions of testicular IF (6-50ul) caused a dose-dependent

stimulation of estradiol production by granulosa cells in vitro, whereasalthough the addition of serum stimulated estradiol produc~thiseffect was not dose-related and was significantly lower at each doselevel than that achieved with IF. In the presence of a maximallystimulating dose of FSH, pregnenolene, but not estradiol, production

increased further with the addition of IF.These data demonstrate (i) the presence of testosterone stimulatory

activity in bFF and (ii) the ability of IF to stimulate granulosa cellestradiol production : and suggest that the stimulatory activity ispresent in the ovary and testis and may represent a common regulator of

steroidogenesis.

Pande. J .1<., ChoYJdhur\l1 S l:i ;) I' " t J "~nc P;ar, t\.3. (191'"<;) J p ····S' uas ,-,up a~ P.Ro1'~ho\'idhL!r·y, A.R",_ 00. roc. oc E>'" Eh 01 1'-' • "1 -,.,e.ddoci,s ~ and (' + ' 11 8 • ./"'. _. _·,ca. ~. 8"'9-9

1i?

T\ _h. ve~cne 1 ,.P. (1936) . - oJ_.g' Europ. Testis Wkshp. Mini~oster rt.:~r&~ .~~' ~OO~~1 :.,••. Widmark, A" Damber. J

IV _:Jt_/"',. le51:15 \,~:snp. t:inipos1:er Fl. . (1986).(3)

(2)

Apart from stimul ati n T . "increaSE I.F. vclume ~ ~ t~he~ret10nl ~C~ treatment has bee~ shown to. . .. ~n~ lS was On r 'lric.ll·,{ tf "t t . .lncrease 1n capillary perro-eao' 'l.t 0f-,::1 ,j 10ugn 0 resulT. fror:. iln"1 c " 1 1 y. IO~fever changes i bl '" 1c.so lJeen shown to be inlportant in tI . n (:,0,_ f ow hc.vl:suggests that testosterone is prefe ~l~.r~~ponse (3). The above datavascular system rather than' I F ren~la y concentrated in thetherefore be affected by b10~~ l' •1 Lev~l: of T measured in I.F. mayanc the r.;hysiolol';cal c:t,,4-e -f 4oehve, S a~a ',nfluenc"d by blood flow rate

~ ~ a~ U .. e ~estlS.

(1)

58

TESTOSTERONE LEVELS IN RAT TESTICULAR INTERSTITIAL FLUID

$. j·laddocks, J. L. ZUPPI $. Sowerb.utts and g. P. Setchell

Department of Animal. SciencE:sl ~/aite A r'Institute.University of Pdelaid Gl aS lcultural Research, e." en smond, South Australia.

Interstitial fluid (LF.) is bei ' .using the "Drip" collection method (l~g.coll~ctea ln many laboratoriesflc,\'t' Tr'oe an incision in the tunica of ln ~hlC~ fluid i: a~lowed tocollection tube for up +0 20 h t 48n solated testls lnto aunphysi~logical techniq~e and '~~~: ~ecen~' We bel!~ve this to be an~.F. uSlng a Push-Pull cannula (2). With ~~.repor~ed the co~lection oflS obtained in-vivo from the tes' . 1S technique a dlluted I.F.pentobarbitone (Nembutal, Abbots~~sT~~ a~~mals ana:sthetised withfrom a drip collection, is taken froml~ ui~1 u~llke t~at obtainedsupply.. The amount of dilution' . t~stls wlth an lntact bloodappropriate radioactive markers ~~a~c~~un ~d for by measuringequil ibrium in the animal rior' C.v~ een allowed to reachwhile the corrected PUSh-P~ll 1:°

ltSamPllng• 'rle have reported (2) that

1 1 resu s for Sodium Pota . deve s are somewhat comparable to those ::. " .sslum an Protein

collections. the result'" obta4ned f T r~port~a prevlously using dripcontrast to those obtai~ed us~ng a ~r. es ~~ter~ne (T) are in directf?unc in LF. obtained using the Pus~2~ Co~ctlon. The lev:ls of Ttnose levels in the testicular ve' (T ~l~ canA~la are.no hlgher than~o\~er: vie have now used the PUSh-;~ll •• la~d._appear.In fact to be: ln ~he testes of rats at various t. cannu~~ ~~ exarr~ne I.F. levels ofsubcutaneous injections of hCG (Si lmes a-:ter ..he an1mals receivedof T in the collected I F fl t gma• 50 1.U.). The corrected levelsthe T.V. but once aqain'a~er~onSecl' tChtalngels in the concentration of T in_ s an y ower:

V.W.K. Lee*, Z.W. Zhang, R.S. Carson and H.G. Burger

Medical Research Centre, Prince Henry's Hospital, Melbourne, Vic 3004.* present address: Division of Biological and Health Sciences, School of

Sciences, Deakin University, Geelong, Vic 3217.

(Reac tion 2)

(Reaction 1)

luciferase .. h \J + FMN + RCOOH (Reaction 3 '-

Principle of bioluminescence assay of oestrogens

FMNH2 + RCHO + O2

EDH = oestradiol dehydrogenaseG6PDH = glucose-6-phosphate dehydrogenase.

FMN-oxidoreductase .. NAD + FMNH2NADH + FMN

(1)

. . . + 3SD at any point) was 0.1 pg in bufferThe sensl.tl.Vl.ty (mean - t f 0 1 ml of ewe plasma.

. th resence of an ether extrac 0 •and 0.5 pg l.n e P . tub ) were added to samples of eweDifferent masses of E2 (0.62-20 pg/ d ~ ether extracts of theseplasma (0.1 ml). Oestrogen me~~~r~hel.:mount added (r = 0.999). Thesamples was highly.c~rrelated Wl.. tion at 26.6 and 69.1 pg/ml were 4.1within-assay coeffl.Cl.ents of varl.a

ff.. nts of variation at 17.1 and

and 4.2% and the between-assay c~~e ~~~: of oestrogen measured was71.8 pg/ml were 11.7 and 5.4%. lasma assayed (0.05-0.2 ml, rhighly correlated with the volume of ewe P

= 0.984). ~ estrogen in samples of peripheralThe assay was used to ~ea~ure °a ewe throughout the oestrous

plasma (0.9 ml) collected dal.ly from b t ' 6 and 16 pg/ml with theh tration fluctuated e ween

cycle. T e concen. da s immediately prior to oestrus.highest concentratl.on on the two ~ t the assay will be most usefulThese preliminary result~ su~gest t a . ing measurement of oestrogenfor studies of reproductl.on l.n sheep requl.rin small samples of peripheral blood.

Boussioux, A.M., Boularan, A.M., Descomps, B.Nicolas, J.C., Paulet, A. Analytical BiochemistrY~: 141-145and Cra:'stes de(1983) •

Figure 1

61

A NEW BIOLUMINESCENCE ASSAY FOR OESTROGENS IN SHEEP PLASMA

J.R. Ford, J.C. Nicolas and A.M. Boussioux

School of Agriculture (Animal science), University ofWestern Australia, W.A. and

INSERM (u.58), Montpellier, France

t f oestradiol-17!3 in the peripheral plasma of ewes isMeasuremen 0 .' loW (1 20 pg/ml). Large volumes

difficult ~~c~us~)t:;ec~~~:n~~:t~~~tl.;adioimm~noassays (RIA) ,and as aof plasma - m . 1 sam les collected frequently hasconsequence measurement of E2 l.n shmal di~ied a specific and sensitive

ible using RIA. We ave monot been pass Fi 1 1) and adapted it tobioluminescence assay ~~rs~:~~r~~~~~e~ o~'pe~iPheral plasma from ewesmeasure E2 or E1 + E2(0.6-1. 2 ml).

Glucose 6-phospM,te \( NADP '\ (E~ (NAD

G6PDH EDH EDH

6- ) "'NADPH) \.E1../ \NADH

Gluconolactonephosphate

EFFECT OF

60

INHIBIN SECRETION FROM RAT GRANULOSA CELLS IN VITROGONADOTROPHINS AND STEROIDS

[1] Lee, V.W.K., McMaster, J.W., Quigg, H. and Leversha, L. (1982)Endocrinology 111: 1849-1854.

[2] Lee, V.W.K. and Gibson, W.R. (1985) Aust. J. BioI. Sci.38 :115-120. .

[3] Lee, V.W.K. (1984) In 'Gonadal proteins and peptides and theirbiological significance', eds. M.R. Sairam and L.E. Atkinson,pp. 141-149. World Scientific Publishing Co.

We have previously reported that pregnant mares serumgonadotrophin (PMSG) treatment of immature female rats and mice leads toincreased ovarian and circulatory levels of inhibin [1,2]. The granulosacells of the ovaries are the source of inhibin since ovariectomy of PMSG­primed rats dramaticallY reduced circulating inhibin levels and thatisolated granulosa cells responded to PMSG stimulation with inhibinproduction [3]. In this study we report the effects of gonadotrophins,steroids and the combined effect of these hormones on inhibin secretionfrom rat granulosa cells in vitro.

Granulosa cells were obtained by puncturing the follicles ofovaries from diethylstilbestrol treated immature female rats. The cellswere washed, suspended in serum-free McCoy's 5amedium, and incubated for24 or 48 h with varying concentrations of PMSG (0-1000 mU/ml), hCG (0-30

U/ml) , rat FSH-RPl (0-900 ng/ml), and steroids (10-5 - 10-9M ofprogesterone (P), oestradiol (E) or androstenedione (A». In addition,hCG or steroids were examined in the presence of varying concentrationsof PMSG. The media were removed, extracted with dextran-coated charcoal(1 mg/ml), centrifuged, and assayed in serial dilutions for inhibinbioactivity (3].

Granulosa cells incubated for 24 or 48 h periods with PMSGsecreted inhibin in a dose-related manner (peak inhibin levels with 100mU PMSG/ml). In a typical 48 h incubation, basal inhibin levels were

8± 4(3) U/ml with peak values of 60 ± 10(3) U/ml. Half maximal responseswere observed with PMSG doses of 30 mU/ml. Rat FSH-RP1 also increasedinhibin production, peaking by about 10-fold higher than basal with FSHdoses of 500 ng/ml. No response was' observed with hCG alone but in thepresence of PMSG (100 mU/ml) hCG at 10 U/ml or higher completelyabolished the PMSG-stimulated secretion of inhibin. E and A wereinactive by themselves but enhanced inhibin production by sub-maximal

«100 mU/ml) doses of PMSG. P at 10-~ or higher inhibited both basaland PMSG-stimulated inhibin secretion.

In conclusion, inhibin secretion from granulosa cells in vitrois stimulated by PMSG and rat FSH'but inhibited by the induction ofluteinization in the form of hCG treatment or a rise in P levels.

62

1. Wide, L. (1985) Acta Endocrinol (Copenh) 109:181-189.2. Wide, L. (1985) Acta Endocrinol (Copenh) 109:190-197.3. Wide, L. (1986) Acta Endocrinol (Copenh) 112: in press.

63

Leif Wide

Department of Clinical Chemistry, University Hospital,S-751 85 Uppsala, Sweden

Human pituitary follicle-stimulating hormone (FSH) exhibits a con­siderable polymorphism. At least 30 different forms of the hormonecan be demonstrated in extracts of individual pituitaries by use ofzone electrophoresis in 0.17 % agarose suspension (1). In additionto this intra-pituitary variation an inter-pituitary variation whichis sex- and age-related was demonstrated for FSH of adults (2). Themedian charge of the forms of FSH was more negative at higher ageand the most negatively charged forms were found in elderly women.A correlation was found between charge of the forms of FSH and theirmetabolic clearance rate (MCR) (3). In the present study the formsof FSH of children were compared with those of adults with respectto median charge and to ratio of in vitro biological (B) to radio­immunological (1) FSH activity (B/I ratio).

The median charge was expressed as median mobility at electro­phoresis in 0.075 M veronal buffer at pH 8.6. The biological FSHactivity was measured in vitro using, as an end-point, the produc­tion of estradiol by cultured Sertoli cells from 10-day-old rats.Extracts of individual pituitaries from 12 boys and 15 girls,aged 2 months to 9 years, 17 men and 26 women, aged 16 years to91 years, were examined.

The median charge of FSH in boys was more negative than that ingirls but less negative than that of men, both young and elderly.In the girls, the median charge of FSH was similar to that of youngwomen. FSH in all 27 extracts of pituitaries from the children hada higher B/I ratio than that obtained for anyone of the 43 adults.The mean B/I ratio of FSH of the girlS was 1.26 times higher thanthat of boys and 2.25 times higher than that of adults. The corre­sponding ratio of boys was 1.79 times higher than that of adults.All these differences between mean values were highly significant.Young and elderly men and'women had almost identical mean B/I ratiovalues.

The results indicate that the forms of FSH are different ingirls and boys and that those of children are different from thoseof adults. The different forms of FSH have different biologicalproperties. The structures of the FSH molecules seem to change inone way from childhood to puberty and then in a different way frompuberty to old age. The first change in structure affects mainlythe biological effect at the target cell while the second changeaffects the MCR of the forms of FSH.

PITUITARY FOLLICLE-STIMULATING HORMONE IN CHILDREN AND ADULTS~ DIFFERENT MOLECULAR FORMS IN RELATION TO SEX AND AGE

Sf)

I , ;) 'i: I , ()(). H:J:().!i

!loom :::.SV

!.O.O±D.li!. ()±lJ. 1

;,~ , f;.;.O • Ii

LIli . 7~'D, ~!

I. C±O. /10, I ±O. J

LI}i.1 • :~ ±! . Ij .!: ±(). r.1. G.±O. :~

1.1),", D! ~ . :~ ± 0 . i:~ ~ ~ • 0 ±0 . ~:~

i . :!t() .:.:: :~.!):tO, ;:;:..: . :':.to, '1 O. :"';Jon, j

Jill.Ylent{th i;;; !he' ,.' - maJ(jf' envu'ot!wHnLal facLot, n~gulatin " >' I'

l'I.'flroduc1.iori ,tn •. !Jf'('p ,!,I" fr' g Sl,asonn lty ufj't J >1'-' .:'. "I' ",1:('1.1,: cd r.,j'I01.opcll'iod ill't: Ihoup;JJt. t' be

nw. III 1'( by Ill" dl.onllJ sr:c!'()tc'tj by th ' I 1 ,(J

illvr'sL;ga1.r:d t.he 'Im:sibiJitv fl.!,: ,r: Pltlea g and, ThIS exper'llUetlt~o 'r 'L' .,' .. ",Id, b/:,thOHllJ ChIHl!..!'t·,": in gonlido1.l'ophin:" c '/~ lun are t;Tltr'ainl'd tu phu1.ope·}T'iodic ['h.vihITI8.' "

,',1\ '\';CT'IlIO r'c'oHsbl'cd "\"r'''' \\'('1'" (lV'" I 'containtno' n8"'Lc,orll't)1 1,-'13" '" ',' .l11(~C ,Ul/IlRnd ,lIid silast.h: im[Jlan1.p

.~ ," r>,.- I, WI'!!'I' Insl'rtr'd t ."'''W~J'l: placed in o:wh of t.Wll C:lllJt t'~);l.' j ,SU.Jr,.uLanulJusly, 1'hr'u(~ animal:ipIHlSI', wil.h <.dtl'l't1'ltin rr If "k' ,fH, "nvH'onrnenL ('oums, 180' {J\J!. or'Hid S!IO!'! dilYS' (H~I 'lf~)\ ';"'I\)\)t'I)::, p~rl'll)ds of long days (lfH.:8D) (I.!»)If ' ,,\ " .'. .,> 101' (), wpek::-; 'I'h/ •

letI' prevailing- phoLoper'in,j ror' '\ r .t f " ;;, ' :,V wc~.r'l: 1.,wn Icdl in1Illtritj(l1l wet'(: COne't'lIll .~. j ,L .. t' ur .ler' ) wenki:>,' lernpf.:,r'atut'p.. and

.", ,. ,vtlC \~'Il\' j tr'ltl4'h ("wi jHanlpim.; We·)t'C' l"tken 'i'l,l . ,.' j .' :, . I pr!J'I()( \,f:lIUIH; PI:JSJlIarr1rdal.oflin ! I).' 1'1';("' 111 ' ,: 'j ... ,d):;;:Il,~C)( . lUt' iur.flir)isillg' hOt'mrme (LH) ann

, Pl ..(. P<,Il',IUJ(:l.r:n; (JT'() I'l"c[wded in Tablt:l.yab!p L !vir'.'Htl 1.11 PI!iS() ft'ec UPtlc'\' (."" , ";"vrd"; (f'.~\') ... , ' 1", I W~), rltllplll.llde (AMI') and bli,sal,,,' "''''l !tIl"USUtr.:d :n plasTllIJ of t.he r!WCH in the' /"

\H.Ek prn'UJl>H [01' each room (J: 'i: scm, n = :i I ' 'oHml t.hree J fi!louJIlI

!lay I UIJ p; LhFrIO, No,' HII:iM]', ng.'lIIlHi\~;, II P; / 1!I I

WiLhtri each r'OUlll all ewes dlspl;J,n:d a marked " . ,il) :lnd ~:ll :)(TllHjS in 11' I' 0 r" \ anatlrlft bet.ween t.he:(['<U.Oi'i). 111' t.ht: . sustair;/)I

dpll ,~c !'C~CPWtl(:.... , tHlll'li! udt·' :-mel bll~:e lr:\'~:ir:;

(1'mal) 1.1) pf~r'i()d 1tI 1{()()llJ I, lhPill'lmlr~t.t,:J'S t.()fJl!/:d to br~ <Jii'i'pf'cn1. ", e fJu[St)in comparison wit.h the pr'evi;), ' LD !I P,Il,I, I.h(Jf:m,I.Y~~It::lj of H LJJ pe!'iod;(!':.7 r ) I) 'f .[.[ J. UH P~,!lOd pulSE) frcquOfWY waH hi<>,J"lor'

"'. dliP l,U{JC' \\'tl';; !nwnr (1'-0 I) •. j 1 " ,..,[~(!'=O,lG) The 'f t. '. ' . -. ,illt 1/-lI-lC 1(~vr'~1 WHH hig~her

, • mg 1 l.IOte nSR In melatonin I,· I" ' "WIl.h II tnr:an ni.,tht. tim(' 1('\'('1 (I' 'rw t,ves WeU> t..vplcal of LDthe sust.ultH'd t!'inal) SLl . :'" II : '"J)L~/!!Ij IltcllTll./lilWd fcJl' Iti hOUI'H. In

, ' PCllur Itt HOOlll .) Lh> JitlSIl at'ypjc~al E,f 11 "I) ,. ,J. ' ~,to P,U HO parlimetel'S wor'p

, . ,J ,H.I lUtJ, In C'HllP"l!'tROn '1 It.penod pub-;(.; frequency IP-OO") .·d '0 WI.) .nepr'nVIOUH Sf)10.)\\'1':1', t.hOlq,;-h i!mrJliil;,J/,' \:'l~ ,J II i;;l,? tJ,l.'d,se It"\'f)!, ,(P=O.02) were both

" . '.u. 0< .-. nC~lUHII,{f' T~. 'h ./DelatoTllct was typic:al of a SD ' 'j '... ,H. eng 1. Luttc: I'.ise in'j 10 I ' . ,porlOr WIth a JIlPan 'hl ', pg TTl! Hlaintained rOl' H hours.' , m.t{ tnne level of'

TIH'se c'8sults Lh' t'. , ,', uug I lmsed on Ii small numb ' " ,l.t~a1. the, ~f~t~r(~tion patl.erns of L1I ir; PW(" • el, ~! anl~lals, indic:al.t,wllh anilclpatc~d changes in da 'ler U . .S rn,~y chdng-(: ltJ accordance,'.Josef" rnlu1.c:d (.0 trw - P!'evt1i1in;~ d;;;'lc:~l~~~atomn secretion, howevet', is

(1) Cut.ler eL 81 (198Gi. PI,'QC' ".usL S R', '" 0 oc. epI'od. BioI. .l7, 38

This work WaH sUpport.ed b ' IT" •

r.:SIRO/TJ niverHit.y of Svd" Cll, .... .1 :-1 rm1.s fr(>TII 1Jw .'\MIlC Ilnd, ,tH,y AJ a"oraLlve Research [~und.

PHOTOPERIODIC ENTRAINMENT OF SEASONAL PATTERNS OFLH SECRETION IN CROSSBRED EWES

S.A. Cll!Jor1 , i;. Kvan::i~, 1< ..1. SCnraTlIuz"Jl'2 ", J I) , ~. ",. " owrl.lng'"1 De~)iu:t.~IHH:1. of ,Animal Ilusbandr',Y, UniversiLy of Svd ' ,

~ U',T!(() DIVIsIOn ql' !\niwlIf J'roduetion l;roHC)('r'1' rNl~,YW' NSWt .... (. • " 'J ~ ...) •

64

OESTROGEN AND PROGESTERONE IN VITRO INFLUENCE PROTEINEPITHELIAL ENDOMETRIAL CELLS SECRETION BY

Loi~ A. Salamonsen, Robert A.. Cherny and Jock K. FindlMed~cal Research Centre, Prince Henry's Hospital, Melb~~rne, 3004.

O:strog:n (E) and progesterone (p), administered' .ovar~ectom~zed ewes, influence overall . ~ toby cultured epithelial endometrial llPr~te~n synthesis and secretionsignificant modification of the ind~:'ds lrom th:se animals and causestudy was designed to determine wheth~ruah pro:e~n~ secreted (1). Thishormones could be reproduced by' . t ese ~n v~vo effects of thecells. ~n v~tro treatment of the cultured

Ovariectomized ewes were treated with' 1prior to hy.sterectomy. Epithelial e ~m~ ants of E for 12 days(1) and cultured (5 x 105 vi bl nldlometr~al cells were isolated

, . a e ce s per dish)' d' 19ta~n~ng 5% (v/v) charcoal d h ' ~n me ~um 9 con-After 48h at 370C d' an eat-treated FCS, with added E or P.( , me ~um was changed to meth' . fminus FCS) with added steroids and 100 C' [35n~~e- r:e ~ulbecco's MEM

four hours later, medium and cells ~ ~ sJ meth~on~ne. Twentydetermine incorporation of 35 S . t wer~lcollected and analysed toSeparate but parallel experimen~~ ~i~~ouul~5 and se~reted pro:ein.content of the cells after 2 d 3 d t S, exam~ned prote~n and DNAexpressed as a % of control (:: adde:Y: of ~ul=ure.~ All results wereanalysed by paired Student's t-test. tero~d - 100% ± SE(N)), and

DNA content of dishes with P (10- 7 -9creased (p < 0 025) b . or 10 M) or E (10-9M) was in-. y compar~son with control d' h f

culture (but not after 2 d ) h ' ~s es a ter 3 days ofsignificant effect. Totala~~o~e~ne~::s E +.p (10-

7 0: 10-9M) had nopared with control dishes. not ~ncreased ~n treatment com-

Treatment of cells with 10-9M P .cellular (69 ± 16% N = 5 P < 0 decreased ~ncorporation of 35S intotreatment (E, 10-li _ 10-7M. P l'OO:~) but not secreted protein; no other

1 35 "M' E + P varying d )a tered S incorporation into cellul' " os:s consistently

Polyacrylamide gel electro h . ar or secreted prote~n.densitometry enabled measurem~n~r~~~~ foilowed by autorad~ography andsecreted maximally with E + Pd' . eve ~ of.a 46K prote~n which isadministration (each 10-7M) . a m~n~strat~on ~n vivo. In vitro, E + Psecreted by a factor of 1 9 ~nOcr4eas(ed_the proportion of the 46K protein

1. . N - 7 P < 0 05) d

tro s. The effect of P (l0-7M) 1 (' . compare with con-ficant (0.05 < P < 0.1) whe ahone :.~ ± 0.2, N = 6) was not signi-either or both steroids at ~~~9Mt e adm~n~stration of E (10-7M) or

In c 1 . ' was w~thout effectone us~on, we have demonstrated" .

cultured ovine epithelial endometrial ~~ v~tro effects of E and P onthe synthesis and secretion of a . d,C:dls. Both DNA synthesis andtrolled in vivo by E + P ~ ~n ~v~ ual protein which is con-

. were st~mulated sugge t' hstero~d hormones on epithel' 1 d .' s ~ng t at the action of. ~a en ometr~al cells d .requ~re the presence of the t . ' oes not nece,ssarily1 . u er~ne stromal cells I .at~on of synthesis of individual ro' . . n v~tro stimu-

a study of the control of th p te~ns w~ll be an important tool insequent purification. ese proteins and their production for sub-

(1) Salamonsen, LAO W S ' D h'.. , , .. , oug ton B. a dF' dlEndocrinology 117, 2148-2159. ' n ~n ay, J.K. (1985).

Supported by the Australian Wool Research Trust Fund.

65

A RADIOIMMUNOASSAY FOR FLUOROGESTONE ACETATE(FGA j CRONOLONE j SEARLE)

Olivia Gaston-Parr~, J.K.E.Nemorin* and T.J.RobinsonDepartments of Animal Husbandry and Organic Chemistry*

University of Sydney, Sydney, N.S.W. 2006

Despite its use for some 20 years for the control ofovulation in sheep and goats, there are no data for plasmaconcentrations of FGA during and after its use. To this end,adouble antibody RIA has been developed.

FGA Deri vati ve: A hemisuccinate derivati ve was impossibleto produce as the OH group on C 11 was sterically hindered. Anoxime der i vati ve 3_0_carboxymethyloxime was anotheroption. Such preparations are more involved but are likely toproduce more speci fic antibodies. In essence, 17 Q( -acetoxy-9 Ot'fluoro-ll ~ -hydroxy progesterone (FGA), dissolved in warmethanol,was added to one equivalent of aminooxyacetic acid'nemi hydrochloride tn water. KOH was added to pH 14 and thesolution was stood for 5h. The volume was reduced on a rotaryevaporator until a precipitate separated. Water was added tocomplete the precipitation and the pH adjusted to neutral. Theprecipitate was dissolved in ethyl acetate and extracted with10% sodium carbonate. Acidification of the aqueous layer gavea colourless precipitate after refrigeration for 2 days.Filtration gave the oxime (FGA-3-CMO) as a mixture of (E) and(Z) isomers (65:35) with M.P.196-199 C. Structure of the oximewas verified using IR, NMR, and mass spectroscopy.

Antigen: FGA-3-CMO (50 mg in 1 ml 50% pyridine-water) wasadded to 50 mg 1-2-ethyl-3 (3_dimethylaminopropyl)carbodiimide, HCl (Calbiochem) in 1 ml 50% pyridine-water andstirred for 30 min.when 100 mg HSA (Sigma,St.LOuis) was addedand stirred overnight. Dialysis was for 2 days, first againstsodium bicarbonate then against several changes of distilledwater. Lyophilization and frozen storage of the FGA-3-CMO-HSA

followed.Antiserum: Antiserum against FGA-3-CMO-HSA was produced in3 New Zealand White rabbits which were injected with 2-4 mg0.9% saline-complete Freund adjuvant emulsion at intervals of2 weeks. Bleeding commenced after 4 weeks.

Labelled FGA: This was 'prepared by Amersham at a specificactivity of 30-60 Ci/m mole and a purity of 95%. UnlabelledFGA (purity 100%) was supplied by G.D.Searle (France).

Standards and Controls: These were prepared from a stocksolution of FGA and made up in wether plasma.

Radioimmunoassay: A standard double antibOdy procedure hasbeen used with goat anti-rabbit globulin (Calbiochem) as thesecond antibody. Use of an extracted assay procedure showed noadvantage over that of an unextracted (r = 0.95). Counting wasin a LKB liquid scintillation counter and the data wereprocessed on a Hewlett-Packard 85 computer, using a weightedlogit-log model.Specificity: Cross reactions werejFGA,lOO% j progesterone,8.5% j 17 ~ hydroxY progesterone, 0.8% j testosterone, 0.7% jl7foestradiol,2.6%j cortisone,0.7% j other steroids, 0.1%.

Sensitivity: Lower limit was 3.1 pg (sd= 0.98 ; n=lO).Accuracy: Determined by a spiking method which gave a mean

recovery of 93% (range 84-98% j n=12).Precision: Mean intra- and inter-assay coefficients of

.. __ ._L' __ . ~ C~ (n_1n' Qnrl In_7% (n=56).

66

EFFECT OF MEDROXYPROGESTERONE ACETATEIN PLASMA OF PREGNANT RATS ON PROGESTERONE AND OESTRADIOL

D.L. Willcox, J.L. Yovich N W BrLaboratory, Leederville WA' 6007 uce and,S.c. McColm: PIVETAustralia, Nedlands, W.A. 6009 and Un~versity of Western

Medroxyprogesterone acetate (MPA) d70ncentrations during the luteal ~~~:ssed plasma progesterone (P) .~n pregnant women did not affe t P, h of the menstrual cycle (1) butplasma until after 20 weeks fC e~t er P or oestradiol-17f, (E) inbound plus free) and free E 0 ge~tation, when both total (protein­this study the effect of MPAwerel epressed significantly (2). Inb on uteal steroid 'y,placental synthesis, was investi ated' ogenes~s, uncomplicatedan~mals were injected i m w' th 0 3 19 ~n pregnant rats. TheC) 2 . • ~ • m peanut oil ( 1

or Omg MPA Suspended in 'I ( contro groups A andtreated on days 7, 8, 9 and ~~lle~est groups B and D). The rats weretreated on days 13 14 15 d k'llon day 10 (groups A and B) orcoincide with maxi~al ~rgan~;ene~ised ~n day 16 (groups C and D), torespectively. an progesterone secretion,

Table 1: Effect of MPA treatment of thetotal and free progesterone (P / 1) plasma concentrations ofin pregnant rats. ' ng m and oestradiol-17, (E, pg/ml)

Group Treatment Total P Free PA Oil 57.4 ± 4.4 (13)B MPA 64.02.9 ± 0.3 (3)± 5.1 (1)C Oil 81.13.9 ± 0.5 (0)± 5.7 (8)D MPA 90.96.3 :t 0.6 (8)± 5.7 (12) 5.4 ± 0.3 (2)Group Treatment Total E Free EA Oil 80.7B ± 7.7 (13) 6.4 :t 0.7 (2)MPA 26.9 ± 3.7 (9) *C Oil 62.52.5 ± 0.4 (9) *± 7.6 (7)D MPA 27.98.1 ± 0.9 (7)± 3.7 (1) * 2.9 :t 0.4 (0) *

Results expressed as mean + S E M ( b* den t " - • . • num er of observat' )o es s~gnif~cantly different (p < 0 001 ~ons .control group. • , unpaired t-test) from

There were no significant differencebetween groups C and D itS between groups A and B, and

, n erms of ovarian weight bcorpora lutea, placental weight . , ' num ers of largeThe f' d' , uter~ne we~ght a d bse ~n ~ngs suggest that i th n num er of fetuseshuman, exogenous MPA perturbsnE senpreg~ant rat, unlike the pregnant'However, decreased Circulating Y thes~s,rather than P synthesis.physiologically-active free E d7~ncent:at~ons of total and .to day 16 of pregnancy. ~ not ~nfluence fetal survival up

1. Johansson, E.B.D. (1971) A2. Willcox et al. (1985) J. cta Endocrinologica 68:779-792

Endocrinol. 107:293-300

67

MATURATION OF THE HYPOTHALAMO-PITUITARY AXIS IN MALE ANDFEMALE SHEEP EARLY IN LIFE : ROLE OF THE GONADS

M.J. D'Occhio, T. Weatherlyt, S. sowerbuttst and B.P. Setchellt

C. S. I. R.O., Division of Tropical Animal Science, Tropical CattleResearch Centre, Rockhampton, Queensland 4702 and t Department ofAnimal Sciences, Waite Agricultural Research Institute, Glen Osmond,

South Australia 5064.

The reproductive endocrinology of the neonatal and prepubertalperiods in sheep has been examined in detail; however, it is yet notpossible to conclude whether there exist differences between ramSand ewes in early maturation of the hypothalamo-pituitary (HP) axisdue to a general lack of direct comparative information. Theobjectives of the present study were to determine if (1) a sexualdimorphism exists in activity of the HP axis of sheep early in lifeand (2) the influence of the gonads in determining HP activity inewe and ram lambs. Luteinizing hormone (LH) responses to exogenousLH-releasing hOIll\one (LHRH; 10 ng/kg BW) were used as a measure ofactivity of the HP axis in entire animals and animals gonadectomizedat 3 days of age. Serum concentrations of LH were measured by RIAand the data were analyzed by ANOVA procedures. Results (mean ±SEM) for peak LH concentrations (ng/ml) following injection of LHRHare presented in the Table.

Age Entire Gonadectomi zed(weeks) rams ewes rams ewes

2 4.8 ± 1.0 5.4 ± 1.7 6.6 ± 0.3 7.6 ± 1.64 12.2 ± 2.2 9.6 ± 1.9 17.7 ± 7.3 12.3 ± 3.56 14.5 ± 2.4 9.8 ± 1.7 17.3 ± 2.0 14.7 ± 3.38 21.5 ± 2.4 11.7 ± 1.8 26.1 ± 8.4 21.6 ± 5.6

12 24.8 ± 4.1 13.6 ± 2.4 27.0 ± 9.2 28.5 ± 3.716 22.6 ± 3.4 11.3 ± 2.4 32.6 ± 13.0 28.6 ± 2.924 12.5 ± 1.6 13.4 ± 2.4 45.8 ± 13.5 36.4 ± 8.2

There was an increas'e (p < 0.05) in peak LH responses to LHRHbetween 2 and 4 weeks in entire and gonadectomized animals of bothsexes and a further increase (p < 0.05) which occurred in entirerams between 6 and 8 ,weeks may also occur in gonadectomizedanimals. Peak LH responses in entire ewes remained consistent from4 to 24 weeks, and from 8 to 16 weeks, were lower (p < 0.05) thanthose in entire rams and gonadectomized rams and ewes, with thelatter three groups not differing. A decline in LH response inentire rams at 24 weeks most likely reflected increasing feedback bythe developing testes. The results indicate that the HP axis inrams and ewes has a similar capacity for maturation early in life;however, in ewes development is suppressed through feedback byovarian factors whereas in rams the testes do not appear toappreciably influence HP activity until after 16 weeks. Thesefindings are consistent, with general earlier onset of puberty inrams.

study sUPpo7"ted in part by a grant from the J.S. Davies Bequest.

68 69

*+

David ~ Handelsman, JA spaliviero, CD scott, RC Baxter.

WEEK 12Male ~

961+93 892+65943+91 595+44

WEEK 6

~ ~713+123 1002+110642+54 530+36

(ng/ml) mean+SEM

WEEK 3

~~179+19 154+18167+18 177+20

* serum IGF-I

~i WEEK.§.. ~.§. WEEK2~! ~~ ~lQ..3Q8+23 300+27 350+43 432+56 691+85 1087+80 900+85299+17 302+17 369+38 304+34 534+69 862+49 644+76

!9-12

10-12CASTRATEINTACT

Expt 2*

Expt 1* !CASTRATE 9INTACT 8

In expt 1, castration of weanling male rats during postnatal week 4had no effect on prepubertal (F=0.02, p=0.88) but enhanced (F=11.8,p=0.004) the pubertal surge of IGF-I levels (F=160, p<O.OOOl). In expt2 neonatal rats were treated With GnRH-A (6 mg/kg/day IP) or salinevehicle on postnatal days 1-6 and castration or sham operation at 3weeks of age. The pubertal IGF-I surge in male and female rats(F=18.3, p<O.OOl) was augmented by castration (F=7.0, p=O.Ol) butneonatal GnRH-A treatment (F=2.1, p=o.16) and sex (F=1.5, p=0.23) hadno effect. In expt 3 the pubertal IGF-I surge was abolished bytreatment with a hypothalamic neurotoxin, monosodium glutamate, (5 x 4g/kg orally alt days) starting on postnatal day 1 but not on day 5 .

HORMONAL REGULATION OF THE PERIPUBERTAL SURGEIN CIRCULATING INSULIN-LIKE GROWTH FACTOR-I (IGF-I)

Therefore both male and female rats undergo a pubertal IGF-I surgeproviding a convenient nonprimate model. The pubertal IGF-I surge is(i) abolished by early postnatal hypothalamiC damage, (ii) enhancedby prepubertal castration and (iii) is unaffected by neonatal blockadeof pituitary gonadotropin secretion. We conclude that, in the rat, thepubertal IGF-I surge is not due to direct gonadal secretion of IGF-Ior other gonadal hormones either perinatallY or during puberty but mayinvolve hypothalamic and/or hepatic programming by events during

prenatal or very early postnatal life.REFERENCES

(1) copeland KC et al. JCEM 55:1198, 1982; JCEM et al 60:1154, 1985

(2) Baxter RC et al. Acta Endocr 106:420, 1984(3) Handelsman DJ et al. Acta Endocr 109:543, 1985

Dept of Medicine, university of sydney and Dept of Endocrinology,Royal Prince Alfred Hospital, sydney, NSW.

puberty in primates is accompanied by a marked growth spurt andincreases in circulating levels of IGF-I (1), a GH-dependent growthfactor, but the relationship to gonadal secretion remains unclear.since IGF-I is produced within the gonad (2,3), the pubertal IGF-Isurge could be due to direct gonadal secretion of IGF-I or indirecteffects via other gonadal hormones. To determine the relationship ofgonadal secretions to the pUbertal IGF-I surge, we have studied theeffects of prepubertal castration (expt 1) or neonatal treatment witha gonadotropin-releasing hormone antagonist [GnRH-Al and prepubertalcastration (expt 2) on serum IGF-I levels in rats bled serially from

before pUberty to maturity.

~ f1gures w~th d1ffer:nt subscr1pts d1ffer significantly, p<0.05response - PGFM (5m1ns. after OT injection) - basal PGFM

+ mean.2:.SEt~

2 NP InfEdxPleri~ent 3 bloo~ samples were taken weekly from 69 pp andewes e up1ns as descr1bed for Expt 2 Re lt h d h

concen~r~ti ons were hi gh (0.90+0.47 ng/m;, •mea~~SE~) s oOnwe

th t adt PGF~partunt10n but declined to 0.08+.03 n Iml b d - e ay 0

pl asma PGFM concent~at~ ons i ncreased t~ 0.66~0.:~sn~;~l P6y I~:I~a~ter~GPF.M Tlhere

lw~s no Sl gm fi cant effect (p<O .05) of nutri ti on on pl a:~:

eve s 1n pp ewes.(1). Fairclough, R. J., Moore, loG., Peterson(1984) Biol. Reprod. ~:271-277 ' A.J., and Watkins, W.B.

Table I. Lupin effect on PGFM (ng/ml) response* to OT in pp ewes

EFFECTS OF OXYTOCIN AND NUTRITION ON PROSTAGLANDIN FMETABOLITE LEVELS IN POST-PARTUM EWES. * -W. Sm1th , R. J. Fairclough+, and A. R. Egan*

Animal Production Section, University of Melbourne ParkvilleDe~artment of Agricu~ture and Rural Affairs, 'An1mal Research Inst1tute, Werribee, Vic.

The effects of nutrition and exogenous oxytocin (OT)13, 14-di hydro-15-keto-Prostagl andi n F (PGFM) , on pl asmastudied in 3 experiments on 108 post-partum (pp) c~~~en1~rat10ns were(NP) Corriedale ewes. non-pregnant

In experiment 1 OT was administered (10 I U . ):306, 010 5and1013 days post partum., Blood samples ·w~;e 1~~ile;~e~ :~~~, " .' '. ' 20, 30 and 60 m1nutes relative to the time f

(~~~ct~~~U1U;Sl n;h~w~~ot~~~~ ~~:eanw:;p~ri~:~~eJ n~oG~~i ng cycl i c a~ima~~oxytocin injection at days 5 and 6 post artum re~ponse to theconcentra~i~ns reaching a maximum 5-10 m1nutes Ppost_in;~;:ionth\/~~~~~~~r::~~ e at 10 and 13 days post partum no detectable resp~nse was

In a more extensive study (Experiment 2) 39 pp and 8 NPwere sampled before and 5 mi nutes fo11 owi n an 0 .,. :wes:~~~~ rt~) ewes ~e;e fed lupins (500 g/head/day) 30x~~~~1~e;~~:cr~~)·and NP ani'ma~~ s~ ore

dand after (HH) parturition. Unsupplemented {Ld

h rve as controls. The results presented in T b1 Is ow that overall there was a significant PGFM response to OT u ~o ~25f

pp (p<O.OOl), but no rise in PGFfvl was found in the NP ewesP

0 1 a~o the ewes from the group 26-35 da . n yresponse to OT stimul ati on. At 20-25 day~S PPth5::

ee a d,etectabl e PGFM

g~ve. a, greater response to OT (p<0~05) t~:n the LH W::e~n i~e LL groupSt1gn1f1cant response to lupins between any other groups o~ ate:~yw~~h~~sage post partum.

Days PP HH HL LH LL TOTAL NP

10-15 1.81.2:.1.16+ .33+.18 .52+.12 .50+.16 1.07+.36 0

20-25 .37+.14ab .28+.16ab .17+.08a 1.07+.32b Oab.39+.10

26-35 0 0 0 .06+.13 .01+.01 0

7071

24

1.50.3

1.20.06

3 5 7 9 111

4 6 8 10 122

wl·thdrawal (ng/mljMean & SEM)after sponge

Hour after sponge withdrawal

4.2 2.8 1.9 1.3 1.50.30.3 0.3 0.30.05 0.6 0.51.3 1.1 1.10.3 4.4 4.Y0.3 0.3 0.20.06 1.1 1.1

2.0 2.30.1 5.3 4.6 2.10.6 0.4 0.3 0.3 0.30.04

0:25 3.0 1.8 1.4 1.3 1.20.08 0.080.02 0.2 0.1 0.07

1.0 0.7 0.7 0.5 0.5 0.41.60.1 0.08 0.08 0.090.3 0.2 0.1

0.3 0.21.0 0.6 0.3 0.3 0.30.060.06 0.07 0.080.3 0.2 0.080.20.4 0.2 0.3 0.31.1 0.7

0.05 0.07 0.07 0.090.2 0.1 0.060.20.5 0.4 0.3 0.31.2 0.7

0.04 0.040.09 0.06 0.06 0.05 0.05

2 successive periods of insertionfor

Beforeinsert

MeanSEM

MeanSEM

MeanSEM

MeanSEM

Data

MeanSEM

MeanSEM

MeanSEM

MeanSEM

Data

* Data pooled

Dose FGA(mg)

Table 2. Plasma FGA

Test 140 (n=6)Test 2A40 (n=5)Test 2B80 (n=5)Test 340 (n=30)~

Dose FGA(mg)

Test 140 (n=6)Test 2A40 (n=5)Test 2B80 (n=5)Test 340 (n=30)*

OF FLUOROGESTONE ACETATEPLASMA CONCENTRA~I~NSEARLE) IN EWES TREATED~;~~ ~G~R~~~~~~NA+ED INTRAVAGINAL SPONGES

livia Gaston-Parry and T.J.Ro~inso~D p~rtment of Animal Husbandry,Unlverslty

e of Sydney, Sydney, N.S.W. 2006

FGA has made possible theThe development of a .RIA ~~r exogenous FGA. This abstractmeasurement of plasma.profl~e~GA during and after treatment ofresents data on pro!lle~ 0 n es with 40 or 80 mg FGA.~noestrous XB ewes Wl th l/V spo g ducted. The first was a

Three tests . have. been c~~ impregnated with 40 mg ~GApreliminary te~t ln WhlCh sPfoonrg 12 days. Plasma conc~ntrat:onwere inserted lnto 6 ewes . the day before lnsertlonof FGA was assaye~ da~ly,cOmm~n~~~gthereafter at intervals ofand· continuing untll ~l thd{at~h' and 24h. The second test was2h until 12h and ag~ln a. was for 10 days and 2 doses ofsimilar except that lnsertlon used in 2 groups,each of 5 ewes.FGA namely 40 and 80mg, were All were treated with spongesThe'third test involved 15 ew~s. On withdrawal,500Lu. PMSGcontaining 40mg FGA for 12 ays. were re-inserted 1, 3, or 5(Intervet) w?s inject:d'deSteO;mgiense the repeatability of t.he FG~days later, In order 0 f 'le and the effect of tlme 0treatment on the. Plas~a o~rfuteal development.insertion of FGA sponge t d in Tables 1 and 2. Plasma

Data for FGA are presen/ el e maintained to day 12. Basalt · of 1 - 2 ng m wer I • hd 1concentra lons . d by 6 _ 8h after Wlt rawa.concentrations were attalne

sponge insertion (ng/mljMean & SEM)Table 1. Plasma FGA during

Day after sponge insertion

J.A. Downing,l D.T. Baird,2 B.K. Campbell 1 and R.J. Scaramuzzi1

CSIRO, Division of Animal Production, P.O. Box 239, Blacktown, NSW,2148 and 2 Department of Obstetrics and Gynaecology, University of

Edinburgh, Edinburgh, Scotland.

The left ovary and its vascular pedicle was autotransplanted to asite in the neck region (1). Seventeen functional transplants resultedfrom the nineteen attempted, six were random bred controls, seven wereBooroola Merinos not carrying the F gene (+/+) and four were BooroolaMerinos carrying the F gene; 1 (F/+) and 3(F/F) animals. The presenceof the F gene was confirmed from ovulation rates determined prior toautotransplantation.

STEROID SECRETION RATES FROM THE AUTOTRANSPLANTED OVARYOF THE BOOROOLA MERINO

The F gene is a gene of major effect on prolificacy. BooroolaMerino ewes carrying a single (F/+) or a dOuble copy (F/F) of the genehave increased ovulation rate compared to ewes not carrying the gene(+/+) or to random bred Merino ewes. This study was conducted toobserve the effect of the F gene on ovarian steroid secretion.

Ovarian and jugular venous bloods were collected every 10 mins fora 4 h period during the luteal phase and from 24-28 h after injectionof a luteolytic dose of Prostaglandin (Estrumate, 125 Ug)ovarian venousplasma was analysed, by RIA, for oestradiol and progesterone, and thesteroid secretion rate calculated from a knowledge of ovarian bloodflow and the haematocrit. The jugular plasma was assayed by RIA, forFSH and LH. The ewes were also monitored regularly for luteal functionand pre-ovulatory LH surges.

Fifteen animals had a functional CL and a preOVUlatory LH surgefollowing PG treatment.These results are presented in the table.

Genotype Reproductive BlOOd flow Prog oestradiol FSHstage (ml/min) (Ug/min (ng/min) (ng/ml)+/+ Luteal 13.6a

5.5aO.60a

1.26aRandom bred11.8a

4.7aO.40 a a1.08

bF/+ or F/F 12.6a

4.2aO.42 a

1.77+/+ Follicular 13.3a b b cO.20b 1.25

b 0.86d

Random bred10.8a

0.2\ 2.60b 0.45F/+ or F/F

11.4a0.15 2.15 O.88c

Wi thin a column values with different superscripts differ at the 5%level of a significance.

AnalYsis of these results show that Booroola Merinos carrying theF gene despite their high ovulation rate have a pattern of steroidsecretion which closely resembles that seen in ewes without the F geneand in control Merinos with an ovulation rate of 1.

(1) GODING, J.R., MCCRACKEN, J.A. and BAIRD, D.T. (1967). J. Endocr.,39, 37-52.

72

It is concluded th t hproper and the a .t.e caput epididymidis is the initial

corpus ep~didymidis is an initial segment. segment

(1) Rouse, G.W. & Robson, S.K. (1985). J. Submicrosc. Cytol. 18:137-152.(2) Pettigrew, J.D. (1986). Science, N.Y. 231: 1304.(3) Martin, R.D. (1986). Nature, Lond. 320:~2-3.(4) Phillips, D.M. & Bedford, J.M. (19Ssr-J. expo Zool. 235: 119-126.

73

ULTRASTRUCTURE OF THE IONOPHORE-INDUCED ACROSOME REACTION INSPERMATOZOA OF THE GREY FLYING FOX, PTEROPUS POLIOCEPHALUS

J.M. Cummins, S.K. Robson, G.W. Rouse and M. GraydonDepartments of Animal Sciences and Production, oral Biology, Zoology and

Anatomy, University of Queensland, st. Lucia 4067.

Semen (c. Iml) was collected by electroejaculation under halothaneanaesthesia, and 'epididymal spermatozoa were collected by maceration orflushing of the vas deferens post mortem. Spermatozoa were centrifugedtwice (500 G, 15 minutes) and re-suspended (10 million/ml) in Tyrode 'ssolution with 1% w/v BSA (Fraction V, Sigma). Acrosome reactions wereinduced with the calcium ionophore A23187 (final concentration 0.01 roM),and samples were fixed for EM at 0, 10 and 120 minutes.

Spermatozoa of Pteropus poliocephalus (Chiroptera: Pteropodidae)show several ultrastructural features reminiscent of the Insectivora and"lower" Primates (1) which, along with anatomical features such as thevisual system (2), lend support to concepts of phylogenetic affinitybetween the Megachiroptera and the Primates (although this iscontroversial - 3). One interesting structure is the extensive apicalsegment of the acrosome, which overlies an anvil-shapedpseudoperforatorium ("subacrosomal material"), flattened in the sameplane of the head, but with lateral extensions. We studied theul trastructure of these sperm components during the course of theinduced acrosome reaction.

The initial phase of the acrosome reaction is marked by swelling ofthe plasmalemma and the acrosomal matrix. Subsequently the matrixcavitates and disperses; the plasma membrane and the outer acrosomal capmeI$rane fuse and fenestrate, forming hybrid vesicles. The equatorialsegment remains intact after loss of the acrosomal cap. The inducedreaction in this Chiropteran thus conforms to the general scheme seen inother eutherian mammals. Completion of the acrosome reaction exposes theinner acrosomal membrane and, rostrally, the anvil-shapedpseudoperforatorium. A similar, though more elaborate structure is seenin the Insectivore Suncus murinus, where it has been speculated to serveas an anchoring mechanism for the apical segment of the giant acrosome(4). Alternatively, this structure could play a mechanical role inpenetration of the zona pellucida.

Initial motility of about 70% decreased to about 10% over 2 hours.The first motile spermatozoa without acrosomal caps were observed after6 minutes, and at 90 minutes about 60% of motile spermatozoa hadcompleted the acrosome reaction (phase contrast microscopy). Theionophore induced other changes in the spermatozoa, notably coiling ofthe axoneme within the plasma membrane, presumably by disrupting normalosmotic balance through calcium influx.Sa-reductase activity

(pmol. T metab./g/h)Head Body Tail

143±1 112±10 17±518±8 22±8 26±751±28 18±7 23+9

Tail1356±82

715±1701002±89

Mass(mg)

Body1050±27

391±83431±36

Head745±72272±45357±30

Benoit~ J. (1926) Arch. Anat. Histol.Holste1n, A.F. (1969) Embryol. 5: 173-412.Stuttgart, pp. 77-91. Z. Abhand. Geb. norm. path. Anat., G. Thieme,Jones, R.C., Hinds, L.A. &Res. ~: 525-535. Tyndale-Biscoe, C.H. (1984) Cell Tiss.Orgebin-Crist, M-C (1973)Fawcett D W & H f'f J. Exp. Zool. 185: 301-310.

, " 0 er ,A (1979) B' 1 ---Robaire, B., Scheer H' &'H k ~o . Reprod. 20: 162-181.Reproduction. Jagi~ll' G ac ey, C. (1982) Bioregu1ators ofpp. 487-498. 0, • & Vogel, H.J. (Eds.). Academic. Press,

G.M. Stone, R.C. Jones and L.A. Hinds

Ve~erin~ry Physiology, University of Sydney'Un~vers~ty of Newcastle; and" ' Biological Sciences,

W~ldl~fe Research, C.S.I.R.O., Canberra.

Since the work of Benoit (1) thregion of the mammalian epidid . eh~s has been recognized as astructure. However, some of ~~~hw ~ch h~s ~ characteristic~sent in the human (2) and tammar a~acte~~~t~c~ of the segment are~s questionable whether the IS' h ()lep~d~dym~s. Consequently, itStudies on the regulation of th~S ~~~dOg~US amongst the mammals (2).contention as it has be h ep~ ~ ym~s may resolve thissupport the IS in the r~b~tO~)that testosterone (T) alone will notleast partly dependent on ~t 1 ~nd rat (5); the segment is atfrom the testis Also . ~ths um1nal fluids transported with sperm

• , 1n e rabbit and rat th "reductase is very much high . e act1v1ty of 5a-th " er ~n the IS than m d' .

e ep1d1dYmis and the activit is d ,or~ ~stal reg~ons offrom the testis (6). y ependent on 1ts luminal fluids

The suggestion that the ca ut " "homologous with the IS f th P ep1d1dym1d1s of the tammar is

. a a er mammals was . .exper1ment summarized in the table belo , exam1ned 1n theand Sa-reductase activity (n=3) f h w wh~ch shows the mass (n=4)epididymis. Animals were anae tho~.t ~ h~ad, bo~y and tail of the(Surital, Parke Davis & C Ans e 1ze w1th sod1um thiamylald d ' 0., n Arbor USA) fOse for slaughter The syst. 1"" or surgery and over-(1), (2) and (3) re~pectivel em1C T evels (ng/ml) for treatments± 0.88 (n=4). y were 1.14 ± 0.59, 0.17 ± 0.02 and 6.02

Treatment

(1) Control(2) Orchidectomized(3) Orchidectomized+T

(1)(2)

(3)

(4)(5)(6)

7475

COMPOSITION OF THE LUMINAL FLUIDS IN THE MALE GENITAL DUCTS OF THEJAPANESE QUAIL, COTURNIX COTURNIX.

J. Clulow and R.C. Jones

tIn t of Biological Sciences, University of Newcastle.Depar en

, d h . al analysis of theSampling difficultie7 havelh~nde~etaICd~~~~ Consequently, only

f l . d of the av~an ma e gen~. d 1luminal u~ s h b n well characterized, an on yfluid from the ductus deferens adS ,ee puncture techniques to collect

. (1) We have use m~cro hin two spec~es " , s in the male genital ducts of t efluid from all the maJor s~teh pIes to determine the majorJapanese quail and analyzed t ese sam

components. . f d by enerqy-dispersive x-rayElemental analyses were per,orme 1 's by-TLC with ninhydrin

' (2) nd amino ac~d ana ys~ dmicro-analys~s a . d termined by the Bradford t in concentrat~ons were estaining an pro e constituents respectivelymethod (3). It was found th~t ~h~S~ osmolalities of fluids from theaccount for 91% and 84% of teo ad f rens' the major constituentsductuli efferentes (DE) an~ duct~: ~leand ~ (Table 1). Ca and Mgwere inorganic.ions, espec~ally l' . rease d;sta1 to the DE. Low

h 'l tein leve s ~nc ~levels decrease w ~ e pro , t d with high levels of Cl.

' unds are assoc~a e 5levels of organ~c compo t d glutamine is only about 1The total concentration of glutamade atn deferens pH measurements

ididYmidis and uc us •roM in the ductus ep (7 3+0 2) indicate thatf the DE (7.3±0.4) and ductus defer~ns . ~ .o . , , t' f the gen~tal flu~ds.there is no ac~d~f~ca ~on 0 "u of s erm is modified much less by

It is concluded that the m~l~e f h P il than by these ducts inthe extratesticular genital ducts 0 t e quamammals.

OVARIAN DEVELOPMENT AND POLYOVULAR FOLLICLES IN THE POTOROO (POTOROUSTRIDACTYLUS, MACROPIDIDAE, MARSUPIALIA).

Maheen Dairi and Suzanne L. Ullmann, Department of Zoology,University of Glasgow, Glasgow, Scotland, U.K. G12 800

The development of the ovary has been studied from birth to day 120 orpouch life. At birth, the ovary is a compact structure consisting ofuniformly distributed germinal and blastemsl 8ells and invested with anirregular mesothelium which lacks a basal lamina. The proliferatingoogonia become peripherally located around day 5.

By day 32, septa ovarii, arising from the newly developed tunicaalbuginea, divide the peripheral oogonial mass into germ cell nests butdistinct germ cell cords are not observed. By this time a cortex andmedulla are distinguishable, separated by a fibrous layer.

Meiosis begins on approximately day 9 of pouch life and continues tillat least day 50. Healthy polyovular follicles containing up to 20oocytes, some of which are polynucleated~ are found during all phasesof folliculogenesis from primary to antral follicles.

Although interstitial tissue formation is histologically recognisableby day 50, there is no detectable hydroxysteroid dehydrogenase (HSD)activity. Lack of functional maturity of the interstitial tissue cellsis confirmed by the absence of well differentiated smooth endoplasmicreticulum and mitochondria.

Two kinds of ovarian development have been described in eutherianmammals, Le. those with "immediate" and those with "delayed" meiosis(1). The lack of germ cell cords, HSD activity and the relativelyprompt entry of the germ cells into meiosis would place the potoroo inthe former developmental category.

Table 1. in plasmas from quail genital ducts.Element concentrations

Na166±11148±5152±5138±6132±5146±2

(1) BySkov, A.G. (1982). In: Germ Cells and Fertilization (Austin &Short, eds). 2nd Edn, p 10. Cambri.dge University Press.

Site

Seminiferous TubuleDuctuli Efferentes .Ductus EpididymidisProx. Ductus DeferensDist. Ductus DeferensBlood Plasma

Element Concentration (roM)Mg CI K

3.6±0.4 155±8 12.7±0.82.7±0.2 l27±4 10.0±0.5O.3±0.1 126±4 24.5±0.9O.3±0.2 124±6 23.1±1.30.9±0.2 l25±4 24.5±1.60.7±0.2 113±3 4.4±0.3

Ca1.5±0.51.4±0.10.2±0.18O.2±0.17O.3±0.172.4±0.3

(1)

(2)

(3)

Lake, P.E. (1966) Adv. Reprod. BioI. !: 93-123. PflUg. Arch.M Dorge, A. & Thurau, K. (1977)Rick, R., Horster, .,

369: 95-98. Biochem. 72, 249-254.Bradford, M.M. (1976) Analyt.

76

~~~ ~~~~G;I~rsMELATONIN ON THE INITIATIO~ OF THE BREEDING SEASON OF

R.T. Gemmell, Department of Anat U· .[ucla, Brlsbane, 4067. omy, nlverslty of Queensland, St.

The rate of change of daylen th h" h .of melatonin from the pineal glandg , w lCbllnfluences the secretionseason of various mammals In thi~ Pfe~um~h y affects the breedingimplants on the initiatio~ of th s u.y e effect of melatoninmarsupial species the possum T ~ ~reedlng season was examined in twobandicoot I d' p~c osupus VuZpecuZa and the

, soo on macpoupus In Queensland thbreeder, the breeding season'beginnin . M he possum i~ a.short-daythrough until September The b d' g l~ arc, and contlnulngfirst occurring in July'and con~~n~~~ot l~.f i~ng-day b~eeder, births

Melatonin implants (0 2) h g u~ ~ e followlng February.~n December produced births'o~ lW ~n admlnlstered to possums (N = 5)March and 27 April, whereas birt~s ~nuarYt llFeb~uary, 6 February, 27from 31 March to 12 April. Bandico~~scon:o anl~als (N ~ 4) Occurredhormonal treatment in May and mo't d(N - 6) WhlCh recelved the samegive birth, however control anim~~so(~ _U~~)Lil late.September, did not2 September. - gave blrth from 26 July to

Exogenous melatonin has advan d thseason in three of the five tre t dce e start of the breedingovulation and pregnancy in all :i~ pos~ums and. has inhibitedwould suggest that photo eriod .tre~ted bandlcootS. These resultsconcentrations may be a ~actor'i~h~~h lS k~OW~ to influence melatonins~ason of both marsupial s eci e regu atlon of the breedingblrths especially with thePban~~~oo~~wever, the gradual bUild-up ofsuch as temperature and rainfall ma W10Ulhd suggest ~hat other factors

y a so ave some lnfluence,

77

HISTOLOGY OF THE GONADS OF TRIPLOID FOWLS

Lin Minjie, M.H. Thorne*, I.C.A. Martin and B.L. Sheldon*

Department of Veterinary Physiology, University of Sydney, 2006*Division of Animal Production, C.S.I.R.O., North Ryde, 2113

From a line of poultry producing a high proportion ·of triploid progenyboth 3A.ZZZ and 3A.ZZW birds have been raised to maturity. Neither typeis fertile and the latter, phenotypically, shows a sex reversal duringgrowth and maturation from the appearance of a pullet to that of acockerel. Gonads and reproductive ducts have been examined from triploidand diploid birds aged from 1 day to over 2 years.

Testes from ZZZ birds were essentially the same as those from ZZ malesin terms of growth and development measured by diameter of seminiferoustubule, cell population in the tubule and ratio of the volumes of tubuleand interstitial tissue. However, in mature birds, derangement ofmeiosis and subsequent maturation of spermatozoa caused accumulation ofspermatocytes and round spermatids. All spermatozoa were morph­ologically abnormal and quantitative histological analysis indicatedthat sperm production rates were lower than for ZZ birds.

Early after hatching, most left gonads of ZZW birds were ovotestes but,by the third week of age, the ovarian component degenerated seriouslyand most of the oocytes had disappeared. Degeneration of ovarian tissuewas accompanied by infiltration of leucocytes into this region andlymphoi d patches coul d also be detected in some parts of the gonaddeveloping as a testis. However, most of the testicular tissuedeveloped sufficiently to produce abnormal spermatozoa but this occurredmore slowly in ZZWs (>25 weeks age) than in ZZs (by 15 weeks). \~hen

birds were over one year of age, a further change in. histology wasobserved when the seminiferous tubules degenerated to contain onlySertoli cells. This was accompanied by an increase in fibrous tissue inthe interstitium but a high number of Leydig cells persisted' and thebirds retained their male secondary sex characteristics.

In the right gonad, an irregularly shaped testis, development wasretarded in comparison with that observed in a normal diploid male. Themean diameter of seminiferous tubules and total cell population pertubular cross-section of 25 week old ZZWs were similar to those of ZZbirds at 15 weeks and some abnormal spermatozoa were produced in birdsolder than 25 weeks. Patches of degeneration of the testicular tissueoccurred in bi rds over one year of age but thi s was never as gen­eralized, or as complete, as in the left gonad.

Partial development of excurrent duct systems occurred for· the rightgonad including the commencement of vasa deferentia containing somespermatozoa. Duct development was either absent or much more primitivefor the left gonads and it is hypothesised that the degeneration of thes~miniferous epithelium is related to the accumulation of spermatids andspermatozoa within the gonad.

7879

We conclude that the earl t . .consistent with the i v'~ rans~ent r~se of P is of luteal origin,remains unknown. n ~ ro data, but the source of the E2 peak

Lutectomy abolished the rise in P~nirnals (1~0.7±56.6 pgjml vs 452.4:~;~ ~t day 5-6 in the sham Lutx~ncreased ~ediately (w'th' 6 . pgjml). In contrast E2h'l ~ ~n h of Lutx) t 16 3w ~ e the sham Lutx had th 0 • ±6. 9 pg/ml (Fig 1)

day 4-5. e expected E2 peak of 12.0±3.6 pg/ml at'Lamt

A NOVEL REPRODll:::TIVE STRATEGY IN THE MALE SHEATH-TAIL BATTaphozous geo:t>gianus

Peak spermatogenic actiVity occurred in February, March andApril when 'all classes of germ cell were abundant. At this time meantubule diameter was 178 ~m and mean thickness of the germinalepithelium was 58 ~m. During the period August to October the meantubule diameter fell to 148 ~m and the germinal epithelial thicknesswas reduced to 34 ~m. Spermatocytes, spermatids and spermatozoa werealmost entirely absent from the testes at this time. In contrast theprostate, ampulla and seminal vesicles were relatively inactive fromFebruary to April and reached their peak between August andOctober. 'n1.e mean volume of the prostate increased from 2.04 to3. 54 ~l and the mean acinar diameter increased from 79 ~m to 98 jlm.The mean volume of the ampulla and seminal vesicles increased from99 \lm to 163 \lm. '!he volume of secretion available in the ampullaand seminal vesicles averaged 1.8 jll from February to April but roseto 10.0 jll in the period August to October. Spermatozoa were presentin the cauda epididymidis in all months of the year but thepercentage of the lu.rnen filled with sperm steadily increased from amean of 35% in November to 88% in JUly.

'n1.e sheath-tail bat is a cave dwelling insectivore occurringthroughout tropical Australia. From May 1985 to April 1986 threeadult males were collected each month from the Springsure district ofQUeensland. The study aimed to establish the seasonal pattern ofreproduction in males of this species. Reproduc tive tracts werefixed in Susa fixative, embedded in paraffin, sectioned at 5 or 7 ~m

and stained with haematoxylin and eosin. Quantitative estimates ofhistological parameters were obtained by stereological analysis ofthe image produced by a video camera mounted on a Zeiss photomicroscope.

These figures indicate that mating occurs from August to October(a finding supported by work on females) and that spermatozoa arestored in the .cauda epididymidis for approximately six months beforemating. Such storage is a common feature of the temperate zone batsand enables them to have sperm ready for immediate use afterhibernation. The prolonged storage of sperm has not been, reportedpreviously in tropical bats however and the significance of theunusual strategy in this species is pUZZling.

Department of Physiology & Pharmacology, University of t;peensland,st. Lucia, Queensland, 4067.

Simon Jolly and Alan W. Blackshaw

I'0 ,

I

•, 1. ;) .,. ~ C '1

P and E2 concent:t'ati~s in tarnmars • , 10RPY. (X. +0.-- 5~""). lutectomised 3 days after

o

ORIGIN OF THE TRANSIENT PEAKS OF POCCURRING EARLY IN' THE OESTROUS Cy~~ESTERONE AND OESTRADIOL-1713WALLABY, MACROPUS EUGENII. AND PREGNANCY OF THE TAMMAR

T. P. Fletcher and M. B. RenfreeDepartment of Anatomy, Monash University,

Clayton, Victoria, 3168.

~?U~ 5 days after reactivation of the '~n~t~ated by removal of th qu~escent corpus luteum (CL)

1 . e pouch young (RPY) th' ,e evat~on of progesterone (P) d ' ere ~s a transientthe peripheral plasma of the tan oestradiol-1713 (E2) measured inc t' . amrnar wallaby (1 2) L

on a~ns s~gnificant amounts f P .." uteal tissueproduction rate at day 5 afte~ RPy

and ~n v~tro there is an increased.quantities of E2 (2 4) b t . (3~ . The CL contains smallt · , u aromatase ~s not d t tabl~SSue at RPY or days 11-25 f e ec e in luteal

d ' a ter RPY (4) Th'es~gned to determine the ori' f . ~s experiment Was

d th g~n 0 P and E2 du 'an e oestrous cycle in the tamrnar. r~ng early pregnancy

Three days after RPY the reactivated CLt~ars and another 5 were sham Lutx Was re~oved (Lutx) from 6da~ly from 2 days prior to RPY t d' The an~mals were bled (5 ml)and 4-6 h after operation at 8 ~ .ay s when blood was taken beforeon day 7 then daily to da; 13 Pl~ntervals from day 3 to 6, twiceradioimmunoassay. . aSma P and E2 was measured by

o

Fig. 1.

,...

(1)(2)(3)(4)

Hinds and Tyndale-Biscoe (1982)Shaw and Renfree' (1984) J. Endocr. 93, 99-107.Hinds, Evans and Tyndal ~: Reprod. Fert. 72, 29-37.

Renfree, Flint, Green ~~ ~::oe(i~983) J. Reprod. Fert. 67, 57-63.P 84) J. Endocrin. '101, 231-240.

8081

RAPID RESPONSE OF THE TAMMAR TO INJECTION OF MELATONIN.

CSIRO Division of Wildlife & Rangelands Res., Lyneham, A.C.T.

C.H. Tyndale-Biscoe & L.A. Hinds

p~oc. Auat. Soc.(1984)

14/2/86 13/3/86 10/4/86 14/5/86

7.75t,0.45 8. 94:!:0.47 8.4Ot.0.66 6.46±0.62

3.49±0.67 5.68±2.93 21. 87±5.47 7.16±2.30

849 ±19 871 :!;34 896 :!;47 788 ±44

4.29:!:0.20 5.17t,0.46 6. 29:!:0.511.05±0.10 1.33±0.24 1.80±0.31

707 :!:18 756 t,18 779 ±17

McGuckin. M.A. and Blackshaw. A.W.Rep~od. BioL •• 16: 23.

(1 )

. b October and accelerated afterTestis size began ~o 1ncrease ~ze during mid-March. Plasma

December; with a maX1mum mean ber and all intact bats showed atestosterone lev.els rose afte: Def:~e March or April consistent withlarge increase 1.n .level~ dUrl~~ 'ty Body weight increased graduallythe time of prol1flc mat1ng aac~av~ i~ the March/April period and thenfrom July onw~rd. reached P < 0.05). The single castrate maledecreased rap1dly to May. (Pb dwight to intact animals (Sep 758 g.exhibited similar changes 1~ ?,Y 8~2 g May 720 g) though plasmaDec 813 g. Feb 904 g. 1 pr(t icall; about 0.5 ng/ml) in this battestosterone level~ were ow yp ration was 6.30 ng/ml.except for mid-Apnl .when t~elco.ncfe~~ation on the relationship betwe~n

These data prOV1?e use u 1~ 0 . e and testosterone levels 1nchanges in body we1g~t, test1s. S~\tiS size and body weight occurindividual oats •. The 1ncrea;;es 1n ein body weight in the castrateconcomitantly alth?ugh th~ 1ncreaS~tosterone levels, would suggest,itmale. in concert w1th ~tat:c lowT~: rise in testosterone concentrat1~nis not due to androgemsat10n. 1 together with large increases 1nduring /lpril in the castrate m\~. adrenal steroid contribution tointact males, sug~ests a ~~SSlwh~n testis size is waning.testosterone secret1on at a 1me

Date

TV (cm3 )TC (ng/ml l

BW (g)

t tof Physiology & Pharmacology. University of Queensland.

Depar men 67St. Luc i a. Queensland. 40 •

1 changes in testis size andWild male fruit b~ts showedF~ef~o~~servations also indicated.the

testosterone concentrat1?n (1). 1e al alteration in body we1ghtpossibil ity of a cons1de~ab\eUd:e:~~n designed to folloW the changes(unpub1i shed) • The. presen s t i ve bats.in these parameters 1n a group of caPt ated male housed in a single

Seven adult mal.es and one cas r 'tored ~onthlY from July 1985large outdoor cage w1th female~o~~~~ ~~~~ until May 1986.Testicularto February 1986 and then me Zal cul ated usi ng the formul a for adimensi ons were measured and vo~u ere obtai ned by venepuncture andprolate spheroid. Blood tsamr~~s Wdetermined by RIA. Animals were1 sma testosterone concen ra 1 )_~e~ mixed fruit ad. Lib. and Complan (Glaxo .-

concentration (TC) and body weight (BWl ofTable 1. Testes volume (TV), testoste~o~e 1985 to May 1986, mean :t s.e.m.seven intact adult male fruit bats from u Y

26/7/85 17/10/85 13/12/85

E PLASMA TESTOSTERONE CONCENTRATIONSEASON:~D C:t~;~EII~;EISJI~AP~ivE MA~E mEV-HEADED FRUIT BATS,

Pte1VJPus pol,wcepha.l,us

McGuckin. L.M. Little and ~.W. BlackshawM.A.

read a melatonin signaltreatment before theyThis rapid response

much slower response(about 4 weeks).

(1) Sadleir, R.M.F.S. and Tyndale-Biscoe, C.H. BioI. Reprod. ~:605-608 (1977).

(2) McConnell, S.J. and Tyndale-Biscoe, C.H. J. Reprod. Fert. 73:529-538 (1985).

(3) Hinds, L.A. and Tyndale-Biscoe, C.H. J. Endocr. ~: 99-107 (1982)

The tammar wallaby breeds shortly after the summer solstice inresponse to decreasing daylengths. The corpus luteum (CL) and blast­ocyst formed after post-partum oestrus are inhibited by the sucking

'stimulus of the pouch young in the first half of the year, but betweenthe winter and summer solstice, increasing photoperiod is the proximateinhibitory factor. The photoperiodic inhibition can be mimicked byan artificial photoperiod of ISh Light: 9h Dark (15L:9D), and exten­sion of the dark phase in the morning or the evening by 3h to 12L:12Dcauses reactivation of the CL and blastocyst and birth occurs 32 dayslater (1,2). Injections of melatonin administered daily for 15 days2.5h before lights out on 15L:9D photoregimen mimic the light changeto 12L:12D since births also occur 30-32 days later (2). The questionaddressed in the following experiment was what is the minimum numberof daily injections of melatonin required to induce reactivation?

Thirty lactating tammars were subjected to the inhibitory photo­regimen of 15L:9D in early March and after two weeks their young wereremoved. No births occurred 26-30 days later and so treatment withmelatonin began. Groups of 5 animals were injected for 0 to 5 success­ive days with melatonin (400ng/kg) 2.75-2.5h before lights off. Bloodsamples were collected for analysis for the early progesterone peakcharacteristic of reactivation in the tammar (3) and checks for birth/oestrus began 26 days after start of treatment.

None of the animals given oil injections for 5 days or a singleinjection of melatonin showed a progesterone peak or gave birth. Twoof the 5 given melatonin for 2 days and 4 of 5 in each of the remainingtreatments showed early progesterone peaks between 4 and 10 days afterthe start of melatonin treatment. All of these animals gave birth21-22 days after their progesterone peak or 26-32 days after the startof treatment.

The results indicate that some tammars canwithin two days, although most need 3 days ofcan interpret the signal and then reactivate.to melatonin in the tammar contrasts with theseen in the sheep (about 6 weeks) and hamster

82

OXYTOCIN SECRETION BY THE OVARY DURING THE OESTROUS CYCLEAND FOLLOWING A CLOPROSTENOL STIMULUS.

R.J. Fairclough1, D.Baird, J.A. Downing2 and R.J.Scaramuzzi2 •

Animal Research Institute, Werribee. 30301CSIRO, Division of Animal Production, Prospect, NSW2

A close temporal relationship between surges of oxytocinor its associated neurophysin (OT-N) and uterine prostaglandin(PG) F2~ has been demonstrated in ewes during luteal regression(1,2). The mechanism regulating oxytocin release at luteolysisis still uncertain although recent evidence suggests thatprostaglandins may playa role. Exogenous cloprostenol has beenshown to provoke endogenous oxytocin release (3) and systemicindomethacin can result in a marked suppression of surges ofplasma OT-N concentrations in ewes during corpus luteumregression (4). This study was undertaken to determine oxytocinlevels in ovarian vein samples collected from recipient eweswith ovaries transplanted to the neck.

Ovaries were transplanted to the neck of 4 Merino ewes2 months before the start of the experiment. Oestrus wassynchronized using a 12-day intravaginal sponge treatmentfollowed by an im injection of clGprostenol at spongewithdrawal. Catheters were inserted into the ovarian andjugular veins using local anaesthesia. Blood samples werecollected hourly from Day 13 to 15 of the estrus cycle. Theewes were then given an injection of saline, followed by an iminjection of cloprostenol. Blood samples were collected at 0,5 j 10, 20 and 30 min after the saline and cloprostenolinjections. Plasma samples were analysed for oxytocin andprogesterone by specific radioimmunoassay.

Plasma progesterone concentrations indicated that therewere functional CL in all ewes over the sampling period.Pulsatile surges of plasma oxytocin concentrations were observedin all ewes over the sampling period. There were 3 detectablepeaks of oxytocin for each ewe. Basal oxytocin levels were2-3.5 ng/ml with peak levels of oxytocin reaching 7.6 to 32.0ng/ml. All ewes showed a significant increase in plasma ovarianoxytocin concentrations in response to the cloprostenolstimulus. However, there was a considerable variation in peakheight with the maximum levels varying from 7-185 ng/ml. Adetectable rise in oxytocin levels in ovarian venous plasma wasfound in 1 of the 4 ewes given saline.

These data, when combined with earlier observations,suggest that an extra-uterine source of PG may be involved inregulating ovarian oxytocin secretion during the latter stagesof the oestrus cycle in ewes.

(1) Fairclough R.J. et al. Prostaglandins. 10 : 199 (1980).(2) Flint, A.P.F. and Sheldrick, E.L. J.Reprod.Fert. 67:215 (1983).(3) Flint, A.P.F. and Sheldrick, E.L. Nature 297 : 58~(1982).(4) Fairclough, R.J. et. al. Proc. ASRB 16 : 3 (1984).

83

EARLY IN THE OESTROUS CYCLEADMINISTRATION OF PROEGAES~T~~O~~FM AND OXYTOCIN-NEUROPHYSINCAUSES PREMATURE REL

2 F S °th1 H R. Tervit11 R J Fairclough, J. • m~ , •A.J. Peterson, •• 3' d W8 Watkins3

L.G. Moore an .'

R ku ra Animal Research Institute~ Hamil~o~, ~.Z21ua tot t Werr'bee V~c~or~a.Animal Research Ins ~ u.e, ., 3

National Womens Hosp~tal, Auckland, N.Z.

'd that oestradiol-178 andThere is now goo~ ~v~o~~c~n regulating the s~nthesis

progesterone playa maJo d' (PG)F The find~ng of a closeand/or release of p:ostaglan ~nlasma ~~~tocin or oxytocin­temporal :elationsh~p b~~~~e~eeels led to the sugge:tion thatneurophys~n (OT-N) a~d potent stimulus for uter~ne PGF 2aOxytocin(~a~)als~x~~en~~Saprogesterone given e~rlY fin therelease , • hown to advance the t~me 0 )oestrous cycle has b~en1~ 14_dihydro-15-keto-PGF2a (P~FM), (3 •intermittent surges ~n, t directly on the uter~ne PGF2aHowever whether prOg7s~7ron~lact~roughthe release of oxytocinsecretory system or ~n ~rec Yd was undertaken to monitoris not known. The present ~tUt~ s in ewes given progesteronelasma OT-N and PGFM concen ra ~on

~arlY in the oestrous CYC~:·synchronized using a 12-dayNine Romney ewes we .. s on es containing MAP. At

treatment with intra-uter~ne p g divided into 2 groups of 4 andsynchronized oestrus the ewes,were t 0;1 was given to 5 ewes

P t ne 50 mg ~n peanu ., 0 . t d5 ewes. rog es ero , cle while 4 ewes were ~nJec eon Days 1 and 2 of the oestrous ~y f the oestrous cycle the eweswith vehicle. On days 7, 81an~ ~2 hours from 0600-1800 hours.were bled at hourly interva s ~r d teet behavioural oestrus.Ewes were run ~ith teaser,ram~GFo ~elease were monitored byOvarian oxytoc~n and uter~ne o2~

measuring OT-N and PGFM respect~velYterone the length of theIn ewes given exogenous pr)oges '0 ificantly lower

f 9 4 + 0 9 (SE days was s~gnoestrous cycle 0 • _. ted ewes of 17.3 ± 1.7 daysthan that found for the untre~ d no significant peaks of OT-N(P<0.05). The con~ro~hew:se:e~w~iven exogenous progesterone ator PGFM. However ~n os 0th PGFM or OT-N was observed.least one detectable peak of e~f ~~-N were observed without aOn day 3 before oestrus peaks 0 t t'ons On day 2 before

do' e in PGFM concen ra ~. 0correspon ~ng r~s 0 of ° ant and concomitant peak ~noestrus there was a s~gn~ ~c trationsboth plasma OT-N and PGFM concen tero~e administered to

These data suggest that irog~Srtens the cycle by inducingewes early in the oestr~u~ ~~CI~t:a~ oxytocin and uterinethe premature release 0 0

prostaglandin F2~' J t 1 Prostaglandins 10:199 (1980).(1) Fairclough, R•• e ado k E L J Reprod. Fert. 67:215(2) Flint, A.P.F. and Shel r~c, •• •

(1983) J C t al 8iol Reprod. 23:1046 (1981).(3) ottobre, •• e • •

8485

CIRCULATING PROGESTINS IN PREGNANTRATS WITH DENERVATED OVARIES

P.J. Roche, G. Jenkin and W.R. Gibson

Department of Physiology, Monash UniversityClayton, Vic. 3168. '

When luteal tissue is t. d 0

progesterone secretion 0 re~te wIth adrenaline or noradrenaline,th JS stImUlated (1) It·

ese catecholamines have a". : IS not clear whetherprogesterone secretion (2) W PhY~IologICal role in regulatingthe ovaries of . e preVIously found that denervatI'orl (If'pregnant rats dProduction of progestins c Id oes not cause abortion (3)The present experiment was deo~ 'd nevertheless, have been affected'

Both ovaries were dene;~;~:d to test. this possibility. .vascular pedicle and the . by brIefly freezing the ovarians 1 ovarIan suspens l'a~p es were collected bet ory Igament (3). Blood

4 7 10 12 . ween 2 and 10 d 1" , ,14,16,18 and 20 f ays ater on daysor 3 days. progeste~on~reg:a~cy. Each rat was sampled on 2(20a-OHP) were measured using spe if. n

~O~-hYdroxypregn-4-en-3_oneProgesterone and 20. c IC radloImmunOassays.

completely denervated ova ai oOHPd

concentrations in rats Wit'lth . r es id not diff' J

ose ~n unoperated or bilater 11 h er sIgnificantly fromstage of pregnancy (Table 1) a ~vas am-operated controls at anylosses also were not affected b 'th d r)an ~eights and reproductiv~

y e enervatIon.TABLE 1. PI

asma co.ncentrations (ng/ml',progesterone and 20a-OHP In pregnant rats. meanis.e.m.) of

DAY OFPREGNANCY

47

101214161820

n

3-46-85-87-106-85-74-62-3

PROGESTERONEUnop. Sham-op. Denerv 20a-OHP

LOW AMOUNTS OF OESTRADIOL-17S REDUCE THE OVULATIONRATE OF MERINO EWES

N.R. Adams and S. Atkinson

Division of Animal Production, CSIRO, Wembley, W.A. 6014.

The mechanism which controls the number of eggs shed by the ewe atovulation is unknown. In particular, the signals from the ovulatoryfo] licle which prevent other follicles from ovulating have yet to beidentified. In this study, we show that relatively low backgroundlevels of oestradiol-17S can reduce the ovulation rate.

Implants were prepared by sealing crystalline oestradiol-17S in601-285 Dow Corning silastic tubing (o.d. 3.18 IllIIl, Ld. 1.57 nnn). Therelease rate of these implants in vitro, estimated using (3H)-oestrad­io1, was 0.58 ~g/10mm/24h. Mature Merino ewes were stratified on liveweight and allocated at random to 3 groups of 43 ewes (Expt 1) and 4groups of 36 ewes (Expt 2). Ewes were fitted ,with oestradiol implantssubcutaneously and with vaginal progestogen sponges to synchroniseoestrus. After 12 days, the sponges were removed and 7 days after thisthe implants were removed and the ovulation rate was determined by1aparoscopy, performed under local anaesthesia.

TABLE 1. Effect of oestradiol implants on ovulation rate.

Implant Estimated Ewes classified by CL OR, Eweslength(mm) 24h release (~g) 0 1 2 ovulating

Expt 10 0 4 26 10 1.28*8 4.5 6 34 3 1.08*

16 9.3 9 30 2 1.06Expt 2

0 0 3 19 14 1.42**1.2 2 30 3 1.09

4 2.3 3 25 8 1.248 4.6 2 26 8 1.24

* **p<(I.OS, P<O.Ol -different from 0 by Chi-square analysis

I.E. (1977) Acta anat. 97:286-2!Hl.H.C. and Gibson, W.R. (1985)

(1) Norjavaara, E., Selstam,.lQQ..: 613-622.

(2) Burden, H.W. and Lawrence(3) Roche, P.J., Parkington'

Reprod. Fert. 75:653-661.'

G. and Ahren. K. (1982) Acta endo(;.

J.

Ovulation rate was significantly depressed in Expt 1 using 8 mmimplants, and -in Expt 2 using implants as small as 2 mm. Thus, lessthan 4~lg/24h oestradiol depressed ovulation rate, presumably by in­hibiting follicle maturation and ovulation. Oestrogen production bythe ovary is estimated to be about 1~g/24h during the luteal phase, andup to 6-8~g/24h during the follicular phase of the oestrous cycle(1,2). The amounts of oestradiol used in this study fall within thatrange. The data therefore support the hypothesis that oestradiol playsa role in the physiological control of ovulation rate in the ewe.

(1) Cox, R.I., Mattner, P.E., Shutt, D.A. and Thorburn, G.D. J.Reprod. Fert. 24: 133-134 (1971).

(2) Baird, D.T., and Scaramuzzi, R.J. J. Endocr. 70: 237-243 (1976).

8687

Table 1. Structure ofluteinizing eel Is at 12 hours

R.T. Swann and N.W. Bruce

STEROID SECRETION FROM WHOLE CORPORA LUTEA IN VITRO, COMPARISON OFMEDIA AND TO IN-VIVO SECRETION RATES IN RATS-AT DAY 16 OF GESTATION.

5.17 ± 0.86

M1990.041 ± 0.007

2.05 ± 0.390.109 ± 0.01812.n ± 2.00

3.20 ± 0.80

1.27

0.0140.730.0232.56l.15

5.20

MEM0.044 ±

2.21 ±0.107n.763.78

4.89 :!.: l.03

KRB

0.066 ± 0.0153.05 ± 0.79

0.081 ± 0.0159.00 ± 1.661.60 ± 0.38

P (ug! 11 per CL)% of in vivo rate20a% of in vivo rateRatio20~PTotal progestagens% of in vivo rate

Values are mean ± s.e.m., n = 11.Since the whole CL still retained a connective tissue capsule and

then' was no recirculation of the media, a secretion rate 5% of thatfound in vivo is quite good. Calculation of this percentage, wheredata were~ilable for direct comparison, reveals most other workersachieve 1-2% with some achieving 7% (2). The ratio of 20a and Psecretion was the reverse of that found in vivo (0.45) and data on 200in vitro limited so no comparisons coul~be made. The present work;;nfirms other findings (2) that culture of whole rat CL maintainsprogestagen secretion rates, better than dispersed cell or tissueslice preparations, and sufficient to compare other measures ofmetabolism such as oxygen consumption measured in vivo (3).(1) Uchida, K. et al. (1970) Endocr. japon. 17~499-507.

(2) Greenwald, ~S~et al. (1984) Biol. Reprod. 30: 93-104.(3) Swann, R.T. and Bruce, N.W. (1985) Proc. A.S;R.B. Abstr. 86.

Many different approaches to in-vitro maintainance of corpora lutea(CL) are used, ranging from dispersed luteal cells and tissue slicesto whole CL. The rate of secretion in vitro has, however, seldom beencompared to that found in vivo. The-aim here was to develop a simplemethod for short term incubation and to relate this secretion rate tothat found in intact animals at Day 16 of gestation, the time ofmaximum secretion (1).

Corpora lutea, with a layer of connective tissue adhering to theirsurface, were quickly dissected from ovaries kept in ice-cold saline.Some were immediately frozen for latter assay of steroid content atzero time (TO)' The remainder were incubated in one of three media,Krebs Ringers bicarbonate (KRB), media 199 (M199) and Eagles minimumessential media (MEM). The latter two were buffered by Repes saltsolution. Incubation was carried out for 2 hours at 370 C in a 5% C02air mixture. Each incubate tube contained 3 CL, about 12 mg, and 2 mlof media and was rotated gently. At the end of the incubation the CLwere removed and assayed for steroids (T2) and aliquots of the mediaalso analysed (M). Progestagens, progesterone (p) and 20(1 hydroxy­progesterone (20a) , synthesised and secreted were expressed as ug ofsteroid per CL per hour and calculated by (M + (T2 - TO))!2.Progestagen production was compared to that found in vivo (2 and 0.9ug! 11 per CL for P and 20a respectively) and expressed as a percentageof the in-vivo production.

Department of Anatomy and Ruman Biology, The university of WesternAustralia, Nedlands, W.A. 6009.

Department of Anatomy dan Hum~n Biology, The UniversitAustral I a. Nedl ands 6009. y of Western

At ov uI at i 0 I II neap laries invade th bc~~~~~~'wiJ~u~i~~a~~~qs~ eel Is adja~:~tr~~at~:a~~~~:aa~:~nt~e thecaH?wever, those eel Is i~Y~~~ ~~~~:ents and luteotrophic stlmul :~medlate~frect blood supply untl I vascula~r~:tihe ~uPtured follicle have noours after ovulation. This stud on s complete a further 24-36

t~e degree of luteinization in th~s:as d~slgned to determine whetherc Ose apposition to a capi II ary n tw cekn. ral cell sis limited unti I

e or IS establ ished.Three ruptured fall iclas from the24 hours of gestati on were process~ eft ovary of 5 rats each at 12 andstandard stereol og Ica I methods d tor el ectron microscopy .Us inorga~el les was collected from 5 ~~~~tltative data on steroidogenic gexaml~ed per corpus luteum. Cells e~ e~ch/rom 2 regions of tissuegranu Osa cell s adJ acent to th am ne from REG ION 1 wereexamined from REGION 2 . e vascular theca Interna C I Id' were I n the t . e s

Istance fram a direct blood sUPPly~en re of ruptured follicles some

Results for eel Is examined at 12eel Is within REGIONS 1 and 2 had hours a:e p:esented In Table 1. Tht~rms of their steroidogenic mamba quantitatively similar structure?s owed a marked increase I :anes. Cells examined at 24 h Inquantitative difference Inn steroidogenic organelles with no ours~;GIO~S examined. Vascular~;~tf~~~eJer measured between the two

e tissue. a only proceeded to REG ION 1 of

THE INFLUENCE ,OF PROXIMITY TOBLOOD CAPILLARIES ON LUTEINIZATION

~~r>iir~.¥_L ..-M~~.c

(Mean+SEM) .

Gytop/ asm vol ume ---B.E~.olLL-Bf~.Q.t:J ZMI tochondr Ial vol ume 510+19 470+32 um3SER vol ume 31+4 29+5 um3Li,pJ.Q-~r>l.um.fL 11+4 9+3 um3Surface area ~;-~~b;~~;;--------2Q±a-------2~2_-.um2Rough Endoplasmic Retlcu/umSmooth Endoplasmic Retlc I 537+32 590+57 um2Inner mitochondrial u urn 327+34 391+53 um2.Q.u1~Lml.:tr>.!;.b.Q.D.Q.c1.Ql 673+26 598+19 um2

-------------~az±~~ ~21±~mZThese results indicate that bgranulosa cells Is not Ilmite~ ~2 ~ours structural luteinization ofsUPply. The leakage of plasma f~ he absence of a direct blood~~~~~~nesis p:oba~ly ensures the~ ~:~t~:fc~~~~ sprouts during luteal

orne stlmul I for luteinization s are not deprived ofstructural requirements ft. . However, despite havinghave restricted synthesiso:n~ eroldogenesis, these central eel Is maytransport mechanisms are estab~~c~e~ion O! steroid unti I propercontact With patent capillar'les.ls e. ThiS would necessitate Intimate

89

ISOLATION uF GRANULATED CELLS FRON THE HETRIAL GLAND OF THE PREGNANT RAT

M.E.D. Cerini and J.D. O'SheaDepartment of Veterinary Preclinical Sciences, University of Melbourne,

Victoria 3052.

Metrial gland tissue was dissected from pregnant uteri of Hooded wistarrats on Days 12 - 15 (Day' 0 = oestrus) ana placed in Hanks' balancedsalt solution (HBSS) at 4oC. Tissues were diced in HBSS, centrifugedand incubated .for 2 hours in HBSS containing 400 u/ml collagenase at37 0C in a shaking water bath. Ouriny ~ncubation tissues were dispersedtwice using a pasteur pipette, ana finally filtered through a sterilecloth (square holes, 150 j1m). The eluate was washed twice with Dul­becco's'modified Eagles medium (UMEM), suspended in OMEM containing 20%foetal calf serum (FCS), and further dispersJP by syringing through 23 ­25 g needles. 1 ml of suspension (- 2 x 10 cells) was layered onto asolution of Ficoll, adjusted to a density of 1.033 with HBSS, and cen­trifuged at 300 g for 30 min. at room temperature. Cells formed twofractions, one on top of the Ficoll and one beneath the Ficol1. Thesefractions were washed twice and resuspended in OMEM with FCS. Samplesof the original suspension and fractions were counted in a haernocyto­meter and categorized as to size with a calibrated eyepiece micrometer.Cell viability was assessed by Trypan blue exclusion. Samples for lightmicroscopy were prepared by cytocentrifugation and stainea with a modi­fied wright's stain (Diff-Quik), and additional samples of metrial glandtissue and free cells were prepared for electron microscopy.

Based on 9 re~esentative preparations, the original cell suspensioncontained _ 10 cells per metrial gland of wnich 11.4 ! s.d.3.8% weregranulated cells. Mean viability was > 9m~, and cells ranged from 7 ­20?m in diameter. Following centrifugation on Ficoll, granulateQ cellswere concentrated in the top fract i on, wh i ch conta i nea - 6 x 10 ce 11 sper metrial gland with 42.4 ! 12.9% granulated cells. Light and elect­ron microscopy confirmed the identity of the granulated cells, and show­ed that the top fraction contained both large (II rnature

ll) yranulated

cells and smaller (ll precursor") cells similar in size and morphology tothose in the original tissue. Binucleate cells made up 2.6% of the gran­ulated cells in the original suspension and 0.7% in the top fraction .

We conclude that this method produces an approximately fourfold concen­tration of the granulated cells from the metrial gland. These cell pop­ulations are now being used for functional studies.

Granulated cells of maternal orlg1n, whose function has not been estab­lished, are present in the pregnant uterus in several species includinyman" In rats these cells form perivascular arrays in the II

metrialgland" within the mesometrium. 'vJe report a method for separation andpartial purification of granular cells from the metrial gland, to permitfunctional studies in vitro.

88

SPACING OF RAT CONCEPTUSES IN THE UAND A "COMPUTERUS" ESTIMATE OF TERINE HORN: EMPIRICAL OBSERVATIONSTHE STRENGTH OF MECHANISMS INVOLVED

N,.W. Bruce, A. Rahima and W.F.C. Blumer

Department of Anat domy an Human Biology, The University of WesternAustralia, Nedlands, W.A. 6009.

In most polytocous species conce tualong the uterine horn Th' h P ses appear to be evenly spaceda definitive estimate ~f "e~s p e~omefnon is seldom quantitated and indeedth veness 0 spacing has 0 1 b

e rabbit (1). Mechanisms hypothesi d . ' n y een attempted forinclude (i) each conceptus 'l se, to expla~n the phenomenon

, h' . on ~mp antat~on sets up f"w~t ~n wh~ch no subsequent conc t ' a zone 0 reJect~onoccurs at random but differ t.ePl us can ~mplant, (ii) implantation

d en ~a growth of the ut 'en ometrium evens out initial discre anc' er~~~,wall orexpansion together with sequent' 1 ~ ~~s (2) and (~~~) blastocystphysically prevents close impla~~at:~~r7t~onalong the uterine hornaddress the essential question f t 1). These proposals do not,osrength of the spa ' ,so prov~de little information on likel b' , c~ng mechan~sm andconceptuses space like soldier d ,Y. ~olog~cal factors involved. Doth s ress~ng to the right d 'f

e average length of their arm? W an ~ so, what ishere. . e attempt to answer such questions

Eveness of spacing was examined i~22 o~ gestation. Each uterine horn was 30 rats at Days 7, 10, 13, 16 ortens~on of 5g and the distances b t stretched in a water bath to anearest mm. An average ' t e ween conceptuses measured to the

, ~n er-conceptus distan d hvar~ation of this distan d' ce an t e coefficient of, ce was eterm~ned for each t

exam~ned. The coefficients of 't' s age of gestationDays 7, 10, 13, 16 and 22 respe~:~~al~on were 28, 32, 19, 35 and 35% atdeviation / mean) less tha ld~~e y. Are these coefficients (standardimplantation sites along thn wou

t,e expected by random selection of

e u er~ne horn?To answer this we developed a "C .

used a random number generator t ~m~uterus 1" basic program whichconceptuses in the same ave ~ pos~t~on the same average number ofthe empirical observations raTghe, ength of uterine horn determined from

h • ~s returned coeffic' t feac stage of gestation examined clear1 in' ,~en s 0 about 100% atto space conceptuses substantiall b t Y dlcat~ng that the rat managesand further, that spacing does ~ ,e ter than a random number generator

A "Computerus 2" program w:o t~mprove with advancing gestation.were allowed to implant at d s en developed ~n which conceptuses

Iran om as before but

c oser than a nominated zone f " no conceptuses could bedifferent values until th 0 lreJectlon. 'I'his zone was accorded

e resu tant coeffi i t f 'approximated those found "1 . c en s 0 var~ationcalculated zone was 5-6 emplr~ca l~ •. At Day 7 of gestation thed' mm or two th~rds the actu' 1 '~st~nce observed, 8.8 ± 0.6mm. It a a mean Inter-conceptusf~ndlng that from a total of 85 ,greed closely wIth the empiricalwere less than that theoret'c IlPosslble distances, only two (4mm each)

... a y expected.Whatever the mechanisms involved in .,' .'

they o~rate over distances of 5mm. Th' spaCIng, tl'llS work shows thateXpanSIOn (1) and favours a zone of ,~s ~learly rul~s out blastocystthe model to test the rejection abil~eJectlon IJ~lenOInen~n. We can now use

" ty of loca" exp',rIP:,'n~al stimuli.

(1) Boving, B.G. (1956) Am. J. Anat. 98, 40'-j'·12) Rogers, PAW I -" ~ . .,J-, •• • • et~. (1982) J. Reproc]. ['P/'.

9091

University, Clayton, Vic. 3168,

EFFECTS OF FORSKOLIN ON GUINEA-PIGSTEROIDS

S. Hall and J.N. Pennefather.Department of Pharmacology Mona hAustralia. ' s

MYOMETRIUM: THE INFLUENCE OF OVARIAN UTERINE PRESENSITIZATION AND EMBRYO SURVIVAL IN BOOROOLA CROSS EHES

D.O. Kleemann, S.K. Walker*, J.R.W. Walkley+, D.H. Smith, R.J. Grimson,P.J. Verhees+, R;W. Ponzoni+, B.A. Stone* and R.F. Seamark*

TABLE 1. Least-squares means (SE) for OR, RR(%), ewes with viableembryos (EVE)(%) and number of viable embryos per pregnancy (NVE/EVE).

Probability values presented (ANOVA) were similar to thoseobtained from an SAS CATMOD procedure with one exception: NVE/EVE valuewas 0.62. Values for treatment x experiment interactions were> 0.31.

Turretfield Research Centre, Department of Agriculture, Rosedale,S.A. 5350 * Department of Obstetrics and Gynaecology, University ofAdelaide, The Queen Elizabeth Hospital, Woodville, S.A., and

+ Department of Agriculture, Adelaide, S.A.

NVE!EVE

U.06

1.70(0.14)2.11(O.16)

EVE n

0.25

44.7(6.1) 2834.9(5.9) 23

RR

0.82

40.9(6.2)42.9(6.1)

OR

0.07

2.73(0.10)2.99(0.10)

n

Probability

Control 63Presensitized 67

No explanation can be given for an increase in OR due to treatmentother than the result was a chance event. At day 35 after inseminationthere was no difference between treatments for the percentage ofpregnant ewes (EVE). However an increased number of viable embryos perpregnancy (NVE/EVE) in presensitized ewes is ~oQ9i~tent withimprovements in litter size in other species ~1)~2). Moreexperimentation is required to substantiate these results.

Reproductive potential of the Merino can be increased by a numberof techniques (e.g. incorporation of the Booroola gene, immunizationagainst steroid hormones). However, this potential is limited by highembryo mortality. Primary immunization or sensitization to paternalantigens has improved embryo survival in laboratory rodents \1) andpigs (2). Two experiments (1 1/2, 2 1/2-5 1/2 year-old Booroola xS.A. t~rino ewes, respectively) were conducted in 1986 to determine ifpresensitization of tre uterus with killed semen, 17 days beforeinsemination with viable semen, improves reproductive efficiency.

Ewes were inseminated into the uterus with approximately 60 x 10 6

killed spermatozoa per uterine horn (in 60 ul) 51 hours followingprogestagen sponge (Repromap, 60 mg) removal and injection of PMSG (400i.u. Pregnecol). Control ewes were not inseminated. All ewes wereresynchronized with progestagen treatment between days 7 and 15 of thesubsequent cycle and inseminated with viable thawed semen. In additionewes were joined with the same ram that contributed semen for bothinseminations. Ovulation rate (OR) on day 4 and rate of return tooestrus (RR) on day 35 after insemination were determined. Ewes wereslaughtered on day 35 and the number of viable embryos counted.

W~ have now examined the effects of f ' ,c~rcular and longitudinal myomet" °brsk~l~n on f~eld-stimulated strips ofM t' 1 r~um 0 ta~ned from ad It ' "yome r~a preparations were taken f " u v~rg~n guinea-Pigs(') rom Dunk~n-Hartley guinea-pigs. '~ untreated, on day 9 or 10 f .

(ii) treated for 14 d ~ the oestrous cycle;ays S.c. w~th oestradi 1 '

beginning on day 9 or 10 of th 0 cyp~onate (20 ~g/kg)(iii) treated as in (ii) then' e oestrous cycle}

oestradiol cypiona~e (20 g~;~n)progesterone (3 mg/animal s.c.) and(Hartley et aI, 1983). ~g g for a further 4 days

Each preparation was stimulated with 'every 100s as described by Hartle et5s1tra~ns of pUlses (60V, 2ms, 30 Hz)forskolin Or isoprenaline by y, a (1983), and exposed to eitherof the drug to establish a lsequent~al addition of increasing concentratio

og concentration_ , nsresponses were expressed as ercent '"' :esponse relat~onship. Thecontractions. p I age ~nh~b~t~on of electrically-evoked

~s :e~orted earlier from this laborator (Hart~nh~b~ted electrically-evoked contra t'y , ley et aI, 1983) isoprenalinelayers of guinea-pig myometrium a dC,~ons ~n both circular and longitudinallongitudinal layer from animals ~ n

t~ts,potency increased in the

maximum in tissues obtained f rea e w~th ovarian steroids, attaining arom group (iii).

Forskolin was also consistently inhibitoobtained from the animals in each f th ry on all myometrial preparationscontrast to isoprenaline th foe three treatment groups In

d . , e e fects of for k l' .an unaffected by steroid treatment. s 0 ~n were longer lasting

The differences observed in the act'to their different sites of a t" ~ons of the two drugs could be attributedd" c ~on. Forskolin t'~rectly by interacting with th " ac ~vates adenylate cyclase

cell 1 e catalyt~c sUbunit th b 'u ar levels of cyclic 3' 5'-AMP ( , ere Y ~ncreasing

exerts its inhibitory action~ via co~~amo~ & D~ly, 1983). Isoprenalineadrenoceptors thus activati d ~nat~on w~th membrane linked 82-in' ng a enylate cyclase and ' d'

crease ~n tissue cyclic 3' 5'-AMP 1 ~n ~rectly causing ancat 1 t' , evels We sugg t th. a y 1C sUbunit of myomet~jal d . es erefore that thesteroids. .. a enylate cyclase is unaffecb"d by ovarian

Har~~~y~3~i~;: Pennefather, J.N. & Story, M.E. (1983) Br.J.Pharmac.

Seamon, K.G. & Daly, J.W.D. (1983) Trendsin Pharmac. Sci., i, 120-123.

(1)

( 2)

Beer, A.E., and Billingham, R.E. (1974) J. Reprod. Fert., Suppl.21: 59-88.Stone, B.A. (1985) PhD Thesis, Univ. Adelaide.

9293

C.S. LEE, F.B.p. Wooding* and M.R. Brandon.

Department of Veterinary Preclinical Scienc ' ,and *Department of Cell B' 1 ' es, Unlverslty of Melbourne

, lO ogy, Agrlcultural and Food ResearchCouncll, Babraham, Cambridge, England.

, Recent studies have shown that the ,ralsed , against trophoblast microvilli fro:onoclonal antlbody, SBU-3,recoglll.se a population of b' 1 the placenta of the sheepsheep placenta (1). The lnuc eate ~ells and the syncytium in thed · , . present studles in t'lstn.bution of the SBU-3- ositive ' ves 19~te the subcellular

technique. p antlgen by USlng the inununogold

Tissues from placentomes and inter 1from Clun Forest or So h p,acentomal areas were obtained

ay seep kllled 'hpentobarbitone sodium at 29 41 I Wlt an overdose ofAll t' , , 87, 123 and 140 d flssues were fixed in 2% 1 ays 0 pregnancyAfter fixation all tissues were e;b::~r~l~ehYde ~t room temperature:mesh uncoated nickel 'd e In Araldlte. Sections on 300

grl s were reacted 'th hfor 30 minutes at room temperat Wl t e monoclonal antibody

, ure. After wash' th 'reacted wlth colloidal gold labelled l~g, e sectlons wereanother 3:) minutes at room t goat antl-mouse antibody for

f b' emperature. To f '1'o lnucleate cells and syncytium some ,acl ltate localisationphosphotungstic acid in 3.5% HC f s~ctlons were stained with 1%

Gold part' 1 ~ or 15 ffilnutes at 60 0 C.lC es were locallsed . 1

bodies and the characteristic .; most_exclusively to the Golgisimilar granules in th ':Jra~u es at the uinucleate cells and

e syncytlum. N th~rganelles, connective tissues or extra . 0 0 er placental celldbove background levels. Quantitati cell.ula,r s~ace showed labellingpercentage of placentomal ve . examlnatlon showed t.hat theSBU 3 " mature blnuclpat 11

- -POsltlve increased rapidly from a 1 -. e ce s that were~regnancy to a plateau of virtually 100% fr:; level (13%) at 29 dayslnterplacetomal binucleate cell 1 41 days to term; whereasThere were no detectable mor r7o;are y showed labelling at any stage.and u:Jlabelled cells. p oglcal dlfferences between labelled

The results confirm that the ant'product. of the binucleat 11 1gen recognised by SBU-3 is aof syncytium formation b~ :igr'ati:eYotl:~ corroborate the hypothesislocal control of the SBU 3 " lnucleate cells and indicate

' - -Posltlve t' .Posslble role in villus formation. an 1gen J:lroduction and lts

A TOPOGRAPHICAL COMPARISON OF IMPLANTING AND DELAYED-IMPLANTINGBLASTOCYSTS FROH NATURALLY I~ATED. RANDor~LY BRED MICE. Ann C. McRae andR.B. Church. Department of Medical Biochemistry. University of Calgary.3330 Hospital Drive N.W .• Calgary. Alberta T2N 4Nl Canada.

The purpose of this study was to determine the morphological stage atwhich blastocyst differentiation is interrupted under the conditions ofdelayed implantation to facilitate investigations of the cellular pro­cesses inducing this interruption. Such studies are part of anelucidation of the factors regulating expression of embryonic anduterine-progestationa" and embryonic prerequisites for implantation.Groups of mice were killed every 3 h from 6 p.m. on day 4 (day 1 =vaginal plug) until 12.01 p.m. on day 5. Delayed implantation wasinduced by bilateral ovariectomy under' nernbutal anaesthesia on theafternoon of day 3. All ovariectomised animals were given 0.5 mgprogesterone in sesame oil. Embryos were recc;vered by flushing exciseduteri with fi1 /<1 containing 2% BSA. Totals of 184 blastocysts from 33control anim~ls and 162 blastocysts from 38 delayed-irnplantirg animalswere examined and photographed usin9 a Zeiss inverted microscope. ThephotOlnicl"ographic images (X32 and X128) WHe scc.lred in accordance \-nth5 predeterlmned morphological febtLirts. n,l';se data pemitted thefoliowing observations.

Firstly. 9 p.m. on day 4 was the earliest time that populations ofimplanting and delayed-implanting blastocysts could be distinguished bythe proportions of each eXhibiting a more differentiated form of eachfeature. That is, by 9 p.m., more contrel blastocysts had lost theirzona (1). had trophoblastic cells which were rounded or ruffled wit~

cvtoplasmic projections and not apparently elongated and smooth (II),had a blastocoele occupying less than the majority, instead of thel1ajority. of the blastocysts ' volume (III), and I:ad an reM whichappeared as a dense cone instead of a thin disc (IV). Furthermore.these blastocysts were significantly more elongated than delayed­implanting ones (V).

Secondly. by 6 a.m. on day 5. individual blastocysts can bedistinguished with confidence as bein9 implanting or delayed-implantingon the basis of the cuter margin of the trophoblast. Fifty-two of 54implantirg blastocysts recovered from 12 animals killed at 6 a.m. orlater exhibited cytoplasmic projections covering varying degrees of theblastocyst s,urface. By contrast. only L of 62 delayed-implantingblastocysts showed any degree of cytoplnsmic blebbing.. Ther8f~re, .blastocyst differentiation appears to be interrupted ~r10r to turmat10nof these cytoplasmic projections by trophoblast cells.

Thirdly. detailed examination of the data suggests several morph?­logical correlates for the projection of trophoblastic cytoplasn. Unesuch correlate. possibly causc:l in nature. is the rounding up of tropho­blast cells which appears to occur as an intermediate stage between theearlier elongated c81ls and the later ruffled.ones. Two .oth~r

correlates, possibly coincidental in nature. ~s that proJect10~ oftrophoblastic cytoplasm occurred exclusively 1n blastoc'yst~ Wh1Ch hadlost their zona and had experienced some degree of growth1n the ICM.

In conclusion. iJ1dividual implanting blastocysts might bedistinauishable from delayed ones, prior to apparent ruffling of thetropho6last cell surface. on the basis of the~r cytoskeletal a~d ~ellsurface propertie~ since this type of geomet~lc changes o~ten 1nd1catesenhanced cytoskeletal mobility. (Alberta Her1tage Foundat10n support.)

Lee, C.S., Gogolin-Ewens K.,Anat. 1.4.Q:565-576 (1985)' vlhite, 'l'.R. and BratldQr" M.H., J.

(1)

9495

, . P and a low level o~ E2 sh~wedAs expected those am rna1s treated

dWll th d increase in uterl ne wel ght

a signific~nt (P < 0.001) buut i~/ype level was significant~y (P <begi nni~g h0; ianbO~:os~aYan~~al s tne:t recei vi ng Ef S~g~~sti~~ a; 1~~~:~~~~~Olih:lgexetra or i~tra-uterine clea:eaennceino the level of c~tosol oranimals. No dramatlC. changets r~~~e Any significant changes were notnuclear receptor for elth~~n~ erowthtemporally related to ute 9 ~h delayed uterine response to PTogether the results indicate thalt. e~ by an increased ability of the

1 1 of E is not exp aln t or any otherand a low eve t t~ P by classic steroid recep orsuterus to concen ramechanism. 6 33

(1979) Endocrinology. 104:2 - .(I), Miller B.G.

OF THE MOUSE UTERUS TO PROGESTERONEDELAYED GROWTH RESPONSE

Christina Murray and Grant Stone

Department of Veterinary Physiology, University of Sydney, ~006o esterone (P) require conslderable

Many physiological responses t,o prthge presence of a very low level o~

time to develop and also reiul~~ the increase in weight and ,l~ngthd ~stradi 01 (E ). One examp e onse whi ch can be mlml cke 1n

~~e mouse utefus ~uring pregndnc~thapr:~~ E (1). To study mec~anismsovariectomized anl~al~ irea~ere~~onse, ovar~ectomized mic~ rec~l~ed ~Oresponsible for th~s e da1yes 3-13 received either 1mg P Wlt~'l1' dn~ h~ng E on day 0 an on (t nt 2) Animals were 1 e(tre&tment 1) or without E25 t~ea9me11 and 13 and uterine weight, D~Aafter i njecti on on days 3 f t ri ne' P and of receptors for P and Ef ~nand pooled tissue levels ~. u e of uterine homogenates were meas~ e fcytosol and nu.clear.frac 110ns1 Data were analysed by analysls 0

1t re 9lVen 1 n Tab e .Resu s a I ltiple range tests.variance and Duncan s,mu ( / DNA) and cytosol and nuclearTable 1:' Uterine welght (mg), PNA)g mgreceptors for P and E2 (pmol/mg D .

Parameter Treatment

CONTINUOUS R~ORDING OF MYOMETRIAL ACTIVITY IN THE RAT BYVIDEO-LAPAROSCOPY

Len Martin and Linda H. Crane

Department of Physiology and Pharmacology,University of Queensland, St Lucia, Qld 4067.

We have used electromyography (EM) in conscious rats to recordmyometrial activity during early pregnancy to elucidate the role ofthe myometrium in embryo-spacing. However, EM gives incompleterecordings as electrodes are often lost from the uterus. It does notdistinguish between longitudinal and circular muscle and electrodes~ay influence myometrial activity. We therefore developed a recordingtechnique using video-laparoscopy.

Female Wistar rats are anaesthetised with xylazine (8 mg.jkg)­ketamine (60 mg/kg) IP, followed by ketamine (30 mg/kg) at 50 minintervals. EM shows that this regime produces transient decreases infrequency of myometrial activity in oestrous rats without altering itsorigin (ovarian or cervical), or propagation.

For laparoscopy, rats are placed on a heated stage ventral sideup, head down at 30° to the horizontal. A 1 cm slit is made in theskin wi th a 2 mm nick through the body-wall, 8 mm behind the xyphoidprocess. A 5 mm diameter trocar/cannula is inserted and held by aclamp. The trocar is removed, a gas line attached to the cannula andthe abdomen inflated with N2• A· 4 mm diameter laparoscope, insertedthrough the cannula to view the posterior 2/3rds of both uterine hornsand cervix, allows one to determine origin/timing of contractions,approximate strength, degree of propagation, synchrony between horns,propagation from horn to horn and nature of contractions, i.e.longitudinal, circular or peristaltic. A rectal thermistor monitorstemperature (37 ± 1°C). A low-light TV camera with timer-display,connected to a monitor, records activity. There is access formechanical/electrical stimulation of the cervix and IV injections.Animals recover and can be examined repeatedly; of 7 laparoscoped atvarious times over days 1-3 of pregnancy, 6 showed normalimplantations on d7.

In unmated oestrous rats (n=5), myometrial activity ispredominantly strong rhythmic longitudinal contractions propagatedtowards the cervix at a frequency of about 1/min wi th 70\ synchronybetween horns. Oxytocin (25-100 mU, IV), induces synchronousbilateral longitudinal contractions and adrenaline (2 ].Jg, IV),quiescence lasting for 2-3 min.

We conclude that video-laparoscopy is a practicable method formonitoring normal in vivo activity of the myometrium and its responseto exogenous drugs in varying physiological conditions.

Uterineweight

P

Cytosol Preceptor

Nuclear Preceptor

Cytosol E2receptor

Nuclear E2receptor

12

12

12

12

12

12

34541

3.53.9

2.02.0

.58

.58

2.01.9

.40

.52

57765

8.24.6

2.02.3

.37.30

2.02.0

.34

.44

Day7

5558

4.01l.5

1.51.4

.32.29

2.01.6

.36

.50

96459

3.96.4

1.92.4

.36

.27

1.81.7

,45.40

118445

4.77.3

2.01.3

.46

.26

2.31.9

.41

.46

138045

3.97.2

3.51.7

.42.27

2.41.5

.48.36

96

THE EFF"OCT OF EXOGENOUS OVARIAN SMORTALITY AND EL"OC'ffiON MICROSCOPY TEROIDS ON EARLY »IBRYONIC

OF THE ENDOMETRIt:M OF THE PIG

D.K.H. Putra and A.W. Blackshaw

Department of Physiology & Pharmacology, U' ,St. Lucia, Queensland, 4067. n~vers~ty of (peensland,

Considerable pre-natal mortalit (pig, and the majority of embry 1 y 35-35%) occurs naturally in thef' 0 osses (30%) t k ,.~rst 40 days of gestation (P,':lpe & First a es place w~ thin the

that intra-muscular injl"ctions f ' 1985). It has been foundpreqnant sows increased li~ter SiZ~ estrone ?lus progesterone intoet al.., 1981). The present stud - at~ term (~~ldt et al.., 1976i de Saduring the first 30 days of y, u~ exam~nes embryonic mortalitywith steroids. preqnancy ~n the pig following treatment

~ group of pregnant sows received d '1 'of 12.5)Jg estrone plus 25 m ' ... a~ y ~~tra-muscular injectionsstarting on day 4 of pregnane; (~:og~s,=-erone ~n 2 ml of peanut oil,consecutive days while t y, - stdnd~nq estrus/mated) for 10

• ' - can rol an).mals we " ,only. Embryo survival rates (ratio of re ~nJected w~th vehiclenumbers) were determined at about da :mbr~os to corpora luteawere fixed and then processed flY 30. ,Uter~ne samples at day 16

or e ectron m~croscopical study.

Th~ results showed that there was " , .embryon~c mortality rates b t no s~gn~f~cant dtfference in(16 4 ) . e .ween treated (20 4%) d• % • :veights of embry - • an control animalssignificantly (treated: 1.71 os at day 30 also did not differsteroid administration plasma g vs. control: 1.63 g). Followingmarkedly throughout th~ in' t' proge~terone concentrations increasedth I I Jec ~on per~od (27 62 18 '

e eve dropped to almost control val f' vs. .16 ng/ml), butues a terwards.

Electron ~icroscopy showed no clear- tin the endometr~um following treatment. cu ultrastructural changes

References

97

EMBRYO - UTERINE INTERACTION USING THEANTERIOR EYE CHAMBER IN THE RAT.

Peter Rogers,Anne Macpherson, Linda Beaton.

Department of Obstetrics & Gynaecology, Monash University,Queen Victoria Medical Centre, Melbourne, Australia.

The anterior chamber of the eye offers a privileged transplantation site whereforeign tissues can maintain a degree of normal function without being rejected.Earlier workers have demonstrated normal uterine responses to peripheralcirculating hormones following transplantation to the eye (1). In this study,small pieces of endometrial tissue were surgically placed over the lower portionof the iris in 3 to 6 month old female albino rats. Following successfulrevascularization of the implant these rats were mated with vasectomized malesto induce pseudopregnancy. On the morning of day 1 of pseudopregnancy (= dayof finding vaginal plug) eight cell embryos flushed from the oviducts of donorrats on the lJ.th day of pregnancy were transferred to the eye chamber containingthe uterine implant. A second series of experiments used non-pseudopregnantrecipients with vascularized uterine implants as controls. So far successfulimplantation has occurred in 19 out of 38 pseudopregnant and 9 out of 26 non­pseudopregnant recipients. Of specimens sectioned for light microscopy 6 out of18 pseudopregnant and 1 out of 7 non-pseudopregnant recipients show whatappears to be a decidual response in the uterine implant. The 6 out of 18pseudopregnant rats showing a decidual reaction all showed a columnar orcuboidal epithelium on the uterine implant. By contrast, the 12 non-decidualizedimplants did not have a demonstrable epithelium. The results demonstrate thatsuccessful uterine-embryo interaction can be achieved in the anterior chamberof the eye. Further studies are required to characterize the decidual responsethat such implantation produces. This model of embryo implantation is beingdeveloped for in-vivo studies of the uterine microvasculature during

implantation.

(1) Markee, J.E. (1932) Am. J. Physio!. 100 : 32-39.

1.2.

3.

P~pe, W. F. and Pi rst, N. L. (1985)Wlldt, D.E., Culver A ~ • ThePiogenoZo9Y, 23: 91-105

(1976) J. Repl'od.'Fe~t·~;.' Morcom, C.B. and Dukelow W'Rd r v/", 48: 209-211. ' ••

e Sa, W.P., Pleumsamran, P( ., Morcom, C.B. and1981) ThePiogenol.ogy, 15: 245-255. Dukelow, W.R.

98

IMMEDIATE INCREASE IN UTERO-OVARIAN OESTRADIOL FROM THE OVAFYDESTINED TO OVULATE AFTER PG ADMINISTRATION

*S. Atkinson, J.R. Ford, C.M. Oldham and D.T. Pearce

Department of Animal S~ience and Production, University ofWestern Australia, and CSIRO Division of Animal Production,

Wembley, W.A.

The time at which a follicle is selected for ovulation is unknown,though it has been identified at 10h (1) and between 30-54h (2) afterprostaglandin administration. The ovulatory follicle secretes theincreased concentrations of oestradiol (E 2 ) that occur prior to thepreovulatory surge of LH (3). This study was undertaken to determinethe' time at which increased levels of E2 were present in the utero­ovarian vein after luteolysis. This timing would then be indicative ofthe selection of an ovulatory follicle.

Four Merino ew~s in midcycle had cannulae inserted in both ute~o­

ovarian veins and in the jugular vein. After a 24h recovery period theewes were administered 125 J.lg of prostaglandin F2 (l analogue (PG) andblood samples were collected for the next / 4h. Samples from the utero­ovarian veins were collected at 30 minute intervals and analyzed for E2content. Jugular blood samples were collected at 15 minute intervalsand were analyzed for LH content. In addition, progesterone concen­trations were measured in jugular samples collected at hourly inter­vals. Laparoscopies were performed at 82h after PG administration todetermine which ovary had ovulated.

Prior to the injection of PG, both ovaries of each ewe secLctedsimilar concentrations of E2 (32.44 ± 3.3 pg/ml, ovulating ovary; 35.07± 2.7 pg/ml, non-ovulating ovary). Thirty minutes after PG adminis­tration, there was a pulse of E2 from the ovary destined to ovulate(mean pulse amplitude = 88.83 ± 8.5 pg/ml). This rise in E2 waspreceded by a pulse of LH in 3 of the 4 ewes. There was no increase inutero-ovarian venous E2 concentrations (x = 35.77 ± 2.1 pg/ml) from theovary that did not produce an ovulation. Peripheral prop,esteroneconcentrations decreased to 50% of the pre-PC levels by 3 hours afterthe induction of luteolysis.

These data suggest that the ovary destined to ovulate can be pre­dicted at the onset of luteolysis through increases in E2 productionand secretion. ,The increased utero-ovarian venous E2 was apparentlystimulated by a pulse of LH which occurred during. the initial phases ofluteolysis when the concentration of progesterone was still relativelyhigh. Further, the rapid response in E:?, presumably from the pre­ovulatory follicle, may indicate that these ovulatory follicles weretruly selected prior to luteolysis, but were only identifiable at theonset of luteolysis.

(1) McNatty, K.P., Gibb, M., Dobson, C., Ball, K., Coster, J., Heath,D. and Thurley, D.C. J. Reprod. Fert. 65: 111-123 (1982).

(2) Driancourt, M.A., and Cahill, L.P. J.Reprod. Fert. 2l: 205-211(1984) •

(3) Baird, D.T. BioI. Reprod. ~: 359-364 (1978).

99

OPHIN ON PERIPHERAL PROGESTAGENEFFECTS OF HUMAN CHORIONIC GONADOTR

ACOMPARISON WITH LUTEAL REGRESSION

CONCENTRATIONS IN CONSCIOUS RATS:

H.M. Cameron and N.W. Bruce

Anatomy and Human Biology, University of Western Australia,Department of Nedlands, W.A. 6009.

d ' r'ly from thein the rat, progestagens are secrete, pr~ma ~ which

During pregnancy CL are exposed to luteotroph~c hormonescorpora lutea (CL). The., d revent early CL regression. Theestablish progesta~en secret~o~ a~hePfine control of progestagenrole of luteotroph~c hormones ln Th' experiment used an exogenous

, ' however less clear. ~sproduct~on,ls, " 'c onadotrophin, hCG) in an effort toluteotrophln (human chor~on~ d

gd t rmine the reserve capacity for

maximally stimulate the CL an so e eprogesterone production.

, , ' t - rterially to chronicallyThe hCG, 25IU, was adm~n~stereddl~ ral~ of pregnancYi Day 1 is the day oncannulated rats (1) at Day 7 an, ay These two days represent low

f nd in a vag~nal smear. . dwhich sperm were ou ecretion (2) supported by pitultary anand high levels of p~ogesteronei:el. Serial blood samples wer~ taken atplacental luteotro~hlns resp:

ct

12a~d 24h after hCG administratlon and

0, 5, 15, 30, 60 m~n, 2, 4, , d 2oa-hydroxypregn-4en-30ne (20~-OHP) byanalysed for both progesterone an

radioimmunoassay.

, 25IU of hCG induced transitory peaks in bothAt Day 7 of gestat~o~ t 4 and 12h. However by 24h both progestagenprogestero~e and 2Da OHP ad t control values. In contrast, at ~ay 16,concentratlons had returne 0 ogesterone concentratlons and25IU of hCG had no stimulatory effect120n24Phr prior to this decline, 20~-

d 1 , of 57% over - • findeed caused a ec ~ne d t 'ed to rise to reach 250% 0OHP increased by 78% over 8-12h an co~ ~nual changes in progesterone and

1 b 24h These rec~proc th t .the original va ue, y • "ar to those described near term, a20~-OHP concentratlons we~e s~m~l To more closely examine normalis, during luteal regresslon ~3). t 'tion serial samples were

trations prlor to par url , D 21progestagen concen 2 4 hourly intervals from aytaken from normal, untreated,rats ~ttheO~ormal progress of lutealthrough parturition. compar~son 0 b hCG revealed that the decline in

. 'th the changes ~nduced Y ",regress10n Wl trations were s~m~lar ~nprogesterone and rise in 20~-OHP conc

et

ne concent-ation preceeded the

the fall in proges eron ~ ft hCGmagnitude, however , b t followed the rise a er -rise in 20~-OHP during normal regress~on u

induction.7 when the CL secrete sub-maximal

These findings suggest that at Day luteotrophins can stimulate at leastamounts of,progester~ne, exoge~o~s whereas at Day 16 when secretion istransient lncreases ~n product~o , 't is apparent. Indeedmaximal, little evidence of a ~eserve capac~ Y to mimic, in part, normalluteotrophin stimulation at th~s stage appears

luteal regression near term.

) J. Endocrinol. 100: 189-193.(1) Bruce, N.W. et ala (1984 I., (1974) Endocrinol 95: 275-279.(2) Pepe, G.J. and Rothchild,(3) Wiest, W.G. ~ ala (1968) Endocrinol. §l: 844-859.

100101

Georgetown

Daniel Dj akiew, Mark A. Hadley, Stephen W. Byers andMartin Dym

Department of Anatomy and Cell Biology,university, Washington, DC. U.S.A.

Huggenvik et. al. (1) proposed that systemic transferrindelivers Fe to Sertoli cells Whereupon the Fe istransferred to de novo synthesized testicular transferrinwhich is either secreted into the lumen of seminiferoustubules and/or taken up by germ cells. In order to testthis hypothesis confluent epithelial sheets of Sertolicells were plated on Millipore filters impregnated withreconstituted basement ~~mbrane and supported in bicameralculture chambers (2). Fe incorporated into human serumtransferrin (520pM) was placed in the basal reservoir ofthe culture chamber in contact with the basal cytoplasm ofthe Sertoli cells for six hours. Media overlying theSertoli cell epithelial sheet was immunoprecipitated withrat transferrin antibody, and found to contain 59Fe .Since rat transferrin antibody does not cross react«0.001%) with human transferrin, the presence 0l959Fe inthe immunoprecipitate indicates a transfer of Fe fromhuman transferrin (basal chamber) to rat transferrin(apical chamber) during transcytosis of the 59Fe acrossthe epithelial Sh~~t of Sertoli cells. The rate oftranscy~osis of Fe was 1. 65pM/hr/culture chamber(3.5xIO cells). Nonspecific binding (normal rabbit serumsUbstituted for the primary antibody) was 26% of totalCPM. Incubation of cultures at 4oC, fixation of Sertolicells in·2.5% glutaraldehyde, and addition of a 100 foldexcess of unlabeled Fe-transfe:o::in to the basal mediumreduced the immunoprecipitable 59Fe-transferrin to belowthe' level of the non-specific binding. When roundspermatids or primary spermatocytes (isolated by unitgravity sedimentation) were incubated overnight on

6top of

the Sertoli cell epithelial sheet (0.5x10 germcells/chamber) and the experiment repeated the next day,the amount of 59Fe-transferrin present in the apical mediawas reduced by 20% and 50% respectively. The prioraddition of erythrocytes, which don't contain transf~9rinreceptors, had no effect. This suggests that the rat Fe­transferrin secreted by Sertoli cells was bound totransferrin receptors on the germ cells.

(1) Huggenvik, J., Sylvester, S.R. and M.D. Griswold(1984). Ann. N.Y. Acad. Sci. 438, p. 1-7.(2) Byers, S.W., Hadley, M.A., Djakiew, D. and Dym.J. Andro11, p. 59-68.

IRON IS TRANSCYTOSED ACROSS SERTOLI CELLS BY TWO FORMS OFTRANSFERRIN, WHEREUPON THE IRON IS SECRETED AS TESTICULARFE-TRANSFERRIN WHICH SUBSEQUENTLY BINDS TO GERM CELLS.

2 3 4 5 61.7+0.1* 1.3±0.1 1.3+0.1

(n=9) 1.3±0.1 1.3±0.2(n=20) (n=11) 1.3±0.1(n-18) (n 9) (n-9)

Group

Ovarian weights (g) and FSH levelsand FSH levels (ng/ml) from days (area units; cm

2) 24 hrs after ULO

2-9 after surgery (mean+s.e.m.)

FSH: 0.-24 h 25 +

Ovarian

INHIBITION OF COMPENSATORY HYPERTROPHYIN EWES BY BOVINE

FOLLICULAR FLUID

B.K. camptiell,l R.J. Scaramuzzi 2 Y C ,2, • ogn~e and J.A. Downing2

Deparim~~~R~f D~i~a~ Husbandry, University of, ~v~s~on of Animal production, Sydney, Sydney, NSW

prospect, NSWUnilateral ovariectomy (ULO) ,

(COH) as determined by , results ~n compensatory h~ncreases in f 11' ypertrophy

and ovulation rate in th . , 0 ~cular growth, ovarian weic"httra ' t ' e rema~n~ng ovary U

ns~en ~ncreases in blood 1 • LO also results indisruption of steroid/inhibin feed:

velsof ~SH, presumably due to

been suggested as an explanation fo ack mechan~sms. 'Ibis FSH rise hasthat COH is due to a locall r COH (1). Another explanation isfollicular fluid (2) 'Ib' Y produced follicle regulatory fact 'd • ~s study examined COH ' or ~noses of charcoal treated bovine foll' 1 ,~n ewes given different

~cu ar flu~d (bFF).

In October, 54 BLM ewes we 1fOllows: Groups 1 and 2 ULO andre a located to 6 groups (n = 9-11) asa?d 4 ULO and sham operated res;:~~i~:;rated respectively; Groups 3(~ •v. ) ; Groups 5 and 6 ULO and sha y plus 1 ml, bFF every 8 hrsbFF every 8 hrs (Lv.). bFF in'e I,m operated respect~vely plus 0.2 mlsurgery at which time all ewes J ct~ons ~ere continued for 9 days aftet~ken at hourly intervals fromw~r~ ov~r~ectomized. Blood samples wer:~~mes daily thereafter. Ovarian wei:::

or~o 24 h after ULO, and three

e Table 1. s an FSH levels are recorded in

weight

* P<0.05 t P<O 1 S' 'f', '. ~gn~ ~cantly differentfrom sham control (Group 2).

~LO ewes exhibited COH in termsassoc~ated transient increase in of ovarian weight and ansurgery. ULO ewes given bFF (at F:H levels in the 24 h following'Ibe. ewes given 1 ml bFF (3 an~ 4) h oth doses), did not exhibit COHoper~~ ~fter surgery. In contrast t~d depres~ed levels of FSH over thehad s~m~lar FSH levels to sham t t e ewes g~ven 0.2 ml bFF (5 and 6)

rea ed ewes.

We conclude from this dataassociated FSH increase H that bFF will inhibit both COH and thpituitary or ovarian le;el ?wev;:, whether this inhibition acts at th:

~s s ~ll open to question.

(1) Findlay J K d ', •• ancumm~ng I A Bi(2) Fry, R.C., Clarke, I.J. a~d ~a~ill 01. Reprod. (1977). 17:178-183.

, L.P. (1985). Proc. ASRB, 12: 52 •

102

REINNERVATION OF THE RAT VAS DEFERENS FOLLOWING VASOVASOSTOMYAFTER VASECTOMY: A LONG-TERM STUDY

R.M. DeGaris and J.N. Pennefather

Department of Pharmacology, Monash University,Clayton, Victoria, 3168.

Loss of catecholamines and response to stimulation of nerve terminalsin the epididymal (proximal) portion of the rat vas deferens, whichpersists for up to 3 months following unilateral vasectomy by medialtransection (1) , can be partially reversed by vasovasostomy four weeks

,after vasectomy. Thus eight weeks later noradrenergic reinnervationis approximately 50% complete (2). In the present study we haveexamined reinnervation occurring (a) fifteen weeks after vasovasostomyfour weeks after unilateral vasectomy, to gauge whether furtherreinnervation occurs and (b) eight weeks after vasovasostomy thirteenweeks after unilateral vasectomy, to determine the influence of theinterval between these two operative procedures upon reinnervation.

Long-Evans rats (300-400g) were unilaterally vasectomised by medialtransection of the vas deferens. The transected halves of theoperated vas deferens in each animal were rejoined either four (groupA) or thirteen (group B) weeks later. All surgery was conducted usinghalothane anaesthesia under aseptic conditions. Rats were killed bycervical dislocation fifteen (group A) and eight (group B) weeks laterand epididymal and prostatic segments of operated and unoperated vasadeferentia fro~ each animal were (i) examined histochemically forcatechol~ine fluorescence or (ii) set up in vitro for recording forcedeveloped in response to noradrenaline and to electrical fieldstimulation (10 pulses, 60V; lms, 0.1-50Hz) as described previously (1,2l.

Rejoined epididymal segments from group A exhibited lessfluorescence than those from unoperated contralateral controlsegments. The level was similar to that observed by us previously intissues taken eight weeks after vasovasostomy (2). Force developed inresponse to field stimulation, and potentiation of the contractileeffects of noradrenaline by inhibiting neuronal uptake with nisoxetine(0.1 prool/l) were no greater than that observed eight weeks aftervasovasostomy. Prostatic segments were relatively unaffected by theoperative procedures. Thus noradrenergic reinnervation of theepididymal segment of the rat vas deferens following vasovas0stomyfour weeks after vasectomy is only partial and apparently plateauswithin eight weeks.

The density of the innervation of rejoined prostatic segments fromgroup B, like that of unanastomosed prostatic segments taken at thesame time (1), was reduced. The extent of reinnervation of rejoinedepididymal segments from this group was decreased compared with thatseen in rejoined epididymal segments from group A.

(1) DeGaris, R.M. and Pennefather, J.N. (1986) Clin. Exp. Pharmacol.Physiol. (in press) '.(2) DeGaris, R.M. and Pennefather, J.N. (1985) Proc. Aust. Soc.Reprod. BioI. 17: 3 (abstract).

103

70 ENVELOPE-RELATEDEXPRESSION OF A RETR0i'~~~~ ~CROSOMAL MEMBRANEGLYCOPRO~:I~oC:Ml~EHUMAN SPERMATOZOA

1 F C McKenzie*, Peter J. Lutjen.Rosemary L. Sparrow, an • •

Human Development, Monash University,Centre for Early. . M d'cal Centre Melbourne

Queen V1ctona e 1 't'*Centre for Cancer and Transplanta. lOn,.

. 'ty of Melbourne, Parkv111e, V1ctona.Umvers1. d by the E43 monoclonalI t in recogmze •

The human-specif~c HULy-M~ ~ ~~~P~~lfs~rface of most nUcleated6n90r;d(i~u~~~antibody (mcAb), 1S expresse 'th molecular weights 60 and .'cellS, and is composed of two :and~a~~d to the gp70 envelope glycoprot~1gs of :heHuLy-M5 antigen appears to e r~ Monke Virus (MPMV) an.d. G1b on peprimate retroviruses Mason ~~1zer d by :_ Yl) the ability of punfled MPM6:~~Leukaemia Virus (G:L~~ ~s ~~A~~c;) polyclonal antisera to ~P~V :~~ mcAbGALV to absorb t e • bl k ~he binding of E4.3 mcAbj 3 t e .precipitate HuLy-M5. and .oc GALV-infected gibbon ape cells (2).recognizes HuLy-M5-llke ant1gen on .

5 I co rotein has been exammed o.nMore recently the expression of the HUL~~i~ ~~ 14.3 mcAb and an FI~C-antl­normal human ejaculated s~e~ma~ozo~, ( hi~ved on fixed sperm prep~ratlons andmouse immunoglobulin). Stam10g 1S on Y aC suggesting that HuLy-M5 IS expressedremains on acrosome-reacted spermatozoaat the inner acrosomal membrane.

. etroviral-related glycoprotein on humanThis is the first. e:lldence lof. a ~l-cell interactions between human gametesspermatozoa. I:S or.lgm and ro e 10 ceare under investlgatlOn•

. (1983) Human Immunol. 7:1-15Sparrow R.L. & McKenZIe, I.F .C. K . 1 F C (1985). Human Immunol.

1. ' 11 D F J & Mc enZle , • . •2. Sparrow, R.L., Purce, •..13 : 83-93.

104105

EARLY PREGNANCY ASSOCIATED THROMBOCYTOPENIA (EPAT) _ A NECESSARYPREGNANCY PHENOMENON

L.M. ADAMSON, J.D. STANGER Y C SMARTUniversity of Newcastle and Ling'ard H ' ~t·Ml· MURDOCH, T.K. ROBERTS.

OSPl a , Newcastle, N.S.W.Apart from Early Pregnancy Factor (EPF)

Thrombocytopenia (EPAT) is the e 1· t f ' Early Pregnancy Associatedpregnancy~~ We have confirmed t~r les orm of maternal recognition ofbe evident in the maternal circul:t~~~u;:~~~e °hf EP~T and shown it topregnancy. Further, a transi 1 ln t.e flrst day of murinesplenectomized mice by admini~~~a~~rombocytopenla was induced in .suggests the presence of a solubl l~n ~f eTbryo culture fluids. Thismediate pla~elet activation and r:mo~~lo~ EPA~-Fa~tor) w~ich acts toBy transferlng the cultured embryos t f ro~ t e.clrculatlon of mice.5 days of culture the time Course of 0 res medlum daily for the firsttored showing a close correlation b 'tEPAT-factor production was moni­EPAT-factor and the circulating Pla~ fe~nl the embryo production offactor offers a model for the inv ~ e . evels of pregnant mice. Thematernal recognition of pregnancy~stlgatlon of embryonic triggers for

EPAT-factor is thought to be related .platelet activating factor PAF a th (if a synthetlc phospholipidactivation, aggregation and lib;r~~· er· f fAF-acether causes plateletsubstances.~) Although to date th lO~ 0 P atelet derived vasoactiveacether-induced platelet aggregat~~~ l~APo specifi~ inhibitor of PAF­~hreshold concentrations) dese ·to' h-acether ltself (at sub-lng ability of PAF-acether was n~l lzes t e plat~let. This desensitiz­ble similarity to EPAT-factor ~~~ ~o fur~h~r l~vestigate its possi-murine platelets in vivo b 3' . .e~ens~tlzatlon was induced indirectly prior to mating a~d dally :nJectlons of PAF-acether (2ug)control group received PBS in:on~~mmlttant EPAT-factor release Thetored on the day prior to mat~ec lOdnsf· The platelet levels we~e moni-dem t t d lng an or 6 days post t·ons ra e the presence of EPAT' th rna lng. Resultsabsent in the PAF treated grou (T ln e control group, however it wasE~AT significantly inhibited the a~~i~tl)·f Further, the removal ofvlable pregnancy. This is the firs~ lbY 0 b~astocysts to establish aplatelet activation, at the time of fO ~~r~atl~n su~ge~ting thatsuccessful implantation. er 1 lzatlon, lS lmportant for

Proo. Aust. Soc. Reprod.(1986)Martin, L. and Crane, L. H.Rial. (this meeting).

1.

Linda H. Crane and Len Martin

Department of Physiology and Pharmacology,University of Queensland, St Lucia, Qld 4067.

We have used video-laparoscopy (1) to compare myometrial activityin unmated female rats and females 6-10 h after mating. Vaginalsmears were taken daily from 8-10 wk old Wistar females housed in 14 hlight: 10 h dark. Laparoscopy was carried out 1-6 h after the end ofthe dark period on the morning of the oestrous smear of the 3rdconsecutive 4 day oestrous cycle. Unmated rats were housed with otherfemales; pro-oestrous females were placed singly with males 16 hbefore laparoscopy and were included in the study if the oestroussmear contained sperm.

POST-COPULATORY MYOMETRIAL ACTIVITY IN THE RATAS SEEN BY VIDEO-LAPAROSCOPY

Patterns of activity in mated rats (n=6) showed consistent largequantitative and qualitative differences. Discrete bursts oflongi tudinal activity were largely replaced by prolonged periods ofcomplex activity involving primarily cirCUlar muscle and includingmul tiple circular constrictions often propagating towards the ovaryand discrete intense peristaltic waves in both directions.Occasionally each horn appeared to "shuffle" its contents.

From our preliminary results with oxytocin and cervicalstimulation, it seems unlikely that either would produce the profoundchanges in myometrial activity seen after mating. We suggest thatthese arise from specific seminal constituents, probably prostanoids.It is significant that oxytocin-induced contractions would be unlikelyto "pump" sperm into the oviduct, whereas those induced by matingwould tend to do so.

Pa tterns of activity in unmated rats (n=5) were as describedpreviously (1), viz. predominantly discrete longitUdinal contractionspropagated from the ovarian end, with little circular contraction orperistalsis. Mechanical/electrical s timulation of the cervix had noreproducible effect, While oxytocin (25-100 mU, IV) stimulated stronglongitUdinal contractions propagating towards the cervix.

Copulation-induced release of oxytocin, mechanical stimulation ofthe cervix during copUlation and seminal prostanoids have been citedas spasmogens that might induce uterine contractions important intransporting sperm to the oviduc t. However, no studies describe theeffects of copUlatory stimulation on myometrial activity in vivo.

6

1.3±0.04 1.3±O.09 I.S±0.081.5+0.07 1.6+0.12 1.7+0.06

J. Reprod. Fert. 73 559.J. Reprod. Fert. 73 567.J. Exp. Med. 143 937.

o

MEAN PLATELET LEVELS (+ SE)DESENSITIZATION WITH PAF/PBS~URING EARLY PREGNANCY AFTER

1.4+0.06 1.0+0.06 1.0±0.07 1 2+0 021.4±O.04 1.4±0~05 1.6±O.05 1:7~0:09

O'Neill, C. (1985a)O'Neill, C. (1985b)Henson, P.M. (1976)

TABLE 1:

TREATMENT--_____________________ PLATELET COUNTS (xl09)

-----D~;-;f-~~;~~~~~;-----------------------2 34 5

(1)

PBSPAF

(2)(3)

106107

PREGNANCY AND PARTURITION.OVINE CIRCULAR AND LONGITUDINAL MYOMETRIUM DURING

OFACTIVITY

(mean + SD··'· 0 05000 _ ,A P < • ,** p < 0.025 *** p < 0 001 0 0 00p < 0.005, control vs grOwth-ret~rded) ., p < .01, p < 0.025,

Department of Physjolo~, Monash University,Clayton, Vic. 3168 Australia.

(1) Harding, R.. poore, E.R .. Bailey. A.. Thorburn. G.D., Jansen.C.A.M. & Nathanielsz. P.W. Am. J. Obstet. Gynecol. 142: 448

(1982) .(2) Parkington. H.C. J. Physiol. 359: 1 (1985).

V. Shepherd, H. Parkington, L-!@J<tl1.. M.M.. Ralph & G.D.Thorburn.

This work was supported by a grant from the NH&M.RC.

Myometrial activity. recorded from the intact ewe during pregnancyand parturition (1). differs from that recorded from the circularcomponent of the myometrium ~vi~~.(2). To investigate the cause ofthese differences, the spontaneous activity of the isolated circular(e) or longitudinal (L) layers of ovine myometrium was studied as wellas whole uterine preparations containing circular and longitudinal

myometrium together with endometrium.A piece of uterus was removed from the pregnant horn of sheep at

(i) 95..114. (ii) 115·134. (iii) 1~-\5·144, (iv) 145-150 (parturient)daYs gestation. Isometric force in strips of C or L myometrium. 7.5mmx 3.5mm. was measured, in the circular or longitudinal planerespectively, by a force displacement transducer. That in wholeuterine preparations. of similar size. was measured in either thecircular (CP) 01' longitudinal (LP) plane. The amplitude (a).frequency (f) and duration at 50% amplitude (d) of spontaneouscontractile activity, the integrated area under the contractileactivity curve (lAUC) , the amplitude of contraction in response to100mM. KCl (K) and the mean a. as a % of the contraction induced byad~~tion of KCl. (~) was recorded, each in the absence or presence of

10 M. indomethacin.Spontaneous contractions of Land l,P produced more tension thanthose of C or CPo Furthermore. tetanic contractions occurred in L butnot in C. K was greater than the amplitnde of spontaneoUS contractionin C, but not in L. K was not, however, significantly altered in anyof the preparations at any stage of gestation. At all stages ofgestation. d and IAUC for L was greater than for C. ~ for L was alsogreater than for C from ii onwards. In ii. and iii. f was greater InC than in L while that of CP and LP was intermediate between C and L.There was an increase in lS.... and a decrease in d. of L at parturition;while an increase in d was observed in C at this time. In LP, but notCP, there was a decrease in f. but all increase in lAUC and K. atparturition. Indomethacin significantly decreased f in parturient CPand LP' but had no effect on any of the parameters recorded in C or L

at this time.Thus the spontaneous activity of C, L. CP and LP differed bothbetween preparations and with the stage of pregnancy. There was,however. no change in the capacity of each preparation to contract.Endometrial prostaglandins may be responsible for changing thefrequency of contractions which occur at term.

-24 + 10110'+ 8947'+ 3033'+ 2913 '+ 713 3:: 7

ControlsUrbi1ical V-A diff.

vein

85 + 45599'+ 149312'+ 81210 '+ 3465'+ 5573:: 13

-34 + 22-94 '+ 690

-55 3:: 43000

-47 + 420

-8.4 "+ 5.5000

-5.93:: 1200

GrcMth retardedUrbilical V-A diff

vein •

79 + 22423 '+ 91282'+ 91358'+ 45*kk90 '+ 19*k88 3:: 24*

Concentrations of amino acids in growth-retarded and control fetal sheep.

Amino Acid

glutamateglycinealaninevalineisoleucineleucine

(unJ1!L)

It is ccnc1uded that the' utilization of' . ..when the grcwth of the placenta was restri:-: a:~~s W1.thm the gravid uterus altereduptake by the fetus. to one of release of ami' W1.t. reversal of the nonnal pattern ofUnder these cireunstances it ~s 'bI no ac~ds fran the fetus to the placenta" • poss~ e that prete' . fl' .pro~de anum acids for IlEtabolism b th 1 rn ~n eta nuscle is ll¥Jbil ised t)'Yep acenta. ..' ,

TIE QUllIHlE.[ARIE) :oom SllPl'LlES ITS_ PI.JaNrA wrm AMIID AC:JIS IN LA'lE~

K. KiJxl J A.. •__,. Owens, J. Falcmer and J. S. Robinscn

Faculty f H:!dicine •o , thiversity of Newcastle, N.S.W., Lnl.

Jmi.no acids are rnaJ' tabol'or rre ~c substrates utilised b hepregnancy, for energy production and tis . 'Y t fetal sheep throughoutt:cm the unbilical circulation consists '~~cre~~:. Fetal uptake of amino acidsW1.th no net transfer. of the acidic amino aci: Y 0 e neu7ral and basic amino acidsto the placenta. When the growth f the 1 s. Glutamate ~s released fran the fetusthe .conc~trations of the neutra: bran~e~:::~nand ?ence ~ fetus. was :estricted,leucme) rn fetal arterial plasma increased and haa:nmo ac~c:s (vahne, ~soleucine,that transport or utilization of amino •d .t .t of glycUle decreased suggestingThis was ex.ami.ned mre close1 ac.~ s W1.thin the gravid uterus had changedplasma fran the unbilical vein~byde:asu:~ng the concentrations of amino acids fufetuses at 116-120 days of cendmg aorta of 5. control and 3 growth-retardedendcmetrial canmcles (po:e:~Y·l ~h retardatwn was induced by excision ofCa

7heters .were implanted into the f:c:;e:~a110a~tacbIlEnt sites) bef~re pregnancy.

anmo ac~ds in plasma sampl ays • The concentrat~ons of fifteen

d. . . es were rreasured by ph halenvat~sat~on and reverse-phase high' rformance . . 0-0 t aldehyde pre-colum

~lasma ccncentrations (;.m:>1!l) of ~ llqu~d chranat?graphy.~reased in the unbi1ical vein (Tabl: neutral branc?ed-charn amino acids weregrowth-retarded fetuses caopared to the 1) an~ descend~ng aorta (not shO\Yn) of the~ vs 27 2:. 15, p < 0.05) and tyr . co(ntro • Concentrations of rrethionine (45 +

ed . os~ne 99 + 16 vs 74 + 13 ) -~r~ rn the descending aorta of the smallfe -' p < 0.025 were alsos~gmhcant uptake of all amino .d t~es. In control fetuses, there was

d ac~ s except serme and t t phan franar: a net secretion of glutamate to the lacent ryp 0 • the placentadlfferences of 12 of the 15 . !'. a. The veno-artenal concentration

. . f' ammo ac~ds rn the growths~gm ~cant1y different fran controls In -retarded fetuses wereamino acids was fran the fetus to the • 1 con(trast to the controls, the net flux of

p acenta Table 1).

Table 1.

108109

Methods in Cellular

46.8 + 10.3 24.2 + 3.755.5 +: 11.8 35.1 +: 8.196.6 +: 3.6 89.9 ! 3.3

t % of control (means of 4 experiments).

0.3 mg/ml* t68.0 + 15.075.8 +: 12.299.8 ~ 2.4

k 8 J. Immunol. lQ2: 1296 (1971).Kasa ura,' Biol Reprod. 25: 77 (1981).Sergerson,E.C., ,:, 8M ceds). SelectedMishell B.B. and Sh11g l , •.Immunol~gy, Freeman & Company (1980).

t that immunosuppression occurs atThese and other (2) data sugges

l' -reJ'ection of the foetus,

l ent materna lmmuno tthe uterine leve to pr:v , UTM or from other sources do nobut that immunosuppres slve fact~rs lnH wever the apparent immuno-pass into the maternal circulatl~n. t °b co~firmed using assay systems

. "t of UTMrema lns 0 e A'd dsuppresslve actlv1 y , ifically than does PH ln ucewhich measure immunosuppresslon more spec

LP.(1)(2 )(3)

* mg protein per ml of medium;

UTM (A)UTM (B)OVX-serum

d 25.0, 12.5, and 6.25 ~l of jugular orWhen cultures containe 65 regnant ewes, LP was 25.1 ! 5.5,

uterine venouS sera from two .Day 5 +P5 4 50.8 + 8.3 and 92.8 ! 16.6 per51.4 + 9.2 and 92.2 ! 14.3 or 23. ,- l' , Hence- site of serum collec­cent ~f the control level, respect:ve y. tl'on~ (UTM) collected from

H r uterlne secre , 'b'tion had no effect. oweve '95 unilaterally pregnant ewes (A,B) ,lnhl 1-

the ligated horns of,two Day nd 2 7 mg/ml) protein concentratlonsted LP (p<0.05) at hlgher (0.9 a, " ed ewe (oVX-serum, Table 1).

d- 'th serum from an ovarlectom1Zcompare Wl , roliferationTABLE 1. Effect of uterine secretlons on lymphocyte P

0.9 mg/ml 2.7 mg/ml

, effects of pregnant sheep sera andThe prese~t study exa~~ne~t;:meroliferativeresponse of lymph-

uterine secretlons on the ~ _v p 1 tinin (PHA) (3). Controlt ewes to phytohaemagg u '1

ocytes from non-pregnan , d 5 x 105 lymphocytes In 0.2 mf 72 hours and conta1ne " (5

cultures were or " A (16 /ml)' [3H-methyllthym~dlne 2.RPMI 1640 medium conta1n1ng PH th~gend ~f cultures. LP was 56.9 !j.lCi/ml) was added 20 hours befored 52 2 + 13 2 per cent of the control

) 45 1 + 10.7 an. .4.3 (mean + s.e., • - 'd 12 5 1 of sera from three eweS atlevel whe~the cultures contalne • tively (Day 0 = oestrus).DayS 0, 15 and 30 of pregnancy, ~e~pec24 9 and 59.5 + 6.0 per cent ofSimilarly, LP was 45.5! 12.3, 6. ! ',' d 12 5 "I-of sera collected

h th cultures conta1ne . ~ ,the control level w en e. .' toml'zed ewes treated dally from

5 d 30 from three ovarlecon Days 0, 1 an N ither pregnancy nor progesterone

1 with 12 mg progesterone. e~~~atment altered the effect of these sera on LP.

IMMUNOSUPPRESSIVE FACTORS DURING PREGNANCY IN THE SHEEP

X. Zhang and B.G. Miller

Department of Animal Husbandry, University of Sydney,Camden, 2570

, sma from regnant women (1) and in uterineSome factor(s) ~n pIa P(2) , hibit in vitro lymphocyte

. f rly pregnant ewes 1n -- ----- 1secret lons rom ea t t the foetus from maternaroliferation (LP) and hence may pro ec

immuno-rejection.

Amara Vinijsanun and Len Martin

Department of Physiology and Pharmacology,University of QJeensland, St Lucia, Qld 4067.

1. Wang, M. et aL (1984) J. Endocro., 101, 95.2. Vinijsanun, A. and Martin, L. (1984) Prooc. Aust Soc. Reprood.

BioL, 16, 31.3. Fantl, v. et~Z: (1982) J. stepoid Biochem., 17, 125.4. Philibert, D. et aZ. (1982) Prooc. 64th Meeting 4m. E,'ndo·')ro.

Soc., ~, 668.

PROGESTERONE DEPRIVATION AND EMBRYO DEVELOPMENT IN MICE

Balb/C mice given 0-5 nMol of anti-P monoclonal IgG llP27 (3) atlOAM on d2 of pregnancy were killed on d4, and embryos flushed/examined as previously (2), or on dl0 and implantation sites counted.Implantation-blocking doses of l1P27 produced nonsignificant increasesin abnormal embryos/embryo-loss. Nevertheless implantation failurecould have resulted from embryo abnormalities. However, the steroidalanti-P RU38486 (4) given to Q3 mice on dl-3, blocked implantation,caused massive embryo loss but nonsignficant increases in abnormalembryos; there was no difference between numbers of "normal" d4embryos and dl0 implantation sites, indicating that the former werecompetent. This was confirmed with another steroidal anti-Po Weconclude that P is not essential for preimplantation embryodevelopment. Why then the large effect of ovariectomy and itsreversal by P? We suggest that it reflects an oviducal inflammationwhich is suppressed by P.

No. d4 embryos dl0 implantsMice Lost Abnormal Normal % Mice

Treatment d4/10 % CL % Total No./mouse No./mouse with

Saline IP 6/10 8±4 11±6 10.1±0.9 8.4±1.1 100P1127 3 nMol 6/7 38±14 35:1:21 6.5:1:2.1 1.4:1:1.4 14

5 nMol 6/4 39±15 15:1:15 5. 8±1. 9 0 0

Arach. oil SC 12/18 4:1:2 15:1:8 12. 5±1. 2 11.0:1:1.3 89RU38486 50 j.lg 8/5 68±13 33±18 4.5:1:2.4 6.0:1:1.5 80

100 j.lg 8/8 70±10 32:1:12 2.6:1:0.9 1.0:1:1.0 13200 JJ;J 8/0 78:1:7 30:1:15 2.9:1:1.1

A report (1) that anti-progesterone IgG given to mice on d2 ofpregnancy increased numbers of abnormal d4 embryos, led us to studyother forms of progestin (P) deprivation. We confirmed our resultswi th CBA/C57 Fl mice (2) in outbred Q3 mice, showing that dlovariectomy significantly increases the proportion of abnormal d4embryos, an effect that is partially reversed by giving P on dl(controls, 9±5%, n=9; sham-ovex, 18±8%, n=7; ovex dl, 83:1:5%, n=12;ovex + Pdl, 38:1:11 %, n=11: results here and in table, means ± SEMs).

. We concluded that P is required for embryo development. However,later experiments led us to question that conclusion.

110111

PLASMA LACTOSE LEVELS AT THE IMOTHERS. NITIATION OF LACTATION IN BREASTFEEDING

RACE/ETHNIC-SPECIFIC PATTERNS OF FETAL GROWTH

lRooth, G. (1980) Lancet, March 22, 639-641.2preventing Low Birthweight. (1985) National Academy Press,Washington, D. C.

Ipacific Biomedical Research Center and Department of Psy­cho1logy, 2School of Public Health, and 3 School of Medicine,University of Hawaii at Manoa, Honolulu, Hawaii, USA

Birthweight differences1among countries reflect the influ­ences of many factors 2including race or ethnicity whichmay have plac~d biological constraints on fetal growth.Race/ethnic-specific patterns of fetal growth for infantsborn in a developed area of the Western Hemisphere (Islandof Oahu, State of Hawaii, United States of America) areexamined in this study. Live birth certificate data forthe Island are analyzed for a five-year period, 1968-1972.Defining the infant's racial/ethnic status as that of themother, the composition of the sample is Caucasian(22,726),Chinese(2,247), Filipino(8,145), Hawaiian(11,175), Japa­nese(10,520). and "Others(689)." The Other group is het­erogenous and consists mostly of Pacific Islanders andSouthea'st Asians. The effect of possible confounding fac­tors such as, (a) complications of pregnancy, (b) condi­tion or illness affecting, but not related to the preg­nancy, (c) education of mother, (d) legitimacy, (e) moth­er's age, (f) parity, (g) previous delivery born dead, (h)total number of prenatal visits, and (i) socioeconomicstatus based on the mother's place of residence,on birth­weightjs examined using step-wise regression for each ges­tational age. The effect of race/ethnicity on birthweightfor each gestational age is then studied with analyses ofcovariances. The Duncan Multiple Range Test is used todetect the individual group differences. After adjustingfor possible confounding factors, there is no differencein birthweight for preterm infants (less than 37 weeksgestational age) except at the gestational age of 35 weekswhere the Hawaiian group is significantly heavier thanthe rest of the samples. Among the term and postterminfants (37-44 weeks gestational age) the Hawaiian and theCaucasian gro~ps are significantly larger than the Fili­pino and Japanese groups. These data indicate ~, thatCaucasia~ fetal growth curves may be applicable for theHawaiian, but' not for the Filipino and Japanese infant atbirth, two, that race/ethnic-specific patterns of birth­weight a;e different for the pre term period when comparedwith the term and postterm periods, and three, that othercharacteristics of fetal growth should be investigated todetermine if those results are concordant with the presentstudy; this possibly may lend support to the formulationof a genetic hypothesis·

David H. Crowell~ Robert I. McGee: Germaine ogasawara;Santosh D. sharma~ Dexter Seto~ and Robin J. Willcourt 3

58(3)44(6)27(7)18(7)24(5)24(5)

±±±±±±

95117162176179186

Milk lactose(mM)

28(3)29(6)7(6)6(7)5(7)3(5)

±±±±±±

524921161411

Milk Sodium(mM)

5.6(4)16(7)30(6)23(7)50(6)70(5)

P.G. Arthur, J. Potter~< and P E H t. . ar mannDepartment of Biochemistr ><Dof Western Australia Nedra' d ePwartment of Pharmacology, UniverSity

, n s, .A., 6009.

Small amounts of lactose a nearbloodstream during pregnan~~ andt~a~~:~,from the milk into the~actose levels OCCur 6 h after the st r lon . In th~ sow, peak plasmaln plasma lactose coincides with an ,a t of f~rrowlng. The riseof lactose in the mammary secret' lncrease ln the concentrationprovided an accurate qualitati l~nd suggesting that plasma lactosethe mammary gland (1) W h ve In ex of synthetic activity of, b . e ave measured pIa 1In reastfeeding mothers at ' "" sma actose levelsa ' 'I lnltlatlon of lact t"

Slml ar relationship occurs in women. a lon to assess whether

Venous blood was collected from moth ."1 to 2 days until 4 to 7 day ft be : s at dally lntervals fromda'l s a er lrth Each h

1 y,a small amount of secret' f . mot er collectedwhen their milk lI came in" L lon rom each breast and recorded 'by b' 1 . . actose was analys d' 1a 10 umlnescent technique (2) d' . e In p asma samplesmethod (1). Sodium was analysed i~nm'~~ ~ll~ samples by an enzymaticBetween days 2 and 3 th . 1 Y lame photometry.

"I ere were lncrease . 1ml k lactose (P<O.Ol, paired t tes· s ln p asma lactose andlevels (P<0.05, paired t test). t), and a decrease in milk sodium

Day~ from Plasma lactose(~M)buth

1 16 ±2 22 ±3 47 ±4 54 ±5 65 ±

6 and 7 79 ±

Mean ± S.D(N)

Plasma lactose peaked at 3 4 d 51 mothers, respectively I' an days after birth for 2 2 and

. . n two mothers pIa I 'contlnued to increase durinth . sma actose levelsof milk lIcoming inll g , e perlod of measurement The feel", I was assOClated with . .. lngIn a I mothers. Milk lactose was ,.lncreaSlng plasma lactosel~ctos~ levels (r=0.487,p<0.001 n=~~)ltlvelY co:related with plasmawlth mllk sodium levels (r=O 857 <0 OOand negatlvely correlated

. ,p . 1 n= 35).These reSUlts demonstrate that there . ,~fter birth in women. This chan ls.an lncrease in plasma lactoseln the synthetic activity of th ge provldes a gUide to changeslactation. e mammary gland at initiation of

(1) Hartmann, P.E., WhitelPhysiol.347: 453-463 y, J.L., and Willcox, D.L.,(1984) J.(~) Arth~ P.G., Po~ter, JBlochem.Soc .1£: 12. . and Hartmann, P.E., (1984) Proc.Aust.

112

GLUCOSE, INSULIN, PROGESTERONE NDLACTOGENESIS IN SOWS. A LACTOSE IN BLOOD PLASMA DURING

M.A.Holmes, J.L.Whitely, G.B.Stokes PDepartment of Biochemistry U' ~ .A.Arthur, P.E.Hartmann

, n1verS1ty of W.A., Nedlands 6009Some commercial piggeries i Wration of feed given to sows ~ .estern Australia reduce the dailylactation. Since glucose is ur1~g late pregnancy and earlyand metabolic pathways in :h major precursor for various synthetica reduction in dietary carbohYdera~:mmary ~lands of. lactating sows,may lead to a decline in 1 ,espec1ally dur1ng lactogenesis

d h g UCose uptake by th 'an t erefore affect the initiation of lactation. e mammary glands

B~ood samples were collected between da s 3b1rth from 7 unrestrained sows (L y -5 before. to 2-4 afterx Large White) housed at a c . alndrace,Large Wh1te, LandraceBullsbrook WA) The 110mm~rc1a piggery (Baconfield Piggeryf ,. . . co ect10n periods we f 24h' ,rom the commencement of bl d re 0 intervals

intervals between day 1 00 sampling to day 1 prepartum' 4-6hintervals from day 1 post~:~~~~tu;t~~d day 1 pos~partum; a~d 24hThe plasma of each blood s 1 the complet1on of sampling1)" amp e was analyzed for 1 (6 .

on y , 1nsul1n, progesterone and lactose. g ucose sows

The sows were fed a ration . .dig~stible energy. During preCgOn:t:c1lllt~ge 15% protein and 12.5 MJ/kgunt1l day 1-2 prepartum when th ~ / were fed 1.8-2. 3kglday~his intake was maintained unt~llnta e was reduced to O. 9kg/day.1ncreased to 1.8kg/day. day 2 postpartum when it was

Progesterone levels decreased fon day 2-4 prepartum to 6.5 ± rom 28.5 ± 3.4]..1g/l (mean ± SEM)The concentration of 1 0.6]..1g/l (mean ± SEM) by day O.15 + 2 64]..1M ( P asma lactose increased in all sows from-. mean ± SEM) on day 2 4± 13.0]..1M (mean + SEM) bet OS - prepartum to a peak of 129

- ween and 2 6 daysows, minimum insulin concent t' . s postpartum. In allw~r~ observed from 24h before :oa ~~~s m(8.66 ± 4.0, SD, unit/ml)m1n1mum glucose concentrations (3. 06 ~ ean O. 4h) after birth andbetween 40h before and 24h ft b" - 0.8,SD,mM) were recordedthe a er 1rth (mean SOh) Hre was no significant (>0 05) , • . owever,minimal concentrations of p .' l' temporal relationship betweenC' 1nsu 1n and glucos d .oncentrat1ons of blood 1 [ e an maX1mumlactogenesis(l) ] . actose a measure of the time of

Although the reduction in feed intakwas associated with '. e of the sows at parturition

d" m1n1mum concentrations of both 1an 1nsul1n, the timing of lacto enesis did not p asma glucoseaffected by these metabolic changes. g appear to be

(1) Hartmann,P.E.,Whitely J Land W'll347: 453-463. ' .. , 1 cox, D.L.,(1984) J. Physiol.

113

THE EFFECT OF CLOMIPHENE CITRATE ON CERVICAL MUCUS SIALYLATION.

Julie Hoy and Peter Lutjen.

Centre for Early Human Development, Monash University,Queen Victoria Medical Centre, Melbourne.

In human cervical mucus there is a marked surge in the ratio of sialic acid toprotein at midcycle. This surge is correlated with peripheral plasma levels of E2and coincides with the LH peak of the ovulatory cycle and maximal spermpenetration through the mucus (1). Clomiphene citrate (CC) is widely used for thetreatment of human infertility, despite its known antioestrogenic properties, whichcause a reduction in sperm transport through the cervix (2,3). The aim 'of thepresent study was to examine the effects of CC on the sialic acid content of humancervical mucus as a possible cause for abnormal sperm transprort.Cervical mucus samples were collected throughout the menstrual cycle from 8spontaneously cycling women (controls) and from 6 women given 50 mgCC/day onDays 5 to 9 or 6 to 10 of their menstrual cycles. Cervical mucus sialic acid andprotein concentrations were determined as described previously (4). Plasma E2 andLH levels were measured by radioimmunoassay.The administration of CC to patients on Days 5 to 9 of the menstrual cycleresulted in the disappearance of the midcycle surge in cervical mucus sialic acidwhich was evident in the controls. Administration of CC beginning Day 6 resultedin a delay of at least 1 day in the appearance of the sialic acid surge in relation topeak LH levels. There was no significant effect of CC treatment on the proteincontent of the mucus throughout the menstrual cycle. In patients receiving CC,cervical mucus sialic acid levels were negatively correlated with plasma E2concentrations (P=0.008), compared to a positive correlation (P=0.003) in controls(1).Whether this reduction in mucin sialylation by CC is due to modification ofcervical epithelial oestrogen receptor or a result of the slightly increasedperipheral P4 levels following CC administration, has yet to be elucidated.These results add further support to the hypothesis that sperm transport throughcervical mucus may be regulated by alterations in mucin sialic acid and that CCmay exert an antioestrogenic effect on the cervical epithelium, resulting in analteration to mucin sialylation. The timing of administration of CC relative toendogenous E2 levels may also be of importance in initiating this effect on mucinsiahylation.

1. Lutjen P et aI, (1985). Aus. Soc. Reprod. BioI 17 :17A.2. Maxson et aI, (1984) 42 (3) 356-359.3. Sharf et aI, (1971) Obstet. Gynecol. 37 : 260-2664. Lutjen P. e't aI, (1985) Gam. Res. 12: 265-274.

114 115

EFFECTS OF CRONOLONE ON THE UTERUS AND ON PREGNANCY

B.G. Miller and X. Zhang

THE EFFECTS OF OVARIECTOMY AND OF EXOGENOUS PROGESTERONE ONPREGNANCY AND ON PLACENTAL PRODUCTION OF PROGESTERONE

Department of Animal Husbandry, University of Sydney,Camden, 2570

Cronolone (Cr) binds with high affinity to the uterine cytosolprogesterone (Pr) receptor in the sheep and rabbit but not in the mouse.However, Cr cannot sustain pregnancies in either mice or rabbits (1).

In the present study, pregnant ewes were ovariectomized on Day 30(Day 0 = oestrus) and pregnancy was maintained until Day 44 in 4/4 and1/4 ewes'given 12.0 and 2.4 mg, respectively, of Pr daily, and in 4/4,3/4 and 2/4 ewes given 10.0, 1.0 and 0.2 mg, respectively, of Cr daily.

To assess the ability of Cr to prime the mouse uterus for thedecidual cell reaction, ovariectomized mice were given 0.1 ~g Oestradiol(E) on Days 1, 2 and 3, followed by 0.01 ~g E plus 1.0 mg Pr (Group P, N= 10) or plus 1.0 mg Cr (Group C, N = 10) on Days 7, 8 and 9; and 1.0mg Pr on Days 10, 11 and 12. Ten ~l of peanut oil were instilled intothe right uterine horn on Day 9. The decidual weight (right horn _ lefthor~) was recorded on Day 13 and Was 17 ~ 3 mg (mean = s.e.) for GroupC, In contrast to 332! 20 mg for Group P (f<O.OOl).

N.W. Moore and Yiqun Xu

Department of Animal Husbandry, University of Sydney,Camden, 2570

The ewe can be ovariectomised as early as Day 50 of pregnancy with­out loss of the pregnancy (1), by which time the placenta is producingsubstantial amounts of progesterone (2).

This experiment was designed to assess the effects of ovariectomyand of exogenous progesterone (12 mg/day by 1M injection) on pregnancyand on placental production of progesterone in Merino ewes •.Ewes wereovariectomised on Day 65 of pregnancy (Group 1), or left entire andgiven progesterone from Days 30-65 (Group 2), or ovariectomised ~n Day30 and given progesterone from Days 30-65 (Group 3). Jugular veIn anduterine vein bloods were collected on Day 65 (24 h after the lastprogesterone injection in Groups 2 and 3) and. their pla~ma progesteronecontents were determined by RIA. In Group 1 Jugular veIn bloo~ wascollected before ovariectomy and uterine vein blood after o~ariectomy,

while in Group 2 uterine vein blood was collected from a veIn contra­lateral to the ovary with the corpus luteum.

The results indicate that Cr ~s a potent progestagen in sheep butessentially inactive as a progestagen in mice. Cr is an effectiveprogestagen in the rabbit, but in this species and probably also in themouse it appears to have side effects which block pregnancy.(1) Zhang, X., Stone, G.M. and Miller, B.G. Proc. Aust. Soc. Reprod.

BioI., ll, 90 (1985).

When pregnant rabbits were ovariectomized on Day 10 (Day 1 =mating), pregnancy was maintained until Day 20 in 9/10 rabbits given 1.0mg Pr daily but in 0/6 rabbits given 1.0 mg Pr plus 1.0 mg Cr daily.When pregnant mi~e were ovariectomized on Day 6 (Day 1 = vaginal plug),pregnancy was maIntained until Day 16 in 15/16 mice given 1.0mg Prdaily, but in only 9/21 mice given 1.0 mg Pr plus 1.0 mg Cr daily (X

28.2, f<O.Ol).

To determine the ability of Cr to induce uteroglobin (Ut) secre­tion, ovariectomized rabbits were given 0.1 mg E daily for 2 daysfollowed by Pr, Cr or oil only (control) for 4 days, and then explantsof endometrial tissue were collected from each animal and incubated induplicate for 4 hours at 37

0C in Eagle's basal medium supplemented with

3H-leucine (250 ~Ci/culture). Approximate rates of Ut in vitro secre­tion were obtained by determining d.p.m. in a fraction Of the incubationmedium eluted from a column of Sephadex G-75 and containing Ut (MW14,000 daltons). Cr and Pr had similar potencies (Table 1).

TABLE 1. Uteroglobin secretion rates (3 H d.p.m./~g tissue DNA! s.e.)

All ewes were slaughtered at Day 95.

**Mean + SD concentrationsof progesterone (ng/ml plasma)Jugular vein Ut vein

6.5 = 2.6 (n=6) 14.8 = 7.54.7 + 2.0 (n=5 ) 8.7 + 4.71.7 + 1.0 (n=6) 5.5 + 3.8

** At Day 65.

4/64/53/6

*Proportion of ewespregnant by Day 95

123

Group

TABLE 1.

* Of those pregnant at Day 65.

In all gr,oups some of the ewes failed to retain their pregnancy toDay 95.

Ovariectomy markedly depressed levels of progesterone i~ per~pheral

(P<O.Ol) and uterine vein (P<0.02) plasmas (Group 1 v 3), whIlst Inentire ewes which had received progesterone from Days 30-65 (Group 2),peripheral and uterine vein levels fell mid-way between those of Groups1 and 3. Hence, it seems that exogenous progesterone can h~ve an effecton progesterone levelS and it may be that its effect is mediated throughplacental secretion of progesterone.

(1) Denamur, R. and Martinet, J. C.R. Soc. BioI. 149: 2105 (1955).

(2) Moore, N.W., Barrett, S. and Brown, J.B., J. Endocr. 21: 187P972).

0.6 mg Cr2

30.0+5.0

6.0 mg Cr2

43.7=1.3

0.6 mg Pr2

29.8~0.9

6.0 mg Pr2

55.1~1.3

Control2

2.4~0.9

TreatmentNo. of animalsSecretion rate

4I

116

117

R.N. Murdoch

GLYCOLYSIS IN THE POST-IMPLANTATION MOUSE UTERUSIN RESPONSE TO ACUTE DOSES OF ETHANOL

Department of Biological SciencesUniversity of Newcastle, '

N.S.W., 2308.

Centrol (9) Canmcle (9)

Oxygen 1.130 + 0.850 0.916 + 0.573(/kg utero-placenta) 0.824 ~ 0.569 0.491 :; 0.412

Glucose 0.169 + 0.059 0.081 + 0.fXJ7'kk(/kg utero-placenta) 0.129:; 0.053 0.fXJ6 :; 0.051*

Lactate -0.092 + 0.033 -o.fXJ8 + 0.031(/kg utero-placenta) -0.058 ~ 0.015 -0.058 ~ 0.025

(mean! SD, *p<0.025, **p<0.OO5 : Canmcle canpared to controls).

Utero-placental constmption of substrates ('llllOl/min)

A close inter-relatiooship exists bet:Yleen fetal and utero-placental netabolism in lategestatioo (Sparks, Hay, M=schia & Battaglia, 1983). The placenta not ooly transfersoxygen and the major fetal mtrient, glucose, to the fetus but also converts glucose tolactate which is then released into the fetal and maternal circulatioos. When placentalgrowth is restricted, the rate of supply and the relative proportioos of thesesubstrates delivered to the fetus may alter. Furthenrore the placenta, normally thecoost.1l!Er of the major fraction of oxygen and glucose taken up by the gravid uterus, maycanpete less for these substrates when its growth is restricted and the feto-placentalweight ratio increases (Robinson, Kingston, Jones & Thorburn, 1979). The aim of thepresent study therefore was to determine the effect of restricted placental growth onutero-placental netabolism of oxygen, glucose and lactate.Nine ewes were operated on before pregnancy and endaretrial canmcles rerooved (carunclesheep). At 110 days gestatioo, catheters were implanted into the fetal faroral artery,tarsal and emm::m unbilical veins, maternal carotid and faroral arteries and jugularand utero-ovarian veins in the caruncle sheep together with nine control sheep. At 121to 130 days gestatien, unbilical and uterine blood flavs were neasured sinultaneouslyby the steady-state transplacental diffusion teclmique with three to six sets of pairedblood 'samples taken at hoorly intervals fran the fetal faroral artery, CCJlIIX)n UIbilicalvein, maternal faroral artery and utero-ovarian vein for neasurerent of oxygen, glucoseand lactate concentratioos. Utero-placental consumptien or production of substrateswas calculated as the difference between uterine and unbilical uptakes.

J. A. <Mens, K. Kind, J. Falconer and J. s. Robinsoo.

Faculty of ~icine, University of Newcastle, Shortland, N.S.W, 2308.

Jones, K.L. & Smith, D.W. (1975) Teratology~: 11-26.

Clarren, S.K. & Smith, D.W. (1978) New Eng.1067. J. Med. 298: 1063-(2)

Glycolysis ~as repre7ented as a major metabolic process in themou~e,uterus dur~ng post-~mplantationpregnancy, as evidenced by the~ct~v~ty.of glycolytic enzymes, the accumulation of glycolytic~ntermed~ates, and the capacity of endometrial preparations to~onsume oxygen and produce lactate during incubation in vitro The~ntraperitoneal administration of teratogenic doses o~ethanoi at3.0 and 6.0 g/kg body weight, but not at 1.5 g/kg body weightday 9 of pregnancy modified glycolysis in the uterus withi 2'h

on

resulting in significant changes in the rates of accumulat~on of~7u~ose, glucose-6-phosphate, fructose-6-phosphate, fructose-l 6-,~p osphate, and,citrate. These effects were not mediated by ~hanges~n redox state s~nce the ratio of lactate to pyruvate remainedunch~nged after inj~ction of ethanol. The increased accumulation ofuter~ne glucose.reg~steredafter treatment with ethanol was related~~ an ac~elerat~on o~ glycogen degradation in the liver and not in

e uter~ne.endometr~um. The results suggest that part of theadverse act~on.that these doses of ethanol exert on fetal develo ment~tem~ from ~nd~rect effects that involve uterine functions relat~ngo t e nurture of the conceptus via glycolysis.

It has been clearly established that alcohol abuse duringpregnancy exerts teratogenic effects in both humans (1 2) dlaborato:y.a~imals (3, 4). The objective of this stud; wasa~o assess~e poss~b~7~ty that alcohol interferes with uterine glycolysis in QS~ce to ~nd~rectly influence fetal development. The post­~mplantation stage of pregnancy was selected for study because itpresents the l~boratory mouse with a period of increased sensitivitytQ the fetotox~c effects of alcohol (3).

(1)

(3)

(4)

Kronick, J.N. (1976) Am. J. Obstet. Gynecol. 124: 676-680.

Chernoff, G.F. (1980) Teratology ~: 71-75.

Utero-plac~tal coosumption of oxygen and particularly of glucose, either as a total orper kg utero-plac:ental tissues tended to decrease when placental growth was restricted,while lactate production was unchanged. Coosequently a greater proportion ofutero-placental glucose consumptien was accoonted for by lactate production in thesmall placenta (42%) canpared to the nonnally gt'CMll. placenta (27%). 'This suggests thatother substrates may be used for lactate production and/or oxidative netabolism by theplacenta when placental grCMth is restricted.Sparks, J.W., Hay, W.W., M=schia, G. & Battaglia, F.e. (1983). Eur. J. Obstet.Gkynecol. Reprod. BioI. 14, 331.Robinson, J.S., Kingstoil,""E.J., Jones, C.T. & Thorburn, G.D. (1979) J. Dev Physio1l,379.

118

Mariann Wide,

Depart:rrent of Zoology, Uppsala University, Box 561, S-75l 22 Uppsala,SWeden

Lead is a world-wide envirol1IOOl1tal fOllutant and has long been recogn­ized to have deleterious effects on reproduction (1). To study theeffect of Ph-exposure at an organogenic stage of develop:rent on futurereproductive ability, pregnant mice were injected i.v. with a seeminglynon-toxic dose of lead on day 8 of gestation (control mice wereinjected with saline). FeJ.tlClles of the Fl-generation were mated at theage of 10 weeks and their uteri examined on day 17 of gestation.Decreased number of inplantations and increased fetal nortality of theF2-generation was found. Sennn levels of oestradiol l7B and progeste­rone were nonnal in the Fl-mice. Histological examination of theirovaries revealed a significant decrease in the number of prinordialfollicles. It was therefore assumed that the lead-treatment of the FO­mice had interfered with the developnent of the germ cells of the Fl­feJ.tlCl1e fetuses (2). To study the fOssible disturbance by Pb of theformation or migration of prinordial genn cells (PGCs), serial sectionsfran whole nouse fetuses were examined on different days of developnent(days 9, 10, 11 or 12) after the Ph-injection (day 8) to the nothers.The PGCs, visualized by histochemical staining for alkaline phosphatase,were found not to have deviated fran the normal migratory pathways, butto be significantly reduced in nmnber at all 4 days of develop:rent.Furthenrore, the staining of the PGCs was weaker in the Pb-exposedembryos canpared with those of control embryos, suggesting an inter­ference by Pb with the production or activity of alkaline Phosphatase(3) •

In conclusion, this study has shown that exposure to Pb at an earlyorganogenic stage of embryonic developnent can interfere with thereproductive ability at maturity. Lead was shown to cause a quantita­tive as well as a qualitative change in the develop:rent of the femalegerm cells. It is assumed that these changes have contributed to theinpaired pregnancy outcane of these mice. A similar finding was recent­1y refOrted also for another heavy metal canpound, namely cadmium'­chloride (4). It is possible that these effects are valid also forother heavy metals and for other rnam:nalian species, including man.

(1) Ran, W.N. (1976) The !>bunt Sinai SChool of Medicine 43: 542-551­

(2) Wide, M. (1985) Teratology 32: 375-380.

(3) Wide, M. Teratology (in press) •

(4) Tam, P.P.L. & Liu, W.K. (1985) Teratology 32: 453-462.

•119

THE EFFECT OF MP A ADMINISTRATION ON THE HORMONAL EVENTS ASSOCIATED

WITH PARTURITION IN SOWS.

J.L. Whitely, G. Bryant-Greenwood*, D.L. Willcox and P.E. Hartmann

U0 °ty of Western Australia, Nedlands, W.A., 6009;*John A.

n~vers~ f H . . H waiiBurns School of Medicine, University 0 awa~~, a .

A . difficulty in elucidating the endocrinology of parturitionan:ai~~togenesis in sows is the rapidity with which these eventsoccur and their close temporal relationship. Therefore weadministered a synthetic progestagen, .med~oxyprogesoterone :~:t~~e(MPA) to late pregnant sows with the obJect~ve of sl~ghtly d .y. gthe time of farrowing and thereby providing mor~ d~OSH~associations between hormonal changes and the term~nat~~n 0

d the initiation of farrowing and lactatlOn ~n thepregnancy, ansow.The MPA was administered orally (140 mg, twice ~ai1Y) pto 8 0 ~~te

day 112 113 and 114 of gestat~on. artun lOnpregnant sows on , . 0 • 200 l'g of Clo-was then induced to occur on day 116 by ~nJect~ng ~ 0 Theprostenol (leI) intra-muscularly on day 115 of gestat~on. of

. h in the plasma concentrat~onsper~-partum c ~nges . 1-17(3 relaxin and 13, 14progesterone,cort~sol, oestrad~o , thesedihydro-15-keto prostaglandin F2a (PGFM) were measured insows together with a further 7 untreated sows.

The estational length for the MPA-treated sows (116.3 ± 0.3 da~s,g + em) was significantly greater (P<O.Ol) compare~ w~th

mean - s .. d' (114 9 + 0 3 days). Plasma concentratlOn oft~~ eUsntt:re:ntee d:co;:ned e~rli~r (P<O.OS) with respect to the timeP

fg t 't o in the treated sows compared with the untr:a~ed

o par ur~ ~on d' ff 0 the t~m~ngAlthou h there was no significant ~ erence ~n .

~~ou:he first gdetectable increase in the concentratio~ of rel~X1~b th the time at wh~ch max~ma

~~~~:~~rat~o:s ~eore g::::is~ed ~nd the tidming of I~he~~e s~~~~~~~~~arlier in the MPA-treate sows.

declineth:erCeon~entration of relaxin increased ~efore t?e decline~ows, t e and in the treated sows the rise ~n relax~n occurred~n proges eron t with the decline in progesterone.either before, or concurren . f PGFMIn the untreated sows the time at wh~ch t~e concent;at~on ~hereasincreased, coincided with the decl~ne ~n proges .erone

- t d with MPA progesterone concentrat~ons began toin sows trea e . h 1 a concentrationdecline before any significan.t increase ~~ ~_~7J as:nd PGFM wereof PGFM. The profiles of cort~sol, oestra ~o ,similar in both groupS of sows.

o f relaxin occurredThe initial rise in the concentrat~.on 0 However,independently of the change in circU~at~~n;e:;:;:s~~ro~~~ time ofthe early decline in Jrogeste~one a;~ected the- subsequent profilefarrowing in the treate so~s~ ::ere may be association betweenof relaxin and suggests ~ a relaxin release. Sincecontinued progestherone d~cll.~~ea~~n~~~~~:~ion of progesterone andthe pre-partum c anges ~n to

. b f . 'ficant increases in the concentra ~onrelaxln occur e are s~gn~ f the luteolytic agent remainsof PGFM in treated sows, the nature aobscure.

120

CELLULAR ORIGINS OF TESTICULAR INSULIN-LIKE GROWTH FACTOR-I

David J Handelsman, Jennifer ~ spaliviero, C D Scott, R C Baxter.

Dept of Medicine, University of sydney and Dept of Endocrinology,Royal Prince Alfred Hospital, sydney, NSW.

The presence of insulin-like growth factor-I (IGF-I) and its bindingprotein in human seminal plasma (1) and perfused rat testis (2)suggests that IGF-I may be synthesized within the testis; however itscellular origin and biological function remains unclear. Since Leydigcells have specific high-affinity IGF-I.receptors (2), IGF-I may besecreted by Leydig cells (autocrine) or by seminiferoustubular/Sertoli cells (paracrine). To examine this question we havemeasured testosterone (T) and IGF-I levels in testicular interstitialfluid (IF), testicular (TV) and peripheral (PV) venous plasma and LHand FSH 4 days after treatment with a Leydig cell toxin, ethanedimethYlsu~Phonate ([EDS] single dose 25 mg/kg IP in DMSO), ago~adotro~1n releasing hormone antagonist ([GnRH-A] 1 mg/kg twiceda1ly SC 1n propylene glycol [PG] for 4 days) or appropriate vehicles.

Interstitial Testicular peripheralFluid Venous Venous

IGF-I T IGF-I T IGF-I T rLH rFSHGroup

EDS/DMSO 789 24 * 911 1 * 895 0.1 * 5.76* 8.62*

DMSO 747 1696 796 259 1140 5.7 0.97 5.03

GnRH-A/PG 756 7 * 908 1 * 853 0.1 * 0.12* 0.85*

PG 892 847 917 253 797 2.8 2.07 5.49

All results (ng/ml) are expressed as means; n=5 rats/group* p<0.05 vs appropriate vehicle control.

We conclude therefore that since IGF-I levels in IF, TV and PV areunalt~red despite virtually total functional ablation of Leydig cellsby e1ther EDS or GnRH-A treatment, Leydig cells secrete negligibleamounts of IGF-I into IF or blood. In conjunction with the observationof specific high-affinity IGF-I receptors on Leydig cells (2) it issuggested that testicular IGF-I may be secreted from the semi~iferoustubules and modulate Leydig cell function.

REFERENCES

(1) Baxter RC, Martin JL & Handelsman DJ (1984) Acta Endocr 106:420(2) Handelsman DJ, Spaliviero JA, Scott CD & Baxter RC (1985) ActaEndocr 109:543

supported by NHMRC

4121

PHARMACOLOGICAL CONTROL OF PENILE ERECTION

E J Keogh, C J Carati, K E Creed*, C M Earle, S Wisniewski, A G STulloch and D J Lord

Impotence Study Group of W A, Reproductive Medicine ResearchInstitute, 2 Verdun St., Nedlands, * School of veterinary Studies,Murdoch University, WA.

The aim of this study was to identify the neurotransmitterswhich regulate penile erection in order to induce erections inimpotent men and to suppress persistent erections (priapism).

Dogs were studied under anaesthesia by electricallystimulating the right pelvic nerve and the sympathetic ch~in.Pressure in the carotid and dorsal penile arteries and the r1ghtcorpus cavernosum was monitored continuously. The effects ofdrugs injected into the dorsal artery of the penis orintracavernosally,+or- electrical stimulation on corpus cavernosumpressure (CCP) were assessed. An increase in CCP was achieved withpapaverine (smooth muscle relaxant, 2-10mg, n=2) but ~o~phenylephrine, isoprenaline, acetylcholine or vasoact1veintestinal peptide (5-100ug, n=3). Stimulation of the pelvic nervecaused an increase in CCP (to 60-90% of mean systemic pressure)which was inhibited by simultaneous stimulation of the sympatheticchain. This inhibition was partially prevented by the en a2

blocker, phentolamine (3-100ug /kg, n=2) and the al blocker,

prazosin (3-100ug/kg, n=5), more so than with an a2 blocker,yohimbine (25-88ug/kg, n=5). The response to electricalstimulation was blocked by phenylephrine (lO-lOOug, n=4) but notby propranolol, (O-120ug/kg, n=3), or ~tropine (lmg/kg, ~=3). Weconcluded that in the dog erect10n 1S evoked by pelv1c nervestimulation and is inhibited by sympathetic chain activity and bya adrenergic agonists. However, blocking the effects of thesympathetic chain does not produce an erection.

34 impotent men were given intracavernosal injections ofpapaverine (10-120mg), phenoxybenzamine (6-9mg), or saline (lOml)in a double blind cross-over trial. This was followed byassessment of their erections (by EK) and self recording of sexualfunction over the ensuing month. 27% of men had adequateerections, but only 20% had intercourse on the day of theinjection-. During the ensuing month intercourse occurred asfrequently in the placebo group as amongst those who receivedpapaverine or phenoxybenz'amine, When given a choi,ce ,17 of, 23 menpreferred phenoxybenzamine. 4 men developed pr1ap1sm, 1n 2 ofthem CCP was 70-90% of systemic blood pressure. Followingwithdrawal of blood and injection of metaraminol (1-4mg, 4 men)into the penis, CCP fell to 10-20% of systemic pressure.

Constant a adrenergic tone maintains the penis in a flaccid

state. a blockers facilitate erections while a agonists preventerections. Thus, mimicking adrenergic control of penile erectionmay help to achieve erections or overcome priapism.

122

REACTIVATION OF FLAGELLAR MOTION OF DETERGENT TREATED HUMANSPERMATOZOA BY ADENOSINE TRIPHOSPHATE

D.Y. Liu, M.G. Jennings and H.W.G. Baker

Medical Research Centre, Prince Henry's Hospitaland University of Melbourne

Department of Obstetrics and GynaecologyRoyal Women's Hospital, Melbourne.

Low sperm motility (asthenospermia) is one of the commonestproblems in men with infertility yet usually the cause is unknown.To determine if disorders of energy generation and lack of ATP insperm cause asthenospermia we have studied the reinitiation ofmotility of demembranated sperm after addition of adenosinetriphosphate (ATP) (1). Washed sperm suspensions (80 x 106/ml) weredemembranated by exposure to Triton X 100 in a K, Mg, Tris/glutamatebuffer pH 7.9 with lmM dithiothreitol and 0.132M sucrose. After 30seconds ATP was added and the proportion of sperm tails with motiondetermined at X 400 magnification. The proportion of sperm withbeating tails decreased with time after addition of ATP and thiscould not be reversed by addition of more ATP. Assessments at 2minutes after addition of ATP were used for all subsequentexperiments. Maximal proportional reactivation occurred with ATPconcentrations between 0.063 and ImM but there was more forwardprogression and high frequency tail beat up to lmM. Thus lmM ATPwas chosen as the standard concentration for testing patients'sperm. The concentration of Triton X also affected results 0.05%appeared optimal. Under these conditions the precision of repeatedmeasurements on the same samples was 5.3%. Percentage flagellarreactivation was lower in groups of men with idiopathicasthenospermia (Mean ~ SD : 33 ~ 15%), oligospermia (17 + 21%),sperm autoimmunity (17 + 12%), vasoepididymostomy (20 + 20%) andidiopathic zero motility (0%) than in that with normospermia(54 ~ 12%). However when expressed as a percentage of the originalsperm motility 84% of motile sperm were reactivated in men withasthenospermia compared with 82% in men with normospermia. Theother groups had lower proportions of motile sperm reactivatedsuggesting a greater sensitivity to Triton X 100. Overall,percentage of reactivated flagella was correlated with percentagesperm motility (r = 0.761), motility index (r = 0.759) and livesperm (eosin Y stain r = 0.602). One man with low motility « 30%motility index < 35) had a high percentage reactivation (> 60%) butthis was not reproducible with other samples from the same man.Thus failure of generation of ATP does not appear to be a commoncause of low sperm motility.

(1) Lindermann, C.B. (1978) Cell 13: 9.

4123

PREPARATION OF SPERMATOZOA FOR ANALYSIS OF INORGANIC ELECTROLYTES.

G.M. O'Brien, J. Clulow and R.C. Jones

Department of Biological Sciences, University of Newcastle.

Electron probe microanalysis is being used in studies of theregulation of electrochemical gradients of inorganic electrolytesacross sperm membranes as potentially the method can distinguishelemental concentrations between compartments of a sperm and betweenintra- and extra-cellular compartments. In initial studies concernedwith establishing reliable procedures for the preparation of spermand analysis of their elemental concentrations fresh samples of humansemen were divided into five treatment groups, T1-T5; after treatmentaliquots from the 5 groups were loaded into Formvar-coated grids andrapidly air-dried (1). Briefly the treatments were: T1 = undilutedsemen; remaining samples were diluted 1:9 in isosmotic solutions asfollows: T2 = artificial seminal plasma; T3 = calcium-free Krebs­Ri~ger solution; T4 = lithium citrate, 100 roM; T5 = mannitol, 296 roM.Relative elemental concentrations of Na, Cl, Mg, S, Ca, K and P inthe nucleus of sperm from each treatment group are presented in theTable (mean, n = 10 sperm counted per treatment).

Treatment Na Cl Mg S Ca K P Na/K

T1 1.98 0.63 0.19 0.49 0.16 0.25 2.38 7.9

T2 2.29 1.19 0.36 0.56 0.04 0.58 1.88 3.9

T3 4.02 2.42 0.28 0.44 <0.01 0.37 2.29 10.9

T4 0.30 0.50 0.04 0.43 0.01 0.01 2.04 30.0

T5 0.85 0.62 0.11 0.46 0.01 0.05 2.32 17 .0

Readily diffusible electrolytes, especially Na and K, moved into(T2, T3) or out of (T4, T5) sperm rapidly when sperm were incubatedin isosmotic media containing elevated levels of the electrolytes(T2, T3) or none of the electrolytes (T4, T5), while the intra­cellular concentrations of the non-diffusible elements, e.g. S and P,

were high and constant.The data in the above Table suggest that there was rapid

equilibration of electrolytes during collection and processing ofsperm. This is consistent with other reports (2) that spermmembranes have very high permeability and with the possibility thatelectrochemical gradients of inorganic ions in sperm may bedifferent from those of such cells as erythrocytes and skeletalmuscle fibres that have low intracellular sodium and high intra­cellular potassium concentrations (2). The results also indicatethat the technique of air-drying cells for·· x-ray microanalysis mayrequire further development before being applied to cells with such

permeable membranes as sperm.

(1) Chandler, J.A. & Battersby, S. (1976) J. Microsc. 107: 55-65.(2) Crabo, B.G., Zimmerman, K.J., Moore, R. & Thornburgh, F.H. (1976)

Biochim. Biophys. Acta 444: 875-885.

•124

125

Standard deviations omitted for clarity. *p<O.Ol, **p<0.05 comparedto control; #p<O.Ol between 4ern implant and 16cm implant groups.

1. Steinberger E. & Steinberger A., Handbook of Physiol. IV.Pt.2

pp.325-345, 19752. Dym M. et al, J.Reprod.Fert.Suppl. 26:175-181, 1979.

EFFECT OF CHRONIC ADMINISTRATION OF TESTOSTERONE ON SPERMPRODUCTION AND PLASMA TESTOSTERONE, FSH, LH, AND

INTERSTITIAL FLUID TESTOSTERONE LEVELS.

Y-T Sun, D.C. Irby, D.M. Robertson, D.M. de Kretser

Department of Anatomy, Monash University, Clayton, Victoria 3168

The view that FSH is necessary for initiation of spermatogenesis orits restimulation after prolonged hormonal withdrawal and that testos­terone is required for maintenance (1) has recently been challenged (2).

This study set out to re-explore the roles of the two hormones onspermatogenesis in the adult rat. Silastic implants, containing testos­terone and varying in length from 1.5 to 16cm, were implantedsubcutaneously in 90 day-oid Sprague Dawley rats which were dividedinto 18 groups (n=5). After 50 days the rats were killed and thefollowing parameters were measured: (i) Serum FSH, LH and testosteroneby radioimmunoassay; (ii) Testicular interstitial fluid testosterone;(iii) Testis weight; (iv) Daily sperm production.

With the smaller sized implants (1.5-6.0ern), LH levels aresuppressed but testosterone levels remain at control levels presumablyby release from the implants. This is supported by the rapid decline ininterstitial fluid T levels reflecting Leydig cell function. Withlarger implants (up to 16cm) , interstitial fluid T levels are still only10% of control levels despite a 3-fold rise in plasma testosterone. Inspite of low interstitial fluid T levels at 16cm implants, daily spermproduction, which was significantly lower with 2-4cm implants, returnsto control levels. A similar pattern is seen with serum FSH levelSwherein a significant suppression is seen with 4cm implants but a riseoccurs subsequently. The cause of this rise and its role in the resto­ration of spermatogenesis with long length implants requires furtherstudy.

Table 1:Testes Sperm T FSH LH Int.FloT

Wt (g) Reserve (ng/ml) (ng/rol) (ng/ml) (ng/ml)

AGE OF RATS (DAYS)(xI06)

1 5 10 15 20 Ocm 3.50 201.5 5.2 4.4 0.87 251.0

V (rom3) 3.00 *6.65 *17.11 *23.592cm *2.24 * 48.5 4.6 *2.7 *0.36 * 15.6

*78.96 * 74.5# 5.8# *1.5 *0.28 * 7.0#D _3(lJ) 6.36 5.92 6.17 6.08 6.65

4cm *1.92#

Na (xlO-3' NO/lJ2) 2.81 *6.20 *9.35 *10.34 3.438cm *2.70 **152.5 *10.8 *3.2 *0.32 * 19.3

Nv (xl06 ' NO/lJ3) 0.38 *0.90 *1.30 * 1.46 0.4512cm *2.53 **128.0 *13.3 *3.2 *0.26 * 29.6

Nt (xl0 ) 1.1505 *5.9518 *22.2909 *34.4414 *35.53216cm -*3.04# 188.3# *17.0# *3.4# *0.39 * 32.1#

Note: Standard deviations omitted for clarity.*p < .001 compared to day 1.

1. Orth,J.M. (1982) Anat. Rec. 203: 485-492.

A QUANTITATIVE ANALYSIS OF SERTOLI CELLS IN POSTNATAL RATSZ.X. Wang, G.P. Risbridger, N.G.M. Wreford, D.M. de KretserDepartment of Anatomy, Monash University, Clayton, 3168

Sertoli cells do not divide in the normal adult rat and thus con­stitute a stable population. Therefore the period when the Sertolicell population expands is important in ultimately establishing thenumbers of Sertoli cells in the adult testis. This study aims toexamine the development of Sertoli cells during the postnatal periodusing quantitative morphometric techniques. Testes from rats (n=5)aged 1,5,10,15,20 days, were fixed by vascular perfusion with caco­dylate buffered glutaraldehyde and embedded in Epon-Araldite. Onemicron sections, stained with toluidine blue, were examined with anOrthoplan microscope using 63x oil objective and lOx eyepieces with441 point graticle. The total number of Sertoli cells per testis (Nt)was obtained using De Hoff's equation: Nv=Na/(l+D), where Nv is MeanSertoli Cell Nuclear No. in one cubic micron of testis, Na is MeanSertoli Cell Nuclear No. in one square micron of testis, and D isMean Average Diameter of Sertoli Cell Nuclei; Nt=Nv x V, based onthe fact that one Sertoli cell has one nucleus.

The results in Table 1 show that testicular volume (V) increasedsignificantly throughout the study. No change in Sertoli cell diam­eter occurred between day 1-20. The mean number of Sertoli cellnuclei/area increased up to day 15 but declined at day 20. Similarlythe mean number of Sertoli cell nuclei/volume showed the same patternof increase up to day 15. However, the total number of Sertolicells per testis rose steadily up to day 15 and remained unaltered onday 20. These data obtained by quantitative analysis showed that thenumber of Sertoli cells increases to day 15 postnatal and remainsconstant between 15-20 days, supporting previous observations byOrth (1). The data provide a baseline for future studies aimed atmanipulating Sertoli cell number.

----------------.-------------126 127

THE EFFECTS OF LIGATION AND CRYPTORCHIDISM ON THE EPIDIDYMISAND EPIDIDYMAL SPERM OF THE GUINEA-PIG

S.J. Gatie, T.D. Glover* and A.W. Blackshaw PROTEIN CARBOXYL METHYLASE IN ASTHENOSPERMIA

H.W.G. Baker and E.J. Cooper

Medical Research Centre, Prince Henry's Hospitaland University of Melbourne

Department of Obstetrics and GynaecologyRoyal Women's Hospital, Melbourne.

Protein Carboxyl Methylase (PCM) methylates the free carboxylgroups (e.g. aspartate) of proteins resulting in rapidly reversablechanges in net charge. This is an important mechanism forcontrolling motility of bacteria during chemotaxis. It was alsothought that the enzyme may have some role in sperm motility, PCMactivity is high 1n sperm tails and very low levels have beenreported in men with immotile sperm (1). The aim of this study wasto determine whether measurement of PCM assists in classification ofdisorders of sperm motility which are frequent but poorly understoodcauses of infertility in men. PCM activity was determine~ ?y thetransfer of tritiated methyl groups from S-adenosylmeth1on1ne togelatin (2). Precision for repeated assays of the same sample andrepeated measurements in different samples from. 7he ~ame man wasbetween 9 and 15%. Fertile men had PCM act1v1ty 1n the range36-235 pmol/Min/109 spermatozoa. Six of 26 men with asthenospermiahad low PCM activity « 36) but the defect in motility was ~otsevere (motility 25-50%). Three had high values (> 235) one . w1thzero sperm motility and sparse mitochondria in the mi~-piec7 .sp1ra~,one following vasoepididyrnostomy and the other wlth 1d1opath1casthenospermia. PCM activity did not correlate with sperm m~tilityor motility index. However it did correlate with the proport1on ofimmature germ cells in the semen (Spearman r = 0.755). Anazoospermic semen sample containing many leucocytes also hadappreciable PCM activity. We conclude the relationship between. PCMand low sperm motility is not simple and other cells al~o contr1buteto overall seminal PCM activity. The function of PCM 1n sperm hasalso been questioned: it may have a role in the modif~cation andrepair of damaged proteins rather than in signal transduct10n (3).

Fertil.

C.w.

Hatch, R., Harvey, S. and Williams-Ashman, H.G. (1985)

Steril. Q: 636.

Murray, E.D.J. and Clarke, S. (1984) J. BioI. Chern. 259: 10722.

Gagnon, C., Sherins, R.J., Phillips, D.M. and Bardin,(1982) New EngI. J. Med. 306: 821.

(3)

(2)

(1)

References

Bedford, J.M. (1975) In Handbook of PhY8io~ogy (ed. R.O. Greep &

E. B. Astwood), Sect. 7, Vol. 5, pp. 303-317,· Washington, D.C.:American Physiological Society.

Gaddum, P. and Glover, T.D. (1965) J. Reppod. Fepti~., 9: 119-130.

The epididymis showed small effects in both studies, the heightof the epithelium was decreased and the cells lost their stereocilia.Ultrastructural study showed a decrease in the cell Golgi bodyfunction and a reduction in the number of mitochondria. The wall ofthe tubules increased in thickness due to hypertrophy of the muscularlayer; the latter effect was more pronounced in the ligationexperiment, as found also by Gaddum and Glover (1965). After 7 daysof either treatment sperm showed only a reduction in rouleauxformation. Within 10 days most of the sperm had become separated dueto degeneration of the matrix between the sperm which by the tannicacid-ferric chloride method was positive for glycoprotein. The sperm,wi thout acrosomes, were contained in a thick secretion containing afew blood cells and degenerated sperm. After 14 days there weresevere effects, most of the epididymal duct was filled with thicksecretion containing blood cells wi th almost no sperm and in the caseof cryptorchidism a few germ cells shed from the seminiferousepi thelium.

The effects of ligation and cryptorchidism on the epididymis andits functions were studied in 18 white New Zealand strain guinea-pigs.Nine animals were used for each treatment and three animals for eachtime period (7, 10, 14 days). Under halothane anaesthesia, bothtestes and epididymis were sutured to the abdominal wall (artificialcryptorchidism) or the terminal segment of the epididymis was ligatedat both ends <ligation). The epididymides were processed for lightand electron microscopical studies.

Department of Physiology and Pharmacology and *Department of AnimalSciences and Production, University of Queensland, St. Lucia, 4067.

Guinea-pig sperm leave the seminiferous epithelium as individualcells which pass through the initial and proximal part of the middlesegment of the epididymis. A few sperm begin to aggregate, formingrouleaux, in the last part of the middle segment and almost all formrouleaux in the terminal segment. Bedford (1975) suggested that

'rouleaux formation was a sign of maturation.

THE ULTRASTRUCT.L:"RE OF SPEK\lATOZOA FRm: ~fE!\ 1,o,"ITH ASTHENOZOOSPHEP.l-lIA

128

After replacement of the pouch young each animal waschecked 4 hourly for 12 hours , to see if the pOlich youngwere attached , daily until day 7 , then less frequentlyuntil day 25 when pouches were again checked daily Ifreactivation had occurred at the time of first removal wewould expect birth or oestrus to occur on days 26-30 .

Birth or oestrus did not occur at the expected time inany of the animals in groups 1-5 except in those animals inwhich the pouch young never properly reattached and weresubsequently lost. In groups 6-9 birth or oestrus occurredat the expected time in 80 % of animals The longestinterval after which successful pouch young replacementprevented reactivation was 72 hours . All those reattachedat 76 hours or longer resumed reproductive activity.

We therefore conclude that it takes longer than 72 hoursfor the maternal endocrine system to become committed toreactivation . This is one day before the earliest measuredreactivation of the uterus , and two days before theearliest change can be recorded in the blastocyst or inperipheral concentrations of progesterone andoestradiol-17B. The actual sequence of endocrine eventswhich trigger reactivation remains to be determined .

129

(1) Renfree,M.B.(1979) Initiation of development ofdiapausing embryo by mammary denervatiion during lactationin a marsupial. Nature 278:549-551.

In the tammar.wallaby , loss or remoyal of the pOllebyoung d,liring the breedin.g season (Feb-'May) results inreactivation of the d:ia~aus!ing CL a,nd blastocyst , withbirth and p.ost.-.paiTtumoestrus occurringabolit .27 d."ayslater. Although it is known that the n'eural .,stimuLus ··from thepouch<youngsuckingon the teat provides tile distal signalfor lactational inhibition in this species (l)t it is notknown how long it takes for the maternal sy.stem toirreversibly react to the loss of the sucking stim1:llus. Thisex.periment was designed to test this .

Pouch young were removed at 08:00 from nine groups offive wallabies carrying pouch young of 20-60 days old andpresumed to be carrying diapausing blastocyst . The day ofpouch young removal was designated as day 0 Replacementyoung of similar ages from donor mothers were re-attachedafter 24hr (group 1), 36hr (group 2), 48hr (group 3), 56hr(group 4), 64hr (group 5), 72hr (group 6), 84hr (group 7),and 96hr (group 8). Pouch young of group 9 were not replaced

Dept. of Anatomy, Monash University, Clayton, Victoria

3168.

K.Gordon , T.P.Fletcher & M.B.Renfree

REACTIVATION OF THE DIAPAUSING EMBRYO AFTER THE TEMPORARYREMOVAL OF THE SUCKING STIMULUS IN THE TAMMAR WALLABY

p<O.Olp<0.01p<0.001p<O.OOlp<0.05

tScore andSignificance Lev~l

3.743.104.556.392.18

7.87 ±0.251. 94 iO.025.22 ±0.543.13 ::to.314.84 ±0.38

Subject MeanFrequency

Normal MeanFrequency

8.84 iO.032.02 iO.018.16 iO.085.38 iO.165.80 iO.60

TABLE 1

AxonemalComponent

Outer DoubletCentral Pa"irOuter Dynein ArmInner Dynein ArmRadial Spokes

The axoneme of the sperDatozoon provides ·the mechanism for motilityand many reports relate ultrastructural abnormalities of the axonemeto a loss of motility. Abnormalities in axonemal structure havebeen detected in patients ..:ith the immotile cilia syndrome v./hosesperm have severely reduced motility. However, no systematic studyof suc~ a~normalities i: available in patients with low sperm motility.TransmlSS10n electron mlcroscopy was utilised to determine thepropurtion of severely asthenozoospermic «20% motility) patientsthat have a structural abnornality as the basis for this disorder.Ten patients were selected on the basis that their semen analyseswere normal in every respect except for the motility parameters.Seventy-five transverse sections from each of the 10 patients werequantitatively analysed and the mean frequency per axoneme of thedoublet microtubules, central pair, outer dynein arms, inner dyneinarms and radial spokes was determined using the counting proceduredescribed by Wilton et al.

Mean frequencies of each axonemal component from individual patientswere compared to the normal mean for that component found by Wilton~ al (1). All of the men with sperm motility less than 20% showeda reduction in at least three of the axonemal components. Themeans of each component·were then pooled and compared as a groupto normal men. Statistical analysis of the grouped data showed thataxonemal components were significantly lower than the normal meanfor all components measured (Table 1.). These ultrastructuraldeficiencies would not have been detected without the use ofquantitative methods, however whether routine EM assessment isjustified requires further examination in view of the time andeffort involved.

1. Wilton, L~J., Teichtal, R., Temple-Smith, P.D. and de Kretser,D.M. (1985) Structural heterogeneity of the axonemes ofrespiratory cilia and sperm flagella in normal men.J. Clin. Invest. 75:825-831 .

A.D. Hancock, J. Johnson and D.~l. de Kretser

Department of Anatomy, ~lC'nash University, Clayton, Victoria, 3168

L...

130 131

THE ORIGIN OF THE GRANULOSA CELLS IN BANDICOOTS

The rete is a deeply staining cell tube which grows in at the hilarend but fails to penetrate far into the ovary. Unless the intra­ovarian rete in the mouse (2) is equated with the medullary cords, therete in the bandicoot cannot be said to participate in follicleformation. Preliminary ooservations on the potoroo, bettong (Macro­pOdidae) and the brush-tailed possum (Phalangeridae) indica~e that therete tubules ex~end deep into the ovary and appear/to contr~bute tofollicle format~on. ~~ ~ ~~~~.~\.~~

It is concluded that the granulosa cells in marsupials mayfrom more than one tissue.

Suzanne L. Ullmann, Department of Zoology, University of Glasgow,Glasgow, G12 8QQ, Scotland, U.K.

The origin of the somatic cells of the mammalian gonad is still acontroversial suOject. Not only is the provenance of the gonqdalblastema disputed but the granulosa cells, surrounding the oocyte hav:ebeen variously derived from the mesothelium, central. blastema or retecells (;1,). . This. study was prompted by a lack of information on thesetopips irJ,.marsupials.

In th~ bandicoot (~eramelidae)sex;ualdif:\erentiation hf the gonadoccurs 1;U'0und di;l.Y 3 o:(powh life ,. when blas.tema cells give :r:ise to twocell typel? The l~ge:r: cells (apparently homolqgous with Sertoli •cells)eventUallY clump to form the medullary cords; the smaller cells formthe fibrous stroma. During week 1 the germ cells settle in the cortexwhich .Oecomes delimited from the medUlla by a zone of fibroblasts.

The medullary corcis extend towards the cortex where they break upand when meiosis commences (week 5), surround the innermost oocytes,giving rise to the granulosa cells. A mesothelial contribution tofollicle formation cannot, however, be excluded.

ROLE OF THE INITIAL SEGMENTS (IS) OF THE EPIDIDYMIS OF THE TAMMAR,MACROPUS EUGENII.

R.C. Jones and J. Clulow

Department of Biological Sciences, University of Newcastle.

The IS is a homologous region of the mammalian epididymis andprobably plays an essential role in sperm maturation. However, itseffect on the luminal milieu in the ductus has not been determinedsinc;:e micropuncture samples from the IS are generally contaminatedwith stereocilia. This report describes a nrlcropuncturestudy on theepididymis of the tanunar which is being used as a model animal sincethe stereocilia in its IS are short and do not contaminate samples(1). In the tanunarthe IS proper corresponds to the caputepididymidis and the corpus epididymidis is also an initial segment(1, 2). Sperm develop full capacity for motility and show allstructural signs of maturation soon after le.aving the IS proper.

The table below shows the elemental composition (3) of micro­puncture samples (mEg/1) collected from tammars (n~5) whileanaesethized with In.actin .(Byk Gulden Pharm. W. Germany).

Sampling Site Na K Ca Mg ~ Cl

Rete testis 120±7 14±1 O.5±O.3 O.4±O.1 O.3±O.2 130±7Epididymis

proximal head 106±7 11±2 O.l±O.l 6.3±l.8 5.8±l. 9 94±7Distal head 84±10 23±2 O.l±O.l 4.4±O.4 21.6±4.0 87±9Distal body 77±7 30±3 O.6±O.3 7.0±l.3 31.5±2.0 91±6Distal tail 51±4 56±3 0.6±O.2 2.9±0.3 13.9±1.2 129±1

Blood plasma 152±3 4±0 2.7±O.1 O.9±l.1 2.5±0.0 114±2

Spermatocrits showed that the ductuli efferentes (DE) reabsorb87% of the fluid leaving the testis and the caput epididymidisreabsorbs 78% of the fluid leaving the DE. The DE had little effecton the luminal concentrations of sodium, potassium, calcium andchlorine, but significantly increased the concentrations of magnesiumand phosphorus. The IS proper reduced the ratio sodium:potassiumfrom 9.3 to 3.6 and significantly increased the concentration ofphosphorus. The ratio of sodium:potassium was further reduced to 2.5in the initial segment.

These data, and our report showing that the initial segmentproper secretes most of the proteins secreted by the epididymis (4),indicate that the IS proper is mainly responsible for spermmaturation in the tanunar.

(1)

(2)

Byskov, A.G. (1986). Differentiation of mammalian embryonicgonad. ~nysiol. Rev. 66: 101.Byskov, A.. G., & Lintern-Moore, S. (:1,973). Folli~~e formation inimmature mouse ovary: the role of the rete ovarJ,J". J. Anat. 116:

207-2J,7.

(1) Jones, R.C., Hinds, L.A.. & Tyndale-Biscoe, C.H. (1984) Cell Tiss.Res. 237: 525-535.'

(2) Stone, G.M., Jones, R.C. & Hinds, L.A. (1986) Proc. 18th Ann.Conf. ASRB, Brisbane.

(3) Rick, R., Horster, M., Dorge, A. & Thurau, K. (1977) Pfuger Arch.369: 95-98.

(4) Jones, R.C., Worrard, K. & Setchell, B.P. (1985) Proc. 17th Ann.Conf. ASRB, Adelaide, p. 102.

394 - 401.(1) Bourne, A. R. (1985) Gen. Comp. EndQcrinol. .5.fr

BIOSYNTHESIS OF ANDROGENS IN THE LIZARD TILIOQARpGOSA

133

A R Bourne, P. A. Huf and T. G. watson

Biological Sciences, Deakin University, Vic. 3217

Epitestosterone (epiT) and te~tosterone (~) hav~ ~eenidentified as the main C19-steroids J.n the male l:J..zi;i;r::dl~.lJ.$la~ (1). The capacity of T rngosa to, synthes~ze.epiT c:S amajor testicular product is unusual. ThJ.s steroJ.d J.S a mJ.norproduct in mammi;ils, and is thought to come from anextratesticular source. The following expe~imen~s are pa~t ~fa study of the enzymic reguli;ition of ano,rogen PJ.os.yntb,e;:;J.s J.n

T rngQsa.Male lizards were killed with an Qverdose of

anaesthetr.ic. Testicularti(3suewasremov~<;l.C,i+J.o,. :i.nct?-b.C,ited .in.v:iA:.rqatr. 32QC (as slices ano,ho'moge?,i;it e (3) w::i..t~~a+:::i..~usradio labelled substrates . Radiometab.oJ.:J..tes were J.o,ent7t+.?dusing thin J.ayer chromatographY',hJ.gh pe;rforman.ce ~J.ql:l.J.dchrOmato,graphy , derivative fQrmatJ.onan<i cJr:ys,taJ.:l.J.s.atJi.On toconstant s.pecific ac,tivity with authentJ.c stano,a r ¥.production of endogenous androgens was als<;> measu:red,us7ngspecific radioimmunQas says, at varJ.ouS J..ncubatJ.Qn

temperatures (18-37Q

C).EpiT was the major testicular prQdu<;:t (poth f~Qm

exogenous and eno,ogenQus substrates). The maxJ..mal, cQnversJ.onQf radiQlabelled substrates, andrQstenedJ.one (A),'dehydrQepiandrQsterone, prQgesterone and p~egnenQ1Qn~ tQ 7PJ.Tand T was approximately 70 and 10% respectJ.vely. A tl.Itle-yJ.eldstudy, using 14 C- A, ~nd~cated ~hat the yi~ld of epiT ,and Treached a maximum wJ.thJ.n 20 mJ.n. IncubatJ.ons of testJ.culartissue with radiolabelled testosteron7 indicated ~ittle or noconversion to epiT or A. BloQd, kJ.dney and lJ.ver showedlittle. if any, capacity tQ synthesize epiT or T. Theendoge~ous prQduction of epiT (unlike T) increased (p<O. 05)with incubatiQn temperature and was apprQximately triple that

Qf T. . 'TThe results indicate that: (i) unlJ.ke mammals., . epJ.

synthesis in T rugOsa is mainly testicular; (ii) as J.r: othervertebrates 'epiT appears to be formed from andr<;>stenedJ.Qn7,~ya 17ci-oxidoreductase (17o(.-OR) rather than an epJ.merase; (J.J.J.)andrQgens can be synthesized by either the 4-ene Qr 5-enepathway'; (iv) the prQduction Qt epiT is temperature dependent,and regulation Qf 17/J...-OR by environmentaJ. temperature ~ay pl?-ya part in the seasQnal prQductiQn of androgens J.n thJ.s

ectothermic animal.

~e reprod~ctive anatomy and biology of the long penis form ofthe httl,e mastJ.ff bat (Mormopterus -elaniceps) was studied fromspecimens (37Q. 37cf) collected in squth-east South AustraliaMaterial was s:udied by serial paraffin or cryostat light mic~oscoPY,and electron mJ.croscopy (SEM and TEM) and radioimmunoassays for sexsteJoids were performed on plasma.

The anatomy of the reproductive tracts of both sexes wasgenerally reminiscent of other Molossidae. However. certain featureswerE7. unique-these included the penis, -which was horizontally bifid,the presence of an unusually long os penis and os clitoridis, andprominent sebaceous para-anal and paravaginal glands. The uterinecorpus was aglandular and its epithelium was stratified and virtuallyindistinguishable from that of the vagina. Deep vaginal fornicessurrounded a prominent cervix. As in other molossids, the bicornuatefem~le tract was dextrally functional and the left ovary was dominatedby J.nterstitial cell tissue and lacked advanced follicular stages.

Mormopterus planiceps was monoestrous and monotocous.Spermatogenesis was initiated in spring (October) and culminated inspermiogenesis in autumn (February - May), during which period plasmatestosterone overtook androstenedione. Thereafter the testesregressed but epididymal sperm reserves persisted. Proestruscommenced in February but ovulation and conception did not occur antillate winter (July/August), after which the epididymides were emptied,the accessory glands regressed and the thick proestrous uterine corpusepithelium was dramatically reduced in width by a massive phagocyticcell invasion. During gestation, plasma sex steroid levels rose toreach t~eir maxima in November/December (progesterone:19 ng/ml;oestrad101:2 ng/ml; oestrone:l ng/ml). The long gestation period(~ntil December/January) may incorporate a short period of embryonicdJ.apause. Growth of the young was slow but the females attained .sexual maturity in their first year.

This study demonstrated that this species has departed from the'normal' temperate molossid pattern of spring coincidence ofs~ermatogenesis and ovulation to conceive during the inhospitablewJ.nter months. The occurrence of conception on stored epididymalspermatozoa is a feature of many rhinolophid and vespertilionid batsbut has not previously been described in a molossid. '

*Currently at Department of Anatomy, University of Arizona, Tucson,Arizona, 85724, U.S.A.

THE REPRODUCTIVE BIOLOGY OF THE LITTLE MASTIFF BAT, MORMOPTERUSPLANICEPS (CHIROPTERA: MOLOSSIDAE) IN SOUTH-EAST SOUTH AUSTRALIA.

Elizabeth ~ Crichton*" and Philip H. Krutzsch

Department of Anatomy/Histology. University of Adelaide AdelaideAustralia and Department of Anatomy, University of Ariz~na TUcso~Arizona. U.S.A. ' •

132

134 135

SATELLITE SYMPOSIUM"ADVANCES IN EMBRYO TRANSFER"

24-25 AUGUST 1985

REPROD1J:fiOR Df FREE AND CAPTIVE StMMER WHITDfG (SILLAGO. CILIAfrA)

R.K. Munro

TABLE 1. The mean number of embryos recovered after treatment with prid

and FSH•

It would se~m that the combination of PRIO and FSH is suitable forthe induction of superovulation, with precise control of the time of

oestrus.

CSIRO, Division of Tropical Animal science,Tropical cattle Research Centre, Bo)C 5545,

Rockhampton Mail centre, Qld. 4702, Australia

The oeSitrOus ano. superovulatory resp<.:mses of 20 Brahm~n and 20Here£ord X Sho;r;t;h.prn (HS) cq'ils were. Obser'\1ed.",ftertreatmentwithfolliCle Sit,~ulatin.g hOPJ\one (Fq}i) •. a.t 4 .do~e. rate,s(~otal dpses 16, 3'2,48 and 64 mg, •.~dministe;r;ed iJ'). ~ ~~ldoseSl ",t12 hour intervals) andintr~va:g.:j.p.~;L ",ciminifitrationofprogestefone (PRIO) for 14. days. Allcows were first observed in oestrus 2~30 hours after PRIO removal. 'ihesuperovulatory response was assessed by non-surgical embryo collection~t O~¥ 6 or. 7 (Day 0 = day ot oe~trus). After completion of the firsttreatmenta,pd collection 16 Brahman and 16 as cows were treated a secondtime in such a way that the members of each dose group contained .Iirepreseptative of each dose group from the first treatment. There wasno significant difference between the numbers of embryos collected ateach dose of FSH nor was there a significant effect -of the dose used atthe first treatment on the number of embryos collected at the secondtreatment (Table 1). significantly more embryos were collected afterthe second treatment than after the first (6.5 ± 1.10 v 4.0 ± 0.78, P <0.05) and about twice as many embryos were collected from Brahman cowsas from HS cOWS (7.0 ± 1.3 V'S 3.2 ± 0.6, P < 0.025). llbe proportion oftransferrable embryos collected was not significantly different betweenbreeds or between the first and second collections but was higher after

16 mg FSH than the other doses (p < 0.001).

'filE supERO'ltJIJ\.'l'ORY RESPONSE OJ!' B. TADRUS ANDB. IRDICUS CA'r'rLE FOLLOIUBG TREATMENT nm FOLLICLE

STIMULA'fiNG. HORICRE AND PROGESTERONEPhamacology, University of QJ.eensland,

Health and NJ.trition, QJ.eensland Institute of

The s1.ll\mer whiting (SiHago '1,' twaters of South-east Queensland Cl,Th'l,a a) .is found abundantly in the

• e aJ.m of this pr' tcompare the reproductive potential of f . . . oJec was tosumm.er whiting and to assess the potentirleefand captive populations ofa or aquaculture.

Both populations of fish -...ere sidprocessed for histology Th h' t lamp e monthly and their gonadsc.ompared and quantita~ve d~ff·1.S 0 ogy of the tTNO populatioJ')$ W!lSi

, 1. erences examined u'tecronques. Each cell tyPe . . .S1.ng. s terologicalaccording to the maturity stag;a:ndSC~ed (female 1-4 and male 1';'5)volume fraction. The weight d s score was used to weight the

ad . e scores were summed t 'repr uctive score which was used a' 0 gJ.veapotential. s an l.ndicator of reproductive

.(1) 'IhFe stafges of oogenesis were similar to those found in• ree emales completed oogenesis with ' other fish

and spawned. However captive females d'd mature eggs belong ovulatedoocytes matured to the vitell . 1. not complete oogenesis. 'Ihe'Ih ogen1.C stage and then b

e reproduc tive score showed th t f ecame atretic.one month earlier than capti' e a

f. hree females started developnent

N v loS reaching peak d 1 .ovem~er whereas the captive fish aked' eve opment l.n

atres1.a became very high in c tpei f,1.n December. 'Ihe levels of

dap ve l.sh as the b d'

procee ed. Spematogenesis was c 1 t d ' ree long seasonobvious qualitative differences. om~e :' t 1.;, both ~pulations with noin the data for male fish since f an 1. ,a love stud1.es showeq. a biaspreparing to spawn and so lack m:

e:1.Sh enter ~e sampling ~rea

s~ages whereas these stages are seen tn t~ the ~arl1.er developmentalf1.sh attained a higher reproduct1.' e capt1.ve male. '!he captive

tai ve score than free fi h bre ned spem and did not sp 'Ih s ecause theypotential as indicated by ~~. us they had a higher reproductiverealised. e score but this potential was not

J.A. Goodall, A.W. Bla~kshaw and *M.F. Capra

Department of Physiology andst. Lucia, 4067.*Department of PublicTechnology, Brisbane.

. Captive fish are non-reproductiveenV1.ro tal f possibly due to some stressful, nme~ actor preventing finalspawn1.ng l.n the males. maturation in the females and

./1.

Wallace, R.A. and Sellman, K. (1981)aspects of oocyte growth in Teleosts.343.

Cellular and dynamicArne1'. Zoo1,., 21: 325-

Breed1st collection 2nd collection

of cow Total dose of FSH (mg) Total dose of FSH (mg) Total

16 32 48 64 total 16 32 48 64 total

HS 1.0 2.6 2.8 3.6 2.5 4.3 1.0 5.0 6.0 4.1 3.2

B 3.4 9.0 6.0 3.6 5.5 7.8 9.8 4.3 13.8 8.9 7.0

Total 2.2 5.8 4.4 3.6 4.0 6.0 5.4 4.6 9.9 6.5 5.1

136137

The e:l".sential role of· d t ' ,supet:'ovl,llation is well dem'o 9tOnatO r?ph1nS 1,n ovulation, andgo d t ' .... •' '. .., ns ra ed 1n hypophysect dna 0 roph1n experimEjnts Th ., ' . ' , omy an exogenous

follicular level remains ~nkno:nP~~~~serole.ofgonadotrophins at thegonadotrqphins can rescue follicl a f Qugh ther';! ar:., suggestidns thator rate of follicles or increa ~~ rom a~res1a, .1ncreas e the growth"ovulable" state.' se e recrU1 tment of follicles to an

FSH preparation OR FSH LH LH!F5H

1(S1 units) (523 units)

1.0 34.02 1.5 32.0

0.26 1 :130

3 4.3 34.00.69 1 :46

4 7.3 32.00.06 1.567

50.D3 1 .1067

12.1 34.0Control 1.0

0.83 1: 41

2. Local ovarian factors

In view of the desirabili t of h"on either a per animal or pe yac ,1e\l1ng a consistent responsewhere different FSH preparat~ogroup bas1s an experiment was conductedfor FSH and LH content and the~sa~~~e,examine~ by r~dioreceptor assayThe resultant ovarian responses ,n~stered 1n equ1-FSH doses per ewerelationship to either FSH t var1e greatly and showed no •con ent, LH content or LH:FSH ratio Table 1.

Table 1. O~ulation rate (DR) in 6-8d1fferent F5H preparations ewes following treatment withand primed with progesterone

FOLLICULOGENESIS AND EGG PRODUCTION

, L.P. Cahill and R.C. Fry,An1mal Research Institute, Werribee.

A major cons~~aint in an e tof eg~s produced per donor (usLlaffy 4:~s;e\9~7ration is the low numberthe h1gh variability of res onse er,1 1zed eggs per cow) andeggs is the result of a comPlex ,between,an1mals. The production offactors acting at the ovary~ 1nteract10n of gonadotrophin and local

The onset of oestrus in superovulated ewes has been'reported as 24-36hr after removal of progestagen sponges or 24-48 hr after prostaglandininjection (5). Whyman et al. (3) reported a median time for the first ovulat­ion of 23.6 ± 0.5 hr after the onset of oestrus in PMSG-superovulated ewes,with a median interval between the first and last ovulation of approximately6 hr. These workers found a similar timing for ovulation in untreatedcontrol ewes (23.5 hr after onset of oestrus). This timing of ovulationseems to agree 'with ,the finding of Evans ~. (4) who reported that mostewes commenced ovulation between 54 and 60 hr after treatment with prosta­glandins or withdrawal of progestagen sponges. These authors also reportedthat insemination close to the time of ovulation resulted in a reduction of

recovery and fertilisation rates.

For ewes treated with superovulatory hormones in embryo transferprogrammes, opinions differ on the optimum timing of intrauterine insemin­ation with respect to synchronisation treatment and/or oestrus, and on theeffect on egg fertilisation rates of varying the dose of spermatozoa.Comparisons between non-superovulated and superovulated ewes in synchronisedoestrus are often made, but may not be valid.

ADVANCES IN SHEEP ARTIFICIAL INSEMINATION

W.M.C, Maxwell

The highest rates of egg fertilisation in superovulated Merino ewes havebeen obtained after intrauterine insemination with pooled fresh semen frommore than one ram 24-28 hr after removal of progestagen sponges (e.g, 5,6).This would appear to be before the expected time of ovulation, but littleinformation is available on the timing or spread of ovulation in superovU­lated Merino ewes. The range of time that fresh spermatozoa retain theircapacity to fertilise ova has been estimated at 24-48 hr (7), and thesuggestion that insemination close to the time of ovulation may interferewith ~va capture by the oviduct due to manipulation of the reproductivetract (4,8), also supports the argument for insemination before ovulation,

Fertilisation failure has been a major limitation following naturalmating or cervical insemination of sheep subjected to superovulation bytreatment with gonadotrophins (particularly PMSG). Fertilisation rateshave beenimproved, however, by surgical deposition of semen in the uterusfollowing laparotomy (1). The adaptation of the laparoscopy technique toinsemination of sheep (2) has made intrauterine semen deposition in embryotransfer, programmes less time consuming, and allows a greater re-use ofdonors due to a lower incidence of post-operative adhesions.

Animal Breeding and Research Institute, Katanning, W,A.

One of the major obstacles to improvement of fertility following art­ificial insemination (AI) of sheep is the near impossibility of passing aninseminating pipette through the cervix of the ewe. The cervix constitutesan initial barrier to the ascent of spermatozoa, and a relatively smallproportion of cells deposited in the cervix reach the site of fertilisation.Higher fertility is .obtained following cervicaldrv'a.ginalinsemf~ationwith fresh diluted semen than with semen stored in a chilled or froz.enstate, due to a reductibh in the transport of stored spierlnatozoathrough the

cervix.

Gonadotrophin action at the ovary.

, The discovery of factors present in f' "speC1es which can have a direct effect ol11cular fluid of manymodulate the gonadotrophin eff t on the ovary and thereforelocal factors may play an impo~~aSthaslle~to the possibility that therate. Hence it is probable that ~h r~ ~ 1n determining the'ovulationof stimulation in terms of the co t e s ate" o~ the ovary at the time~umber of large follicles and then ent,o! ~ol11cular fluid or the:-nfluence the eventual ovulation r,:~~s1t1v1ty to the ,local factors1f there are many large foIl' 1 • Several stud1es have shown thatstimulation the response in ~~u~:t~re5entt a~ the time of gonadotrophin10n ra e 15 low.

1 •

138139

In non-superovulated e~es, pregnancy rates of 63-76% have been obtainedusing fresh semen doses for laparoscopic insemination ranging from 12.5 to100 million total spermatozoa (9). Using frozen-thawed semen in N.S.W., dosesranging from 10-80 million motile spermatozoa resulted in lambing rates of58-66% (10). In Western Australia, doses of >20 million are required toachieve the same result (8).

ESTABLISHMENT OF A SHEEP E~RYO BANK

J. Eppleston

School of Wool and Pastoral .Sciences, University of N.S.W.,Kensington. N.S.W.

• rob s is routine in many overseasEmbryo banking of frozen. m~ce e ryo strains and .as genetic

laboratories for the preservat~on of unused . . of the best. (1) While Australia possesses some. .

control populat~ons . d mbryo freezing. r these techno10'gJ.esexpertise in sheep supero:vu:ah~a:~ s~eepgenotyp.e'S.have not beem used to b~ ~po ed b the-Rural Credits Development

A co-operative proJe:t r f~d Neil Moore (Uni. Sydney), EuanFund of the Reserve Bank, ~nvolvJ.ng (Monash) was initiated to

( . N S W ) and Alan Trounson, , . . .Roberts , Un~. ..•. k' and to provide th~s serv~cedemonstrate the viability of embryo ~an ~n:to those,interested in the preservat~on of. f economic importance .

(a) Rare breeds of sheep no lon~er 0 insurance against natural(b) Present day commercial stra~ns a.s disease or selection of

disasters, introduction of exot~c

faulty sires d but which may be required(c) Selection lines not currently use ,

later, t 1 lines for the measurement of genetic progress(d) Genet~c con ro , tead of maintaining costly

in selection programmes l.ns

randomly bred f1~ckS. ise and training in .embryo freezi~gMr. Roger Bilton provl.ded expert. d t demonstrating the viabill.ty

. th first programme al.me aprocedures durl.ng e d d d seventy-seven embryos wereof the techniques used. One hun reo an

th'ng 43% 'developed in culture

collected from 42 donors and follow:n9 aw~ T'able 1 lists the f10·oksamb ' recipl.ent ewes.

and 45% developed to 1 s l.nb

k d and the results obtained.from which embryos have been an e

conservation 20 41 (2.1)Camden parkMerinos of Historic

Breed(4.9)

Genetic 101 495Hyfer

control Flock

16 59 (3.7)Stud Insurance

Merino Against Loss

~: Embryo Banking Programmes

In superovulated ewes, Walker et ale (11) detected no differencesbetween doses of 2 and 20 million motile spermatozoa for fertilisation ofova. Type of semen (fr~sh or frozen) did not affect conception rate in theseewes, but the proportion of ova fertilised was lower for frozen than forfresh semen. This agrees with the findings of J.P. Ryan and N.W. Moore(unpublished data), who obtained ova development rates of 395/457 (86.4%)and 46/141. (32.6%) for superovulated ewes inseminated intrauterine withfresh and frozen semen respectively. In most embryo transfer programmes,donors are inseminated with an "excess" number of spermatozoa in order toobtain optimum fertilisation of ova, and doses greater than 100 millionmotile spermatozoa are often used.

Further research is required to investigate the time and spread ofovulations in animals subjected to oestrus synchronisation and superovulat­ion with gonadotrophic hormones, and to determine more accurately theappropriate time for intrauterine insemination, particularly when frozensemen is used, in order to maximise ova fertilisation rates.

(1) Trounson, A.D. and Moore, N.W. Aust. J. BioI. Sci. 27: 301-304 (1974).(2) Killeen, I.D. and Caffery, G.J. Aust. Vet. J. 59: 9S;(1982).(3) Whyman, D., Johnson, D.L., Knight, T.W. and MoQ;e, R.W. J. Reprod.

Fert. 55: 481-488 (1979).(4) Evans,~., Holland, M.K., Nottle; H.B., Sharpe, P.H. and Armstrong,

D.T. In: Reproduction in Sheep, Australian Academy of Science andAustralian Wool Corporation, pp. 313-315 (1984).

(5) Moore, N.W. In: Mammalian Egg Transfer, CRC Press, pp. 119-131 (1982).(6) Ryan, J.P., Bilton, R.J., Hunton, J.R. and Maxwell, W.M.C, In: Reproduction

in Sheep, Australian Academy of Science and Australian Wool Corporation,pp. 338-341 (1984).

(7) Dauzier, L. and Wintenberger, S. C.r. Seance Soc. BioI. 146: 660-663 (1952).(8) Maxwell, W.M.C. Anim. Reprod. Sci., in press (1985).(9) Davis, I.F., Kerton, D.J., McPhee, S.R., Grant, I. and Cahill, L.P. Proc.

Aust. Soc. Reprod. BioI. (1984).(10) Salamon, S., Maxwell, W.M.C~ and Evans, G. Proc. Aust. Soc. Reprod.

BioI. (1985).(ll) Walker, S.K., Smith, D.H., Little, D.L., Warnes, G.M., Quinn, P. and

Seamark, R.F. In: Reproduction in Sheep, Australian Academy of Scienceand Australian Wool Corporation, pp. 306-309 (1984).

Frozen Embryos - An Exportable Proposition?

T.D. Heath

Flock

FlockMerinos

purpose of,Storage

Demonstration

DonorsTreated

42

Embryos StoredTotal (Mean)

177 (4.2)

Fate

43% developedfully in cultureand 45% developedto lambS

2/12 (17%) deve-loped to lambs.Remainder stored

Stored at BerryA.I. centre

20 transferred.Remainder stored

Only title supplied by author.

(1) The Freezing of Mammalia EmbryoS.

Amsterdam. 1977.

Ciba Foundation Series, Elsevier,

--------------------------140

141

EARLY Pl-U':UNANCY DETEC'1'lON

Peter A. W. ROJers

Department of Cbstetr ics am GynaecolOJYMonash University

Queen Victoria Medical centre1'<Elboume Victor ia

Culture Media: Quality Centrol

f Obs le'ricg and GynaecologyDepar~ment 0 ,~'"

UniverSIty ot SydneyRoyal North Shore Hosp~~al

St Leonards NSW 2000

Table 1 Percentage Pregnancy per Transfer; z...'JCOash IVF PrOJrarnme

Pregnancy data frc::m 1432 errbryo transfers at the r.bnash IVF prOJrarrare CNerthe past two am a half years reveals a large nonth by m<nth variatien insuccess rate (Table 1).

~ believe a large part of this variability is generated in the errbryoculture system; in particular in the culture media. l\e use a mcilifiec1Whittingham's T6 medium, although a number of other media are in use,including Hams F-IO am Earles. Culture medium prdJlerns are usuallyindicated by reduced fertilization a~ cleavage rates, increased embryofragmentatiOl and reduced survival to the 8-cell stage am beyond.Quality cOltrol procedures should include a thorough clean am check ofthe water system used to prciluce the water for the culture media, or inthe case of bottled ultra-pure water an alternative supply should beassessed. Chemicals fran which the media is made should be assessed forcOltamination or alternatively new batches should be started. Osrnorretersam pH rreters used in making the media should be checked for accuracj, andall glassware should be tissue culture washed alx1 steriliZed with theutmost care. Culture media should be routinely tested in an anirral S'jstemsuch as the rrouse, with the embryos shoving high survival rates [rom t.'1e1-cell to the blastOC}'st stage. A more de fini te test of cu ItLlre med iaquality can be arranged by borroving S~ prepared :redia frOG ,:tfl ,,~mi)r.lolaboratory with goo:1 results am running the two sets of me-J :'J :-,jje biside.

" ' and monit.or embryonIcI"rlabI'..l'l'ty to detect fert.IIIzat.lon. ' ecies severJy

'1'he. nan (' yin a 0 mest 1 C S P , ,b 1 i t Y t h r 0 ugh 0 U t. ear 1 y pre g , ' , ,han i ma is" I,' a .I 1 e ci

VIa I nt optIons of sue . "f tst.r icts the manageme , t lr't u have a slgnr lcan,re I ,bryonlc mor a J b

fertilization and ear y em, t of supprovulation and em ryoa ffect on fecundity. The adverhl, 'hI' r'ght~d this problem. The

1 's have Ig, 'istransfer tech~o ogle . for early pregnancy deteCLIO~ ,development or methods , " f reproduction researcL.thirefore. an important prIorIty or .

, , .' " alor placental protning naveb of pregnancy-assoCIatea an, 's While many of

Anum er " 'variety of domestIC specIe,. .'. 1 earbeen descrIbed for a " bly det.ect pregnancy. all aLPthese have been shown to re~laanancy by which time currentto' do so relativelylate,lll pre~ 'p'hY 'appears to be Just as

h real time ullrasonogramet.hods suc as ' .satisfactory,

R Soe Lond i:lJ Reprod Fert 73:567-77,

( i) 0' Ne ill C. (1 98 5 ) 1 V'7 6 I P l' 0 ('V Clunlc GJA. I .(2) Mor1.on H. Hegn ,

193:41~1-19.

".' n have been elusive. in recentMeans of early pregnancy deteC~lo h siology have been sho~n.toyears alterations to materna~ .p ~e lion ana early pregndncY·

"ur as sale consequences or concl

P-observations that theOl-e. these were tIe '. 'atelC'!Prominent among , d . d a factor whlctt eauser! PL .,.,

.i plantation embryo pro uce ,t' of a putatIvepre ,.m - (1) ana the produc Ion .,activatron '. factor 1n early pregnancy (2).ImmunosuppressIve

, " offers hope of. ternal pnVSlologyV iMonitol'ing ,such changes to . m: y within llourg of concep1.ion anlroutinely diagnosing pregn~~ 'irt~ throughout the prean~

1 t () rIllgem b r von 1 C V1 a b1. IN hi 1(' t, 11 e em br yon I Cman ~ . of pregnancy. " ' 'z'dt\ e r] . imp.i ant. a 1. j 0 II S tag Il h p'n ,: n~ 0 mE' n ~ h a v e bee n c h a r act e r I :. e .~ "ble for t ese .~' .' P1. ocenfactors responsl .' 1'0'" thcJr delcct_Iun haVtl not y:. theserobugt assay systems ". the tr'ue potentralol'. Assessment ofaeVl-loped. ." it these assays.observalions must KWH

23.823.827.913.314.019.2

19851984

a20.521..113.520.822.414.627.614.318.415.19.7

o11.9

5 .• 028.027.518.98.7

15.835.016.313.513.8

1983

JanFebMarAprMayJuneJulyAugSepto::tNovDec

EMBRYO TRANSFER IN THE SHEEP.

harnes. G.J.Vl .. Quinn P .. Sea;;.a.rk· R.F.XI, us t, Ve t. J. 6.1: 1 U5 - 1 0 6 (1 98 5 ) .

142

S.K. Walker, R.J Ashman. G.M. Warnes. D.H Smith*, P.Quinn. Depan:.ment of Obstetrics and Gynaecology,University of Adelaide, T~e Queen Elizabeth Hospital.Woodville S.A. 5011 and Turretfield Research Centre.Department of Agriculture, Rosedale, S.A.

Factors affecting the success of embryo transfer insheep have been examined in numerous reports (eg 1).Embryos aFe usually transferred by surgical procedures toeitner the oviduct or uterine horn depending on the stageor embryo development .. A recent development has been the~ransfer o~ emoryos by laparoscopy, with potential savings1n t1me and costs.

Most data published on this procedure is from smallnumber of sheep. Pregnancies have been obtained in 3/6(2), 2/5 (~) and 2/3 (4) recipients to which 1-2 embryoswere trans! erred', In these studies, the uterine horn ofeach sheep was cannulated and embryos were introduced intothe lumen using an appropriately sized catheter. However,time taken (15 - 30 min per ewe) was no quicKer than thatrequired for transfer by laparotomy.

A quicKer method (2 - 4 min per ewe) of transfer bylaparoscopy without cannulation has been reported by thisla90ratory (5). The number of pregnant ewes aftertransfer of 2 morulae per ewe was 4U.7% (22/54) and therewere 1.6 foetuses or lambs per ewe. This study has nowbeen extended and pregnancy rates of 40.0% (14/35), 33.9%(20/59) and 44.4% (4/9) were obtained in ewes to which 2embryos were transferred on day 4, 5 and 6 respectively(day 0 = day of oestrus). Pregnancy rates were higherwhen morulae were transferred to the oviduct by laparotomy(d5.7% 6/7) than when transferred to the uterus bylaparoscopy (33.3%, 5/15).

Transfer by laparoscopy involves relocation ofembryos with1D the uterus (eg from the terminal 1/3 of theuterine horn to the body of the uterus) and this may bedetrimental to embryo survival. Determination of theoptimal site of transfer within the uterus may beworthwhi le. Whatever the a"venues or future research. aviable alternative to transfer by laparotomy will need tobe .equally succes5!ul and more rapid1. Arffi5trong. D.T and Evans. G. Theriogenology ~~: 31­

42 (1983).2. SChiewe. M C.. Bush. M.. Stuart, L.S. and Wildt.

D.E. I'11eriogenology 22: 675-682 (1984).3. r'JcKe1-"ley, WAC. and·-Robinson. J.J. The Vet. Rec.

J. l ?'2 3 u (1. 9b 4) .4. l"Jl1i:.lga. E R an::} .5c,,~er. A.A. The Vet. Rec. 115: 401

4U2 (198.;).5. \\3 L\.er, S. K..

ar,d 'smith. D.H

143

EMBRYO SEXING WITH X-LINKED ENZYMES

T.J. Williams

C51 RO, Division of Tropical Animal Science,Tropical Cattle Research Centre, Box 5545,

Rockhampton Mail Centre, Qld. 4702, Australia

A potential tool for embryo sexing which has received little attention isthe X-linked enzyme. Prior to X-inactivation, the levels of X-linked enzymesare dependent upon the number of X chromosomes. From the early morulastage to the late blastocyst, females (X X) have double doses of X-linkedenzymes whereas males have single doses. Discussed here are the results ofsexing mouse blastocysts prior to transfer using a simple and rapidcolorimetric test for the X-linked enzyme, glucose 6-phospate-dehydrogenase(G6PD).

The assay utilizes the vital blue stain, brilliant cresyl blue (BCB) whichbecomes colourless when reduced by NADPH. In the presence of substrate,glucose 6-phosphate (G6P) and coenzyme, nicotinamide adenine dinucleotidephosp'hate (NADP), the enzyme G6PD converts G6P to 6-phosphogluconate andliberates NADPH. The rate of reduction of the blue stain to a colourless stateis a semi-quantitative measure of G6PD activity.

Blastocysts were collected on day four post-mating from superovulatedB6D2 F1 females in a substrate-free medium (Dulbecco's PBS + 0.3% BSA) andstained for 20 minutes in .05 mg/ml of BCB. After 30 minutes at 37°C in areaction mixture of 0.5 mM G6P and 0.5 mM NADPH, embryos were visuallyscored on a scale of 0 to 5 for amount of BCB remaining (0 = no stain: highlevels of G6PD; 5 = dark stain: low levels of G6PD).

Distribution of embryos according to G6PD content appeared bimodalwith 10%, 22%, 18%, 19%, 22% and 9% (n = 759) scored as 0,1,2,3,4 and 5,respectively. 50% scored as females, i.e., 0, 1 or 2; 50% scored as males,i.e., 3,4 or 5. Transfer of sexed embryos according to G6PD score resulted ina decrease in the proportion of females born as G6PD decreased (13/16 (81 %),32/42 (76%), 17/28 (61 %),23/48 (48%), 18/44 (43%), 0/3 (0%) scored as 0,1,2,3, 4, and S, re.spectively). The combined percentage of females born fromgroups 0, 1 and 2 was 72% (62/86) and percentage of males born in groups 3, 4and S was 57% (54/95). Overall, 64% (116/181) or about two-thirds of theembryos were correctly sexed prior to transfer. There was a slight decline inbirth rate from sex embryos (35%, 181/516) compared to controls (46%,39/84).

Conservation of the X-linkage of the G6PD locus in most mammalssuggests that this sexing method could be applied to embryo transfer in manylivestock species.

4

AUSTRALIAN SOCIETY FOR REPRODUCTIVE 8I0L0GY

Minutes of the Annual Student Meetingheld at 1.00 pm Thursday 29th August 1985

Attendance - approximately 40 students

1. All students found the Annual Dinner too expensive and 90% of thestudents at the meeting did not attend.

2. It WBS reported that there were too many post~rs in one session and thatovercrowding around the posters was a problem.

1. Concern was raised re the timing of the Annual General Meeting each year,i.e. just prior to the Annual Dinner. Those students who did attend thed i.nner found ther~ to be n,) time to get changed etc between the end ofthe meeting and the departure of buses for the dinner. It was proposedthat perhaps an alternati.ve ti.me could be arrallged for tne AnnualMeeting, e.g. one lunch-time.

4. As approximately 50% of the students present were Endocrine Society (ESA)members in addition to ASRB members, some concern was felt re ESA holdingtheir annual meeti.ng in Canberra in 1988 in conjunction with theFederation of Australian Societies for Experimental Biology. The overallopinion was that there was always much to gain from the ESA meetingespecially the Harrison Lecture. The students felt that it would bedifficult financially to attend two interstate conferences in one year.

5. It was suggested that a social evening should be organized for the secondnight of the Annual General Meeting when, there is usually no eventscheduled. The majority of students could get to know one another; theminority felt that it should be an ASRB/ESA function.

6. The students felt that there should be some post-conference excursionsorganized by the students in the particular state where the meeting isheld e~ch year. Again it was suggested that this would enable studentsto meet one another.

7. It was suggested that early registration would be more appropriate thanat the'Opening Ceremony.

8. Concern was raised re the lack of space at the Opening Ceremony.

9. It was proposed that a programme for the ASRB and ESA meetings should beenclosed in satchels (or displayed on a board) to remove the necessity ofbuying the conference proceedings, e.g. where students are not financialmembers of ESA.

10. It was brought to the attention of those at the meeting that a newstudent ASRB representative would be elected at the 1986 Annual StudentMeeting in Brisbane.

"#iII AUSTRALIAN SOCIETY FOR REPRODUCTIVE BIOLOGY

Minutes of 17th Annual G~neral Meeting

Held at ~:30 pm, University of South Australia

17.1 Attendance and apologies. Approximat'"ly 100 Hembers ,'1ttend~d. Ap,)logieswere received from B.S. Bellinge, L.P. Cahill, J.K. Findlay, J.U. O'Shea,T. Stelmasiak, A.D. Trounson and C.H. Tyndale-Siscoe. As the Chairman(J.K. Findlay) was unable to be present, B.G. Miller pr~sided as DeputyChairman.

17.2 Minutes of 16th A.G.M. The l1inlltes were accepted (proposed 'l'ihite,seconded Temple-Smith).

17.3 Business Arising. J. Cummins (Convenor, Local Organising Committee)announced that tne 1986 Annual Meeting would be held from September 1-3,1986, at the University of Queensland. Venues and accommodation had beenreserved, and Australian Convention and Travel Services Pty Ltd (ACTS)had been engaged as Conference Organisers.

17.4 Treasurer's Report. The Treasurer (C.D. Nancarrow) presented his Report,and indicated that there were currently 483 financial and new Members.He proposed that sale of the current Proceedings should be set at $15.00.In discussion it was noted (Scaramuzzi) that membership lists had beensold to publishers, and the Treasurer agreed to enquire what use, if anyhad been made of this sale. The Report was accepted (moved Martin,seconded Galloway).

17.5 Chairman's Report. B.G. l1iller read to the meeting the Chair~an's

Report, in which he apologised for his absence, and thanked retiringmembers of the Committee. Principal items in his report wer~ theConstitutional changes to be debated (see 17.9 below); the possibleIncorpora t ion of the Soc iety; the appointment of a periuancnt bus inesssecretariat (see 17.10 below); the possibility of joining tha Federationof Australian Scientific and Technologicai Societies (FASTS: see 17.12,below) j the activities of the Pituitary Hormone Distribution Committee;the organiz'ltion of the current Annual t1eeting, and society support ofsatellite meetings such as those on the Epididymis and on Marsupials andLactation.In discu,ss ion the quest ion of Incorporation was raised. The Committeereported that a sub-committee of B.M. Bindon, I..J. Clarkt~ and J. Falconerhad heen asked to investigate and to make recommendations to the nextA.G.M. The Report was accepted (proposed Bindon, seconded De Kratser).

17.6 Programme Organising Committee Report. D.R. Lindsay (convenor) reportedthat in 1985 the P.O.C. had moved from Melbourne to Perth, and had beenincreased from 4 to 6 members. One hundred and twenty one papers hadbeen submitted for the A.G.M., an increase of 11%. Three had beenrejected: two for topic area and one for incorrect for~at. Two paperswere withdrawn, leaving a total of 116. Candidates for the JuniorScientist Award where possible, were given priority for Oral sessions.The card system for notification was poorly obeyed in general.

In discussion some disquiet was expressed at the uneven distribution andlack of time available for the poster sessions, as well as at the poorcoordination between A.S.~.B. and E.S.A. sessions. Members urged fullercollaboration and communication between P.O.C.'s in the future, andstressed the advisability, if possible, of stationing the two Committeesin the same city. The Report was accepted (proposed Wales, secondedBindon) •

17.7 New Hembers.~1embership.

The Treasurer announced that 51 persons had applied rorThese ware duly elected.

a

T.J. RobinsonC.W. EmmensD.M. de KretserN.W. MooreJ.K. FindlayB.M. Bindon

P.J. LutjenR.J, Rodger & C.B. GowS.P. FlahertyC. O'NeillB.J. Waddell

C.W. EmmensT.J. Robinson

Life Members

Junior Scientist Award

19811982198319841985

1969-19731973-19771977-19811981-19831983-19851985-1986

8 ~~)

'rS

Permanent Business Secretariat. The Act ing Chain\an l:2ported that theCommittee had eKamined four bids from interested groupS. It had h,.~endecided to recommend the submis~ion of A.C,'r.S., who had based theirsubmission on a budget of $10.00 per member per year. The Tr,~asurerstressed that this was fur a trial period of one year, and pointed Olltthe advantages of basing a secretariat in Canberra. After discussion,the meeting voted to ratify this decision of the Committee (proposedBindon, seconded Galloway), and the Treasurer and Secretary wareempowered to contact A.C.T.S. and to draw up a contract.

Constitutional Changes. Proposed changes to Ordinary Rules 1, 4 and 5had been circulated two months prior to the A.G.M. After discussion toclarify the nature or the changes they were moved (proposed Flaconer,seconded Wales) and carried with one abstention. The Secretary agreed tocirculate the rev ised Const itut ion to all members with the neKt

Newsletter.

Elections. The Secretary reported that only one nomination had beenrece~for the position of Chairman, and B.M. Bindon was thereforedeclared elected. C.D. Nancarrow was likewise nominated and electedTreasurer. Three Co~nittee ~embers were nominated ror three vacancies:J.e. Rodger (second term), L. Hinds and 1.J. Clarke.

17.13 Bicentennial meeting of Federation of Australian Societies forExperimental Biology, Canberra 1988. The Acting Chairman reported onbehalf of the Committee that A.S.R.B. would be looking to holding asatellite meeting in Canberra as part of the Bicentennial meeting.

17.14 Future Meetings. Provisional venues were: 1986 Brisbane; 1987 Sydney;1988 Newcastle; 1989 Melbourne and 1990 Perth. In 1988 the ESA would beholding their A.G.M. in February as part of the Bicentennial meetingabove and there would be no joint meeting in that year.

17.12 Federation of Australian Scientific and Technological Societies (FASTS).As delegated Committee member, J.C. Rodger spoke in favour of A.S.R.B.joining FASTS, and pointed out the need for scientists to act in aconcerted way to protect their proression by means of a centralisedlobby. B.G. Miller reported on behalf of the Committee that, while itwas broadly sympathetic to the ideals of FASTS, it would prefer to urgecaution for the time being until the activities and financialrequirements of the organization were more clearly defined.

17.11 Membership Fees. In the light or the decision made in item 17.10, theTreasurer proposed that rull Membership fees be raised to $30.00. Thisproposal was agreed upon by the meeting (proposed Martin, seconded

Rogers).

17 .10

17.9

17 .8

17.15 "Human Embryo Experimentation Bill 1985". The Chairman of A.S.R.B. hadissued a public statement of opposition to the proposed Bill, and theSecretary offered to send a copy to any interested Member.

17.16 General Business. H.W.G. Baker announced that the Australian FertilitySociety would be meeting in Melbourne from November 15-17 this year. L.Hinds announced (on behalf of H. Tyndale-Biscoe) that a Boden Conferenceon Lactation and the Physiological Development of Young Marsupials(sponsored in part by A.S.R.B.) would be held from February 3-5, 1986.In final discussion members urged better facilities for posters in 1986,and more rigorous refereeing of abstracts', The meeting closed at 6.45 pm.