J. Embryol. exp. Morph., Vol. 11, Part 4, pp. 637-648, December 1963Printed in Great Britain

Homograft Implants of Young Testes, Ovariesand Ovotestes in the Intact Female Chick

by ALAN E. ERICKSON and GREGORY PINCUS1

From the Worcester Foundation for Experimental BiologyShrewsbury, Mass.

ATTEMPTS to control differentiation of the reproductive system of vertebrateshave been made by use of hormone treatment, grafting, and parabiosis ofembryos, larvae and neonates (for reviews, see Domm, 1939; Burns, 1949;Willier, 1952; Witschi, 1961; Burns, 1961). True sex reversal by use of steroidcompounds has been accomplished only in certain amphibians (Witschi &Chang, 1950; Chang & Witschi, 1955) and in more primitive forms. Amongbirds and mammals such manipulations have modified components of thereproductive system but have not resulted in ovulation and fertilization of ovafrom the cortex of feminized gonads of the male. Spermatogenesis in thehypertrophied right rudiment of a small percentage of hens ovariotomizedprior to 30 days has been reported by Domm (1929) but in only rare instanceshas it occurred in birds similarly treated at an older age (Domm, 1927). Nochicks have hatched from eggs of hens artificially inseminated with spermderived from such experimentally modified birds.

Pincus & Erickson (1962) described the results of dipping the fertile chickegg, between 0-5 days of incubation, into solutions of diethylstilbestrol.Differentiation of the male reproductive system was modified in a mannercomparable to that seen following injection of estrogens (Domm, 1929; Willieret ah 1937). A cortex developed on the left gonad, the genital papilla wasreduced to the form characteristic of the female, and portions of the Mulleriansystem were often retained. Apparently only Domm (1939) has reportedpersistence of some cortical material in adult male gonads following estrogentreatment limited to embryonic stages. Boss & Witschi (1947) reported per-sistence of the cortical portion of ovotestes which were induced by estrogentreatment of herring gull embryos on the 3rd, 6th or 9th day of incubation.Injections were continued beyond hatching and, as a result of this supportivetreatment, cortical elements persisted up to 4 years in treated males. Otherswho have reported on the subsequent fate of feminized gonads of male chicks

1 Authors' address: Worcester Foundation for Experimental Biology, Shrewsbury, Massa-chusetts, U.S.A.

41

638 ALAN E. ERICKSON AND GREGORY PINCUS

have described persistence of some portions of the medullary component butregression of the cortex (Wolff, 1935; vanTienhoven, 1957; Have, 1959).

This paper reports on the results of one approach which has been made tothe problem of maintaining the cortex of the feminized testis in the chick.Cortical and medullary development of ovotestes implanted in intact femaleswas compared with cortical and medullary development of the host ovary andimplants of normal testes and ovaries, to determine if isolation from an appa-rently unmodified right testis combined with residence in the female organismmight present an endocrine environment more favorable to the persistence ofthe cortical elements. We were further interested in the fate of such earlygonadal grafts—looking for an advantage similar to that described by Cannon&Longmire (1952) and Cannon (1954) who reported that the highest percentagesof successful skin homografts to chicks were made immediately after hatchingwith diminishing success as implantation was delayed.

MATERIALS AND METHODS

Merryknoll chicks were used in all experiments. These birds are a cross ofRhode Island Red males with Barred Plymouth Rock females to give a sex-linked white feather spot on the head of the male (which persists in spite ofsteroid treatments used to date) in contrast to the black head of the female.Ovotestes were obtained from newly hatched (0-3 days of age) males whichdeveloped from eggs that had been immersed at 96 hr. of incubation for 10 sec. in40 mg. per cent, diethylstilbestrol dissolved in 100 per cent, isopropyl alcoholheld at 10-12°C. Whole ovotestes, or longitudinal thirds to halves of ovaries ortestes, were implanted. The hosts were 1-3 day sex-linked females. They wereanesthetized with 0-03-0-04 ml. of Nembutal (60 mg./ml.) intraperitoneally.The feathers were plucked from the left side over the ribs and anterior marginof the thigh. An incision approximately 2 cm. long was made through theskin. Connective tissue between the body wall and the iliotibialis muscle wasteased free and the last intercostal space located. A puncture, made with thetips of fine forceps, was spread to allow introduction of rib retractors. Under adissecting microscope at 15 x the implant was placed beneath the peritoneumoverlying the anterior margin of the left kidney, or in some cases under thethin fold of peritoneum which is reflected over the surface of the ovary. Theopening between the ribs was closed with two to three sutures of 6-0 braidedblack silk and the skin approximated with a piece of adhesive tape. Mortalitywas associated more with the anesthesia than with post-operative complications.Wounds and instruments were washed with 1/1000 Zephiran Chloride. Instru-ments were washed between implantations.

Implants of testis, ovary and ovotestis were removed from hosts at 14, 30,60, 90, 120, 150, 180 days and compared with normal controls and with theleft gonad of other male chicks of comparable age which had also been treated

HOMOGRAFT IMPLANTS OF YOUNG TESTES 639

at 96 hr. incubation with diethylstilbestrol. Histological examination wasmade on Bouin-fixed tissue cut at 10/x and stained with hematoxylin and eosin.Note was also made of the reproductive status of the host.

RESULTS

The recovery data for implants have been grouped according to type of tissueimplanted. Table 1, sections A, B and C, has been arranged to indicate thenumber of implants which had been made for recovery at each age, the numberof implants actually recovered, condition of cortical elements (presence offollicles and/or oogonia in cords or nests), medullary elements (immature versusmature tubules producing spermatozoa), a note if lymphoid elements pre-dominated over gonadal components in the implanted tissue, and the numberof ovulating hosts among the birds sacrificed. The last column gives the per-centage of implants made which contained recognizable medullary and/orcortical elements.

Forty-six per cent, of the testis implants, 20 per cent, of the ovarian and29 per cent, of the ovotestis implants were recovered. The most persistentelement of the implants was the medullary tubule in which both germinal andSertoli elements could be readily identified. The number of follicles whichpersisted in the ovarian implants was always considerably below the numbersseen in section of host ovary, and even in cortices containing almost normalnumbers of follicles the largest follicular diameter was less than 50 per cent,of that of the host ovary. The medullary component of the ovotestes wasidentified in all thirty-one of the recovered organs but in only one 14-dayimplant was a cortical remnant identified. Enough males which had beentreated at 96 hr. incubation were left intact to permit sacrifice of three to fourfor each group. The summary of characteristics of their left gonads is found inTable 2. It will be realized that while all ovotestes were evaluated for presenceof a continuous cortex by low power observation before they were used forimplantation, there is no way of knowing the original extent of cortical develop-ment in the left gonad of birds included in Table 2. The most frequently en-countered characteristics of the intact ovotestes were lacunae and cords of'fat-laden' cells (Nonidez, 1924). These also appear in the normal ovary asmodifications of the medullary tubules. Portions of the surface of the ovotesteswere often simple cuboidal epithelium, comparable to that of the ovary ratherthan a fibrous capsule such as is seen in the testes. In some of the ovotestesthere were two further modifications which were not common to either the testisor ovary. The first was the development of a rather broad intermediate ortransition zone of loose connective tissue located between the cortical oogonialnests and whatever normal medullary components remained. This transitionzone, seen so distinctly in ovotestes of 1-day chicks, persists long after the dis-appearance of cortical gonia. The second modification was the appearance of

TA

BL

E

1

Surv

ival

of

impl

ants

to

0-3

-da

y-o

ld fe

mal

e ch

ick

host

s

Age

at

sacr

ific

eda

ys 14 30 60 90 120

150

180

No.

im

plan

tsm

ade

10 11 9 9 10 9 9

Impl

ants

rec

over

edK

Wit

h go

nada

lel

emen

ts

5 8 5 2 6 1 4

Lac

k go

nada

lel

emen

ts

2 2 1 0 0 0 0

Cor

tex

Fol

licl

es

0 0 0 0 0 0 0

Oog

onia

A.

TE

ST

IS

0 0 0 0 0 0 0

Med

ulla

rytu

bule

sA

Imm

atur

e M

atur

e

5 8 5 2 5 0 0

0 0 0 0 1 1 4

No.

wit

hly

mph

oid

elem

ents

pred

omin

ant

5 6 4 0 1 0 0

No.

wit

hm

atur

eho

stov

ary

0 0 0 0 0 1 (w

ithm

atur

ete

stis

)8

(one

imm

atur

eho

st w

ithim

mat

ure

impl

ant)

Per

cen

tim

plan

tsre

cove

red

wit

hgo

nada

lel

emen

ts

50 73 56 22 60 11 44

> 2 tn W JO o 00 O % > 0 0 JO w 0 o JO . > •< c 00

Tot

al67

3146

17 30 60 90 120

150

180

Tot

al 14 30 60 90 120

150

180

11 13 8 12 10 11 10 75 10 14 12 13 12 820

15

8 4 1 0 1 1

B.

OV

AR

Y

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0 0

C.

OV

OT

EST

IS

1 0 0 0 0 0 0

6 3 8 4 4 1 0

5 1 1 0 1 1 0 5 3 4 1 0 0 0

0 0 0 0 0 1 (h

ost

to i

ntac

tim

plan

t)10 0 0 0 0 0 0 13

(tw

om

atur

eim

plan

tsin

im

mat

ure

host

s)

73 31 13 0 10 9 0 20 60 21 66 31 33 38 15

X o o o > H i—i

& •-d r H O K; o c 0 H hrl

UJ 00 H W

Tot

al89

3128

Age

at

sacr

ific

e(d

ays)

14 30 60 90 120

150

180

No.

of

anim

als

3 3 3 3 3 4 4

Fol

licl

es0 0 1 0 0 1 0

Cor

tex O

ogon

ia2 2 0 1 0 0 0

TA

BL

E

2

Cha

ract

eris

tics

of

inta

ct

Med

ulla

Imm

atur

e3 3 2 3 3 2 2

Mat

ure

0 0 1 0 0 2 2

ovot

este

s

Tub

ules

1 1 2 1 1 1 2

Inte

rmed

iate

Fat

-lad

ence

lls

1 1 0 2 2 2 2

char

acte

rist

ics

Mod

ifie

dco

nnec

tive

tiss

ue2 3 2 2 2 1 0

Lac

unae

2 1 2 2 2 1 1

w w ICK CO O > M /»\

ffl

O o G CO

HOMOGRAFT IMPLANTS OF YOUNG TESTES 643

sterile cords or primitive tubules immediately beneath the surface of the gonadand generally close to the intermediate zone if one was present. These structureshave been previously described (Wolff & Hampe, 1950) as being the result of asecondary proliferation of sex cords in the ovotestes following the initial effectof estrogenic stimulation of typical cortical cords. The cords referred to appearto be similar to those which formed in remnants of partially extirpated ovariesand in hypertrophied right rudiments of castrate females (Domm, 1927).Regression of the intact ovotestes paralleled that described by others (Wolff,1935; vanTienhoven, 1957; Haye, 1959). The most persistent gross modifica-tion noted was the flattening and wrinkling of the involved testes. Normalmedullary tubules with germ cells were present in all specimens examined.

DISCUSSION

It is apparent that the attempts to prolong survival of cortical portions ofthe ovotestes were unsuccessful. Since the cortex of control ovarian implantsalso failed to survive, there was no evidence to indicate whether the potentialof the oogonia of the ovotestis is less than that of natural oogonia.

The homograft reaction, as evidenced by lymphoid involvement (Tables 1 &2), was apparently responsible for loss of the majority of implants since thetissues were positioned to render accidental dislodging unlikely. Seven addi-tional ovarian implants not included in the tables, were made to hormonallybursectomized female hosts prior to learning that cell lines derived from thethymus, not the bursa, are apparently responsible for immunological responsesto tissue grafts in the chick. At 30 days four of the seven implants were re-covered. All showed some lymphoid involvement, three contained follicles,and one of the three closely resembled the host ovary in number of folliclesbut these were not equal in size to those of the host. The results were similarto those obtained with untreated hosts.

Cock (1962) reported that ovarian implants made to fully castrated males ofvarious ages retained both endocrine and reproductive capacity to a far greaterextent than did those implants made to incompletely castrated males. Heobserved that 'the presence of testicular tissue, particularly in large amountsduring the initial period, prejudices the survival and normal development ofthe (ovarian) graft'. This corresponds to our conclusions that the remainingmedullary tissue of the ovotestis may be the factor responsible for corticalregression rather than an inherent inadequacy of the cortical gonia or follicles.Parrott (1960) reported that endocrine capabilities are maintained better thanreproductive capacities in stored and transplanted mouse ovaries. Beber (1957)who cultured 15-18-day embryonic rabbit ovaries and testes individually andin combination, found that testes grew better than ovaries and that in com-bination the ovaries ceased development or developed into sterile intersexes.

644 ALAN E. ERICKSON AND GREGORY PINCUS

Holyoke (1956) implanted embryonic rabbit testes and ovaries to adult rabbitkidney and found ovarian survival depressed in presence of a companion testisalthough sex of the host did not appear to play a role.

The poor survival of cortical elements of the implants might be related to(1) establishment of vascularity, (2) superfluous nature of the implants from anendocrinological point of view of the host, or (3) inherent instability of thecortex—at least in early stages of ovarian growth. Experimental evidence indi-cates that early vascularization may be a mixed blessing—serving to supportnormal metabolism of the tissue but affording more immediate access of thetissue to host immunological mechanisms. Parrott (1960) found that ovarianimplants to the subcutaneous site were in an immunologically privileged loca-tion in comparison to those made orthotopically in the rat. The tendency forautografts and homografts of mammalian ovaries to be deficient in oocyteshas been shown by Buyse (1935), Jones & Krohn (1960), and Mussett & Parrott(1961). Greenwood (1924-5) reported that chick embryo ovaries survivedtransplantation to the chorioallantoic membrane less well than did testes.Taber (1956) reported poor oogonial survival in perirenal implants of em-bryonic chick ovaries made to bilaterally ovariotomized or caponized hosts.The number of oocytes lost during the first week of orthotopic implantation inrats has been determined to be 65 per cent, (minimal) and tends to be evenhigher in ovaries taken from progressively older donors (Mussett & Parrott,1961). It is possible that the embryonic or neonatal ovary may not be suffi-ciently stable for transplantation and that the shock to homeostatic mechanismswithin the implant may result in regressive changes not seen in implants ofslightly older ovaries. The marked difference in ability of dissociated embryonicchick testes and ovaries to reaggregate in culture (Abraham, 1960) may be areflection of such inherent instability.

There are several points to be made based on this report and reports ofothers. (1) The use of newly hatched chicks as hosts did result in retention ofsufficient numbers of implants to demonstrate the far greater viability ofmedullary than of cortical components of the chick gonad. (2) The chickchromosomal constitution shows the male with ZZ and the female with ZOcomplement of sex chromosomes (although the cataloguing of chick chromo-somes is not complete). This suggests that implants of testis and ovotestis suchas made in our experiment are not faced with the complication of host intoler-ance to a sex chromosome such as described for homografts of male (XY)mammalian tissue to female hosts (Eichwald & Silmser, 1955; Lustgraaf et ah,1960). The experimental work with mammalian embryos versus chick embryoshas shown a distinct difference in in vivo studies of differentiating gonads. Six-to eleven-day left or right gonads (Wolff, 1946-7) and left or right medullarycomponents (Mintz & Wolff, 1954) from female chick embryos will inducecortical development of the differentiating left gonad of the male when thetwo are approximated in vivo. Heterosexual pairs of rat gonads have been

HOMOGRAFT IMPLANTS OF YOUNG TESTES 645

implanted to the subcapsular site of castrated adult hosts (Macintyre, 1956).In such a combination the ovary was inhibited or transformed but the testiswas unaffected. Inhibition of the testis occurred only in a small percentage ofcases in which 13-day anlage were placed adjacent to 16-19-day differentiatedovaries (Macintyre et ah, 1959). The development of the right rudiment inpartially or completely ovariotomized hens has already been referred to.The contrast of endocrine potential of the embryonic ovary in which bothcortex and medulla, left and right gonads, feminize versus the aspect of themore mature ovary in which without inhibitory control of the cortex themedulla will undergo some degree of masculinization with regard to bothstructure and function is most interesting. Domm's report (1927) of featherand sex skin changes following ovariotomy and subsequent hypertrophy of themedullary rudiment on the right side with return of feathers to henny-typeplumage but persistence of masculine comb and wattles underscores the dualendocrine role of the medullary tissue in the female.

Further investigation is being made to determine the survival potential ofimplants of cortical material alone. At the present time we are studying thestability of heterotopic autografts of ovarian cortex made at varying agespost-hatch. If such grafts can be made successfully, heterotopic homograftsof cortex from ovaries and ovotestes will be followed by attempts at ortho-topic cortical implants to hosts in which greater efforts will be made to develophomograft tolerance.

SUMMARY

1. Implants of 1-3 day chick ovary, testis and ovotestis were made to intactfemale hosts of the same age to determine the effect of the host's endocrineactivity on survival of normal ovarian cortex, testicular medulla and the cortexof feminized testes (ovotestes).

2. Isolation of the ovotestis from an apparently unmodified right testis andrelocation to the endocrine environment of the female organism did not en-hance persistence of normal follicular development of ovotestes. Corticaldevelopment of transplanted ovaries, however, was also severely inhibited.

3. Medullary tubules were the most persistent elements in implanted tissues.

4. Spermatogenesis was complete in seminiferous tubules of a number ofovotestis and testis implants recovered from both ovulating and non-ovulatinghosts.

5. The significance of proximity of the cortex of the ovotestis to persistingmedullary components, the homograft reaction, and the possible instabilityof embryonic and neonatal ovaries is discussed.

6. The medullary components of testis and ovotestis implants survived witha much greater frequency than did portions of the implanted ovaries.

646 ALAN E. ERICKSON AND GREGORY PINCUS

RESUME

Homogreffes de jeunes testicules, ovaires et ovotestis chez la poule intacte

1. On a greffe des ovaires, des testicules et des ovotestis de poulet de 1 a 3jours sur des hotes femelles du meme age pour determiner Faction de l'activiteendocrine de l'hote sur la survie du cortex ovarien normal, de la medullatesticulaire et du cortex de testicules feminises (ovotestis).

2. L'isolement de l'ovotestis d'un testicule droit apparemment non modifie,et son transfert dans le milieu endocrinien de 1'organisme femelle, n'ont pasaccru la persistance du developpement normal des follicules des ovotestis.Neanmoins, le developpement cortical des ovaires transplanted a ete, egalement,fortement inhibe.

3. Les tubules medullaires ont ete les elements les plus persistants dans lestissus implantes.

4. La spermatogenese s'est accomplie dans les tubules seminiferes d'uncertain nombre d'ovotestis et de testicules repris sur les hotes ou ils avaientete implantes, que ceux-ci soient ou non en periode d'ovulation.

5. On discute la signification de la proximite du cortex de l'ovotestis sur lapersistance des constituants medullaires, la reaction d'homogreffe, et l'insta-bilite possible des ovaires embryonnaires et neonataux.

6. Les constituants medullaires des implants de testicules et d'ovotestis ontsurvecu beaucoup plus frequemment que ne l'ont fait les portions d'ovairesimplantes.

ACKNOWLEDGEMENT

We wish to acknowledge the assistance of Raymond Alie whose technical skill contributedso much to the completion of this project. The work was aided by a grant from the NationalFoundation.

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{Manuscript received 21st March 1963)