0VARlA.N IN A · ACKNOWLEDGMENTS 1 would like to express my sincere thanks to my advisor Dr. B.L....
190
EFFECTS OF EQUINE RELAXIN ON THE ACTIVITY OF EQUW 0VARlA.N STROMAL CELLS IN VITRO A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by LIFU SONG In partial Nfilhent of requirements for the degree of Doctor of Philosophy January, 1998 O Lifu Song, 1998
Transcript of 0VARlA.N IN A · ACKNOWLEDGMENTS 1 would like to express my sincere thanks to my advisor Dr. B.L....
EFFECTS OF EQUINE RELAXIN ON THE ACTIVITY OF E Q U W
0VARlA.N STROMAL CELLS IN VITRO
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
LIFU SONG
In partial Nfi lhent of requirements
for the degree of
Doctor of Philosophy
January, 1998
O Lifu Song, 1998
National Libmty Bibliothèque nationale du Canada
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ABSTRACT
EFFECTS OF E Q ~ E RELAXIN ON THE AcnvIn OF EQUME OVARIAN
STROMAL CELLS IN VITRO
Lifb Song University of Guelph, 1997
Co-advisors: Professor B.L. Coomber Professor R.M. Liptrap
This thesis examined the effects of equine relaxin (ERXN) on proliferation of
equine ovarian stromal cells (EOSC) , the production of gelatinase A (MMP-2) and
gelatinase B (MMPa), tissue inhibitors of matrix metdloproteinases (ïïMPs), urokinase-
type plasminogen activator @PA), tissue-type plasminogen activator (PA) and
plasminogen activator inhibitor- 1 (PM- 1) by EOSC in vitro. Equine ovaian stromal cells
were isolated from stroma at the apex of growing follicles. Equine relaxin inhibited 3 ~ -
thymidine incorporation by EOSC and decreased proliferation. Equine relaxin stimuiated
the production of gelatinase A, gelatinase B, uPA and tPA. Equine relaxin dso stimuiated
the production of TIMP-1, TIMP-2, and PAI-1. Evidence is presented that ERXN
performs its effects in a paracrine manner in equine ovary. Equine relaxin contributes, in
part, to the connective tissue remodeiing required for equine foliicle growth by inhibithg
the proliferation of stromai ceiis, by stimuiating gelatinases and plasminogen activators
and by stimdating production of TIMP-1, TIMP-2 and PAL1 to prevent excessive
extracellular ma& degradation. Transforming growth factor-p and phorbol 12-myristate
13-acetate were found to influence EOSC activity and modulate the production of
gelatinases A and B, TIMP-1 TIMP-2, uPA, tPA and PAI-1.
13-acetate were found to influence EOSC activity and modulate the production of
gelatinases A and B, TIMP-I T m - 2 , uPA, tPA and PM- 1.
ACKNOWLEDGMENTS
1 would like to express my sincere thanks to my advisor Dr. B.L. Coomber for her
guidance, support and encouragement throughout rny studies. My gratitude and
appreciation are extended to CO-advisor Dr. R.M. Liptrap and the members my advisory
cornmittee, Drs. W.A. King from the Department of Biomedicd Science, and A.M.
Gibbins from the Department of Animal and Poultry Science for their assistance during
my study and in the preparation of this thesis.
I would like thank Dr. P.L. Ryan, post-doctoral fellow in the late Dr. Porter's
laboratory, for his suggestions and Friendship; and thank Ms Carol Wasnidge and Ms
Patsy Huether.
Thanks are also extended to Kanwal Minhas and Carol Galligan. We shared the
sarne laboratory and they made our laboratory a happy place.
I would like aiso to extend a speciai thanks to the late Dr. David G. Porter.
1 also thank the personnel at Barton Feeders, Owen Sound, Ontario, for helping
collect equine ovarïes.
This research was supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Ontario Ministry of Agriculture, Food and Rural
Main (OMAFRA) and the Dynasty Equine Trust.
DECLARATION OF WORK PERFORMED
1 declare that, with the exception of the items below, al1 work reported in this
thesis was performed by me.
Bovine pulmonary artery endothelial cells were isolated and cultured by Dr. B.L.
Coomber.
Equine relaxin was isolated, purified and quantified by Dr. P.L. Ryan, Ms Carol
Wasnidge, and Ms Patsy Huether.
TABLE OF CONTENTS
. . DECLARATION OF WORK PERFORMED ............................................................ i i
LIST OF FIGURES ........................................................................................................ vi
LIST OF ABBREVIATlONS ........................................................................................... x
INTRODUCTION .............................................................................................................. I L) REVIEW OF LITERATURE .......................................................................................... 3
Molecular Features ........................................................................................................ 3 Relaxin Receptors ......................................................................................................... 4 S ynthesis In Follicles .................................................................................................... 5 Relaxin In The Horse .................................................................................................... 6 Effects On Connective Tissue Remodeling .................................................................. 7
Effects Of Relaxin On Cellular Proliferation . .......................................................... 8 E ffects On Extracellular Matrix ................................................................................. 9 Effects On Proteolytic Enzymes ...................................... .... ...................................... 9 EEect On Proteolytic Enzyme Inhibiton ................................................................. 1 1
Anatomy Of The Ovary ............................................................................................... 12 Horse Ovary ............................................................................................................... -1 2
...................................................................................... Stroma1 Tissue In The Ovary 13 ...,.....................*........ ............*...*................*...*........ Extracellular Matrix ., ..,........ 1 3
............................................................................................................ Stroma1 Cells 14 Enzymes in Extracellular Matrix Degradation .......................................................... 15
Tissue Inhibitors of Metailoproteinases ...................................................................... 2 1 .............................................................................................. Plasminogen Activators 2 5
Urokinase-Type Plasminogen Activator .................................................................. 25 Tissue-Type Plasminogen Activator ........................................................................ 27 . .
Plasminogen Activator Inhibitors ............................................................................... -3 O Transforming growth factor-p and Phorbol 12-myristate 13-acetate .................... 3 1
MATERIALS AND METHODS .........................................,... 3 4 S o m C e C e ....................................................................................................... 34
q - Immunocytochemistry And Immuno fluo rescence ........................................................ J 3 . . ........................................................................................... Thymidine Incorporation 3 6 Ce11 Number Determination ........................ ,. .............................................................. 3 7
Preparation Of Conditioned Medium ............................................................................. 37 Collection Of Ce11 Extracts ................................. ,. ......... 38 Collection Of Extraceilular Matrix ................................................................................. 38 Protein Quantification .................................................................................................... -39
.............................................................. SDS-PAGE ....................................................... 39 .................................................................................................................... Zymography 40
...................................................................................................... Reverse Zymography 40 .......................................................................................................... Fibrin Autography 41
............................................................................................ Reverse Fibrin Autography 42 ........................................................................................................ Chromogenic Assay 42
Image Processing And Densitometric Analysis ............................................................ 43 ........................................................................................................ Statistical Analysis -44
RESULTS ....................................................................................................................... 45 Characteristics Of EOSC ................... ,., ....................................................................... 45
General Features Of EOSC ......................................................................................... 45 ................................................................................................. Immunocytochernistry 45
3 ~ - ~ h y m i d i n e Incorporation And Proliferation ....................................................... 51 Changes In Activity Of The Gelatinases ................................................................................. 55 - *
Characteristics Of Gelatinase Activity ....................................................................... -33
Effects Of Different Doses Of ERXN On The Gelatinase Activity ............................ 57 ..................... Time Course Of Gelatinase Production By EOSC Exposed To ERXN 61
Effects Of Different Doses Of TGF-P Or PMA On Gelatinase Production Of .......................................................................................................................... EOSC -62
Effects Of ERXN In Combination With TGF-P Or PMA On Gelatinase Production By EOSC .................................................................................................. 65
Activity Changes Of Tissue Inhibitors Of MetalIoproteinases ................................. 68 Characteristics Of TIMPs Produced By EOSC .......................................................... -68 Effects Of Different Doses Of ERXN On TIMP Production By EOSC ..................... 70 Time Course Of TIMP Production By EOSC Exposed To ERXN ............................. 71 Effects Of Different Doses Of TGF-P Or PMA On TIMP Production By EOSC ...... 71 Effects Of ERXN In Combination With TGF-P Or PMA On TIMP Production By EOSC .................................................................................................................. -76
Activity Changes Of Plasminogen Activators .......................................................... -77 Characteristics Of PA Produced By EOSC ............................................................... -77 Effects Of DifEerent Doses Of ERXN On PA Production By EOSC ........................ .81 Time Course Of PA Production B y EOSC Exposed To ERXN ................................ -83 Effects Of Different Doses Of TGF-P Or PM A On PA Production By EOSC .......... 87
Effects Of ERXN In Combination With TGF-B Or PMA On PA Production By EOSC ........................................................................................................................... 91
Activity Changes Of Plasminogen Activator Inhibitor ......................................... ...94 Characteristics Of PAL 1 Produced B y EOSC ..................... ... ......... .... ......... 94 Effects Of Different Doses Of ERXN On PAL1 Production By EOSC ..................... 95
............................. Tirne Course Of PAI-1 Production By EOSC Exposed To ERXN 95 .... Effects Of DifXerent Doses Of TGF-P Or P M On PAL1 Production By EOSC 100
Effects Of ERXN In Combination With TGF-P Or PMA On PAL1 Production By EOSC ................................................................................................................... 100
.................................................................................. ............................ DISCUSSION ,., 103
Characteristics Of EOSC ........................... ,, .............................................................. 103 Characteristics Of ERXN Effect ............................................................................ 1 0 4 Gelatinase Production By EOSC ............................................................................... 108 Production Of TIMP By EOSC .................................................................................... 113 Production Of PA By EOSC ...................................................................................... 1 1 8 Production Of PA[-1 By EOSC .................................................................................. 124 Effect Of Erxn On Proliferation Of EOSC .................................................................. 129
SUMMARY .................................................................................................................... 133
CONCLUSION ............................................................................................................ 136 LITERATURE CITED ............................... ... ............................................................... 137
APPEmm 1 SOURCES OF SUPPLIES AND MATERIALS .................................................. 163
APPENDIX iI RECIPES FOR SOLUTIONS AND BUFFERS ................................................. 167
APPENDIX STANDARD CURVE OF GELATIN ZYMOCRAPHY ................................... 171
APPENDm STANDARD CURVE OF REVERSE GELATIN ZYMOGRAPMY ................... 172
APPENDrX VI STANDARD CURVE OF REVERSE FIBR~N AUTOGRAPHY ........................ 174
LIST OF FIGURES
FIGURE 1
FIGURE 2
FIGURE 3
FIGURE 4
FIGURE 5
FIGURE 6
FIGURE 7
FIGURE 8
FIGURE 9
F I G W IO
FIGURE 11
FIGURE 12
Phase contrast micrograph of cultured equine ovarian stroma1 ................................................................................... ............. cells.. .. 46
Growth curve of equine ovarian stromal cells in 10% FBS MEM .................................................................................................. 47
Survival ce11 numbers of equine ovarian stromal cells in senim-free .......................................................................................... medium. ..A8
Immunostaining for u-smooth muscle actin and procollagen type I .................................................................................................. 49
Imrnunostaining for Von Willebrand Factor ..................................... 50
Effects of equine relaxin, porcine relaxin and transforming growth factor+ on the 3~-thymidine incorporation and ce11
............................................................................................. numbers 52
Viability of cultured equine ovarian stromal cells in serum-free MEM after exposure to equine relaxin (0.0 1 - 100 ng/rnl) for
Detection of gelatinase activity present in conditioned medium ............................. of equine ovarian stromal cells by zymography -56
Gelatinase activity in conditioned medium of equine ovarian stromal cells treated with equine relaxin (0.0 1-100 ng/ml), as detected by gelatin zymography .................................................... 59
Gelatinase activity in ce11 extracts of cultured equine ovarian stroma1 cells treated with equine relaxin (0.0 1-100 ng/ml), as detected by gelatin zymography ........... ... ............................... 60
Gelatinase activity in conditioned medium of equine ovarian stromal cells treated with equine relaxin (100 ng/mi) for different times, as detected by gelatin zymography ......................... 63
FIGURE 13
FIGURE 14
FIGURE 15
FIGURE 16
FIGURE 17
FIGURE 18
FIGURE 19
FIGURE 20
FIGURE 21
Gelatinase activity in ce11 extracts of equine ovarian stromal cells treated with equine relaxin (1 00 ng/ml) for different times, as detected by gelatin zymography ......................................... 64
Gelatinase activity in conditioned medium of equine ovarian stromal cells treated with transforming growth factor-p (1 - 10 ng/ml) or phorbol 1 Zmyristate 13-acetate (5-20 @ml), as detected by gelatin zymography ................................................... 66
Gelatinase activity in conditioned medium of equine ovarian stromal cells afler exposure to equine relaxin (100 ngfml) in combination with transforming growth factor-p (5 ng/ml) or phorbol 12-myristate 13-acetate ( 10 ng/ml) .................................................... 67
Detection by reverse zymography of tissue inhibitors of metalloproteinases produced by cuitured equine ovarian stromal
............................ cells, as detected by reverse gelatin zymography.. 69
Activity of tissue inhibitors of metalloproteinases-l and -2 in conditioned medium of equine ovarian stromal cells treated with equine relaxin (0.0 1 - 100 ngfml), as detected by reverse
...................................................................................... zymograp hy .72
Activity of tissue inhibitor of metailoproteinases-3 in extracellular matrix of cultured equine ovarian stromal cells after exposure to equine relaxin (0.0 1-100 ng/ml), as detected by reverse
.................................................................................... zymography.. .73
Tissue inhibitors of metalloproteinases-l and -2 in conditioned medium of equine ovarian stromal cells treated with equine relaxin (100 ngfrnl) for diEerent times, as detected by reverse zymography .......................................................................... .74
Tissue inhibitor of metdoproteinases-3 activity in extracellular matrix of equine ovarian stromal cells after exposure to equine relaxin (100 ng/ml) for indicated times, as detected by reverse
.......................................................... zymography ................... .... .75
Activity of tissue inhibitor of metalloproteinases-1 and -2 in conditioned medium of equine ovarian stromal cells treated with transforming growth factor+ (1 -10 ng/ml) or phorbol 12-myristate 13-acetate (5-20 ng/ml), as detected by
........................ ....*...............*....................... reverse zymograp hy ....... 78
FIGURE 22
FIGURE 23
FIGURE 24
FIGURE 25
FIGURE 26
FIGURE 27
FIGURE 28
FIGURE 30
Activity of tissue inhibitors of metalloproteinases-1 and -2 in conditioned medium of equine ovarian stromal cells after exposure to equine relaxin (100 @ml) in combination with transfonning growth factor-p (5 ng/ml) or phorbol 1 Zrnyristate 13-acetate
....................................................................................... ( 1 O ng/ml). -79
Identification of plasminogen activator activity in conditioned medium and ce11 extracts of equine ovarian stromal cells as
........................................................... detected by fibrin autography .80
Identification of plasminogen activator activity in extracellular matrix detected by fibrin autography in the presence or
........................................................... absence of amiloride (1 0 mM) 82
Fibrin autograms of urokinase-type plasminogen activator activity in conditioned medium and ce11 extracts of equine ovarian stromal
........................... cells treated with equine relaxin (0.0 1 - 1 00 nglml). 84
Plasminogen activator activity in conditioned medium and ce11 extracts produced by equine ovarian stromal cells treated with equine relaxin (0.0 1 - 100 @ml), as detected by chromogenic
............................................................................................... assay.. .8 5
Plasminogen activator activity in extracellular matnx of equine ovarîan stromal cells treated with equine relaxin (0.0 1 - 100 ngfml), as detected by fibrin autography ........................................................ 86
Urokinase-type plasxninogen activator activity in conditioned medium or ce11 extracts of equine ovarian stroma1 ceils treated with equine relaxin (1 00 ng/rnl) for different
............................................................................................... times.. -8 8
Plasminogen activator activity in conditioned medium or ce11 extracts of equine ovarian stromal cells exposed to equine relaxin (100 ngh l ) for different times, as detected by chromogenic
................................................................................................ assay ..a9
Plasmlliogen activator activity in extracellular matrix of equine ovarian stromal cells treated with equine relaxin (100 ng/ml) for îndicated times, as detected by reverse zymography ................... 90
FIGURE 3 1
FIGURE 32
FIGURE 34
FIGURE 35
FIGURE 36
FIGURE 37
FIGURE 38
Plasminogen activator activity in conditioned medium of equine ovarian stromal cells treated with transforming growth factor+ (1 - 10 ng/ml) or phorboi 12-myrktate L 3-acetate (5-20 nghl) , as detected by chromogenic assay ............................... 92
Plasminogen activator activity in conditioned medium of equine ovarian stromal cells after exposure to equine relaxin (100 ngh l ) in combination with transforming growth factor+
.................. (5 ng/ml) or phorbol 12-myristate 13-acetate (1 0 @ml) 93
Detection of PAL 1 in conditioned medium, ECM and ce11 extracts ....................................... from EOSC by reverse fibrin autography.. .96
Plasrninogen activator inhibitor-1 activity in conditioned medium of equine ovarian stromal cells treated with equine relaxin (0.0 1-1 00 ng/ml), as detected by reverse gelatin
...................................................................................... zymography .97
Plasminogen activator inhibitor-l activity in conditioned medium of equine ovarian stromal cells treated with equine relaxin (1 00 ngml) for different times ......................................................... 98
Plasminogen activator inhibitor- 1 in extracellular mauix of equine ovarian stromal cells treated with equine relaxin (0.0 1 - 100 ng/ml) or treated with equine relaxin (1 00 ng/rnl) for different tirnes, as detected by reverse gelatin
...................................................................................... zyrnograp hy .99
Plasminogen activator inhibitor- 1 activity in conditioned medium of equine ovarian stromal cells treated with transforming growth factor-p (1 -5 ng/ml) or phorbol 12-myristate 13-acetate (5-20 @ml), as detected by reverse fibrin
...................................................................................... autography. 102
Plasminogen activator inhibitor-l activity in conditioned medium of equine ovarian stromal cells after exposure to equine relaxin (100 ngh l ) in combination with transfomiing growth factor-p (5 ng/ml) or phorbol Il-myristate 13-acetate (1 0 ng/ml) ................ 103
LIST OF ABBREVIATIONS
a-SMA
Ab
APMA
BSA
DMSO
ECM
EDTA
eCG
EGF
EGTA
EOSC
ERXN
FBS
HBSS
IGF-I
LH
MEM
MMP
PA
PAI- 1
a-Smooth muscle ce11 actin
Antibody
4-aminophenylmercuric acetate
Bovine serurn aibwnin
Dimethyl sulfoxide
extracelluIar matrix
Ethylenediaminetetraacetic acid
Equine chorionic gonadotropin
Epidermal growth factor
Ethylene glycol-bis(2-aminoethyl ether) N,N,Nf?N tetraacetic acid
Equine ovarian stroma1 cells
Equine relaxin
Fetal bovine semm
Hank's balanced sait solution
Insuiin-like growth factor4
L uteinizing hormone
Minimum essential medium
Matrix metalloproteinases
Plasminogen activator
Plasminogen activator inhibitor-l
PBS
PDGF
PMA
PMSG
PMSF
P R X N
RXN
SDS
TGF-P
TIMP
tP A
uP A
uPAR
Phosphate buffered saline
Platelet-derived growth factor
Phorbol 12-myristate 1 3-acetate
Pregnant mare serum gonadotropin
Phenylmethylsulfonyl fluoride
Porcine relaxin
Relaxin
Sodium dodecyl sulfate
Transforming growth factor-p
Tissue inhibitors of metalloproteinases
Tissue-type plasminogen activator
Urokinase-type plasminogen activator
Urokinase-type plasminogen activator receptor
INTRODUCTION
Relaxin 0, a peptide hormone, has been identified in the ovaries of several
species, including pigs (Bagnell et al., 1987) and humans (Wathes et al., 1986). Recently,
RXN protein was identined in equine ovarian follicular fluid, thecal and granulosa cells
and in the corpus luteum (Ryan et al., 1997). Accumulated evidence indicates that RXN
may act locally in the ovary. For example, RXN induces ovulation in the perfused rat
ovary in vitro (Brannstrorn and MacLennan, 1993). Administration of RXN antibody
partially suppresses gonadotropin-induced ovulation in immature rats and this effect can
be reversed with RXN treatment (Hwang et al., 1996a). In addition, RXN promotes DNA
synthesis in both porcine theca and granulosa cells and their proliferation in vitro (Zhang
and Bagnell, 1993; 1994).
The actions of RXN on ceMx and uterine ligaments are linked to remodel
comective tissue in its target organs (Bryant-Greenwood and Schwabe, 1994). The
mechanisms underlying this effect are related to its regdation of severai proteolytic
enzymes. Recombinant human RXN modulates the synthesis and secretion of interstitial
collagenase (matnx metalloproteinase-1) in human skin and lung fibroblasts (Unemori et
al., 1990; 1996), and human cervical stromal cells (Hwang et al., 1996b). Recombinant
human RXN stimulates interstitial collagenase activity in cultured uterine c e ~ c a l ceils
fiom guinea pigs (Mushayandebvu and Rajabi, 1995). In the ovary, RXN modulates
proteolytic enzymes required in comective tissue rernodeling during follicular growth
and ovulation. Relaxin stimulates rat grandosa cells to synthesize interstitial collagenase,
plasminogen activator and proteoglycanase (Too et al., 1984); and stimulates grandosa
and thecal-interstitial cells to produce gelatinases A and B (Hwang et al., 1996a). ovarian
follicles are embedded in stromal tissue, which is composed of stromal cells and ECM.
During follicle development and ovulation, the stromal matrix undergoes dramatic
remolding to allow follicle expansion and migration to the surface of the ovary. Several
proteolytic enzyme systems are involved in breaking d o m ECM of stromal tissue. In the
ovary, two enzyme systems have been identified. They are the matrix metailoproteinases
(MMP) and tissue inhibitors of rnetalloproteinases (TIMPs); and the plasminogen
activators (PA) and plasrninogen activator inhibitors (PAI). These two systems cooperate
to perform ECM remodeling. Normally, these enzymes and their inhibitors are in kinetic
balance and are precisely coordinated to regulate follicular connective tissue remodeling.
In the ovary, it h a been demonstmted that g r d o s a and thecal cells can
synthesize gelatinases, TIMPs, PA cornponents and PAI, but there are few studies about
production of these enzymes by ovarian stromal cells. Without doubt, ovarian stromal
cells should synthesize these enzymes during follicle growth and ovulation.
The main objectives of this study were based on the hypotheses: (i) ovarian
stromal cells produce gelatinases, TIMPs, PAS and PAL1 and (ii) RXN acts on these
ovarian stromal cells to regulate production of these enymes. The experimentai approach
taken was: first, isolate equine ovarian stromal cells fiom the stroma at the apex of
growing follicles in ovary; second, determine whether these cells are able to synthesize
proteolytic enzymes and third determine how RXN influences the production of these
enzymes.
REVIEW OF LITERATURE
Relaxin
Relaxin (RXN) is a peptide hormone widely produced by many mammals,
including mouse, rat, rabbit, horse, elephant, pig and human being (Shewood, 1994). An
important role of RXN is its involvement in the maintenance of pregnancy and in the pre-
partum softening of the ligaments that surround the pelvic cavity (Porter 1979, 1981 ;
Bryant-Greenwood, 1982). In other reproductive tissues, RXN stimulates marnmary
gland growth (Winn et al., 1994; Kuenzi et ai., 1995) and infiuences sperm mobility in
vitro (Sarosi et al., 1983). In the central nervous system, RXN causes the release of
vasopressin (Mumford et al., 1989) and oxytocin (Surnrnerlee et al., 1984). Porcine
relaxin increases heart rate and blood pressure in nonpregnant rats (Parry and Sumrnerlee,
1991). Recently, effects of R X N on remodeling of connective tissue have drawn great
interest. - Relaxin was discovered in the 1920s (Hisaw, 1926), but large-scde purification
was not achieved until 50 years later (Sherwood and OYByrne 1974). The amino acid
sequence and the complete covalent structure of procine relaxin (PRXN) was detennined
f5st (Schwabe and McDonald, 1977). Subsequently, relaxins fiom several mammals were
isolated and their primary structure was detennined. Later, a chernical synthesis was
developed and many RXN derivatives were synthesized (Büllesbach and Schwabe, 199 1).
Relaxin is derived from a large single chain precursor, preprorelaxin, from which
prorelaxin is formed by removal of the signal peptide. Then RXN is converted to a double
chain structure after the connecting peptide is cleaved (Gast, 1982, 1983). In mature
RXN, the A chain (24-29 amino acid), containing an intradisulfide Iinkage, and the B
chah (29-33 amino acid) are linked by two disuifide bonds at positions A7 and A19
(James et al., 1977). Studies have shown that the B chain carries the receptor-binding site
and rnediates the biological responses (Büllesbach and Schwabe, 1991). However, the B
chain alone is not active and does not inhibit the binding of intact RXN to its receptor. It
is the A chah that most likely confers the biologically active component to the whole
molecule by steric effects (Büllesbach and Schwabe, 1994).
Relaxin is viewed stnicturally as a member of the insulin-like growth factor
family because of the similar A and B chain lengths and the position of the disulfide
bridges (Schwabe and McDonald, 1977). However, there is ody about 25% amino acid
sequence homology between RXN and insulin (Issacs and Dodson, 198 1). The arnino
acid residues needed for insulin differ or are not present in RXN. Relaxin and insulin do
not share the same receptor-binding domain (Mercado-Simmen et al., 1 982a; Olefshy et
al., 1982) and do not share immunological determinants (Sherwood et al., 1975).
A receptor for RXN has not been isolated. However, studies at both the whole
animal and tissue Levels have demonstrated that RXN binding site are present in various
tissues in different species, including rat utenis, cervix, brain, and heart atrium (Osheroff
et al., 1990, 1992), and the mouse pubic symphysis and uterus (Yang et al., 1992).
Evidence for a RXN receptor based on a biological response has been obtained fiom
porcine grandosa cells (Yang et al., 1992). Relaxin binding sites are regdated by several
hormones and growth factors. For instance, RXN binding sites in rat and pig uterine
tissue were reported to be elevated during the estrogen-dominant stages of the estrous
cycle and after estrogen administration to ovariectomized rats and pigs (Mercado-
Simmen et al., 1 W a , 1982b).
As a hormone, d e r its synthesis by specific organs or tissue, RXN is released
into the circulation and carried to other tissues to act on target cells. At least one organ
determines the RXN level in the bloodstrearn: this organ or tissue is called the primary
source. Secondary sources of RXN are those organs or tissues which do not detennine the
RXN level in blood, although they are able to synthesize RXN (Sherwood, 1994). The
ovary is the primary source of the RXN in the peripheral blood in some species, such as
pig. By using sensitive techniques for the identification of RXN and its rnRNA, it has
been found that the ovary is a secondary source of RXN in severai other species.
Secondary sources appear tdikely to contribute appreciably to RXN levels in blood but
may have local physiological fùnctions (Bagne1 et al., 1993).
Relaxin has been found in ovarian follicuiar fluid of the pig (Bryant-Greenwood
et al., 1980; Matsumoto and Chardey, 1980) and humans (Wathes et al., 1986). In pig
ovarian follicles, RXN has also been localized to the theca interna layer of developing
follicles by immunoreactive studies (Evans et al., 1983; Bagnell et al., 1987). Relaxin
mRNA is aiso identified in the theca interna (Bagnell et al.. 1990a). In the human
preovulatory follicle, RXN gene expression and irnrnunoreactive RXN have been
localized to the theca cells, identiSing them as a source of the hormone (Lee et al., 199 1).
Relaxin has been localized by molecular and imrnunocytochemical techniques in the
corpus luteurn in the pig (Fields and Fields, 1985; Bagnell et al., 1989, 1993; Denning-
Kendall et al., 1989) and in the corpora lutea in the hurnans (Ive11 et al., 1989; Stoelk et
al., 199 1). Gonadotropins, which modulate the development of the follicle, are capable of
inducing the production of RXN. For instance, luteinizing hormone (LH) stimulated the
gene expression of RXN in the porcine follicle (Bagnell et al., 1990b) and the secretion of
RXN from cultured porcine preovulatory granulosa cells (Loeken et al., 1983).
Ho=
Equine relaxin (ERXN) was fist purified and characterized by Stewart and
Papkoff (1986) and its sequence has been determined (Stewart et al., 1991). Relaxin
isolated nom horse placenta has six isoforms and their molecular masses range from
5,170 to 5,570 kDa. Its specific bioactivity was far lower than that of porcine relaxin, as
determined in the mouse interpubic ligament assay (Stewart and Papkoff, 1986).
Relaxin in equine peripheral blood during pregnancy mallily cornes fkom the
placenta (Stewart et ai., 1992). Penpherai blood RXN levels graduaDy increase then
decrease during pregnancy followed by a surge a few hours before birth untii afier the
placenta is expelied (Plunkett et al., 1960).
Recently, ERXN was identified in ovarian thecal cells and the corpus luteum
using an antibody against ERXN and a cDNA probe for human RXN. Therefore, the
ovary could be a secondary source of RXN in hoaes. Relaxin is present in follicular fluid
at concentrations of 1-10 ng/ml (Ryan et al., 1997), very low compared with RXN
concentrations in penpheral blood during horse pregnancy (Plunkett et al., 1960). Unlike
the production of PRXN in the porcine follicle, which is increased by treatment of
gonadotropins (Bagne11 et al., 1990b; Loeken et al., 1983), the production of ERXN in
equine thecal and granulosa cells is not altered by follicle stirnulating hormone (FSH) and
LH in vitro (Ryan et al., 1997). In the pig, the follicle is the primary source of RXN; its
production is increased before ovulation (Evans et al., 1983). In contrat, the level of
ERXN in equine follicle tluid does not show a significant increase with the development
of the follicle (Ryan et al., 1997).
cts Of RXN On Comect ssue R e m o d e b
Connective tissue is composed of fibroblasts and extracellular matrix (ECM). The
components of ECM include glycoproteins, proteoglycans, collagen, elastin: and
glycosaminoglycans (Albert et al., 1989b). Fibroblasts are able to synthesize components
of ECM. Fibroblasts also have the ability to produce several proteolytic enzymes, which
degrade components of the ECM. In general, fibroblasts also synthesize factors that may
regulate proteolytic enzyme activity by inhibition or stimulation. Therefore, the effects of
RXN on fibroblast proliferation, on production of ECM components, on proteolytic
enzymes and their inhibitors will be discussed regarding t ie effects of RXN on
connective tissue remodeling.
Effects Of Relaxin On Cellular Proliferation
Because RXN is capable of regulating ce11 growth, it has been considered to be a
growth factor as well as a hormone. Many studies on the effects of RXN on ce11 growth
have been performed, but the results are inconsistent. Porcine relaxin (1 - 1000 nglml) was
reported to promote the proliferation and DNA synthesis of porcine thecal and granulosa
cells in various stages of follicular development and to have synergistic effects with
insulin-like growth factors and insulin (Bang et al., 1993, 1994). However, Beck et al.
(1 992) reported that only extremely high concentrations (100 pg/ml) of PRXN stimulûted
DNA synthesis. Relaxin (1-1000 ng /ml) inhibited the proliferation of rat 3T3-LI
fibroblasts (Pawlina et al., 1989, 1990) and human MCF-7 breast-cancer cells (Sacchi et
al., 1994). Several studies have s h o w that RXN has a biphasic effect on some ceil types.
For example, PRXN stimulates at a low concentration (0.1 @ml), but inhibits at a high
concentration (3 pglrnl), 3~-thymidine incorporation into the DNA of guinea pig
marnmary gland fibroblasts in vitro (Shefied and Anderson, 1984). One study found that
PRXN (1-100 ng/ml) had no effects on the proliferation of normal cultured cervical
stromal cells fiom women undergoing hysterectomy for a variety of uterine disorders
(Hwang et al., 19960).
Effects On Extracellular Matrix
Relaxin c m cause relaxation of the pubic ligaments and softening of the ceMx.
The changes in these organs include the extensive dissolution and disorientation of
collagen fibers. Human RXN inhibits procollagen mRNA levels and the collagen
synthesis and changes the collagen secretory phenotype by cultured human dermai
fibroblasts and sclerodenna fibroblasts (Unemori et ai., 1990, 1992). In a rodent mode1 of
fibrosis, hurnan RXN in a purnp, which was implanted into rat demis, was able to
decrease collagen accumulation surrounding the osmotic pumpo suggesting that human
RXN appears to be able to decrease collagen synthesis (Unemon et al., 1993). Porcine
relaxin decreased ' ~ - ~ r o l i n e incorporation by approximately 40% in both hurnan cervical
and uterine comective tissue, suggesting that collagen message synthesis in the two
tissues is decreased (Wiqvist et al., 1984). In ovariectomized gilts, RXN and progesterone
CO-treatment resulted in significantly less collagen density in the stroma1 tissue of the
mammary gland than in untreated gilts (Winn et al., 1994). Recombinant human RXN,
given to piglets via an osmotic purnp, caused changes in their skin expansion cuves,
compatible with a relaxin-induced change in structural collagen (Kibblewhite et al.,
19%).
Effects On Proteolytic Enzymes
Regulatory effects of RXN upon proteolytic enymes in sofiening of the cervix
and elongation of the pubic synthesis have been proposed. Accumulated evidence
indicates that relaxin plays a role in connective tissue remodehg by stimulating
production of proteolytic enzymes.
The ovary has become the mode1 for study of collagenolytic activity. It was
shown that exogenous recombinant hurnan RXN (10 pg/ml) induced ovulation in
perfùsed ovaries, in vitro, fiom equine chorionic gonadotropin (eCG)-primed immature
rats and that this effect was comparable to 100 ng/rnl ovine LH (Brannstrom and
MacLennan, 1993). Intraovarian bursal injection of a monoclonal antibody specific for rat
RXN but not for PRXN partially suppressed hCG-induced ovulation in rats; moreover,
this inhibitory efiect of antibody was reversed by concomitant administration of PRXN
(Hwang et al, 1996a). The underlying mechanism(s) of RXN-induced ovulation is linked
with connective tissue remodeling. Studies have shown that RXN may modulate the
production of mûtrix metalloproteinases (MMPs). Porcine relaxin stimulates an increase
in collagenase levels in cultured rat ovarian cells. Since type I collagen was used as the
substrate, this collagenase is most likely interstitial collagenase (Too et al., 1984). In a
study on human follicular tissue, segments were incubated with PRXN and the
collagenolytic activity in the medium was shown to be increased significantly in 4 hours
(Norstrom and Tjugum, 1986). Recently, PRXN has been s h o w to stimulate gelatinase
production in conditioned medium by culhued thecal-interstitial cells kom eCG primed
rats (Hwang et al., 1996a). There is also evidence that RXN takes part in modulation of
the plamiinogen activator (PA) system. For example, RXN stimulates production of
plasminogen activator by rat grandosa cells, in vitro (Too et ai., 1982, 1984).
The fetal membrane is another object of study as a target site for the
collagenolytic activity of decidual and placental RXN (Koay et al., 1986). Changes in
levels of chorion and decidua mRNA for the MMP-1, 2, 3, and 9 were quantified. The
results led the authors to partition the collagenolytic events in the fetal membranes into
two phases. The fint phase is a slow and controlled loss of type III collagen. The second
phase is a more rapid cascade of collagenolytic events and results in the detachment of
the decidualplacenta fiom the uterine wall (Bryant-Greenwood and Schwabe, 1994).
Recently, it was confirmed that recombinant human RXN induced the changes of MMP-
1, -3 -9, and tissue type plasminogen activator (PA) at the mRNA and protein levels in
chorion and decidua of human beings (Qin et al., 1997a; 1997b).
In cultures of normal hurnan skin fibroblasts, human RXN caused a dose related
increase of interstitial collagenase at both the mRNA and protein levels (Unemori et al.,
1990). Recombinant human RXN also stimulated MW-1 activity in cultured primary
uterine cervical cells from guinea pigs (Mushayandebvu and Rajabi, 1995).
However, there are conflicting reports about effects of f on proteolytic
enzymes. Porcine relaxin has been shown to inhibit collagenolysis in the involuting
postpartum rat utems (Adams and Frieden, 1985). This inhibition might result from
inhibition of interstitial collagenase activity because PRXN inhibited interstitial
collagenase activity in the rat cervYt (Too et a!., 1986). in another relevant study, PRXN
prevented postpartum collagenase-mediated resorption of the guinea pig pubic symphysis
(Wahl et al., 1977).
Effect On Proteolytic Enzyme Inhibitors
Proteolytic enzymes involved in ECM remodeling are under the tight control of
several inhibitors. Enzymes and inhibitors work together to make proper ECM
degradation occur. Ovulation is an excellent example of this tight control on ECM
degradation. Because RXN is able to induce ovulation in the rat model, it is possible that
RXN also regulates these enzyme inhibitors in the ovary to ensure that the follicle wall
breaks down at its apex. However, there is little study in this area. Unemon et ai. (1 990)
reported that recombinant human RXN moderately inhibited production of tissue
inhibitors of metalloproteinases (TIMP) mRNA in human skin fibroblasts while
stimulating production of interstitial collagenase mRNA. Another report showed RXN
had no effect on production of TIMP- 1 mRNA in fetal membranes (Qin et al., 1997a).
Anatomy Of The Ovary
Primordial follicles are embedded in stroma1 tissue and are composed of a single
layer of granulosa cells surroundhg an oocyte. After the follicle enters the development
stage, the grandosa cells proliferate and the oocyte enlarges. These follicles have no
antrum and are classined as primary follicles. As primary follicles mature, a fluid filled
cavity is formed. Coincidentally, the thecal layes are formed also. The selected follicle
continuously grows with enlargement of the antnim of the follicle and proliferation of the
granulosa and the thecal cells. When the mature follicle approaches the stage of
preovulation, it moves close to the surface of the ovary. The follicle wall becomes thin,
breaks down, and the oocyte is released (Ham, 1974).
Horse Ovary
The equine ovary provides an unusual opportunity for the study of stromal
remodeling in the ovary. Fust, the equine ovary is large enough that its cellular
components can be easily isolated, especially the stromal cells. Second, unlike the ovaries
of many other adult mamrnals, the equine ovary has specific anatornic features that
Facilitate study. The region identified as cortical tissue in other species is located in the
center of the equine ovary, while the medullar portion is superficial. The ovary is almost
covered by a layer of strong connective tissue, the tunica albuginea, except at hilus or
ovulation fossa. In the process of development, the selected follicle expands fiom 100 nm
in diameter at the primordial follicle stage to 5-7 cm in diameter at the mature stage.
Unlike random ovulation taking place in any other mammals, the equine follicle
migrates/expands toward the hilus of the ovary, the only site that a mature oocyte can be
released fiom the ovary (Ginther, 1992). Thus the ovarian stromal tissue surrounding the
growing follicles undergoes extensive tissue remodeling in favor of the expansion and
migration of the follicles on a cyclic basis.
Stroma1 Tissue In The Ovary
The primary function of the mammalian ovary is to produce fertilizable oocytes
regularly during the reproductive cycle. The development of small follicles into large
preovulatory follicles is characterized by dynamic alterations in morphology as weli as
the biochernical properties of the follicular ceils. The degradation of connective tissue
surrounding the preovulatory follicle occurs, in part, by the action of proteolytic enzymes
(Lemaire, 1 989).
Extracellular Matrix
Stromal tissue is the connective tissue of the cortex in the ovary and contains a
high proportion of cells to ECM. Collagen is an abundant protein in most compartments
of the ovary. The ovarian capsule contains collagen types 1, III, and VI, while type IV is
located in the ovarian surface epithelium, the walls of atretic follicles and of capillaries
(Kaneko et al., 1984; Auersperg et al., 1991). The tunica albuginea and follicular theca
extema contain abundant collagen fibrils of the interstitial types 1 and III (Palotie et al.,
1987). In preovulatory follicles of the hamster, collagen fibrils fan out around the
follicular wall to provide mechanical support (Martin and Miller-Walker, 1983). The
meshwork becomes depleted in human mature follicles, particularly around the apex as
the time of ovulation approaches (Okamura et al., 1978, 1980). Ovarian surface epithelial
cells secrete collagen types I and III as well as keratin and laminin. OC these proteins, the
fibrous protein collagen is the major structural element that provides tensile strength and
anchors the developing follicles (Auersperg et al., 199 1).
Stromal Cells
Ovarian stromal cells are active and influence the fiinction of follicles. Basement
membrane separates the &randosa cells nom stromal ceus. One study has shown that
ovarian stroma1 cells were essential for the organization of the foUicle basement
membrane in rat ovary at the neonatal stage, suggesting their roles in the formation of
primordial follicles (Rajah et ai., 1992). During the development of the follicle, stromal
cells surrounding the follicle transform into the thecal ce11 layers when the primary
follicle expands into the stromal tissue. These thecal cells synthesize androgens, which
are transferred to the granulosa cells for the synthesis of estrogen and progesterone
(Barbieri et al., 1986). Ovarian stroma1 cells are able to produce several growth factors,
such as epidermal growth factor (Maruo et al., 1993), insulin and inhibin-like growth
factor4 (Jih et al., 1993). Stromal cells can be influenced by granulosa and thecal cells.
For instance, oxytocin-binding sites on stroma1 cells in the guinea-pig ovary have been
identified (Zhang et al., 1991). Ovarian stromal cells express receptors for epidermal
growth factor (Maruo et al., 1993), insulin (Samoto et al., 1993) and androgen. A role for
ovarian strornai cells in ECM remodeling has been suggested, since they synthesize PAS
and PAIS (Peng et al., 1993), and TIMPs (Vigne et al., 1994).
Enzymes in Extracellular Matrix Degradation
Extracellular matrix plays a central role in maintaining the structural integrity of
multicellular organisms. In addition, ECM is able to influence basic cellular processes
such as proliferation, differentiaûon, migration and adhesion (Alberts et al., 1996b). The
early notion that the ECM is an inert and stable structure has been dispelled.
Parenchymal and connective tissue cells contribute to the slow ECM remodeling
by a continuou process of synthesis and degradation of ECM components. Extracellular
matrix remodeling is considered a very important biological process involved in many
physiological phenornena, such as angiogenesis, attachrnent of the placenta,
embryogenesis, growth of the marnmary gland, wound healing and ovulation. Nomally,
ECM remodeling is in a dynamic equilibriurn: a balance between synthesis and
breakdown of ECM. In pathological situations, this balance is disturbed. Relatively more
synthesis of the ECM will cause diseases such as liver fibrosis (Takaharû et al., 1995) and
scleroderma (Kirk et al., 1995). Excessive arnounts of proteolytic enzymes will
excessively degrade ECM, as seen in rheumatoid arthntis (Ishiguro et al., 1996) and
formation of abdominal aortic aneurysms (Knox et al., 1997; Reilly, 1996).
As a part of remodeling, degradation of the ECM, or extracellular proteolysis, is
perfomed by many different enzyme systems: serine proteinases, metalloproteinases and
cysteine proteinases. In addition, endo- and exoglycosidases contribute to ECM
degradation. Matrix metalloproteinases and PA have been studied widely and have been
well characterized.
Matrix metalloproteinases, PA and their inhibitors are precisely coordinated to
regulate follicular comective tissue remodeling. The site and rate of synthesis OF both the
enzymes and inhibitors are important in this concept. If the enzyme and inhibitor are
produced by the same cells, an increase in enzyme activity might precede an increase in
inhibitor activity, or the concentration of tbe enzyme could exceed that of the inhibitors to
allow comective tissue degradation. in order to prevent premature and uncontrolled PA
activity or MMPs and consequent activity at inappropriate sites, t h e - and site-specific
inhibition of PA and MMPs are extremely important. Therefore, plasminogen activator
inhibitors (PAI) and T[MPs are also located in the ovary. The net activity is determined
by the balance of enzymes and inhibitors, but various factors rnight complicate the
interaction between inhibitors and the enzymes. Therefore, a direct correlation with
enzymelinhibitor ratios and activities is not dways observed.
Al1 members of the MMP family share amino acid similarities and are zinc2'-
dependent endopeptidases. They are secreted in an inactive proenzyme f o n and require
activation to exert proteolytic activity. Tissue inhibitors of rnetalloproteinases specifically
inhibit al1 MMPs (Matrisian, 1992).
The MMPs can be classed into four groups: (1) collagenases, including interstitial
collagenase (MMP-1), neutrophil collagenase (MMP-8) and a recently characterized
collagenase (MMP-13). Their activity is restricted to the cleavage of fibrillar types 1, II,
III collagen; (2) stromelysin, including stromelysin-1 and -2, and matrilysin; (3)
gelatinases and (4) membrane-bound type MMPs (Matrisian, 1992).
Both interstitial collagenase and neutrophil collagenase are able to cleave the
alpha chah of types 1, II, and III collagen. producing fngments such as gelatin. Although
these enzymes may degrade other components of ECM, the cleavage of intact, fibrillar
collagen is specifically limited to these enzymes. Once the higher order structure is
destroyed, many proteases, including other MMPs, can degrade the denanired substrates
(Matrisian, 1992).
Stromelysins have a relatively broad substrate specificity. Their natural substrates
appear to be the proteoglycans and glycoproteins such as fibronectin and laminin
(Matrisian, 1992).
Recently, several membrane-bound type MMPs (MT-MMP) have been found.
Their fünction is thought to relate to the activation of other MMP mernbers. For example,
a MT-MMP has been found in tumor cells and has a fwictional transmembrane domain
required for activation of latent gelatinase A (Sato et al., 1996).
The gelatinase family contains gelatinase A (MMP-2, 72 kDa type IV
collagenase) and gelatinase B (MMP-9,92 kDa type IV collagenase). They readily digest
denatured fibrillar collagens, but also degrade a nurnber of extra ECM components,
including collagen IV, V, VI1 and XI, elastin, fibronectin, laminin, aggrecan core protein
and cartilage link protein (Emonara and Grimaud, 1990; Matrisian, 1992). However,
arnong dl members of the MMPs, only gelatinases can degrade type IV collagen.
Gelatinase A is the most widely distributed of al1 MMPs and is produced constitutively
by many ce11 types in culture. Gelatinase B is also produced by many types of cells,
especially by tumor cells (Matrisian, 1992). Gelatinases are secreted rapidly as latent
forms, or the progelatinases. In vitro, only small amounts of fiee latent gelatinases are
present in conditioned medium. Most progelatinases are associated with TIMPs forming
progelatinase A/TIMP-2 and progelatinase B/TIMP- 1 complexes. Pro gelatinase A
interacts preferentiaily with TIMP-2 and progelatinase B interacts preferentially with
TIMP-1; there is no cross interaction (Goldberg et al., 1989).
The progelatinase amino acid sequence contains five domains: (i) a signal
sequence that directs the translation product to the endoplasmic reticulurn; (ii) an N-
terminai propeptide which confers latency on the proenzymes and is cleaved during the
activation; (iii) a catalytic domain which has a zinc2*-binding site; (iv) a dornain that has
hornology with fibronectin type II; and (v) a C-terminal domain that has a hemopexin-
and vitronectin-like domain and takes part in the complex formation with TIMP-1 or -2.
Latency of the progelatinases is thought to be retained through the cosrdination of the
zinc2+ ion at the active site and the SH group of the cysteine residue in the highly
conserved PRCGNPD sequence of the propeptide (Birkedai-Hansen et al., 1993). During
the activation, the cysteine residue in the propeptide is coordinated with the active site
zinc ion, followed by proteolytic cleavage of the propeptide domain (Wilhelm et al..
1989; Springrnan et al., 1990; Van Wart et al., 1990).
Activation of the progelatinases is a crucial step for the expression of their
enzymatic activity. The mechanisms of activation in vivo are not hlly understood.
Progelatinase A binds to the ce11 surface through a membrane-type MMP and is activated
by the membrane-type MMP (Sato et al., 1996) or by autoactivation (Bergrnann et al.,
1995). In vitro, progelatinase A is resistant to activation by many proteinases including
kdlikrein, plasminogen activators, plasmin and cathepsins (Okada et al., 1 !BO), but can
be activated by organomercuric compounds (Howard et al., 199 1 b). Gelatinase B may be
activated by kallikrein, plasminogen activators, plasmin, cathepsins and organomerc~c
compounds. There are reports that progelatinase B can be actîvated by stromelysh
(Shapiro et al., 1995) or gelatinase A (Fridrnan et al., 1995). Activated gelatinase A alone
is more proteolyticdly active than the gelatinase AmMP-2 complex, but has a shorter
half life than the enzyme complex due to autolysis (Kleiner et al., 1993).
In progelatinase/TIMP complexes, only the C-terminal region of progelatinases
bind to the C-terminus of TIMPs. Progelatinases in complexes can stiil be activated since
TIMP does not interfere with the catalytic domain of progelatinases. Mer activation, the
catalytic site of the gelatinases reacts with the N-terminai domain of TlMP and forms a
tighter binding with it. In this way activated gelatinases are inhibited by TIMP (Itoh et al..
1995; Ogata et al., 1995). Progelatinase/TiMP complexes have been characterized fiom
conditioned medium of several ce11 types including human melanoma cells (Stetler-
Stevenson et al., 1989), uterine cervical fibroblasts (Itoh et al., 1995) and lung fibroblasts
(Goldberg et al., 1989). They are stable, even in the presence of 1.0 M NaCl or 2.0 M
urea. Progelatinases dissociate from TIMP in the presence of sodium docecyl sulfate
(SDS). The activated gelatinase/TIMP complexes lose gelatinase activity, but remain
intact. If activated gelatinases are separated nom TIMPs, the activity can be recovered
(Wilhelm et al., 1 989; Stetier-Stevenson et al., 1989; Goldberg et al., 1992).
Since proteolytic enzymes play roles in follicle growth and ovulation, gelatinases
are of great interest. Interstitial collagenase activity is present in Graafiian follicles of
rabbit ovary (Tadakuma et al., 1993) and is responsible for degrading type I and III
collagen. However, it cannot degrade type IV basement membrane collagen. In addition,
many studies codïrm that gelatinolytic activity is present in ovarian foliicdar fluid
(Puistola et al., 1986; Palotie et al., 1987; Hirsch et al., 1991). Another line of evidence
supporting gelatinase involvement in ECM remodeling in the ovary cornes fioom the
changing levels of gelatinase activity during ovulation. Activity of gelatinases in thecal-
interstitial cells of rat ovary begins to increase 12 hours before ovulation and reaches a
peak just before ovulation and then declines dramatically after ovulation (Palotie et al.,
1987; Reich et al., 199 1).
n i e source of gelatinases in the ovary is localized in residue tissue with the use of
a human gelatinase probe, while the residuai tissue includes thecal-interstitial cells and
stromal cells (Reich et al., 1991). Later, thecal cells were proven to be one source of
gelatinase. However, stromal cells in the ovary might also be a source of gelatinase. It is
Iikely that gelatinases are not synthesized by granulosa cells since gelatinolytic activity
has not been detected as protein or mRNA in these cells (Palotie et al., 1987; Curry et al.,
1992; Hurwitz et al., 1993). However, Hwang et al. (1996a) showed that rat thecal-
interstitial and granulosa cells in vitro produced gelatinases as detected by zymography.
In order to degrade the ECM, latent foms of gelatinase must be activated. In one study,
only the latent form of gelatinase was found in the medium nom cultured rat follicle
(Palotie et al., 1987). In another study, the ratio of the latent to the active form of the
enzyme in follicular fluid diminished as ovulation approached (Cuny et al., 1992).
Fully activated gelatinases can be inhibited by interactions with inhibitors, such as
armacroglobulin and TIMPs. Of central importance are the TIMPs.
Three distinct types of TIMPs have been identified on the basis of their molecular
sizes and biological activities. The individual TIMPs show about 40% sequence identity,
but they share considerably higher structual similarity. They are essentially
interchangeable in their ability to inhibit MMPs (Murphy et al., 1993). TiMPs are found
in many ce11 types, body fluids and tissue extnicts. In ce11 culture, TIMP-1 and -2 are
present in conditioned medium, whereas TIMP-3 binds to ECM and is not present in
conditioned medium (Pavloff et al., 1992; Wilde et al., 1994). The UMPs are stable to
acid and heat, retaining partial activity at pH 2 or when heated at 100°C for 30 min.. They
are inactivated by alkylation (Cawston et al., 1981). Recently, a new member of this
farnily, TIMP-4 has been cloned from a human heart cDNA library. The predicted
structure of TIMP-4 shares 37% sequence identity with TIMP-I and 51% identity with
TIMP-2 and -3. The rnRNA of TIMP-4 is reported as absent fiom human ovarian tissue
(Greene et al., 1996).
Al1 TIMPs are able to act on ail MMPs, including collagenase, gelatinase and
stromelysin (Cawston et al., 198 1; Murphy et al., 1985; Fridman et al., 1993). They form
a tight complex with active forms of MMP as nonconvalent bimolecular complexes in a
ratio of 1 :1 (Fridman et al., 1993) and inhibit MMP activity. As mentioned before,
progelatinase A interacts preferentially with TIMP-2 and progelatinase B interacts
preferentially with TIMP-1 (Goldberg et ai., 1989). One study showed that at half-
maximal inhibition, TIMP-2 was over 10-fold more effective than TIMP- 1 in inhibiting
fully activated gelatinase A and over 7-fold more effective in inhibiting gelatinase B
(Howard et al., 1991b). In contrast, TIMP-1 was found to be over 2-fold more effective in
inhibiting interstitial coliagenase than TIMP-2 (Willenbrock et al., 1993).
The mechanism for TIMP specificity in inhibition of MMP function is not fully
clear. It has been found that TiMPs bind to the active site, as cornpetition has been
observed between TIMP- I and low molecular weight synthetic inhibitors that are directed
at the catdytic zinc2+ (OtShea et al., 1992). Another midy found that TIMP- 1 bound to a
site of progelatinase A, which stabilizes the molecular structure of gelatinases. By this
mechanism, TIMP-1 might prevent the autoactivation of gelatinase A (Howard et al.,
199 1 a). Progelatinase A/TIMP-2 complexes are able to inhibit activated gelatinase B,
suggesting that the TIMP-2 domain that interacts with the gelatinase B is not involved in
binding to progelatinase A (Kolkenbrock et al., 1991).
A third TIMP domain has been suggested (Ernmert-Buck et al., 1995). By this
binding site, progelathases A /nMP-2 complexes may bind to the ce11 surface (Strongin
et al., 1995). The significance of TIMP binding to the ce11 surface is that cells malr
actively participate in tissue remodeling and still be protected fiom extensive degradation
of ce11 surface molecules by gelatinases (Bertaux et al., 1991; Emmert-Buck et al., 1995).
As well as the activity inhibithg eEect, nMPs have other effects on gelatinases.
It has been suggested that TIMP-2 participates in the activation of progelatinase A on the
ce11 surface. The active gelatinase AITIMP-2 complexes are markedly resistant to
autodegradation, whereas activated, TMP-2 fiee gelatinase undergoes rapid
auto pro teolytic degradation to inactive fragments (Kleiner et al., 1 993).
Aside fiom the function of regulating MMPs, other biological functions have been
identified for TIMPs. TIMP-1 and TIMP-2 possess ce11 growth-promoting activity for a
wide range of celis in vitro (Hayakawa et al., 1992, 1994). This occurs in a
dose-dependent manner via a CAMP-dependent mechanism (Corcoran and
Stetler-Stevenson, 1995).
Various ce11 types of different species can synthesize gelatinases and TIMPs
sirnultaneously, such as hurnan heart fibroblasts (Tyagi et ai., 1996), bovine corpora lutea
(Goldberg et al.. 1996), mouse placental trophoblast giant cells (Alexander et al., 1996)
and rabbit aortic smooth muscle cells (Fabunrni et al., 1996).
inhibitor activity for MMP was first detected in the rat ovary at the time of
ovulation (Curry et al., 1986). More recently, 'MMP-I has been found in the ovaries of
cow (Freudenstein et al., 1990), sheep (Smith and Moor, 1991), and humans (Chun et al..
1992). Messenger RNA for TIMP-1 has been localized to rat granulosa cells by Northem
blot analysis (Curry et al., 1990; Smith et al., 1993) and by in situ hybridization (Chun et
al., 1992). Interstitial and thecal cells also produce TIMP-1 (Reich et al., 1991). Levels of
TIMP expression increase in the granulosa cells of non-ovulating antral follicles,
suggesting a role for these inhibitors in preventing premature ovulation (Chun et al.,
1992). The ovary is the tissue with the highest level of TIMP-1 mRNA among al1 the
tissues examined in the adult mouse (Waterhouse et al., 1993). Messenger RNA for
TIMP-1 increases in amcjunt toward the time of ovulation, and declines &er ovulation
(Chun et al., 1992).
Messenger RNA for TLMP-2 is expressed by the thecal layer of preovulatory
follicles and its expression is not increased by the preovulatory gonadotropin surge. In
contrast, the expression of TIMP-2 mRNA is maximal during the early luteal phase and is
greatest in large luteal cells (Smith et al., 1995).
Human CG increases the arnount of TIMP-1 secreted by thecal and granulosa
cells in culture (Chun et al., 1992). The TIMP- 1 mRNA Ievel in granulosa cells reaches a
maximal increase 6-9 h after stimulation by hCG. Progesterone participates in
maintainhg the homeostasis of the comective tissue matrix in the uterine cervix by
augmenthg both TIMP-I and TIMP-2 production dong with having suppressive effects
on proMMP-1 and proMMP-3 production (Imada et al., 1994).
The stringent balance of MMP and TIMP activities is very important to ensure
normal tissue structure and function. In various tissues, MMP and TIMP can be expressed
simultaneously. In human kidney, skin, liver, lung and heart, MMP and TIMP have been
found in the sarne location by in situ hybridization and show the sarne trend of increases
(Tyagi et al., 1995). Human osteoblasts synthesize gelatinases A and B, and TIMP-1 and
-2 (Meikle et al., 1995). Aithough the preovulation increase in inhibitor activity parallels
the collagenolytic changes, follicular degradation may occur only at the apex of the
follicles due to an excess of the enzyme in relation to the inhibitor.
en Activato~
The primary component of the plasminogen system is the broad-spectmn
protease plasmin, which is able to dissolve fibrin clots, digest ECM proteins and activate
latent MMPs. Plasmin is converted fiom abundant plasminogen by two specific
plasminogen activators: tPA and uPA. The uPA and tPA are products of different genes
but are about 40% homologous on the basis of their amino acid sequences (Belin et al.,
1985; Pennica et al., 1983). These two activators may be secreted by the same cells and
participate in the regulation of several physiological processes by initiating a cascade
reaction leading to the formation of active plasmin. Tissue-type PA is regarded as an
important mediator of fibrinolytic activity in the circulation, while uPA has a role in cell
migration, inflammation, comective tissue remodeling, wound healing and ovulation.
Uro kinase-Ty pe Plasminogen Activator
Urokinase-type PA was first found in urine (Williams, 195 1; While et al., 1966).
Actually a variety of tissues rnay produce uPA, including placenta, uterus and ovary.
Urokinase-type PA is synthesized, secreted and present in the extracellular fluid as a
single chah molecule, termed pro-uPA, with little intrinsic activity (Petersen et al..
1988). Catalytic arnounts of plasmin may convert single-chah pro-uPA to an active two-
chah form and enable an autocatalytic acceleration of uPA formation (Saksela, 1985). By
this mechanism, initial traces of piasmin catalyze the production of active PA, which
leads to the formation of more plasmin. Trypsin and kallikrein may also activate the
single chain uPA by converting it to a two-chah molecule (Eaton et al., 1984; Ichinose et
al., 1986). The active twothain form consists of an A chain and a B chain, linked by one
disuifide bond. The catalytic domain resides in the B-chah (Ichinose et al., 1986).
Two-chah uPA is able to bind to uPA recepton (uPAR) through the EGF domain
of the uPA molecule with high affinity and retains its catalytic activity (Appella et al..
1987; Blasi et al., 1986). Usually ceils containhg uPAR also express receptors for
plasminogen on the celi surface. The receptor-bound two chain LPA may activate
plasminogen, which simuitaneously binds to the ceU surface. This mechanism provides a
potent cell-surface plasmin generation system and enables cells to express site-directed
proteolysis (Stephens et al., 1989). The uPAR possesses a glycosyl-phosphatidylinositol
plasma membrane anchor at the carboxyl-terminal domain, which may facilitate its laterai
movement in the plasma membrane (Estreicher et al., 1990; Ploug et al., 1991). Thus,
receptor-bound two chah uPA is c m e d rapidly to areas where an increased proteolytic
activity is required and away from areas where it is no longer necessary. Receptor-bound
two chah uPA still has the ability to bind to PAL1 and to be inhibited by this molecule
(Cubellis et al., 1989).
It has been hypothesized (Andreasen et al., 1994) that a continued high levei of
surface plasminogen activation is dependent on the clearance of PM-inhibited uPA from
uPAR in order to allow new rounds of pro-uPA binding, conversion to active uPA and
subsequent plasminogen activation. The rnechanism for clearance of PAI-inhibited uPA
has been elucidated. When receptor-bound two-chah uPA f o n s complexes wiih PAL 1,
they simultaneously cross-link to low density lipoprotein receptor-related protein (LRP)
and are intemalized (Nykjaer et al.. 1994b). Low density lipoprotein receptor-related
protein is expressed on cells and is responsible for mediating the cellular intemalization
of many ligands, including proteinases, proteinase-inhibitor complexes and lipoproteins
(Strickland et al., 1995). Within the endosome, the ligand dissociates fiom the receptor
(Kounnas et al., 1993). Recent studies have shown that uPA/PAI-2 complexes are not
intemalized upon binding to the uPAR in cells. Instead, these complexes are cleaved into
two fragments by cells; the 70 kDa fiagment is ïntemaiized and the 22 kDa fiagment is
still present on the cell surface (Ragno et al., 1995).
Single pro-uPA is able to bind to uPAR and remains on the ce11 surface for long
periods of time (Blasi, 1993). A study has shown that uPAR may protect pro-uPA From
LRP-intemalization (Nykjaer et al., 1994b). Single pro-uPA can also bind directly to
L W , but the binding is much weaker than that of uPA/PAI-1 complexes (Kounnas et al.,
1993).
Tissue-type plasminogen activator
Tissue-type PA is a glycoprotein secreted as a single chah molecule, named pro-
tPA (Pennica et al., 1983). Plasmin converts single chain pro-tPA to active two-chain tPA
with increased catalytic activity (Ranby et al., 1982). Specific sites in the heavy chain of
tPA mediate the binding to certain compounds, such as fibrin, fibrinogen fragments,
polylysine heparin or denatured proteins and these compounds markedly stimulate tPA
catalytic activity (Dano et al., 1985). Because plasminogen also binds to fibrin, the
juxtaposition of plasminogen and tPA on the cornmon ligand results in enhanced
production of plasmin (Dano et al., 1985). The heavy chain of tPA contains an EGF-like
domain responsible for binding the enzyme to ce11 surface receptors (Orth et al., 1994).
Recent studies show that tPA or tPA/PAI-1 complexes may also bind to L W and be
internalized (Snickland et al., 1995).
Since a close correlation between the appearance of PA activity in the ovary and
ovulation has been reported (Beers, 1975; Strickland and Beers, 1976; Liu et al., 1987), it
was proposed th& PAS produced by the ovarian cells rnay activate plasminogen in the
foilicular fluid, giving rise to plasmin. Plasmin rnay in turn act on procollagenase to
generate active collagenase in a proteolytic cascade causing the destruction of the follicle
wall (He et al., 1989). Follicle stimulating hormone (FSH), a gonadotropin triggering the
development of follicles, elevated the PA activity in conditioned medium and on the
grandosa ce11 surface (Knecht, 1986). Similarly, Too et al. (1984) found that FSH
stimulated the PA activity after 20 hours of incubation with rat granulosa cells. In
addition, injection of serine protease inhibitors into the ovarian bursa of gonadotropin-
stimulated immature rats decreased the sizes and nurnber of antral follicles, as well as the
ovulation rate (Pellicer et al., 1988). Recently, a mouse with deficient uPA, tPA and
PAL1 gene functions was developed. This mouse was still able to ovulate and the
progeny appeared normal at birth (Carmeliet et ai., 1993, 1994). However, the ovulation
rate in such mice was reduced by 26% (Leonardsson et al., 1995). Tnese results suggest
that the plasminogen system is not the only proteolytic system involved in the ovary and
may not be as important during ovulation as previously proposed.
The activation cascade leading to the formation of plasmin can be controlled by
either inhibition of plasmin or PA activities by proteinase inhibitors (PAI-I), some of
which may function specificaiiy against oniy plasrninogen activators. Plasminogen
activator inhibitors belong to the serine proteinase inhibitor superfady (Hill and Hastie,
1987). The inhibition of the enzyme activity is accomplished by the formation of 1 : 1
covalent complexes between the enzymes and the inhibitors. Plasminogen activator
inhibitor4 is one of the three different, effective and functionally rapid PAIS.
Plasminogen activator inhibitor-1 is secreted by the cells as a 46-54 kDa single-
chah glycoprotein that was initidly characterized as an endothelid cell PA inhibitor
(Van Mourik et al., 1984). Newly synthesized PAL1 is in an active f o n and has a short
half-life of about 2 h in the ce11 culture medium because of rapid conversion to an
inactive variant caused partly by the binding of PAL1 to uPA or P A . This forms higher
molecular weight complexes (Hekman and Loskuto ff, 1988; Kooistra et al., 1 986).
Plasminogen activator inhibitor-1 npidly and specifically foms inactive complexes with
one- or two-chain tPA and two chain uPA, but not with single chain uPA (Andreason et
al., 1986). These complexes resist dissociation by denanuing agents such as urea,
guanidine or SDS; however, these denaturing agents regenerate activity of the bound
enzyme (Hekman and Loskutoff, 1985,1988).
In non-ovulatory follicles, PAL1 was mainly produced by thecal-interstitial cells
and stroma1 cells, as detected with in situ hybridization, northem blot anaiysis, gelatin
zymography and in situ zymography (Liu et al., 199 1). The PAI-1 activity increased
toward the t h e of ovulation and declined after ovulation, showing a similar trend as PA.
For example, ovaries fiom PMSG-primed rats express low levels of PAL1 mRNA.
Treatment with hCG triggea the synthesis of PAL1 mRNA. The induction of PAL1
mRNA is rapid and maximum levels are obtained 8-10 h af'ter hCG treatrnent, then the
levels of PA14 dramaticaily decrease in the ovary (Liu et al., 199 1). The production of
PAL1 is affected by agents similar to that of the PAS.
T r a n s f o w o w t h factor-? and Phorbol 1 2-rnyristate 13-acetato
Transforming growth factor+ (TGF-P) is a homodimeric peptide structwally
related to the inhibins, activin and Mullerian inhibiting substance (MIS). Transforming
growth factor-p is a conserved structure across species. Only one arnino acid difference
has been found between simian, human and rnouse sequences of the mature TGF-P
(Laiho and Keski-Oja, 1989). Transforming growth factor+ acts at multiple levels
affecting protein synthesis, including stimulation of transcription, increased stability of
mRNA, and enhancement of translation. Transforming growth factor+ has been found in
the ovary. In ovarian tissue, theca-interstitial cells of the rat, cow and pig secrete TGF-P
in vitro (Skinner et al., 1987; Gangrade and May, 1990). Immunocytochemical studies
using specific antibodies also localize TGF-P to the theca-interstitial cells (Thornpson et
al., 1989; Chegini and Flanders, 1992). Furthemore, human ovarian stroma1 cells were
immunostained for TGF-P (Che@ and Flandee, 1992). Transforming growth factor-p is
thought to be an intra-ovarian factor involved in follicle development.
Transforming growth factor-p differentially regulates DNA synthesis and ce11
proliferation in vitro. Transforming growth factor-p is considered a growth inhibitor for
epithelial cells, but a growth-stimulatory factor for rnesenchymal cells. However, many
studies have shown that TGF-P inhibits proliferation of fibroblasts. For instance, TGF-P
markedly inhibited the proliferation of chicken fibroblasts (Yang and Moses, 1990).
Transforming growth factor- p may also inhibit proliferation of granulosa cells.
Mondschein et al. (1988) demonstrated that hurnan and porcine TGF-P signincantly
inhibited 3~-thymidine incorporation into DNA by porcine granulosa cells, while ce11
numbers were reduced an average of 35% by 10 n g h l TGF-P in short term cultures.
These workers also showed that TGF-P inhibited the epidennal growth factor (EGF) and
PDGF stimulatory effects in . '~-th~midine incorporation.
Phorbol ester can substitute for diacylgycerol as a cofactor for protein kinase C
production and can activate protein kinase C for phosphorylation of substrate proteins.
The mechanimi of action of Phorbol 12-myristate 13-acetate (PMA) appears to be a
facilitation of ca2' binding (Albert et al., 1989a), because PMA is somewhat analogous in
stereochemical structure to diacylglycerol. In many snidies of proteolytic enzymes in
vitro, PMA has been used to examine regulatory mechanisms, although it is a non-
physiological factor (Albert et al., 1989a).
RATIONALE
Relaxin (RXN) has been identified in the equine ovary and is thought to have local
effects. Previous studies show that RXN may regulate the production of several
proteolytic enzymes by the cells in ovanan tissues as well as granulosa cells in the
follicle. Ovarian follicles are embedded in stromal tissue. During their development,
stromal tissue alters to provide space for the expanding follicles. Whether ovarian stromal
cells are able to produce proteolytic enzymes and whether RXN regulates these enzymes
is not known.
The present snidy was designed to examine the production of several proteolytic
enzymes by equine ovarian stromal cells (EOSC) and evaluate the effects of RXN on the
activity of these enzymes in vitro. The study was conducted using EOSC isolated fiorn
ovaries containing follicles larger than 3 cm in diameter. The effects of RXN on the
proliferation of EOSC was assessed using 3~-thyrnidine incorporation. The effects of
RXN and other factors on gelatinases, tissue inhibitors of metailoproteinases (TIMP),
urokinase-type plasminogen activator @PA) and plasminogen activator inhibitor-l (PAI-
1) in conditioned medium, cell extracts and extracellular ma& were assessed using
zymography, reverse zymography, fibrin autography, reverse fibrin autography and
chromogenic assay. These five techniques are specifically known to detect these enzymes
or their inhibitors.
MATERIALS AND METHODS
Details on the commercial sources of reagents are listed in Appendix 1. Recipes
for bufTers and solutions used in this study are listed in Appendix II.
7
Ovaries from mature, cyclic mares were collected fiom a local abattoir (Barton
Feeders, Owen Sound, ON) during the breeding season fiom April to September. Ovaries
were transported to the laboratory on ice in sterile Hank's balanced salt solution (HBSS)
with gentarnycin (5 pg/ml).
The outer surface of the ovaries was rinsed with 70% alcohol, followed by two
washes with HBSS. The ovaries were bisected longitudinally within 4-6 hours of
collection. Eight ovaries with follicles 3-4 cm in diameter fiom eight horses were chosen
for the isolation of stromal cells. Only stromal tissue around follicles judged as healthy
were used in this study. Stromai tissue at the apex of developing follicles (3-4 cm in
diameter) was collected and minced into 1 mm3 pieces. The tissue was digeçted by
shaking in collagenase type I (125 U h l ) in senun-fiee minimum essential medium
(MEM) at 37OC for three hours. During the last half hour of digestion, DNase i (50 U/ml)
was added to the solution. After the tissue pieces were allowed to settle, crude debris was
removed. Then, the supernatant containhg equine ovarian stromal cells (EOSC) was
centrifuged at 250 g. The supernatant was discarded and the ce11 pellet was resuspended
with HBSS. Viability of the EOSC was estimated by &pan blue dye exclusion. Equine
ovarian stroma1 cells were grown in MEM supplemented with 10% (v/v) fetal bovine
serum (FBS), 2 mM L-glutamine, penicillin, streptomycin and gentarnycin at 37O C in a
5% CO2 atmosphere. After cultures were established, EOSC isolated fiom the eight
animals were pooled together in order to have enough cells for the whole study and to
ensure that cells used in each experiment were fiom the sarne pool. Finally, EOSC were
aliquoted into cryovials at a density of 1 x 1 06/ml in media containing dimethy lsul faxide
(IO%, V/V) and FBS (20%, v/v) and were fiozen and stored in a liquid nitrogen tank. Cells
between passage 3 and 8 were used in these experiments.
Jmmunocpcherriism nnd hlxul9fluorescem
In order to characterize the isolated cells, and confirm their identity as EOSC, u-
smooth muscle ce11 actin (a-SMA), Von Willebrand Factor and type I procollagen were
chosen as markers. Cells were cultured on sterile coverslips in MEM with 10% FBS
ovemight, were then rinsed with HBSS solution, dried completely at room temperature
and fixed in cold acetone for ten minutes. Cultured EOSC were immunostained as
follows.
For expression of a-SM& cultured cells on slides were immersed in PBS for 6
min and then were blocked with 10% rabbit serum solution. Primary antibody
(monoclonal, mouse) against a-SMA was applied to cdtured cells. M e r incubation for
1 h at roorn temperature, sections were washed with PBS and incubated with biotinylated
secondary antibody for 10 min. Streptavidin-peroxidase conjugate was applied for 6 mio.
The color reaction was develo ped with a substrate-chromogen mixture (hydrogen
peroxide and DAB), monitored under microscopy. Slides were counterstained with
hematoxylin.
For expression of type I procollagen, acetone-fixed cells on coverslips were
immersed in PBS for 6 min. Cells were then blocked with 3% BSA solution at 4OC
ovemight. Primary antibody against procollagen type 1 @olyclonol, rabbit) was applied to
cultured cells. Afier incubation for 1 h at room temperature, cells were washed with PBS
and incubated for 30 min with a secondary antibody conjugated with alkaline
phosphatase.
For expression of Von Willebrand Factor, primary rabbit antibody against human
Von Willebrand Factor @olyclonol, rabbit) was applied to acetone-fuced EOSC on
coverslips for 1 hour, followed by incubation with a second antibody (goat anti-rabbit)
conjugated with FITC for another 30 min. Sections were observed under a fluorescence
microscope.
This assay was performed according to the method of Sheffield and Anderson
(1984) with some modifications. Bnefly, EOSC were seeded into 96-well plates at a
density of 2 x 1 0 ~ cellslwell in MEM with 10% FBS. Medium was changed every 48
hours until cells reached confiuence. After 24 hours in 0.5% FBS MEM, the EOSC were
exposed to various treatments for 48 hours and with 3~-thymidine (3 pCi/ml) added
during the last 12 hours of incubation. Cells were detached nom wells with 0.5% trypsin-
EDTA and collected onto glass fiber Nters (Wallac Oy, Turku, Finland). Radioactivity
was measured with a liquid scintillation counter (1205 beta plate, LKB). The results were
expressed as the percentage of thymidine incorporation, compared to the control groups.
CelI number det- . .
Equine ovarian stromal cells were plated with 10% FBS MEM into 35 mm dishes
at a density of 1 x 1 0 ~ cellddish. M e r reaching confluence, EOSC were cultured in 0.5%
FBS MEM for a fùrther 24 hours and then exposed to PRXN or ERXN at concentrations
of 0.01 to 100 @ml. Fresh medium and treatments were added after 48 hours and
incubation was continued for another 48 hours. Cells were treated with 0.5% trypsin-
EDTA until attached cells could no long be found on culture dishes when inspected by
phase contrast microscopy. Cells were collected and counted with a hemocytometer. The
viability of the EOSC in each group was also estimated by the trypan blue dye exclusion.
P r e p d o n of c ~ ~ ~ o n e d IIlahm . .
Equine ovarian stromal cells at a density of 5 x 1 0 ~ viable cells were seeded into 60
mm dishes. nie medium was changed every 48 hours until the cells became confluent.
The cells were cultured in serum-fiee medium for another 24 hours, followed by various
treatments. The conditioned medium was a s p h e d fiom 60 mm culture dishes and
centnfuged at 250 g to remove dead cells and debris. The supernatant was decanted and
stored at -80'~. The conditioned medium was lyophilized and resuspended with ddHzO.
The fmal solution was 20-fold concentrated. The protein content in concentrated
conditioned medium was detennined by the Bradford method (1 976). Concentrated
conditioned medium was aliquoted in 500 pl vials and stored at -80'~.
C s
M e r collection of the conditioned medium, cells in the dishes were washed with
HBSS three times to remove the medium. Then 5 ml 1% Triton X-100 were added to
each dish to lyse the cells. The resultant solution was collected and agitated vigorously
After centrifugation at 250 g, the supernatant was collected, fiozen and lyophilized. Afier
resuspension with ddHzO ai a 20 fold concentration, the protein content was determined
and specimens were stored at -80°C.
Collection of extracelldar matrirr
The ECM in petri dishes was prepared as described previously (Hampton et al.,
1995). Briefly, after collection of conditioned medium, the confluent ce11 monolayers
were washed three times with PBS containhg 5 m M ethylenediaminetetraacetic acid
(EDTA)/S mM ethylene glycol-bis(2-aminoethyl ether) N,N,Nf,N tetrmcetic acid
(EGTA), then incubated at 37OC in 5 ml of PBS with 5 mM EDTNS rnM EGTA until
the cells began to detach fiom the surface of the dishes. The dishes were washed several
times with PBS until ceils could no longer be found on culture dishes when inspected by
phase contlast microscopy. The remaining ECM was washed three times with plain PBS,
followed by ddH20 to remove the salts. Then, ECM was collected by scraping the dish
bottom with a hard rubber spanila in 10 pl of 5x gel loading buffer without reducing
agent and 40 pl of ddHZO. The volume of collected bufYer was adjusted with ddH,O to 50
pl and samples were used immediately for reverse zymography or were stored at -80°C,
Protein concentrations in the conditioned media and ce11 extracts were measured
using the Bradford (1976) method. Bovine s e m albumin was diluted in ddH20 and used
as the standard. Ten pl of each sarnple were added into wells of a 96-well plate with 10 pl
of dM2O and 200 pl of dye reagent. For standard curves, 10 p1 of BSA solution at
different concentrations were added into wells in the sarne plate with 200 pl of dye.
Absorbantes were measured at 6 10 nm with a microplate reader (Multiskan MCC/340.
Du Pont, USA). The protein concentrations in the samples were determined from the
BSA standard curves.
SDS-PAGE
Samples were examined using SDS-polyacrylamide gel electrophoresis (Laemmli.
1970). Samples were mixed with nonreducing loading buff'er (5x) and heated at 100°C
for 3 min and loaded into gels. Gels were subjected to electrophoresis at 4OC with
nuining buffer. When the bromphenol blue dye front reached the bottom of the gels, the
current was stopped and the gels were processed for staining with Coomassie Brilliant
Blue G-250 for 30 min followed by destainhg with a solution of 5% methanol and 7%
glacial acetic acid. Gels were then washed with ddHZO. Gels were photographed using a
Gel Pint 2000i apparatus (Bio/Can Scientific) and digitized images were saved for
M e r densitometric analysis.
ZvmoeraDhv
Active and latent foms of gelatinase A and B were detected by zymography, as
described by Heussen and Dowdle (1980). Sarnples were loaded with non-reducing
loading buffer in a ratio 1:4 into acrylamide (7.5% v/v) gel polyrnerized with 0.2% (wh)
calf skin gelatin. After electrophoresis, gels were washed with 2.5% Triton X-100 (2 x 30
min), then nnsed in ddH20. Afier washing in the incubation buffet for 30 min. gels were
incubated with the incubation buffer at 370C over night. Gels were stained with
Coomassie Bnlliant Blue G-250 for 30 min and destained in 5% methanol/7% glacial
acetic acid. Since gelatinase A and B degrade gelatin present in the acrylamide gel, clear
lysis bands indicate the absence of gelatin and the presence of gelatinases. In order to
identiQ lùrther the gelatinolytic activity in conditioned medium of EOSC, two proteinase
inhibitors, 10 mM ethylenediaminetetraacetic acid (EDTA) and 1 m M
phenylmethylsulfonyl fluoride (PMSF), were incorporated into the incubation buffer.
Gels were photographed using a Gel Print 2000i apparatus and digitized images were
saved for m e r densitometric analysis.
Reverse nmpecaehr
Tissue inhibitors of metailoproteinases were analyzed with reverse zymography
(Hampton et al., 1995). Bnefly, 12% SDS polyacrylamide gels were made with 0.2% calf
skin gelatin and crude gelatinase A purchased from University Technologies International
Inc. (Calgary, Al). Samples were loaded with non-reducing loading buffer in a ratio of
1:J. After electrophoresis, gels were washed with 2.5% Triton X-100 (2 x 60 min),
followed by rinsing in ddH20. Gels were washed in incubation buffer for 30 min, then
incubated with incubation bufTer at 37OC oves night. Gels were stained with Coomassie
Brilliant Blue G-250 for 30 min and destained in 5% methanoV7% glacial acetic acid.
The activity of TIMPs resulted in the presence of dark blue bands on a cleared
background. Purified mouse TIMP-1, -2 and -3 were run in each gel as standards. Gels
were photographed using a Gel Print 2000i apparatus and digitized images were saved for
M e r densitometric analysis.
. . ibnn a i l t o m
Plasminogen activator activities were cxarnined with fibrin autography (Monge et
al., 1989). Samples were loaded with nonreducing loading buffkr in a ratio 1 :4 in wells of
10% acrylamide gels. M e r electrophoresis, gels were washed in 2.5% Triton X-100 (2 x
30 min), followed by rinsing in ddH20. Fibrin-agar indicator films were made as follows.
Low molecular weight agarose (100 mg) was dissolved in a 10 ml solution (0.14 M NaCl,
0.01 M Na?HP04, - pH 7.2) at 100°C and the resulting agarose solution was kept in a
waterbath at 50°C. Fibrhogen (final concentration 20 mgld), human plasrninogen (final
concentration 3 mg/ml), and thrombin (finai concentration 1 Ufml) were added to the
agarose solution. ïhe agarose solution was then poured into a prewarmed (50°C) mini-
gel apparatus. Then the acrylamide gels were dried with paper towels and overlaid on the
indicator films. Gel and films were incubated at 37OC in a humidified chamber overnight.
Plasminogen activator activity was indicated by developrnent of lysis bands. In order to
distinguish uPA fiom PA, amiloride (100 mM), a specific inhibitor of uPA activity, was
added to several indicator films. Gels were photographed using a Gel Pnnt 2000i
apparatus and digitized images were saved for further densitometric analysis.
Plasminogen activator inhibitors (PM) were quantified with reverse fibrin
autography (Enckson et al., 1984). Samples were loaded with nonreducing loading buffer
in a ratio 1:4. AAer electrophoresis, gels (10% acrylamide) were washed in 2.5% Triton
X-100 (2 x 30 min), followed by rinsing in ddHzO. Then the gels were overlaid on fibrin-
agar indicator films which had the same components as those used in fibrin autoçraphy
(described above), plus hurnan uPA (final concentration 1 IU/ml). Gel and film were
incubated at 370C in a hurnidified chamber for an appropnate period. Plasminogen
activator inhibitors were indicated by opaque lysis-resistant zones. Gels were
photographed using a Gel Print 2000i apparatus and digitized images were saved for
fùrther densitometric analysis.
Plasminogen activators were measured with the assay described by Coleman and
Green (198 1) with some modifications. Wells of 96-well plates were blocked by adding
300 pl of 3% BSA solution containing 0.5% (vlv) Tween 20 and incubating at 37OC for 1
hour. Then, the blocking buffer was removed and wells were washed 3 tirnes with 0.05%
Tween-PBS and dried completely at 37OC. Ten pl of PBS containhg Tween (0.05%) and
gelatin (2.5%), plasminogen (2 mg/ml) and sarnples were added into the wells. After the
mixture was incubated for 60 min in a humidified charnber at 37OC, a 200 pl solution
containing 5,s'-dithiobis[2-nitrobenzoic acid] (1 1 nM) and Na-CBZ-L-lysine theiobenzyl
ester (1 0 RM) was added into each well. The absorbance was determined at 4 10 nm using
a microplate reader (Multiskan MCCf340, Du Pont, USA) after incubation at 37OC in a
humidity chamber for 1.5 hours. Human kidney uPA in a serial dilution was added to
wells on the same plates as a standard. The PA concentration in sarnples was obtained
fiom a standard curve based on the human kidney uPA values.
Gels fkom zymography and reverse zymography, and gels with indicator film
from fibrin autography and reverse fibrin autography were processed with a Gel Print
apparatus (mode1 2000i; BioKan Scientific, Mississauga, ON). This is composed of a
video camera, a computer and a thermal printer.
Digitized images of gelatinolytic bands of gelahases A and B nom gelatin
zyrnography, gelatin bands corresponding to TIMP-1, -2 and -3 from reverse gelatin
zymography, fibnnolytic bands of uPA or tPA fiom f i b ~ autography, and fibrin bands
representing PA14 fiom reverse f i b ~ autography were analyzed on a Macintosh
computer equipped with the NM software "Image" (version 1.60). The integration area of
each band was meamred, and values were expressed as a ratio of the control area (no
additional treatment set to unity). Results were thereby standardized for each gel, and
were expressed in dimensioniess uni&. Densitometry results f?om different replicate gels
could be compared, and means were calculated for each experimental system.
. . atlsfical
Most experiments were repeated at least three times. Ali data are presented as the
mean * SEM. The software, Statistix (V4.1), was used for statistical analysis. The
significance of the differences among the rneans within each experiment were determined
by two way ANOVA. Differences were considered significant at p < 0.05.
Characteristics of EOSC
c Under phase contrast microscopy, cultured EOSC showed a fibroblast-like
morphology in 10% FBS MEM (Figure 1). Occasionally, the cells tended to form
localized rnultilayered clumps after they reached confluence. There were no apparent
morphological changes from passage 1 to passage 8. Cells of the primary culture showed
the same morphology as the cells of Iater passages. The population doubling time of
EOSC in 10% FBS MEM was about 20 hours (Figure 2). They survived in serum-fiee
medium for up to 7 days without any obvious il1 effects, and cell numben appeared not to
change significantly (Figure 3).
Because antibodies against equine a-SMA, type 1 procollagen, and Von
Willebrand Factor are not commercially available, anti-human a-SMA antibody, anti-
human procollagen type I antibody, and anti-human Von Willebrand Factor antibody
were chosen for the purpose of identification of EOSC. Verification that these antibodies
cross react with equine antigens was done, using appropriate controls (cultured equine
uterine smooth muscle cells for a-SMA, equine skin for type 1 procollagen, and cultured
equine endotheliai cells for Von Willebrand Factor). Strong labeling for a-SMA was
observed in the cytoplasm of most EOSC, but occasional negative celis were also seen
Figure 1. Equine ovarian stroma1 cells at passage 5 in 10% FBS MEM, under phase contract microscopy Magn. 85x.
O 1 2 3 4 5 6 Incubation time (days)
Figure 2. Growth cuve of equine ovarian stromal ceiis (EOSC) that were seeded in 12- well dishes with 10% FBS MEM. At certain time points, EOSC were dispersed by trypsin treatment and counted with a hemocytometer. Values shown are mean I SEM, N=3.
1 2 3 4 5 6 Incubation time (days)
Figure 3. Numbers of equine ovarîan stromal cells grown in senim-fkee medium. Equine ovarîan stromal cells were seeded with 10% FBS MEM and incubated for 48 hours. Cells were then incubated with serum-fiee MEM for up to 6 days. Equine ovarian stroma1 celIs were dispened by trypsin treatment and counted with a hemocytometer. Values shown are mean SEM, N=3.
la)
Figure 4. Immunostaining for a-smooth muscle actin (a-SMA) or type 1 procollagen. (a) Equine ovarian stroma1 cells stained with a monoclonal mouse anti-human a-SMA antibody followed by secondary antibodies conjugated with hoaeradish peroxidase. Occasional negative ceiis were seen (arrow). (b) Equine ovarian stromal cells stained with a polyclonal rabbit anti-human type 1 procollagen antibody, followed by secondary antibodies conjugated with alkaline phosphatase. Magn. 120x.
Figure 5. Immunostaining for Von Wiebrand Factor. (a) Cells incubated with FITC conjugated secondary antibody only. @) Cells incubated with anti Von Willebrand Factor and secondary antibody. Note that in both fields, cellular staining is weak and diffuse, indicative of non-specific (negative) staining. Magn. 120x.
(Figure 4a). Strong labeling for type 1 procollagen was also observed in the cytoplasm of
most EOSC (Figure 4b). Staining for Von Willebrand Factor was negative in EOSC
(Figure 5).
' ~ - ~ h ~ r n i d i a e incorporation and proliferation
In order to detemine whether ERXN, PRXN or TGF-P influences DNA
sy nthesis, 3~-thymidine incorporation into EOSC DNA and cell numbers were
determined. Compared with a control group prepared by culture in 0.5% FBS MEM
without any additional treatment, either ERXN or PRXN at concentrations of 10 nglml
significantly inhibited thymidine incorporation into cultured EOSC by about 30.35%
(Figure 6). They also inhibited ceil proliferation by 30935%. There was no significant
difference in ce11 numbers between ERXN- and PRXN-treated cultures. The latter data
are consistent with the results of 3~-thymidine incorporation. Transfonning growth
factor+ significantly Uihibited the '~-thynidine incorporation and proliferation of
cultured EOSC (Figure 6). Cultured EOSC were exposed to ERXN in senun-fiee medium
for 48 hours to test whether ERXN influenced the viability of EOSC. The results showed
that ERXN did not have any obvious cytotoxicity, since the viability of the EOSC after
exposure to different concentrations of ERXN was not signincantly different (Figure 7).
The inhibitory dose-dependent effects of ERXN on the EOSC were also tested. The
maximum inhibitory effects were induced at a concentration of 10 ng/rnl and higher.
Equine relaxin also inhibited cell proliferation in a dose-dependent manner (Figure 8).
Viable Cell number Thymidine incorporation 4 2 (X10 /cm ) (% of control)
O 0.01 o. 1 1 10 1 O0 ERXN (ng/ml)
Figure 7. Viability of cultured equine ovarian stroma1 ceiis in serum-free MEM after exposure to equine relaxin (ERXN, 0.01-100 ng/ml) for 48 hours. Ce11 viability was estimated by the trypan blue exclusion test. Values show are mean k SEM, N=3.
u
Control ERXN PRXN TGF-P
w - -
Control ERXN PRXN TGF-P
Figure 8. Effects of equine relaxin (ERXN, 10 ng/ml), porcine relaxin (PRXN, 10 ng/rnl) and transforrning g o wth factor-6 (TGF-P, 5 nglml) on: (a) ~-th~midine incorporation and (b) ceii numbers of EOSC. Cells were exposed to ERXN, PRXN or TGF-P for 48 hours. Results are expressed as percent of the control group (no treatment). Values shown are mean * SEM, N=6. Bars with different letters indicate significant ciifference. p<0 .O 5.
Changes in activity of the gelatinases
. . . ics of w e activitv
In conditioned medium collected fiom EOSC cultured without any additional
treatment, two major bands indicating gelatinolytic activity were obtained, as detected by
gelatin zymography. The gelatinolytic activity at approximately 72 kDa was
predominant, whereas gelatinolytic activity at approximately 92 kDa was relatively weak
(Figure 9a). According to their molecular masses, gelatinolytic activity at 72 kDa
represented latent gelatinase A, and gelatinolytic activity at 92 kDa represented latent
gelatinase B. In the presence of 1 m M 4-arninophenylmercuric acetate (APMA), which is
a substance used in vitro to activate latent foms of gelatinases, two extra faint bands at
68 and 82 kDa appeared, as detected by gelatin zymography (Figure 9b). The
gelatinolytic activity at 68 and 82 kDa represented active gelatinase A and B respectively.
In the presence of 10 m M EDTA, gelatinolytic activity at bot ' 72 and 92 kDa was
completely inhibited (Figure 9c). The presence of 1 mM PMSF, a serinekysteine protease
inhibitor, but not a MMP inhibitor, had no inhibitory effect on the gelatinolytic activity in
conditioned medium from EOSC (Figure 9d). In order to determine whether the enzymes
were gelatin specific, fibrinogen or a-casein were used as substrates for zymography,
instead of gelatin. For the substrates tested, the enzyme activity in EOSC samples was
absent on fibrinogen or a-casein zymography (Figure 9e, 99, suggesting that the
enzymes in conditioned medium causing gelatinolytic bands were gelatin specific.
Extracts of untreated EOSC were assayed for gelatinases A and B. A gelatinolytic
band at 72 kDa corresponding to latent gelatinases A was dominant, a band at 92 kDa
Figure 9. Detection of gelatinase activity present in conditioned medium of equine ovarian stroma1 ceus by zymography. Equal amounts of total protein (0.5 pgllane) were loaded into the wells of a 7.5% acrylamide gel. (a) Gelatin was added as substrate. (b) Sample was incubated with 10 mM 4-aminophenylmercuric acetate (APMA) for 4 hours prior to electrophoresis. (c) Gelatin zymogram was incubated in the presence of 10 mM ethylenediaminetetraacetic acid (EDTA). (d) Gelatin zyrnogram was incubated in the presence of 1 m M phenylmethylsulfonyl fluoride (PMSF). (e) Fibrinogen or (f) a-casein were used as a substrate of zymography respectively .
conesponding to latent gelatinase B was faint. Nevertheless, both latent gelatinase A and
gelatinase B activity in extracts was low compared with the activity of gelatinase A and B
activity in conditioned medium. Activity of both latent gelatinases A and B was
completely inhibited by 10 rnM EDTA, which was added to the incubation buffer, but
was not inhibited by 1 m M PMSF (data not show). When the substrate used in
zymography was fibrinogen or a-casein, these bands corresponding to gelatinases A and
B were not detected.
Effects of d i f f e r f i s e activip
The conditioned media of EOSC treated with one of five doses of ERXN (0.01 - 100 ng/ml) were subjected to gelatin zymography in three experiments. An example of a
gelatin zymograrn fiom one experiment (Figure 10) showed that the two main unstained
bands, corresponding to latent gelatinase A (72 kDa) and latent gelatinase B (92 kDa)
were increased by treatment with ERXN (Figure 10a). The bands corresponding to active
gelatinase A (68 kDa) were also dramatically increased by ERXN (1-100 nghl). Since
bands representing active gelatinase A (68 ma) could not be separated cleady from
bands representing latent gelatinase A (72 kDa) by the PAGE with 7.5% acrylamide,
densitometric analysis in Figure 10b actuaiiy showed the total activity of latent and active
gelatinase A. Equine relaxin signifïcantly increased total gelatinase A activity at a
concentration as low as 1 ng/ml. The greatest increase in total gelatinase A activity was
about 2.5-fold at the highea concentration of ERXN (100 ng/ml), compared to the control
without treatment. The effects of ERXN on total gelatinase A activity in conditioned
medium by cultured EOSC were dose-dependent.
Both latent and active forms of gelatinase B in conditioned medium of EOSC
were increased by treatment of ERXN (Figure 10a). However, only those bands
representing latent gelatinase B (92 D a ) activity were quantified by densitometric
analysis. Equine relaxin significantly increased the latent gelatinase B activity even at the
lowest concentration tested (0.01 nglml). At the highest concentration tested (100 @ml),
ERXN induced a 2-fold increase of latent gelatinase B activity. Densitometric analysis
showed that ERXN increased latent gelatinase B in conditioned medium by cultured
EOSC in a dose-dependent manner (Figure 1 Ob).
From the gelatin zymogram, it is noted that ERXN at concentrations of 1-100
ng/ml also induced the gelatinolytic bands at 45 kDa. It is possible these bands were
caused by active interstitial collagenase, because this collagenase is able to degrade
gelatin in this assay and the molecular mass after activation of latent interstitial
collagenase (50 kDa) is 43-45 kDa.
The ce11 extracts of EOSC treated with five doses of ERXN (0.01-100 ng/ml)
were subjected to gelatin zymography in three experirnents. An example of a gelatin
zymogram fiom one experiment (Figure 11) showed that two gelatinolytic bands at 72
and 92 kDa were displayed, correspondhg to latent gelatinase A and latent gelatinase B.
Gelatinolytic bands at 92 kDa were very faint (Figure 1 la). Treatment of ERXN
increased the latent gelatinase A activity, but it appears that ERXN at 1-100 ng/ml
reduced latent gelahase B activity. At a concentration of LOO g h l , ERXN also slightly
m i
5 4 'C) a> N 3 .- E ; 2 m " 1
O O 0.01 0.1 1.0 I O 100
ERXN (nglml)
Figure 10. Gelatinase activity in conditioned medium of equhe ovaian stroma1 cells treated with equine relaxin (ERXN, 0.0 1-1 00 nghl), as detected by gelatin zymography. (a) A representative gelatin zymogram. Equal arnounts of total protein (0.5 pg/lane) were loaded into the weiis of 7.5% acrylamide gels. (b) Densitometic analysis. Each column represents the mean density (* SEM) of samples from 3 separate experirnents. Bars with different letters indicate signincant difference. p<O.OS.
O 0.01 0.1 1.0 10 100 ERXN (nglrnl)
Figure 11. Gelatinase activity in ceil extracts of cultured equine ovarian stroma1 cells treated with equine relaxin (ERXN, 0.0 1 - 100 nglml), as detected by gelatin zymography. (a) A representative gelatin ymogram. Equal amounts of total protein (1.5 pg/lane) were loaded into the wells of 7.5% acrylamide gels. (b) Densitometric analysis. Each column represents the mean density (* SEM) of sarnples fiom 3 separate experiments. Bars with different letters indicate signincant ciifference. ~ 0 . 0 5 .
stimulated gelatinolytic activity at 68 kDa in ceIl extracts of EOSC, which represented
active gelatinase A. Densitometric analysis showed that ERXN significantly increased the
level of latent gelatinase A activity in ce11 extracts in a dose-dependent manner (Figure
1 lb). Equine relaxin failed to significantly increase the latent or active forms of
gelatinase B in ce11 extracts.
se orodiiction bv EOSC rxaosed-
The conditioned medium of EOSC treated with ERXN (100 ng/ml) for varying
times was subjected to gelatin zymography in three experiments. An example of a gelatin
zymograrn from one experiment (Figure 12) showed that in conditioned medium of
control groups without any treatrnent, latent gelatinase A activity was detected at the 12
hour point, and its accumulation increased until48 hours. Latent gelatinase B activity was
present in fow amounts at 12 hours and increased during the whole period of snidy.
Similady, in conditioned medium from treated cells, ERXN continuously stimulated an
increase in latent gelatinase A and gelatinase B activity starting fiom the 12 hour time
point until 48 hours (Figure 12a). Densitometric analysis demonstrated, within treatment
period, a maximal stimulatory effect on the activity of latent gelatinase A and gelatinase
B was induced by ERXN at the concentration of 100 n g / d at 36 and 48 hour tirne points
compared to the control group. Densitometric analysis showed that ERXN signincantly
increased latent gelatinase A activity at 36 and 48 h o u time points and significantly
increased latent gelatinase B activity during the whole treatment penod when compared
to control (figure 12b).
Extracts of EOSC treated with ERXN (100 nglml) for various times were
subjected to gelatin zymography in three experiments. An example of a gelatin
zymograrn from one expenment (Figure 13) showed that in ce11 extracts of the control
group without any treatment, the bands corresponding to latent gelatinases A and
gelatinase B activities were very faint at 12 and 24 hour time points. At 36 and 48 hour
time points, latent gelatinase A activity was present in great amount in extracts; in
contrast, only a very srna11 amount of latent gelatinase B activity was present in extracts
of both groups (Figure 13a). Densitometric analysis showed that ERXN significantly
stimulated latent gelatinase A activity at the 36 and 48 time points, compared with the
control group without any treatment at the sarne time points (Figure 13b). These
gelatinolytic bands corresponding to latent gelatinase B activity in cell extracts were very
weak and not suitable for densitomehic analysis.
ent doses of TCtF-B or PMA on
The conditioned medium of EOSC treated with TGF-P (1 -1 0 @mi) or PMA (5-
20 @ml) for 48 hours was examined by gelatin zymography in three experiments. An
exarnple of a gelatin zymograrn from one experiment (Figure 14) showed that either
TGF-P or PMA increased gelatinolytic bands correspondhg to latent gelatinase A and
latent gelatinase B activities (Figure 14a). In addition, TGF-P at concentration of 5 @ml
increased the accumulation of active gelatinase A activity, and PMA increased the
accumulation of active gelatinase B activity. Densitomehic andysis demonstrated that
increasing concentrations of TGF-B resulted in a signincant increase of latent gelatinase
Control Relaxin
12 24 36 48 12 24 36 48
Control
*
O 12 24 36 48 Incubation time (hour)
Figure 12. Gelatinase activity in conditioned medium of equine ovarian stroma1 cells treated with equine relaxin (ERXN, 100 ng/ml) for different thes, as detected by gelatin zymography. (a) A representative gelatin zymogram. Equal amounts of total protein (0.5 pg/iane) were loaded into the weUs of 7.5% acrylamide gels. (b) Densitometric analysis. Each points represents the mean density (I SEM) of samples fiom 3 separate experirnents. An asterisk indicates significant difference from control. p<O.OS.
Contrd Relaxin
12 24 36 48 12 24 36 48
12 24 36 Incubation time (hour)
Figure 13. Gelatinase activity in ce11 extracts of equine ovarian stroma1 cells treated with equine relaxin (ERXN, 100 ng/ml) for different times, as detected by gelatin zymography . (a) A representative gelatin zymogram. Equal arnounts of total protein (1.5 &lane) were loaded into the wells of 7.5% acrylamide gels. @) Densitometric analysis. Each column represents mean density (* SEM) of samples from 3 separate experiments. An asterisk indicates signincant merence fiom control. p<0.05.
A and gelatinase B activities (Figure 14b). Densitometric analysis also showed that
stimulation by TGF-P at a dose of 5 @ml caused a 1 .éfold increase of gelatinolytic
bands corresponding to latent gelatinase A activity compared to control values.
Stimulation by TGF-P at a dose of 10 n g h l resulted in an increase of gelatinolytic bands
representing latent gelatinase B activity by 3.5-fold. The phorbol ester PMA significantly
increased latent gelatinase A activity at the lowest concentration of 5 nglml and continued
to cause significant M e r increases at 1 O and 20 @ml. Phorbol ester PMA at 20 ng/ml
caused a 2.2-fold increase in latent platinase A activity compared to that of control.
Phorbol ester PMA also caused a significant increase in latent gelatinase B activity.
However, the maximal stimulatory effects of a 4.5-fold increase in latent gelatinase B
activity was induced by PMA at concentration of 5 nglml. With increased concentrations
from 5 to 20 &ml, PMA induced accumulation of active gelatinase B. Densitomeüic
analysis for active gelatinase B (86 kDa) was not performed due to the lack of detectable
active gelatinase B activity in the control group.
th TGF-B or PMA on
The effects of ERXN in combination with TGF-P (1-10 ng/ml) or PMA (5-20
nglml) on gelatinase production by EOSC conditioned medium were exarnined with
gelath zymography in three experiments. An example of a gelatin zyrnogram fiom one
experiment (Figure 15) showed that ERXN, TGF-P or PMA increased gelatinolytic bands
corresponding to latent gelatinase A and latent gelatinase B activities (Figure 15a). but
densitometric analysis showed that there were no significant dif5erences between
(a) TGF-P PMA
O 1 5 10 5 10 20
O 1 5 1 0 TGF-p (nglml)
O 5 I O 20 PMA (nglml)
Figure 14. Gelatinase activity in conditioned medium of equine ovarian stroma1 celis treated with transfomiing growth factor+ (TGF-P, 1-10 nglml) or phorbol 12-mfistate 13-acetate (PM& 5-20 ng/ml), as detected by gelatin zymography. (a) A gelatin zymogram. Equal amounts of total protein (0.5 pg/lane) were loaded into the wells of 7.5% acrylamide gels. (b) Densitometric analysis. Each column represents mean density (* SEM) of samples fiom 3 separate experiments. Bars with dBerent letters indicate significant Merence. p<O.O5.
Figure 15. Gelatinase activity in conditioned medium of equine ovarian stroma1 ceus d e r exposure to equine relaxin (ERXN, 100 nglmi) in combination with transiùrmîng growth factor-B (TGF-P, 5 ng/ml) or phorbol 12-myristate 13-acetate (PMA, 10 nglrnl). Lane 1: control; lane 2: ERXN; lane 3: TGF-P; lane 4: P M , lane 5: ERXN + TGF-P; lane 6: ERXN + PMA. (a) A representative gelatin zymogram. Equal amounts of total protein (0.5 pg/lane) were loaded into the weils of 7.5% acrylamide gels. (b) Densitometric andysis. Each column represents mean density (* SEM) of sarnples fiom 3 separate experiments. Bars with Merent letters indicate signincant dïfference. p ~ 0 . 0 5 .
treatments of TGF-P and of ERXN plus TGF-p, or between treatment of PMA and of
ERXN in combination with PMA (Figure 15b), in terms of gelatinolytic bands
corresponding to latent gelatinase A and latent gelatinase B activities.
Activity changes of tissue inhibitors of metalloproteinases
. . ctenstics of m r o d u c e d by EOSC
Reverse gelatin zymography revealed that cultured EOSC secreted three factors
that inhibited gelatinase activity (Figure 16a). Two inhibitors were present in conditioned
medium and their molecular masses were very close to those of mouse TIMP-1 and -2.
The third factor was bound to ECM and its molecular mass was similar to that of mouse
TIMP-3. These three factors remained partiaily active at pH 4.5, or when heated to 90°C
for 70 min (Figure 16b, c). M e r incubation in the presence of EDTA (10 mM), reverse
gelatin zymogram failed to show any bands (Figure 16d). This provided evidence that
bands on reverse gelatin zymograms were caused by inhibited degradation of gelatin.
Therefore, these three bands represented undegraded gela& and rneant that gelatinases
added into the gels as part of the reverse zymography protocol were inhibited by these
three factors. In SDS-PAGE gels without gelatin and gelatinase, EOSC conditioned
medium and ECM did not contain visible proteins in the range of 21-30 kDa (Figure
16e). Therefore, bands at these locations in reverse gelatin zymography were indeed
caused by gelatinases present in the electrophoresis gel, suggesting TIMPs. Two
inhibitors in conditioned medium were about 28 and 21 D a in size respectively; while
the third one in ECM was about 24 kDa in size (Figure 160.
Figure 16. Detection by reverse zymography of tissue inhibitors of metailoproteinases (TIMPs) produced by cultured EOSC. Lane 1 : conditioned medium. Lane 2: extracellular mat& Lane 3: purïfïed mouse TIMP-1, -2 and -3 as standards. Ail gels were stained with Coomassie B f i a n t Blue (3-250. (a) Samples and standards without heating and at pH 7.5. (b) Samples and standards were acidifïed to pH 4.5. (c) Samples and standards were heated at 90°C for 70 min. (d) Reverse gelatin gel was incubated in the presence of 10 rnM EDTA. (e) Samples in SDS-PAGE without gelatin and gelathases. The nght lane was molecular marker. (0 Samples in reverse gelatin zymogram. The right lane was molecular marker.
cts of different doses of ERXN on TlMP ~roduct
The conditioned medium of EOSC treated with five doses of ERXN (0.01-1 00
nglrnl) was subjected to reverse gelatin zymography in three experiments. A
representative reverse gelatin zymogram from one expenment (Figure 17) showed that
inhibitory activity at two bands, corresponding to TIMP-1 (28 kDa) and TIMP-2 (21
kDa), were increased by treatment with ERXN (Figure 17a). Densitometric anaiysis
dernonstrated that ERXN at the lowea concentration tested (0.01 ng/rnl) slightly
increased TIMP-1 activity, but the increase was not significant over control. Compared to
the control without treatrnent, a maximal 2.5-fold increase of TIMP-1 activity was
induced by ERXN at the highest concentration tested (100 ngfml), while the maximal 2.2-
fold increase of TIMP-2 activity was caused by ERXN at concentration of 1.0 ng/ml
(Figure 1%).
The ECM of EOSC treated with varying concentrations of ERXN (0.01 -1 00
ng/ml) was used in reverse gelatin zymography in three experiments. A representative
reverse gelatin zymogram demonstrated that TIMP-3 activity was present in ECM
(Figure 1 8a). In addition, one rninor band with molecular mass of appropriate 28 kDa was
present in ECM, and is Iikely TIMP-1. Densitometric anaiysis showed that ERXN at
doses of 0.01 to 100 @ml had no significant effect on TIMPJ activity in ECM
compared to control (Figure 18b).
Time course of TIMP production bv EOSC exposed to E m
The conditioned media of EOSC treated with of ERXN (100 ng/ml) for varyïng
times were subjected to reverse gelatin zymography in three experiments. A
representative reverse gelatin ymogram fiom one experiment (Figure 19) showed that
TIMP-1 and TIMP-2 activity were present in conditioned media. Densitometric analysis
showed that TIMP- 1 and TIMP-2 activity in the control group increased from the 12 hour
time point and reached a maximum after 48 hours (Figure 19a). M e r exposure to ERXN,
accumulation of TIMP-1 and TIMP-2 activity in EOSC conditioned medium increased
from 12 hours, reached a maximum at 36 hours and declined by 48 hours (Figure 19b). At
36 and 48 hour tirne points, ERXN significantly increased the TIMP-1 activity compared
to the control group at the same time points. At 24, 36 and 48 hour time points, ERXN
also significantly increased the accumulation of TIMP-2 compared to the control group at
the sarne time points. In a time course study of TIMP-3 in ECM, there were no
significant differences between ERXN-treated and control groups in TIMP-3 activity at
12,24,36 and 48 hour time points, as analyzed by densitometry (Figure 20).
rent dases of TCTF-B or PMA on TTMP produc
The effects of increasing concentrations of TGF-P (1-10 nglml) or PMA (5-20
nglml) on TIMP-1 and -2 activity in conditioned medium were assessed by reverse
gelatin zymography. Equine ovarian stroma1 ceils were treated with TGF-P or PMA for
48 hours. A representative reverse gelatin zymogram shows two bands representing
TIMP-1 and TIMP-2 activity in conditioned medium of EOSC (Figure 21). Increasing
O 0.01 0.1 1.0 10 100 ERXN (nglml)
Figure 17. Activity of tissue inhibiton of metalloproteinases-1 and -2 (TIMP-1 and -2) in conditioned medium of equhe ovarian stromal cells treated with equine relaxin (ERXN, 0.01-100 nghl) , as detected by reverse zymography. (a) A representative reverse gelatin zyrnogram. Equal amounts of total protein (0.5 pgllane) were loaded into the welis of 12.5% acrylarnide gels. (b) Densitometrie anaiysis. Each column represents mean density (k SEM) of samples fiom 3 separate experiments. Bars with different letten indicate sipnincant merence. p<O.OS.
O 0.01 0.1 1.0 10 100
ERXN (nglml)
Figure 18. Tissue inhibitor of metalloproteinases-3 (TIMP-3) activity in extracellular matrix (ECM) of cultured equine ovarian stromal cells after exposure to equine relaxin (ERXN, 0.01-100 ng/ml), as detected by reverse zymography. (a) A representative reverse gelatin zymognun. Equal amounts of sample (15 pl/lane) were loaded into the wels of 12.5% acrylamide gels. Far right lane contained p d e d moue TIMPs. (b) Densitometnc analysis. Each column represents mean density (k SEM) of sarnples î5om 3 separate experirnents. There is no signifïcant clifference (p0.05) between treatrnent and control.
Control Relaxin 12 24 36 48 12 24 36 48
48 Incubation time (hour)
Figure 19. Activity of tissue inhibitors of metalloproteinases-l and -2 (TLMP-1 and -2) in conditioned medium of equine ovarian stromd cells treated with equine relaxin (ERXN, 100 ng/ml) for different times, as detected by reverse zymography. (a) A representative reverse gelatin zymogram. Equal amounts of total protein (0.5 @lane) were loaded into the wells of 12.5% acrylamide gels. (b) Densitometric analysis. Each points represents mean density (* SEM) of samples fkom 3 separate experiments. An asterisk indicates signincant Merence fiom control. p4.05.
(a) Control Relaxin
12 24 36 48 12 24 36 48
1 $'":j Relaxin 1
Incubation time (hour)
Figure 20. Tissue inhibitor of metalloproteinases-3 (TIMP-3) activity in extracellular matrix (ECM )of equine ovarian stroma1 cells after exposure to equine relaxin (ERXN, 100 ng/ml) for indicated times, as detected by reverse zymography. (a) A representative reverse gelath ymogram. Equal amounts of sample (15 p h n e ) were loaded into the wells of 12.5% acrylamide gels. Far right lane contained mouse purïfied TIMPs. (b) Densitometric analysis. Each colurnn represents mean density (* SEM) of samples from 3 separate experiments. There is no sigaificant difference (pi0.05) between ERXN treatment and control.
TGF-P concentration resulted in a increase in TIMP-1 activity in a dose-dependent
marner. Densitometric analysis showed that TGF-P at 5 and 10 n g h l or PMA at al1
concentrations tested significantly increased the accumulation of TIMP- 1 activity over
control values in conditioned medium of EOSC. Maximum increases of 3.3 and 3.6 fold
in TIMP-1 activity were caused by TGF-P at 10 @ml and PMA at 20 nglml respectively,
compared to control values. Transfoning growth factor-p at concentrations of 5 and 10
nglml, and PMA at concentrations of 5 and 10 ng/ml also caused slight increases in
TIMP-2 activity. However, these increases were not statistically significant compared to
control values.
. . n c o m o n th TGF-B or PMA on TIMP ~rociuct
The effects of ERXN in combination with TGF-P (1-10 @ml) or PMA (5-20
ng/ml) on TIMP-1 and -2 production in conditioned media from EOSC were exarnined
with reverse gelatin zymography. Equine ovarian stromai cells were exposed to ERXN
(100 ndrnl) in combination with TGF-P (5 ng/ml) or PMA (10 @ml) for 48 hours. An
example of a gelatin zymogram fiom one experiment is shown (Figure 22a). Bands
corresponding to TIMP-1 and -2 fiom three experiments were quantined by densitometric
analysis. Ali these reagents (ERXN, TGF-B or PMA) stimulated significant increases of
TIMP-1 and TIMP-2 activity compared with their control group as seen previously.
Densitometry showed that ERXN had no additive eEects with TGF-P or PMA on T[MP-
1 activity; and failed to show additive effects with them on TIMP-2 activity, compared to
control values with treatment of ERXN alone (Figure 22b).
Activity changes of plasminogen activators
C h a r a c t e ~ c s of PA . .
nroduced bv FOS€
Fibrin autography was used to detect PA activity in EOSC conditioned medium
and extracts. One band demonstrating fibrinolfic activity corresponding to a protein of
approximate molecular mass of 50 kDa was detected in conditioned medium fiom EOSC.
Another band demonstrating fibrinolytic activity corresponding to a protein of about 23
kDa in extracts of EOSC was detected (Figure 23a). These two bands were totally
inhibited when 10 mM amiloride was added to the indicator films (Figure 23b, c).
Arnilonde is a specific inhibitor for uPA, but not for tPA. Therefore, these fibrinolytic
bands were attributed to high molecular weight uPA (50 kDa) and low molecular weight
uPA (23 kDa). There were no detectable levels of tPA (70 kDa) in the conditioned
medium or extracts of cultured EOSC examined by fibrin autography (Figure 23a, b, c).
Mer extended incubation, a band demonstrating fibrinolytic activity corresponding to a
protein of approximate molecular mass of 95 kDa was detected in EOSC conditioned
medium (Figure 23d). This band was also inhibited by 10 mM amiloride (data not
shown). Therefore, the protein corresponding to this fibrinolytic activity was considered
to be uPA/PAI-1 complexes which have an expected molecular mass of 95 D a . Figure
23d demonstrates that a very small amount of uPA/PAI-1 complex activity was present in
EOSC conditioned medium compared with the amount of uPA activity.
Fibrinolytic activity in the ECM of EOSC was also examined by fibrin
autography (Figure 24). Two major fibrinolytic bands corresponding to proteins of
approximate molecular mass of 70 kDa and 105 kDa were demonstrated These hhro
TGF-p PMA
O 1 5 1 0 TGF-B (nglml) PMA (nglrnl)
Figure 21. Activity of tissue inhibitors of metalloproteinases-1 and -2 (TZMP-1 and -2) in conditioned medium of equine ovaian stroma1 cells treated with transforming growt. factor+ (TGF-P, 1-10 ng/ml) or phorbol 12-myristate 13-acetate (PMA, 5-20 ng/ml), as detected by reverse zyrnography. Equal amounts of total protein (0.5 pg/lane) were loaded into the wells of 12.5% acrylamide gels. (a) A reverse gelatin ymogram. (b) Densitometric analysis. Each column represents mean densïty (k SEM) of sarnples nom 3 separate experîrnents. Bars with different letten indicate signincant ciifference fiom control. ~ 0 . 0 5 .
Figure 22. Activity of tissue inhibitors of metdoproteinases-1 and -2 (TIMP-1 and -2) in conditioned medium of equine ovarian stroma1 celis afler exposure to equine relaxin (ERXN, 100 ng/ml) in combination with transforming growth factor+ (TGF-P, 5 nglml) or phorbol 12-myristate 13-acetate PMA, 10 ng/ml). Laue 1 : control; lane 2: ERXN; lane 3: TGF-P; lane 4: PMA; Iane 5: ERXN + TGF-P; lane 6: ERXN + PMA. (a) A representative reverse gelatin zymogram. Equd arnounts of total protein (0.5 pg/lane) were loaded into the wells of a 12.5% acrylamide gels. (b) Densitometrîc anaiysis. Each column represents mean density (* SEM) of samples of the correspondhg lane fkom 3 separate experiments. Bars with different letters indicate signincant ciifference. px0.05.
Figure 23. Identification of plasminogen activator (PA) produced by equine ovarian stromal ceils as detected by fibrin autography incubated overnight. (a) Fibrin autograms. Lane 1: conditioned medium; Lane 2: ce11 extract; Lane 3: human kidney uPA; Lane 4: human single-chah tissue-type plasminogen activator (@A). (b) S ample of conditioned medium or (c) sample of ceil extract in the presence of 10 rnM amiloride. (d) Reverse gelatin autogram of conditioned medium sample. This autogram was incubated for 60 hours*
bands were not inhibited by amilonde (10 mM) added to the indicator film (Figure 24).
Therefore, the band corresponding to a protein of 70 kDa likely represents PA, and the
other band corresponding to a protein of 105 kDa represents tPA/PAI-1 complexes. A
third minor fibrinolytic band corresponding to a protein of 95 kDa was also present in
ECM. This third fibrinolytic band was identified as uPA/PAi-1 complexes because it was
inhibited by 10 m M axniloride.
nt doses of E&CN on PA nroduct
The conditioned medium and extracts of EOSC treated with five doses of ERXN
(0.0 1 - 100 @ml) were subjected to fibrin autography and chromogenic assay. An
exarnple of a fibrin autogram fiom one experiment showed ERXN increased fibrinolytic
bands corresponding to high molecular weight uPA (50 ma) activity in conditioned
medium, and bands corresponding to low molecular weight uPA (23 kDa) activity in ceIl
extracts (Figure 25). No fibrinolytic band corresponding to tPA activity was detected in
conditioned medium or in ce11 extracts. Chromogenic assays were used to further
characterize PA activity in conditioned medium and ce11 extracts. Equine relaxin at a
concentration of 0.1-100 n g / d significantly stimulated an increase of PA activity
produced by EOSC in conditioned medium (Figure 26a). Equine relaxin at a
concentration of 1-1 00 n g h l also significantly stimulated an increase of PA activity
produced by EOSC in ce11 extracts (Figure 26b). Since there was no detectable level of
tPA in conditioned medium or cell extmcts, the increase of PA activity in conditioned
medium or ceIl extracts might be caused by uPA. However, since the chromogenic assay
Amiloride (-) Amiloride (+)
1 2 3 1 2 3
Figure 24. Identification of plasminogen activator (PA) in extracellular matrix @CM) of EOSC as detected by fibrin autography in the presence or absence of amilonde (10 mM). Lane 1 : sample of ECM; lane 2: human kidney urokinase-type pIasmùiogen activator (d'A); lane 3 : human single-chah tissue-type plasminogen activator (PA).
measures total PA activity (both uPA and tPA) activity, and is more sensitive than fibrin
autography, further examination should be done to identify whether tPA was present in
conditioned medium and ce11 extracts.
The ECM of EOSC treated with five doses of ERXN (0.01 -100 ngfml) was also
subjected to fibrin autography, and fibrinolytic bands fiom three experiments were
quantified by densitornetric analysis (Figure 27). An example of a fibrin autogram fiom
one experiment showed that ERXN (0.01-100 nglml) induced fibrinolytic bands at 70
kDa and enhanced fibrinolytic bands at 105 kDa. As mentioned above, fibnnolytic bands
at 70 kDa and 105 kDa are likely due to tPA and tPAPAi-1 complexes respectively.
Densitornetric analysis showed ERXN treatment resulted in a stiitistically significant rise
in the fibrinolytic activity present in ECM at 70 and 105 kDa (Figure 27b). The maximal
induction of fibrinolytic activity at both 70 and 105 kDa was caused by ERXN at a
concentration of 1 ng/ml.
The conditioned medium and extracts of EOSC treated with ERXN (100 ng/ml)
for varying times were subjected to fibrin autography and chromogenic assay. An
example of a fibrin autogram fiom one experiment shows that uPA activity was not
detectable by 12 hours, either with or without ERXN treatment (Figure 28). Equine
relaxin treatment increased fibrinolytic bands corresponding to high molecular weight
uPA (50 kDa) in conditioned medium, and those corresponding to low molecular weight
uPA (23 kDa) in ce11 extracts in a the-dependent manner within 48 hours of treatment,
Figure 25. Urokinase-~.pe plasminogen activator @PA) activity in conditioned medium and ce11 extracts of equine ovarian stromal cells treated with equine relaxin (ERXN, 0.0 1 - 100 ng/ml), as detected by fibrin autography. Equal amounts of total protein (1.5 pgflane) were loaded into the weiis of a 10% acrylamide gel. (a) Conditioned medium. @) Ce11 extractS.
O 0.01 0.1 1 10 100 ERXN (nglml)
O 0.01 0.1 1 10 100 ERXN (nglml)
Figure 26. Plasminogen activator (PA) activity produced by equine ovarian stroma1 ceils treated with equine relaxin (ERXN, 0.01-100 ngfml), as detected by chromogenîc assay. Values shown are mean SEM, N=3. Bars with different letters indicate significant merence. pc0.05. (a) Conditioned medium. (b) Ceil extracts.
O 0.01 0.1 1.0 10 100 ERXN (nglml)
Figure 27. Plasminogen activator (PA) activity in extracellular matrix (ECM) of equine ovarian stroma1 cells treated with equine relaxin (ERXN, 0.01-100 nghl), as detected by fibrin autography. (a) A representative fibrin autogram. Equal amounts of samples (15 p h n e ) were Loaded into the wells of 10% acrylamide gels. (b) Densitomeaic analysis. Each column represents mean density (* SEM) of samples nom 3 separate experiments. Bars with different letters indicate signincant ciifference. PQ).05.
compared with control groups (Figure 28). As revealed by chromogenic assay, ERXN
promoted increases in total PA activity in both conditioned medium and ce11 extracts by
24 hours, and this continudly increased until 48 houn when the difference was
statisticdly significant (Figure 29). In contrast, PA activity in conditioned medium of
control cultures started accumulahg from 36 hours, but did not increase at 48 hours. In
ce11 extracts, a similar phenomenon was also found (Figure 29).
The ECM of EOSC treated with ERXN (100 nglml) for different times was
subjected to fibrin autography, and fibrinolytic bands from three experiments were
quantified by densitometric malysis (Figure 30). A representative fibrin autogram is
shown in Figure 30a The fibrinolytic activity at 70 and 105 kDa in ECM of EOSC was
significantly increased by ERXN treatment, as demonstrated by densitometric analysis
(Figure 30b).
n bv EOSC
The effects of different doses of TGF-P (1-10 ng/mi) or PMA (5-20 ngM) on
total PA activity secreted by EOSC into conditioned medium were assessed with
chromogenic assay. Transforming growth factor-p at concentrations of 1-10 n g / d caused
a significant increase in PA activity compared to control values (Figure 3 1). The 2-fold
increase of PA activity was obtained at concentrations of 10 n g / d TGF-B. Phorbol 12-
myristate 13-acetate at the lowest concentration (5 nglml) caused a signifcant decrease in
PA activity compared to that in control cultures. Increasing the concentrations of PMA
(10 and 20 ng/mi) faiied to renilted in M e r decreases of PA activity in conditioned
Control Relaxin
(b) Control Relaxin
Figure 28. Urokinase-type plasminogen activator @PA) activity produced b y equine ovarian stroma1 ceUs (EOSC) treated with equine relaxin (ERXN, 100 ng/ml) for different times, as detected by Fibrin autography. Equal amounts of total protein (1.5 pgllane) were loaded into the weUs of 10% acrylamide gels. (a) Conditioned medium. (b) CeH extractS.
-1 Relaxin l nConvo' l
-1 Relaxin l I
12 24 36 48 Incubation time (hour)
Figure 29. Plasminogen activator (PA) activity produced by equine ovarian stroma1 cells after exposure to equine relaxin (ERXN, 100 ndrnl) for different times (h), as detected by chromogenic assay. (a) Conditioned medium. (b) Ceil extracts. Values shown are mean * SEM, N=3, p<0.05. An asterisk indicates a significant ciifference fkom control at the same thne point.
Control Relaxin 12 24 36 48 12 24 36 48
5 ; ' .
- - - 1 0 5 k D a 17.. -
r] Controt ERXN
Incubation tirne (hour)
Figure 30. Plasminogen activator (PA) activity in extracellular matrix (ECM) of equine ovarian stromal ceiis treated with equine relaxin (ERXN, 100 nglmi) for iodicated tunes. (a) A representative fibrin autogram. Equal amounts of samples (15 pVlane) were loaded into the weiis of 10% acrylarnide gels. (b) Densitometric analysis. Each point represents mean density * SEM) of samples fiom 3 separate experiments. An asterisk indicates significant merence from control. p<0.05.
medium.
n corn c
Plasminogen activator activity in conditioned media of EOSC, which were
exposed to ERXN (100 ng/ml) in combination with TGF-P (5 ng/mi) or PMA (10 @ml)
for 48 hours, was examined (Figure 32). A representative fibrin autogram showed that
either ERXN or TGF-P stimulated an increase of uPA activity. Equine relaxin did not
have additive effects with TGF-P on this activity when compared to the control group
exposed to ERXN alone (Figure 32a). Fibrin autography also showed that the
combination of ERXN (100 nglml) with PMA (10 ng/ml) completely antagonized the
stimulatory effects of ERXN on uPA activity released into conditioned medium. In
addition, PMA induced fibnnolytic activity corresponding to uPA/PAI-I complexes with
a molecular mass of 95 kDa. In support of the results of fibrin autography, chromogenic
assay of the same samples showed that ERXN or TGF-P significantly increased, while
PMA signincaatly decreased, PA levels produced by cultured EOSC (Figure 32b).
Consistent with fibrin autography, ERXN did not show additive effects with TGF-P on
PA activity in conditioned medium of EOSC; while PMA antagonized the stimulatory
effects of ERXN.
TGF-B (nglml)
PMA (nglml)
Figure 31. Plasminogen activator (PA) activity in conditioned medium of equine ovarian stromal cells treated with transforming growth factor+ (TGF-P, 1 - 10 ng/mi) or phorbol 12-myristate 13-acetate (PMA, 5-20 nglml), as detected by chromogenic assay. Values shown are mean * SEM, N=3. Bars wîth different letters indicate significant difference. pO.05.
Figure 32. Plasminogen activator (PA) activity in conditioned medium of equine ovarian stroma1 cells after exposure to equine relaxin (ERXN, 100 ng/mi) in combination with transforming growth factor+ (TGF-P, 5 ng/ml) or phorbol 1Zmyristate 13-acetate (PM& 10 nglml). (a) Fibrin autogram of uPA activity in conditioned medium. Equal arnounts of total protein (1.5 pgilane) were loaded into the welIs of 10% acrylamide gels. Lane 1: control; lane 2: ERXN; lane 3: TGF-P; lane 4: PMA; lane 5: ERXN + TGF-p; lane 6: ERXN + PMA. (b) Plasmhogen activator activity detected by chromogenic assay for the same treatments. Values shown are mean * SEM, N=3. Bars with different letters indicate significant clifference. P<O.OS.
Activity changes of plasminogen activator inhibitor
Charactemttcs of PA14 ~roduced bv EOSC . .
Reverse fibrin ymography revealed that cultured EOSC secreted a factor into
conditioned medium that inhibited the activation of human plasminogen by human
kidney uPA in vitro, when this was added into the indicator films. The molecular mass of
this inhibitor was approximately 50 kDa, similar to the molecular mass of hurnan
recombinant PAI-1, a positive control used in reverse fibrin autography (Figure 33). This
inhibitor was also detected in ECM, but was not detected in cell extracts of EOSC by
reverse fibrin autography (data not show). Because reverse fibrin autography
specifically detects PM, the inhibitor produced by EOSC is a member of PAI farnily. The
stability of this inhibitor in the presence of the denaturant SDS is consistent with its
identification as PM-1, because this characteristic is not shared by other members of the
PA1 family (Hekrnan and Loskutoff, 1987). In EOSC conditioned medium, PA14 formed
complexes with uPA, and in ECM from EOSC, it formed complexes with PA. However,
PAI-1 in both uPA/PAI-1 and tPA/PAI-1 is not able to be displayed by reverse fibrin
autography. Rather, the presence of these complexes cm be revealed indirectly by fibrin
autography (Figures 23,24).
Plasminogen activator inhibitor-l was also detected in ECM of EOSC by reverse
fibrin autography (Figure 33). The PAL1 in ECM had the same molecdar mass as that
seen in conditioned medium. In contrast, PM-1 was not detected by reverse fibrin
autography fiom celI extracts of EOSC (Figure 33).
Effects of different ~QSCLQERXN on PAI-I production bv EOSC
The conditioned medium of EOSC treated with five doses of ERXN (0.01-100
ng/ml) was subjected to reverse fibrin autography in three experiments. A representative
reverse fibrin autogram is s h o w in Figure 34a. Densitometric analysis showed that
ERXN significantly increased PAI-1 activiv in conditioned medium of EOSC at doses of
10 and 100 ng/ml (Figure 34b). A maximal effect of a 2-fold increase of PM-1 activity in
conditioned medium was achieved by treatment of ERXN at the highest dose (100
ng/ml), compared to the control value.
The effects of different doses of ERXN on the PAL1 in ECM of EOSC were
examined and it appeared that ERXN did not influence the accumulation of PAI-1 in
ECM, demonstrated by densitometric analysis (Figure 3Sa).
course of PA1 OSC exposed to
Equine ovarian stroma1 cells were incubated in the presence or absence of ERXN
(100 ng/ml) for 12, 24, 36 and 48 hours and their PA14 production was evaiuated by
reverse fibrin autography. A representative reverse fibrin autograph is s h o w in Figure
36a. Densitometric analysis showed that ERXN caused an accumulation of PAL1 in
conditioned medium in a tirne-dependent manner (Figure 36b). Equine relaxin treatment
signincantly increased PAI-1 activity in conditioned medium at the 24, 36 and 48 hours
compared to the control value.
Plasminogen activator inhibitor-l activity in ECM was also examhed. Results
showed that PA14 activity was present in ECM. The PAL1 activity in ECM of ERXN
Figure 33. Detection of PA14 in conditioned medium, ECM and ce11 extracts fiom EOSC by reverse fibrin autogmphy. Lane 1: sample of conditîoned medium; lane 2: sample of ECM; Iane 3: sample of ce11 extracts; lane 4: human PM-1.
O 0.01 0.1 1.0 10 100 ERXN (nglml)
Figure 34. Plasrninogen activator inhibitor-l (PAI-1) activity in conditioned medium of equine ovarian stroma1 cells (EOSC) treated with equine relaxin (ERXN, 0.0 1 - 100 nghl), as detected by reverse gelatin zymognphy. (a) A representative reverse fibrin autogram. Equal amounts of total protein (1.5 pg/lane) were loaded into the wells of 10% acrylamide gels. (b) Densitometric analysis. Each column represents mean density (* SEM) of samples fiom 3 separate experiments. Bars with different letters indicate significant ciifference. P<0.05.
Control Relaxin
Figure 35. Plasminogen activator inhibitor4 (PM-1) in extracellular matrix (ECM) of equine ovarian stromal cells, as detected by reverse gelatin zyrnography. Equal amounts of samples (15 p h n e ) were loaded into the 7.5% acrylarnide gels. (a) Extracellular matrix of equine ovarian stromal cells treated with equine relaxin (ERXN, 0.01-100 nghl ) . (b) Extracellular matrix of equine ovarian stromal cells treated with equine relaxin (ERXN, 100 ng/ml) for difFerent times (h).
Control Relaxin 12 24 36 48 12 24 36 48
Controi -1 ERXN
Incubation tirne (hour)
Figure 36. Plasminogen activator inhibitor-1 (PM-1) activity in conditioned medium of equine ovarian stromal cells treated with equine relaxin (ERXN, 100 ng/ml) for different times. (a) A representative reverse fibrin autogram. Equal amouats of total protein (1.5 p,g/lane) were loaded into the wells of 10% aqlamide gels. (b) Densitornetric analysis. Each point represents mean density (* SEM) of samples fiom 3 separate experiments. An asterisk indicates signincant ciifference from control. P<0.05.
treated EOSC appeared lower than that in controls (Figure 35b).
Effects of different doses of TGF-B or PMA on PM-1 production by ~ O S C
Plasminogen activator inhibitor-l activity in conditioned medium of EOSC
treated with transforming growth factor- p (TGF-P, 1 - 1 0 nglml) or phorbol 1 2-mynstate
13-acetate (PM& 5-20 @ml) for 48 hours was examined by reverse zymography. A
representative reverse fibrin autogram is show in Figure 37a. Both TGF-P and PMA
significantly increased PAL1 activity in the conditioned medium, demonstrated by
densitometnc analysis. Transforming growth factor+ at a concentration of 10 ngmi and
PMA at 20 n g h l induced the highest PAL1 activity for each agent, of the doses tested.
(Figure 37).
To explore M e r the effects of ERXN in combination with TGF-P or PMA,
ERXN (100 ng/ml) with TGF-P (5 ng/ml) or PMA (10 ng/rnl) was added to EOSC
cultures for 48 hours. A representative reverse fibrh autogram is shown in Figure 38a.
Densitometric analysis demonstrated that ERXN, TGF-P, or PMA alone caused a
signifïcant increase in PM-1 activity in conditioned medium compared to the control
value. Equine relaxin combined with TGF-P or PMA did not show any significant
additive increase in PA14 activity compared to ERXN, TGF-P or PMA aione (Figure
3 8).
TGF-B PMA
TGF-p (nglml) PMA (nglml)
Figure 37. Plasminogen activator inhibitor4 (PAL 1) activity in conditioned medium of equine ovarian stroma1 cells treated with transforming growth factor-p (TGF-b, 1-10 ng/mi) or phorbol 12-myristate 13-acetate (PM& 5-20 ng/ml) as detected by reverse fibrin autography. Equal amounts of total protein (1.5 pg/lane) were loaded into the wells of 10% acrylamide gels. (a) A reverse gelath zymogram. (b) Densitometric analysis. Each c o 1 m represents mean density (* SEM) of samples fiom 3 separate experiments. Bars with different lettea indicate signincant difference. Pc0.05.
Figure 38. Plasminogen activator inhibitor-l (PAL 1) activîty in conditioned medium of equine ovarian stromal cells d e r exposure to equine relaxin (ERXN, 100 ng/ml) in combination with tmsforming growth factor+ (TGF-P, 5 ngfml) or phorbol 12-rnyristate 13-acetate (PMA, 10 nglml). Lane 1: control; lane 2: ERXN; lane 3: TGF-P; lane 4: PMA; lane 5: ERXN + TGF-P; lane 6: ERXN + PM.. (a) A representative reverse fibrin autogram. Equivalent amounts of total protein (1.5 pg/lane) were loaded into the welis of 12.5% acrylamide gels. (b) Densitomeûic analysis. Each column represents mean density (k SEM) of samples given the same treatments fiom 3 separate experiments. Bars with different letters indicate signincant merence. P<0.05.
DISCUSSION
Characteristics of EOSC
The results of this study have demonstrated that cells obtained fiom stromal tissue
at the apex of developing follicles in the equine ovary are fibroblastic in shape and
express type 1 procollagen and a-SMA. Bovine ovary stromal cells are reported to show
fibroblast-like morphology in culture (Vigne et al., 1994). Other authors have reported a-
SMA positive cells in the stroma of human ovaries (Czemobilsky et al., 1989; Santini et
al., 1993). While the EOSC can form multilayered aggregations under crowded culture
conditions, EOSC cells in this study did not display the classic "hi11 and valley"
morphology highly characteristic of smooth muscle cells in vitro which is seen in rny
cultures of equine uterine smooth muscle cells. Although EOSC are strongly positive for
procollagen 1 irnmunostaining and have a fibroblast-like morphology, they showed
neither the expected spindle shape and motile morphology, nor the contact inhibition of
"typical" connective tissue fibroblasts (Freshney, 1994). Based on these charactenstics, 1
postulate that the EOSC used in this study are most likely myofibroblasts, a ce11 type first
identified in 1971 (Gabbiani et ai., 1971) and reported to express characteristics of both
smooth muscle cells and fibroblasts (Grinnell, 1994; Kirk et al., 1995). Myofibroblasts
are typically found in connective tissues undergohg remodeling, such as in the human
coronary artery (Shi et al., 1996). In addition to their contraction function, myofibroblasts
have been found to take part in tissue remodeling by producing several proteolytic
enzymes. For instance, u-PA, PAL1 and u-PAR have been found by
immunohistochemistry in myofibroblasts of human breast cancer (Christensen et al..
1996). Thus, myofibroblasts in equine ovary may take part in remodeling required for
follicle growth. EOSC were able to produce gelatinases A and B, TIMP-1, -2 and -3,
uPA, tPA and PM-1, supporthg the hypothesis that ovarian stroma1 cells are involved in
ECM remodeling during follicle growth and ovulation.
Histological and biochemical changes have been observed at the follicular apex.
An electron microscope study documented the disappearance of the collagen bundles in
the apex wall of preovulatory follicle (Espey et al., 1981). Degradation of ECM in this
apical region is significantly enhanced, compared to ECM degradation in basal tissue of
ovarian follicles (Murdoch and McCormick, 1992). In rabbit, tissue at the follicular apex
contains more MMP-1, demonstrated by immunohistochemistry, than other areas of
follicle wall (Tadakurna et al., 1993). These results indicate that cells within the follicle
apex are possibly more important for follicle expansion, migration and ovulation than
cells elsewhere in the follicle wall. Due to the unique morphology of the mare ovary, the
preovulatory follicle must migrate thtough dense connective tissue and rupture at the
ovulation fossa (Ginther, 1993).
Characteristics of ERXN effect
In this study, the effects of ERXN on EOSC were not dependent upon prior
steroid exposure. This finclhg is consistent with previous snidies in which PRXN
modulated the proliferation of porcine grandosa cells (Zhang and Bagnell, 1993) and
gelahase was produced in rat thecal-interstitial cells (Hwang et al., 1996a), without
pr-g with estrogen. Equine ovarian stromal cells were induced by ERXN to produce
TIMP- 1 and -2, uPA, tPA and PAL 1 without prior estrogen exposure.
Relaxin binding sites have been reported in rat ovary (Yang et al., 1992).
However, which ce11 type expresses RXN receptor has not been determined. It is possible
that ERXN directly binds to its receptors on EOSC and induces a series of events. It is
also possible that other factors, such as insulin-like growth factor4 (IGF-I), mediate the
effects of ERXN, since RXN is able to induce the production of IGF-1 (Ohleth and
Bagnell, 1995).
It has been suggested that RXN produced by secondary source does not enter the
blood Stream; however it influences target cells by a paracrine a d o r autocrine manner.
Porcine relaxin has been identified in porcine ovary, small porcine follicles are very
sensitive to RXN, and RXN is able to regulate the proliferation of granulosa and thecal
cells (Zhang and Bagnell, 1993, 1994). Therefore, those investigators proposed that IU<N
fkom the larger preovulatory porcine follicles or from the corpus luteum may perfiise
through stromal tissue and influence small follicles (Zhang and Bagnell, 1993). A
previous study demonstrated that ERXN was not detected in equine stromal tissue, but
was detected in thecal and granulosa cells, corpus luteum and in follicular fluid (Ryan, et
al., 1997). It seems reasonable that, in vivo, ERXN secreted by thecai cells perfuses into
stromal tissue surrounding the follicle and modulates the functions of the stromal cells
during follicle growth. The follicle is the ody known source of RXN in equine ovary
when the mare is not pregnant. It is likely that RXN in the developing follicles is
produced by components of the follicles. However, further study is needed to determine
whether or not RXN perfuses into ovarian stromal tissue.
The effective concentration of ERXN associated with EOSC growth and enzyme
production in this study ranged fiom 0.1 to 1 00 @mi. These doses compare favorably
with the physiological level of RXN reported in equine follicular fluid, which ranged
from 1 to 10 nglml (Ryan et al., 1997). Early studies of PRXN at 1 - 1000 ng/mI resulted
in the increased proliferation of porcine granulosa cells in vitro (Zhang and Bagnell,
1993). Studies using physiological levels of recombinant human RXN (1-100 ng/ml)
showed a dose-dependent increase of collagenase and gelatinase activity by human
c e ~ c a l stromal cells in vitro (Hwang et al., 1996a). Similarly, physiological
concentrations of recombinant human RXN stimulated the production of collagenase
mRNA and the activity of collagenase from hurnan skin and lung fibroblasts in vitro
(Unemon et al, 1990, 1996). Usually, when a hormone is present in excess, the number of
active receptors decreases. However, high supraphysiological concentrations of PRXN
(1-20 pg/d) still caused an increase in gelatinase production in rat granulosa and thecal-
interstitial cells in vitro (Hwang et al., 1996b). The reason for this is not clear.
Equine relaxin used in this shidy was extracted from equine ovaries in the
laboratory of Dr. David Porter according to the method of Shenvood and O'Byme (1974);
it eluted out following HPLC with a similar profile to purified ERXN (provided by Dr.
Demis Stewart, University of California at Davis, CA). Although this ERXN isolated in
our laboratory has not been tested for its bioactivity with standard methods such as the
mouse interpubic ligament bioassay, these experiments have shown ERXN has
comparable bioactivity to PRXN with respect to equine ovarian stromal cells in vitro.
Equine relaxin was purified more than ten years ago (Stewart and Papkoff, 1986)
and its protein and mRNA sequences have been elucidated (Stewart et al., 1991). In
contrat, dmost nothing is known about the biological functions of ERXN. Just as
recombinant human RXN and PRXN have been proven to regulate ce11 proliferation and
synthesis of enzymes, results fiom the present study show that ERXN has similar
activities. Thus, results suggest that RXN in the home has a similar activity to that
reported in several other species.
Since many previous results were obtained using PRXN, PRXN was also
examined in this study for its effects on EOSC proliferation in order to compare the
bioactivity between PRXN and ERXN. Although bioactivity of PRXN is reported to be
approximately 100 times stronger than the bioactivity of ERXN, measured with the
mouse interpubic ligament bioassay (Stewart and Papkoff, 1986), fmdings in this study
showed that porcine and equine relaxin at the same concentrations had similar effects on
EOSC in vitro. A possible explanation for this rnight be that these two RXN molecules
share about 62% homogeneity of amino acids in their peptides; this percentage is much
higher than that of RXN homogeneity between the pig and the human being or between
the pig and the rat (Sherwood, 1994).
Stroma1 tissue has to accommodate the growiag follicle. In the equine ovary, the
follicles in the cortex are surrounded by thick stromal tissue. Before ovulation, equine
ovarian stromal tissue separating the foilicle and germinal epithelium to tdy disappean.
This is the result of migration andor a decrease in prolûeration of equine ovarian
stroma1 cells and increased degradation or decreased production of ECM. Stroma1 cells
are regulated by many hormones, growth factors and cytokines.
Most previous studies of the effects of RXN on connective tissue remodeling in
vitro did not examine ce11 proliferation and enzyme regulation in the same experiment.
Therefore, the relationship between ce11 proliferation and enzyme production has not been
clear. In this study, both aspects were examined. Equine relaxin decreased the
proliferation of EOSC and increased the activity of gelatinases and PA as well as that of
their inhibitors. The results in this study suggest that by inhibiting stromal ce11
proliferation and increasing the activity of proteolytic enzymes, ERXN plays an
important role in influencing the ECM surrounding the growing follicles to allow for
follicle expansion.
Gelatinase production by EOSC
Equine ovarian stromal cells were shown to produce gelatinolytic activity at 72
and 92 kDa in conditioned medium, as well as gelatinolytic activity at 68, 72 and 92 kDa
in ce11 extracts, as detected with gelatin zymography, a technique used for identification
of gelatinases A and B. The gelatinolytic activity was not found when fibrinogen and u-
casein were used as substrates for zymography indicatbg that the enzymes in conditioned
medium were gelatin specific. The enzymes which possessed gelatinolytic activity were
characterized as metalloproteinases, and not serinekysteine proteinases, since EDTA,
which chelates with zinc2+ at the active site of MMP and leads to the inhibition of MMP
activities, inhibited the activity; whereas PMSF, a serinekysteine protease inhibitor but
not a MMP inhibitor, had no inhibitory effect on the gelatinolytic activity in conditioned
medium of EOSC. These result suggest that gelatinase activity at 72 kDa represents a
precursor form of gelatinase A (latent gelatinase A); while that at 92 kDa represents a
precursor form of gelatinase B (latent gelatinase B). Together, these observations are
consistent with results obtained using the same approach to examine gelatinases in rat
cultured granulosa cells (Hwang et al., 1996b).
It has been shown that whole rat ovarian dispersates secrete gelatinases into
conditioned medium in vitro (Hurwitz et al., 1993). To identify which ce11 type in the
ovary has the ability to produce gelatinases, residual tissue (containing thecal and
adjacent stromal tissue) was separated from the granulosa cells in PMCG-primed
immature rat preovulatory follicles. Application of a human gelatinase cDNA probe
revealed gelatinase mRNA to be present in the residuai tissue, but not in the granulosa
cells (Reich et al., 1991). This finding was supported by others who found that cultured
rat thecal anil adjacent stromal tissue of preovulatory follicles secrete gelatinases in
conditioned medium, as detected by gelatin zymography (Hirsch et al.? 1993).
Furthemore, a shidy using a monoclonal antibody against human gelatinase A
demonstrated the presence of gelatinase antigens in cultured thecal-interstitial cells
(Hwang et al., 1996b). In accordance with these results, the current study shows that
horse ovary also produces gelatinase A and gelatinase B and M e r indicates that stromal
cells, at least in part, contribute to the total gelatinase content in the horse ovary.
Gelatinases A and B in conditioned medium and ce11 extracts were shown to be
latent forms based on their relative molecular mass and activation by
aminophenylmercuric acetate (APMA), an organomercuriai compound that is widely
used to activate precursor forms of MMP to their active forms. The presence of latent
forms of gelatinase in medium fkom cultured rat follicles was reported by Palotie et al.
(1987), although which type of gelatinases was not identified. Only a latent form of
gelatinase was present in rat whole ovary extracts examined by zymography (Cuny et al.,
1992). Gelatinases obtained in vitro From other ceil types, including human umbilical
vein and microvascular endothelial cells (Hanemaaijer et al., 1993), human keratinocytes
(Sarret et al., 1992), and several tumor ceIl lines (Brown et al., 1990), have al1 been
present as latent forms. Large amounts of latent gelatinases with very little active enzyme
present may indicate that the gelatinases are binding to substrate and/or to inhibitors and
therefore their activity is difficult to recover by SDS-PAGE (Curry et al., 1985).
The mechanisms controlling the activation of MMP from a latent to an active
form in the ovary are still not clear. Enzyme activation may be regulated by multiple
pathways including protein activation of the latent form. Addition of plasmin to extracts
from follicles or ovaries collected after hCG stimulation increased activation by 2-4 fold;
addition of the serine proteinase inhibitor PMSF to ovarian extracts diminished gelatinase
activity by 16%, suggesting that a s e ~ e proteinase may activate ovarian gelatinase
(Curry et al., 1992).
In the curent study, both latent (72 ma) and active forms (68 kDa) of gelathase
A were extracted fiom cells with Triton X-LOO solution. Therefore gelatinase activity in
ce11 extracts was not likely to have been contaminated by conditioned medium, since the
cells were washed severai times before collection of ce11 extracts. Recently. a new
member of MMP, membrane-type MMP (MT-MMP), was identified, which is localized
on the ce11 surface, and possesses a transmembrane domain (Sato et al., 1996). Latent
gelatinase A is able to bind to MT-MMP and can be obtained fiom the ce11 surface
through washing with Tris solution (Sato et al., 1996). It is possible that gelatinase A in
EOSC extracts was derived from the ce11 surface and was bound to MT-MMP. It is also
possible that gelatinases A an B in EOSC extracts were partially derived From ECM
during the extraction with Triton X-100, because gelatinases A and B contain a
fibronectin-like domain (Matrisian, 1992) and gelatinase A is capable of binding to
collagen through this fibronectin-like domain (Banyai and Patthy, 1991). However.
fuaher evidence for these possibilities is needed.
It has been well established that RXN affects collagen in the pubic symphysis and
ceMx in a variety of mammalian species, leading to extensive dissolution and
disonentation of the collagen fibers in the pubic ligament of guinea pigs (Chihal and
Espey, 1973) and the porcine uterine ceMx (Winn et al., 1993). The mechanisms
whereby RXN promotes these changes in collagen are linked with collagenase. Studies
have demonstrated that RXN treatment elevated the level of pubic symphyseal
coilagenase in estrogen-primed mice (Weiss et al., 1 979) and the interstitial CO llagenase
content in cultwed human skin fibroblasts (Unemori et al., 1990). Since interstitial
collagenase is not able to degrade the denatured type I collagen, gelatinases must be
involved in the complete degradation of type I coilagen, as well as type IV collagen in
basement membrane. Several studies have demonstrated that RXN indeed plays a role in
the increased activity of gelahase. Hwang et al. (1996b) demonstrated that PRXN
stimulated 71, 76 and 92 kDa gelatinase activity produced by rat granulosa and thecal-
interstitial cells in a dose-dependent manner in vitro. Qin and CO-workers (1997a, 1997b)
showed that recombinant human RXN induced the production of gelatinase B by human
chorion-decidua. In the present study, ERXN was able to induce the activity of
gelatinases A and B in conditioned medium and ce11 extracts of EOSC, supponing the
concept that RXN regulates gelatinases.
At the DNA level, the promoter of the gelatinase A gene contains an AP-2
binding site and two Sp- 1 sites but no 12-0-tetradecanolphorbol- 1 3-acetate-responsive
element (Huhtala et al., 1990). In the promoter region of gelahase B, as well as
interstitial collagenase (MMP-I), strornelysin-1 (MMP-3) and matrilysin (MMP-7), a 12-
O-tetradecanolphorbol-13-acetate-responsive element is present and binds the AP-1
transcription factor complex (Sato and Seiki, 1993). Therefore, PMA can indirectly
induce the production of gelatinase B, but not gelatinase A. However, PMA is able to
activate gelatinase A through a protein kinase C-dependent mechanism, as well as by the
participation of membrane-type MMP (Hanemaaijer et ai., 1993; Lohi and Keski-Oja,
1995; Foda et ai., 1996). For example, PMA increased gelatinase B mRNA and protein
levels, while gelatinase A was efficiently cleaved to the 62 kDa f o m in human
embryonic lung fibroblast cultures treated with PMA Gohi et al., 1996). There is a report
that calcium ionophores decreased the expression of gelatinase B in both untreated and
PMA-treated HT- 1080 cells and decreased the gelatinase A activation, niggesting that in
some way expression of gelatinase B and activation of gelatinase A are linked. In this
study, PMA stimulated gelatinase B activity, consistent with previous reports. An
activation of latent gelatinase A was aiso observed in this study, but the gelatinase A
activity at 68 kDa could not be separated fiom latent gelatinase A at 72 kDa by gelatin
zymography .
Production of TIMP by EOSC
The present experiments demonstrate that EOSC secrete two substances in
conditioned medium and one in ECM with inhibitory activity toward gelatinases. as
detected by reverse gelatin zymography, a technique that is used specifically for detection
of TIMPs. These three substances had similar characteristics and activities to those
reported for TIMP in other species and other biologicai systems. Al1 these inhibitors were
heat- and acid-resistant, consistent with the TIMP characteristics described by Cawston
and coworkers (1 98 1). The two inhibitors in conditioned medium had molecular masses
of 28 and 21 kDa respectively, consistent with those of mouse TIMP- 1 and TIMP-7. The
third one with a molecular mass of 24 kDa was found exclusively in ECM and had the
same molecular mass as mouse TTMP-3. Thus, TIMP-1 and -2 appear in conditioned
medium and TIMP-3 in ECM produced by EOSC. To my knowledge, this is the first
report showing that equine cells can synthesize TiMPs and that they have similar
characteristics with their counterparts in other species.
In ECM extracts one band had a molecular mass sirnilar to that of TIMP-1. Tissue
inhibitor of rnetalloproteiuases-l was unlikely to be present because of contamination
frorn the conditioned medium during the collection of ECM since the ECM was washed
several times before collection. Although gelatinases A and B contain a fibronectin-like
domain (Matrisian, 1992) and are capable of binding to collagen through this fbronectin-
iike domain (Banyai and Patthy, 199 l), it is unlikely that TIMP-1 in ECM was from
gelatinase BmMP-1 complexes bound to ECM, since only TIMP-1, not both TIMP-I
and -2, was found in ECM.
Studies have identified TIMP in whole ovary extracts of hurnans (Chun et al.,
1992) and rats (Curry et al., 1989; Reich et al., 199 1). Ushg a human TIMP cDNA probe,
TIMP-1 rnRNA was detected in rat grandosa cells and ovarian residual tissue (Reich et
al., 1991). Ovarian residual tissue contains at least thecal cells and stromal cells. Thus
these previous study did not show whether stromal cells produce TIMP, as has been
demonstrated in the horse. The present studies showed TIMPs were present in
conditioned medium and ECM of cultured EOSC, and extends previous observations in
human beings and rat by demonstrating that cells in equine ovary can aiso synthesize and
secrete TIMPs.
There are only two reports to date which examine the efCects of RXN on the
production of TIMP. One reports shows that recombinant human RXN (0.25-25 ng/mI)
failed to induce changes in TIMP-1 and -2 mRNA, protein levels and activity, as detected
by Northem blot, Western blot and reverse zyrnography respectively, in human chorion-
decidua of humans (Qin et al., 1997a). The second report shows that recombinant human
RXN decreases the TIMP-1 mRNA produced by human skin fibroblasts. My results show
that ERXN stimulates the activity of TIMP-1 and -2, as detected by reverse gelatin
zymography. This differs fiom the results in the two previous studies. This conflicting
effect of RXN on TIMPs might be due to the source of RXN and ce11 types being used.
Ovulation involves cooperation OP gelatinases and TiMPs. Relaxin can trigger ovulation
in a pemised rabbit mode1 (Brannstrom and Mademan, 1993), and there is no
significant difference between ovulation rates induced by RXN and LH. LuteiniPng
hormone is able to increase the TIMP-1 production during the penod of ovulation. These
observations support indirectly the concept that RXN takes part in the modulation of
TIMPs in order to prevent excessive degradation of the ovarian wall and other structures
in the ovary.
It has been well documented that gelatinase A interacts with TIMP-2
preferentially and that gelatinase B interacts with TIMP-1 preferentially to form
complexes. and that there is very little fiee gelathme in conditioned medium of other ce11
systems. In the complexes, TIMP-1 and -2 do not prevent latent gelatinases from
activation. Instead, these complexes may actually provide a rnechanism for the activation
of latent gelatinases. A recent sîudy showed that complexes of nMP-2 and latent
gelatinase A c m bind to the ce11 surface and thus latent gelatinase A is activated
(Corcoran and Stetler-S tevenson, 1995). In the current study , ERXN stimulated the
production of gelatinases A and B; it is therefore reasonable that ERXN also stimulated
the production of TIMP-1 and -2 to combine with the gelatinase B and gelatinase A.
Relaxin stimulated the production of gelatinases and TIMPs. The same
phenornenon may be induced by other hormones and growth factors required in follicle
growth. Human CG stimulates production of gelathases in the rat ovary (Curry et al.,
1992) and also stimulates the synthesis of TIMP-I mRNA by granulosa and thecal cells
during the periovulatory period (Mann et al., 1991). Other studies have also s h o w that
hCG stimulates TIMP and gelatinase activity in the rat ovary (Curry et al., 1989; Reich et
al., 1991). It is reasonable that stimulation of RXN results in parallel increases of
gelatinases, TIMP-I and TIMP-2 activity and subsequently results in the formation of
such gelatinaseA'IMP complexes.
There are several reports showing that TGF-P stimulates both gelatinase and
TIMP production in the same culture system. For example, Marti et al. (1994)
demonstrated that in human glomenilar mesangial cells in vitro TGF-P stirnulated the
mRNA and protein production of gelatinase A at a higher concentration (>5 ng/rnl), while
it stimulated mRNA and protein production of TIMP-1 at a lower concentration (1
nghl) . In the present report, TGF-P stimulated gelatinase A, gelatinase B and TIMP-1
activity at sarne concentration, in accordance with previous studies.
The results in this study showed that TGF-P only slightly stimulated the increase
of TIMP-2 as detected by reverse gelatin ymography, consistent with the results of
Graham and CO-workers (1994) who demonstrated that TGF-P was able to upregulate the
level of TIMP-2 mRNA in normal human trophoblasts. In conflict with the reports of
stimulatory effects of TGF-P on TIMP-2 production, several reports showed inhibitory or
no effects on TIMP-2 expression. For example, TGF-B suppressed the TIMP- mRNA
expression and protein synthesis in human tumor ce11 lines (Stetier-Stevenson et al.,
1990) and caused a modest decrease of TIMP-2 levels while it enhanced the expression
of TIMP-1 and -2 mRNA in cultured osteoblasts (Rydziel et al., 1997). The reasons for
these differences are not known, but could be due to different ce11 types being used.
Within the promoter region of TIMP-1, a binding site for transcription factor AP-
1 has been implicated in TIMP-1 activation by PMA (Edwards and Mahadevan, 1997). In
support of this view, several studies have demonstrated that PMA induced TIMP-1
mRNA expression and protein synthesis by human umbilical vein endothelial cells
(Hanemaaijer et al., 1993), HT-1080 cells (Lohi and Keski-Oja 1995) and human
embryonic lung fibroblasts (Lohi et al., 1996). The result in this study showing that PMA
induced TTMP-1 activity in the conditioned medium of EOSC is consistent with those
previous studies. The implication of an AP-1 site in the promoter region of TIMP-1 may
also explain the CO-expression of gelatinase B and TMP-1 by PMA stimulation. The
expression of TIMP-2 has been found in cells which also express TIMP-1 (Hanemaaijer
et al., 1993; Lohi and Keski-Oja 1995; Lohi et al., 1996), but PMA did not influence
TIMP- rnRNA and protein production in those studies. However, one study has shown
that PMA is capable of inducing slight increases of TIMP-2 by cultured mouse fibroblasts
(Leco et ai., 1994). The mechanism is not clear. A recent study has suggested TMP-1
may play a direct or indirect modulatory role in TIMP-2 and TIMP-3 expression
(Nothnick et al., 1997).
Production of PA by EOSC
Results in the present study showed that EOSC were able to produce both uPA
and PA, in agreement with the previous studies. Granulosa ce11 proliferation and ovarian
follicle growth are accompanied by the dissociation of the surrounding cellular matrix
and tissue rernodeling which may involve uPA and PA. Both tPA and uPA have been
identified in the follicle (Ny et al., 1985). Studies in the rat suggested that tPA and uPA
originate from the granulosa and theca cells, respectively (Canipari and Strickland, 1985).
Other investigations in the rat found that both uPA and P A were produced by the same
granulosa and theca cells with 80.90% of total activity corning fiom the granulosa cells
(Hettle et al., 1986; Reich et al., 1986). Immature rat ovaries were found to express uPA
in the granulosa after PMSG treatment; subsequent hCG treatment caused the expression
of tPA in the granulosa and uPA in the theca, but the granulosa of large follicles
expressed uPA at the t h e of the LH surge (Park et al., 1991). Primate follicles contain
both uPA and tPA but the approach of ovulation is associated with an increase in tPA
(Reinthaller et al., 1990; Liu et al., 1991). In addition, uPA was identified by in situ
hybridization in ovarian stroma1 cells surrounding the developing follicles (Bacharach et
al., 1992) and in the oocytes of preovulatory follicles (Canipari et al., 1987; Sappho et
al., 1989). However, both uPA and tPA production by ovarian stromal cells at the protein
level has not been reported.
in conditioned medium of EOSC, a band at 50 kDa was demonstrated by fibrin
autography. Amiloride confirmed it represented uPA activity. The molecular mass was
close to the reported size of uPA found in equine plasma and platelets (Coilatos et al.,
1994). Latent uPA (single chain) can be cleaved by SDS in the process of fibrin
autography and become active two-chain uPA. Therefore, uPA in conditioned medium
was likely the latent form of uPA. In ce11 extracts of EOSC, a band representing uPA was
also demonstrated and was confirmed by amiloride. However, the molecular rnass (23
kDa) was much smaller than that of uPA in conditioned medium of EOSC or uPA in
equine plasma and platelets. One explanation is that high molecular weight uPA is
present in conditioned medium while low molecular weight uPA is in ce11 extracts. It has
been found that the amino-terminal portion of uPA can be deleted without any apparent
loss in enzymatic activity in vitro (Stoppelli et al., 1985). Both the high and low
molecular-weight uPA have been identified in various ce11 systems (Sakcela, 1985; Dano
et al., 1985; Latrance et al., 1993). In humans, these two isoforms share an identical B-
chain, which contains the active site, but differ in motecular weight because of the limited
proteolytic cleavage of the 24 kDa A chain of the high-molecular weight uPA, leaving a
2 1 amino-acid polypeptide attached to the 30 kDa B chah (Sakcela, 1985).
Another explanation for the difference between conditioned medium and ce11
extracts is associated with uPAR. Mer binding to uPAR, uPA/PAI-1 complexes are
cleaved into two fragments. The 70 kDa hgment containing PM-1 is internalized via
low density lipoprotein (LDL) receptor-related protein (Hen et al., 1992). The 22 kDa
fragment corresponding to most or ail of the B chah of uPA remains on the ce11 surface.
After extraction with a solution containing Triton X-100, the 22 kDa fiagrnent can be
demonstrated by fibrin autography due to the catalytic site of uPA in the B chah (Gross
and Sitrin, 1990; Ragno et al., 1995).
A proteolytic band with molecular mass of 95 kDa was occasionally seen in
ECM. Amiloride inhibited the activity of this band. On the basis of these observations,
this band represents uPA/PAI-1 complexes. Because no evidence has been found that
uPA is able to bind to ECM, the binding of uPA/PAI-1 complexes to ECM would be due
to PAI-1 in these complexes. Plasminogen activator inhibitor-1 is able to bind to ECM
through its vitmnectin-domain (Lawrence et al., 1994), while vitronectin, an adhesive
glycoprotein, is present in ECM of cultured cells (Declerck et al., 1988).
Two additional predominant fibrinolytic zones representing plasminogen
activation in ECM were observed. Amiloride was not able to inhibit their activity.
Therefore, these two bands were caused by tPA in ECM. Their molecular masses were 68
and 105 kDa respectively, very close to the molecular masses of tPA (70 kDa) and
tPAPAi-1 complexes (1 10 D a ) found in equine plasma and platelets (Collatos et al..
1994). Nevertheless, further identification for tPA and tPA/PAi-1 is needed, such as the
use of specific antibodies against PA. Tissue-type plasminogen activator and tPA/PAi- 1
complexes were not detected in conditioned medium or in ce11 extracts. The difference in
this distribution between uPA and tPA in EOSC system might be due to the fact that tPA
has a binding site for fibronectin in ECM, but uPA does not contain any binding site for
components of ECM. Accumulation of tPA in the ECM has been well investigated in
other systerns in vitro, such as human skin keratinocytes (Koli and Keski-Oja, 1993) and
rat granulosa cells (Knecht, 1988). In cultured rat grandosa cells, the incorporation of
P A into the ECM increased with t h e and remained in the ECM for an extended time;
the haif-life of tPA in ECM was about 5-7 hours (Knecht, 1988).
Relaxin has been reported to stimulate the production of PA activity in
conditioned medium of cultured rat granulosa cells, as detected by chromogenic assay;
however, which type of PA was not indicated (Too et al., 1984). In the present study,
ERXN stimulated the accumulation of PA activity in conditioned medium and in ce11
extracts, as exarnined by a chromogenic assay, consistent with the study of Too et al.
(1 981). Because only uPA was detected in conditioned medium and ce11 extracts by fibrin
autography, the increased PA activity in conditioned medium and ce11 extracts was likely
caused by uPA. The possibility that stimulation by ERXN of tPA activity in ECM by
EOSC is supported by another study in which recombinant human RXN at a
concentration of 25 @ml stimulated the production of tPA mRNA and protein by human
fetal membranes (Qin et al., 1997a).
After latent uPA is secreted in vivo, it binds to the ce11 surface through uPAR. On
the ceIl surface the activation of uPA is facilitated. The activated uPA can be inhibited by
PAL1 (Cubellis et al., 1989), and then the uPA/PAI-1 complexes can be internalized. In
extracts of EOSC treated with ERXN, no uPA/PAI-1 complexes were demonstrated by
fibrin autography, either due to the very low production of uPA/PAi-1 complexes on the
ce11 surface, or due to the lack of surface uPA/PAI-1 complexes, because they were
internaiized. It has been observed in HT-1080 fibrosarcoma cells and in normal
fibroblasts that endogenous uPA and PA14 do not colocalize. Urokinase-type PA are
located at sites of celi-cell and fibronectin-fiee substratum contacts (Pollanen et al., 1988;
Hebert et al., 1988). It has been proposed by Cubellis and CO-workers (1989) that binding
of uPA and plasminogen to uPAR on the celi surface will lead to extracellular proteolysis
and to the local sevenng of ceil-cell and cell-substrate connections; this region of the ce11
is therefore free to move, but the movement will transpose uPA to a region in which PAI-
1 is present. The PAL1 can then inactivate uPA and in the absence of local proteolytic
activity the ce11 will form new connections with matrîx. In extracts of EOSC treated with
ERXN, the uPA activity was increased, indicating ERXN rnay facilitate the migration of
cells by increasing the level of active uPA on the ceIl surface.
Urokinase-type plasminogen activator and tPA have been proposed to play roles
in ECM remodeling during follicle growth and ovulation, but their precise mechanisms of
action are not known. A recent study showed that uPA mRNA is mainly expressed by rat
ganulosa cells and ovarian residual tissue in growing follicles, and tPA rnRNA is
expressed m d y by rat granulosa cells and ovarian residual tissue in the preovulatory
stage, supporting the concept that uPA plays a role during follicle growth and tPA plays a
role in ovulation (Li et al., 1997). In the present study, ERXN stimulated activity of both
uPA and tPA by culhired EOSC, suggesting ERXN takes part in the ECM remodeling in
the follicular growth stage, as well as during ovulation.
The mechanism via which RXN regulate uPA and tPA production is not yet
known. Relaxin binding sites have been identified in many tissues, including cells in rat
ovary (Yang et al., 1992). It is possible that RXN binds to its receptors on ovarian
stromai cells and regulates uPA production directly. Altematively, there is evidence
indicating that RXN may regulate uPA and tPA by the mediation of IGF-1. Porcine
relaxin significantiy stimulated IGF-1 secretion by cultured porcine granulosa cells
(Ohleth and Bagnell, 1995). Receptors for IGF-1 have been identified in rat ovxian
stromai cells (Jih et al., 1993). Insulin-like growth factor4 is able to stimulate uPA
secretion by murine marnmary adenocarcinorna (Guena et al., 1996), and to stimulate
tPA production by human retinal pigment epithelial cells (Grant et al., 1990). Based on
these observations, an hypothesis is made that ERXN might stimulate the secretion of
IGF-1 by stromal, thecal or granulosa cells first, and then this IGF-1 modulates uPA and
tPA production by ovarian stromal cells and other cells in an autocrine/paracrine manner.
However, IGF-1 and its receptors have not been reported in equine ovarian stromal cells.
Phorbol ester has been s h o w to increase levels of uPA mRNA and protein in
various cell types, such as macrophages (Stacey et al., 1995) and myeloid cells (Nusrat
and Chapman, 1991). However, fibrin autography and chromogenic assay in this study
showed that uPA activity in conditioned medium and ce11 extracts of cultured EOSC was
diminished upon ERXN treatment. As there is no report in other systems that PMA
inhibits uPA production, it is not likety that PMA inhibits uPA production in EOSC. It
should be noted that uPA activity in conditioned medium and ce11 extracts c m be
influenced by other factors. One possible explanation for this reduction of uPA activity is
that PMA caused an increased production of uPA in conditioned medium, accompanied
by activation of uPA. Then, PM-1 and active uPA in conditioned medium formed
complexes, which resulted in the decrease of uPA activity in conditioned medium. This
explanation is also supported by the fact that treatment of PMA resulted in enhanced
fibrinolytic activity representing uPA/PAI-1 complexes (95 D a ) in conditioned medium,
as detected by fibrin autography. This explanation is supported by the study of Alitalo
and CO-workers (1989). They found that in the human chronic myeloid leukemia ce11 line
k562, PMA induced secretion of latent uPA polypeptide, which was processed into the
active f o m in conditioned medium. Subsequently, active uPA formed complexes with
excessive PAL1 induced also by PMA and was removed from conditioned medium. In
addition, increased number of uPAR on the ce11 surface may also take part in this
reduction of uPA activity. Since PMA stimulates the expression of uPAR in various ce11
types (Nykjaer et al., 1994a; Shetty et al., 1995; Wang et al., 1994) and uPAR mediates
the intemalization of uPA by cells (Nykjaer et al., 1994a), the reduction of uPA activity
in conditioned medium of EOSC exposed to PMA might be caused by increased
expression of uPA receptors, which subsequently increases the intemalization of uPA.
Production of PA14 by EOSC
As a major regulator of ECM remodeling catalyzed by the plasrninogen-plasmin
system, PAL1 mRNA has been found within thecal-interstitial cells and granulosa cells
(Chun et ai., 1992; Peng et al., 1993), and PM activity towards PA has been detected
previously in conditioned medium of rat (Liu et al., 1995). In this study, PAI activity was
shown to be present in the conditioned medium of EOSC, as detected by reverse gelatin
zymography. The stability of this inhibitor to the denaturant SDS is consistent with its
identification as PAI-1, because this characteristic is not shared by other memben of the
PA1 family: PAL2 and PAL3 (Hekman and Loskutoff, 1988). However, other methods
such as Western blot should be used to further veri@ its identity.
Plasminogen activator inhibitor4 activity was also found in ECM of cultured
EOSC. Since conditioned medium was aspirated, foliowed by washing and EOSC were
removed fiom dishes under conditions that did not involve significant ce11 lysis, PA14
was not likely to have been absorbed ont0 the ECM, or contaminated From ce11 extracts.
The matrix-associated PAI-VuPA or tPA complexes demonstrate unusual stability in
SDS-PAGE during electrophoresis in the Laemmli gel system (Knudsen et al., 1987).
Therefore, the matrix-associated PA14 in this snidy did not likely occur from
dissociation of matrix-associated PM-l/uPA or tPA complexes. As was first noticed
during the study of the mechanisms of pencellular proteolysis, PAL1 was distributed
widely on the growth the of substratum of cultured human HT-1080 sarcoma cells
(Pollansen et al., 1988). In support of this finding, snidies of other tissues have reported
the presence of PAI-1 in the ECM of human lung fibroblasts (Laiho et al., 1986a), rat
alveolar epithelial cells (Gross et al., 1991) and human endothelial cells (Levin and
Santell, 1988).
Further study demonstrated that PM-I binds to ECM through vitronectin and
retains its capacity to inhibit uPA or tPA (Salonen et al., 1989). The vitronectin binding
domain within PA14 has also been characterized (Lawrence and Sane, 1994). In human
fibroblasts and fibrosarcorna cells, PA14 is bound to the ECM in a homogenous carpet
under the cultured ceils, at sites in which uPA is absent (Pollanen et al., 1988). The
distribution of PAL1 between ECM and conditioned medium has not yet been reported.
Some researchers suggest that ECM is a storage pool of PAL1 (Gross et al., 1991), since
exogenously added uPA released PA14 fiom ECM of HT-1080 cells (Laiho et al., 1987).
The physiological signincance of the matrix-associated PAL1 has not been M y
determined. Recently, PA14 bound to vitronectin in the ECM has been shown to block
the binding of integnns (Stefansson and Lawrence, 1996) and the uPAR (Deng et al..
1996) to vitronectin, and this interaction was shown to inhibit ce11 adhesion and migration
on vitronectin. In addition, PAL1 in ECM is thought to protect tissue against proteolytic
degradation by inhibithg local plasmin generation and by scavenging uPA or tPA in
ECM via PAI- lhPA or P A complexes (Levin and Santell, 1988).
A study reported that PAI-1 was detected by irnrnonoprecipitation in extracts of
rat alveolar epithelial cells, but at very low level (Gross et al., 199 1). In this study, PAI- 1
activity was not found in extracts of cultured EOSC, possibly due to the relatively lower
sensitivity of reverse fibrin autography compared to imrnunoprecipitation.
The PAL1 has been found in both a iatent and an active forrn. The latent fonn of
PAL1 can be partially converted to an active form by treatment with protein denaturants?
such as SDS (Hekman and Loskutoff, 1985). Therefore, the latent forrn of PAL1 is able
to be revealed by reverse f i b ~ autography. On the other hand, active PAL1 combines in
equimolar amounts with uPA or tPA to form complexes, which cannot be separated by
SDS (Andreason et al., 1986), and the activity of these compacts cm not be dernonstrated
by reverse fibnn autography. So, the PA14 activity in conditioned medium and ECM of
EOSC, detected by reverse fibrin autography, is caused by SDS-activated latent PAI-1.
nie relative molecular mass of the latent PAL1 is similar to the reported size of latent
PM-1 (50 kDa) in equine plasma and platelets (Collatos et al., 1994). Since active PAL1
bound to uPA or tPA is not able to inhibit the activation of uPA or tPA by SDS, active
PM-1 activity can be revealed indirectly by fibrin autography. In conditioned medium of
EOSC, active PAL-l/uPA complexes were found. The molecular mass (95 kDa) is
consistent with the PAI-lluPA complexes found in equine plasma and platelet (Collatos
et al., 1994).
A small amount of PAI-l/uPA cornplex was dso found in ECM. It is possible that
vitronectin binds to PAL1 in these complexes. In ECM, a fibrinolytic band at 105 kDa
was demonstrated by fibrin autography. Since this band is not inhibited by amiloride, it is
caused by P A . The molecular mass of tPA in conditioned medium and ECM is about 70
D a . Therefore, it represents active PAI-l/tPA complexes. The use of solid-phase binding
assays to quantitate the PM-llvitronectin interaction has suggested that only active PAL 1
binds to vitronectin (Seifiert et al., 1991), however other investigators have reported no
apparent difference in the binding of active and latent PAL1 (Kost et al., 1992).
Modulation effects of RXN on PA synthesis and activity have been reported
previously in rat granulosa cells (Too et al., 1984; Qin et al., 1997b), but whether RXN
regulates PAL1 production is not known. To my knowledge, the current study is the first
one showing that ERXN may modulate PM-1 activity, in vitro. However, the mechanisrn
used by ERXN to increase the latent PAL1 activity in EOSC conditioned medium was
not defined. There are several possibilities. Firstly, ERXN stimulates the production of
PAi-1, dong with uPA, by EOSC to ensure proper ECM remodeling. Secondly, the level
of latent PAI-1 depends on the level of active uPA; or latent or active tPA, because PM-1
is secreted as an active fonn and converted to a latent form rapidly unless it complexes
with uPA or PA. Only single-chah uPA but no detectable tPA was present in EOSC
conditioned medium, possibly resulting in active PAI-1 conversion into a latent fom,
and accumuiation in conditioned medium. Thirdly, ECM been suggested as a storage
pool for PAI-I (Gross et al., 1991). If ERXN reduced the ECM capacity for PAI-I.
relatively more PAL1 would be released into conditioned medium. To test this
explmation, ECM examined. Results showed that treatment of EOSC with ERXN
seemed to reduce PAL1 activity in ECM. The decrease of PAL1 in ECM could result in
increased ECM degradation and subsequent ce11 migration.
The mechanism via which RXN acts on target cells and induces PAi production
has not been determined. It is possible that RXN binds to its receptors and influence the
production of PA1 by target cells directly. It is also possible that RXN modulates the PAL
1 production via IGF-1. As mentioned before, RXN is able to increase production of IGF-
I (Ohleth and Bagnell, 1995), while IGF-I is able to increase mRNA and protein levels of
PAI-I in human hepatic cells (Anfosso et al., 1995; Padayatty et al., 1993). It is possible
that ERXN stimulates the secretion of IGF-1 by stromal, thecal or granulosa cells, and
then IGF-1 modulates the production of uPA and tPA in an autocrinelparacrine manner.
Thirdly, ERXN might first have induced an increase of PA, which in tum resulted in an
increase in PM-1 in the EOSC. Such a mechanism for PAL1 production has been
demonsated in hepatic and endothelial ce11 culture (Fujii et al., 1990).
As an important regulator in ECM remodeling, TGF-P is reported to stimulate
both uPA and PAL1 production. For example, TGF-P increased the activity of uPA and
PAL1 in human skin fibroblasts (Laiho et al., 1986b), rat microvascdar endothelial cells
(Sankar et al., 1996) and osteoblast-like cells (Ailan et al., 1991), and in cultured normal
and transformed human mammary epitheiial cells (Stampfer et al., 1993). The result in
the current study that TOF-P increased both uPA and PAL1 activity produced by cultured
EOSC is in agreement with these previous studies.
Interestingly, PM-I and uPA activity produced by EOSC c m bey in parallel.
stimulated by ERXN or TGF-P. It is speculative whether the observed in vitro changes in
protein expression parallel the function of ERXN in vivo. However, one c m envision that
increased expression of protease inhibitors rnight represent an adaptive regulatory
mechanism for containing and focusing proteolytic activity or, as suggested by Sankar
and CO-workers (1996), that high levels of API-1 may be needed to cause a constant
change of uPA receptor position.
Effect of ERXN on proliferation of EOSC
Proliferation of ovarian stromal cells in species other than the horse has been
studied previously. Accumulated evidence indicates that the proliferation of ovuian
stromal cells is inhibited as the follicle matures during the ovarian cycle. For example, as
detected by autoradiographic assessrnent of DNA synthesis, 3~-thymidine incorporation
by mouse ovaian stromal cells declines drarnatically as the follicles approach ovulation?
while 3~-thymidine incorporation by grandosa cells does not decrease (Li, 1994). During
the stage of foliicle development, many hormones are involved. Gonadotropins may
stimulate proliferation of ovarian stromal cells, as shown by Snowden et al. (1 989), who
examhed the efTects of LH and FSH in vitro on proliferation of ovarîan strornd cells
isolated fiom the human ovary, in an attempt to elucidate the etiology of ovarian stromal
hyperplasia after menopause. They found that gonadotropins increase the proMeration of
ovarian stromal cells. Since ovarian stroma hyperplasia is not found in women before
menopause, it is suggested that inhibitors might be present in the ovary to prevent the
proliferation of ovarian stromal cells in the presence of gonadotropins. Since ERXN
inhibited the proliferation of such cells in this study, it is possible RXN might be one
candidate responsible for this inhibition of ovarian stromal cells. Other mechanisms of
inhibiting proliferation of ovarian stromal cells are possible. Ovarian stromal cells are
regulated by many other factors. Epidermai growth factor (EGF) is a potent mitogen for a
variety of cells. Epidermai growth factor receptor expression on hurnan ovarian stromd
cells was not present when the follicle was in the antral and preovulatory stages (Maruo
et al., 1993). In contrast, the EGF receptor was intensely expressed by stromal cells
around the corpus albicans. Also, insulin receptors are expressed by hurnan ovarian
stromal cells sunounding follicles throughout the cycle, although the expression is
stronger in the primordial follicle stage and declines towards ovulation (Samoto et al..
1993). The rnechanisms underlying this phenornenon are no? ciear.
In the present study, 1 measured the thymidine incorporation by EOSC in MEM
supplemented with 0.5% FBS, attempting to minimize the effects of serurn derived
growth factors and hormones on EOSC. Equine relaxin inhibited the proliferation of
EOSC triggered by growth factors in senim. For the subsequent studies of enzyme
activity, it was important to determine the mitogenic effect of RXN on ovarian stromal
ceUs in culture. There was no significant change in ce11 nurnbea at ERXN concentrations
of 0.01-100 ng/ml over a 48 hour exposure of confluent celis cultured in senun-fiee
medium. This finding is consistent with the results of Mushayandebvu and Rajabi (1995)
and Hwang et al. (1996b) in other reproductive tissues. They found that PRXN did not
influence ce11 numbers when guinea pig uterine cervical cells and rat granulosa and
thecal-interstitial cells were incubated in serum-free medium. The dissociation between
mitogenic activity and enzyme activity makes it easier to examine enzyme production by
cells. However, it is not known whether the disassociation is due to RXN effects or not.
The ce11 proliferation sîudy showed a dose-dependent decrease in EOSC numbers
in response to increasing concentrations of ERXN, which correlates with the fact that
RXN induced a decrease in '~-th~rnidine incorporation by EOSC. Relaxin inhibits
proliferation of rat 3T3-L 1 fibroblasts (Pawlina et al., 1989, 1990) and human MCF-7
breast-cancer cells (Sacchi et al., 1994). However, several reports suggest that RXN can
stimulate proliferation of fibroblasts and parenchymal cells fiom rat and rabbit mammary
glands (Winn et al., 1994; Kuenzi et al, 1995). In the porcine ovary, RXN stimulates the
growth of theca and granulosa cells (Zhang and Bagnell, 1993, 1994), but the overall
ovarian mass and protein content are unaf5ected by RXN treatrnent in vivo (Ohleth et al.,
1996). Relaxin caused a reduction of fibroblast numbers in the demis, as well as a
decline of collagen in another study of remodeling (Unemori et al., 1993). In the
mammary gland, while stimulating the growth of mammary parenchyma, RXN also
caused a reduction in collagen levels in the stromal tissue surrounding the alveolar lobule.
These observations implicate RXN in the stimulation of both the growth of mamrnary
parenchyma and reduction of stromal tissue (Winn et al., 1994). Therefore, it is very
likely that RXN is involved in the growth of follicles and decreases the growth of other
ovarian components, such as stromai cells. In this way, the follicle is allowed to grow and
expand into the surrounding strornal tissue.
In order to understand the roles of RXN in matrix remodeling during equine
follicle growth, the effects of ERXN on cell activity of EOSC, including proliferation, the
production of gelatinase A and gelatinase B, TIMPs, uPA, tPA and PAI-1, were
examined in vitro. In addition, effects of TGF-P and PMA on the production of gelatinase
A and gelatinase B, TIMPs, uPA, tPA and PAL1 by cultured EOSC were also studied.
Equine ovarian stroma1 cells were isolated fiom the apex of developing follicles
(larger than 3.5 cm in diameter) during the breeding season. These cells exhibited a
fibroblast-like morphology. They were a-SMA and procollagen positive, and Von
Willebrand Factor negative as revealed by immunostainhg techniques.
In vitro, EOSC were able to secrete gelatinase A and B, which accumulated in the
conditioned medium and in ceII extracts. Treatment with ERXN enhanced the latent
gelatinase A and B activity in conditioned medium and in ce11 extracts. Equine relaxin
also induced activation of latent gelatinase A in conditioned medium and ceIl extracts.
Both TGF-P and PMA stimulated the activity of latent gelathases A and B in conditioned
medium. Phorbol ester PMA also induced the activation of both gelatinase A and B in
conditioned medium.
Equine ovarian stroma1 cells produce TIMP-1 and -2 in conditioned medium and
TIMP-3 in ECM of EOSC. Activity of TIMP-I and -2 in conditioned medium was
significantly increased by treatment of ERXN. However, ERXN failed to stimulate an
increase of TIMP-3 activity. Transfomiing growth factor-p and PMA stimulated
significant increases of TIMP-1 activity in a dose-dependent rnanner. In contrast, both
TGF-P and PMA just slightly stimulated the TIMP-2 activity.
Urokinase-type PA was found in conditioned medium and ce11 extracts. However
uPA in conditioned medium might be high molecular weight uPA (50 kDa); uPA in cell
extracts might be either low molecular weight uPA (23 kDa), due to the loss of the
amino-terminal domain, or a fragment of uPA/PAI-1 complexes. In contrast, tPA was
found in ECM. This different distribution may result fiom the fact that tPA has a
fibronectin-binding domain, but uPA does not. Treatrnent with ERXN significantly
increased the uPA activity in conditioned medium and ce11 extracts. Activity of tPA in
ECM was significantly increased by ERXN. Transforming growth factor-p induced uPA
activity. In contrast, uPA activity in conditioned medium was reduced, probably due to
the activation of uPA and the increase of PAI-1 activity caused by PMA.
Equine ovarian stroma1 cells were able to synthesize PA14 (50 kDa), which was
found in conditioned medium and ECM. A small quantity of uPA/PAI-L complex was
found in ECM, probably resulting fiorn the binding ability of PAI-1 to vitronectin.
Treatrnent with ERXN increased the PM-1 activity in conditioned medium. Deposition
of PM-1 in ECM seems to be reduced by treatment with ERXN. Additiondly, the
formation of tPA/PAI- 1 complexes was also significantly increased b y ERXN treatment .
Both TGF-P and PMA stimulated PAI-1 activity in conditioned medium.
Both ERXN and PRXN decreased '~-thymidine incorporation by EOSC in a
dose-dependent manner. The inhibition was about 35% lower than that of contrds
(without treatment of m. Relaxin also inhibited ce11 proMeration in a similar dose-
dependent rnanner. Botb ERXN and PRXN exerted the same degree of effect regarding
the inhibition with an effective range of 0.1 - 1 00 ngfml. Transfo rming gro wth factor- p
inhibited the incorporation of 'H-thpidine into DNA by EOSC and inhibited their
proliferation.
CONCLUSION
Equine ovarian stroma1 cells take part in the ECM remodeling during the growth
of the follicle by producing gelatinases A and B, TIMP-1, -2 and -3, uPA, tPA, and PM-
I . Equine relaxin is able to modulate the activity of EOSC. The altered activity facilitates
ECM remodeling and the migration of EOSC in the equine ovary during follicle growth.
It is possible that RXN, synthesized by thecdgranulosa cells under stimulation of various
hormones and growth factors during the growth of the follicle, perfuses the stromal tissue
and acts upon the stromal cells. It is likely that, as the follicle expands, RXN inhibits the
proliferation of stromal cells surrounding the follicle and at sarne time enhances the
movement of stromal cells. Equine relaxin modulates the PA activity, which may activate
plasminogen originating nom the plasma via capillaries. In tum, plasmin degrades the
ECM directly and, more importantly, activates MMP components, such as gelatinases A
and B. Equine relaxin also stimulates the activities of gelatinases A and B in vitro.
Celatinases A and B may m e r degrade denatured collagen and cleave the type IV
collagen in the basement membrane. Equhe relaxin also appears to play an important role
in the control of activity of PA and gelatinases A and B by stimulating production of
PAL1 and TIMPs. Increased PAL1 and TKMPs may prevent premature and excessive
degradation of the ECM, ensuring that the ovary is not destroyed and that the follicle may
grow properly to ovulate at the right time and site.
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APPENDIX I SOURCES OF SUPPLIES AND MATERIALS
Materials Acrylamine: bisacrylarnide (40%, w/v)
4-aminophenylmercUnc acetate
Agarose low EEO
Anti-human a-smooth muscle Actin antibody (monoclonal, mouse)
Anti-human procollagen type I antibody (polyclonal, rabbit)
Anti-human Von Willebrand Factor (polyclono1, rabbit)
Anti-mouse IgG (goat, conjugated with horseradish perioxidase)
Anti-rabbit IgG (goat, conjugated with aikaline phosphatase)
Amiloride
Ammonium persulphate
Bacterial collagenase type 1
Bovine serum albumin
Source GIBCO BRL. Life Technologies Inc., Buriington, ON
Sigma Chemical Company St. Louis, MO, USA
Fisher Scientific, Fair Lawn, NJ, USA
Sigma Chemical Company St. Louis, MO, USA
Cedarlane Laboratories, Homby, ONT
DAKO Corporation, Carpinteria, CA, USA
Zymed Laboratories Inc., South San Francisco, CA, USA
Sigma Chemical Company St. Louis, MO, USA
Sigma Chemical Company St. Louis, MO, USA
Fisher Scientific, Fair Lawn, NJ, USA
Sigma Chernicd Company St. Louis, MO, USA
GIBCO BRL. Life Technologies hc., Buriington, ON
Calcium chloride Fisher Scientific, Fair Lawn, NJ, USA
Coomassie Brilliant Blue G-250
D imethy 1 sulfoxide
5,S-dithiobis (Znitrobenzoic acid) @TNW
DNase type 1
EDTA
EGTA
ERXN
Fetal bovine semm
Fibrinogen (Type IV, fkom bovine plasma)
Fungizone
Gel drying film
Gelatin
Gentamycin
Glaciai acetic acid
Glycine
Hanks' balanced salt solution ( 10x1
Bio-Rad Laboratories, Richmond, CA, USA
Fisher Scientific, Fair L a m , NJ, USA
Sigma Chemical Company St. Louis, MO, USA
Sigma Chemical Company St. Louis, MO, USA
J.T. Baker Chemical Co., Phillipsburg, NJ USA
Fisher Scientific, Fair Lawn, NJ, USA
Department of Biomedical Sciences, W C , University of Guelph, Guelph, ON
GIBCO BRL. Life Technologies Inc., Burlington, ON
Sigma Chemical Company St. Louis, MO, USA
GIBCO BRL. Life Technologies Inc., Burlington, ON
Promega, Medison, WI, USA
Fisher Scientific, Fair Lawn, NJ, USA
GIBCO BRL. Life Technologies Inc.. Burlington, ON
Fisher Scientific, Fair Lawn, NJ, USA
Fisher Scientific, Fair Lawn, NJ, USA
GIBCO BRL. Life Technologies Inc., Burlington, ON
ICN Radio chemicals Inc., M e , CA
Kit for reverse zymography
Na-CBZ-L-lysine thiobenzyl Ester
Methanol
Minimum essential medium (witho ut L-glutamine)
Minimum essential medium (without L-glutamine and phenol red)
Minimum non-essentid amino acid solution (1 0x)
Mini protein II dual slab ce11
Mouse TLMP- 1 ,-2 and -3
Phenylmethylsulfonyl fluoride
Phorbol 1 Zmyristate 13-acetate
Plasminogen activator inhibitor-l (recombinant active human)
Tissue culture plates and dishes
Porcine relaxin
University Technologies International Inc. Cakary
GIBCO BRL. Life Technologies Inc., Burlington, ON
Sigma Chemical Company St. Louis, MO, USA
Fisher Scientific, Fair Lam, NJ, USA
GIBCO BRL. Life Technologies Inc., Burlington, ON
GIBCO BRL. Life Technologies Inc., Burlington, ON
GIBCO BRL. Life Technologies Inc., Burlington, ON
Bio-Rad Laboratories, Richmond, CA, USA
University Technologies International Inc. C a k w
GIBCO BRL. Life Technologies Inc., Burlington, ON
Sigma Chemical Company St. Louis, M0,USA
Sigma Chemical Company St. Louis, MO, USA
American Diagnostics Inc. Greenwith, CT, USA
Becton Dickinson Labware, Frankin Lakes, NJ, USA
Departrnent of Anatomy, University of Bristol, Bristol, UK
Resolving gel buffet concentrate
SDS-PAGE molecular weight standards (B road range)
SDS-PAGE molecular weight standards (rainbow)
Sodium docecyl sulfate
Stacking gel buffer concentrate
S treptomycin
Thrombin
Tissue-type plasminogen activator (double chain)
Tissue-type plasminogen activator (single chain)
Tris
Triton X- 100
Trypsin-EDTA
Tween 20
Urokinase plasminogen activator (human)
GIBCO BRL. Life Technologies Inc., Burlington, ON
Bio-Rad Laboratories, Hercules, CA, USA
Bio-Rad Laboratories, Hercules, CA, USA
Fisher Scientific, Fair Lawn, NJ, USA
GIBCO BRL. Life Technologies, Inc., Gaithersbug, ND, USA
GIBCO BRL. Life Technologies Inc., Burlington, ON
Park-Davis Div., Warner-Lam bert Canada Inc., Scarborough, ON
Biopool AB, Umea, Sweden
Biopool AB, Umea, S weden
R & D Systems Inc. Minneapolis, MN1 USA
Fisher Scientific, Fair Lawn, NJ, USA
BDH inc., Toronto, ONT
GIBCO BRL. Life Technologies Inc., Burlington, ON
ICN Biomedicals, Inc., Aurora, Ohio, USA
Sigma Chernical Company St. Louis, MO, USA
Zinc chioride Fisher Scientific, Fair Lawn, NJ, USA
APPENDIX II RECIPES FOR SOLUTIONS AND BWF'ERS
Recipes for gels
Bring up to 50 ml with ddH20. Stored at 4'C
10% gelatin 100 pl Resolving buf5er (fom GIBCO, BRL) 1.25 ml 1 0% Ammonium perd fate 60 pl 40% Acrylamine-bisocrylamide 963 pl (form GIBCO, BRL) ddH20 2.65 ml
Total volume is 5 ml, enough for one 0.75 mm gel.
J î . 5 % R e s o l m e l for reverse gdafb -- 10% gelath 100 pl Resolving buffer (fom GIBCO, BRL) 2 2 5 ml 10% Arnmoniumpersulfate 60 pl 40% Acrylamine-bisacry~de 1.3 ml (form GIBCO, BRL) MMP solution 500 p1 ddH20 1.8 ml
Total volume is 5 ml, enough for one 0.75 mm gel.
Resolving buffer (fonn GIBCO, BRL) 1.25 ml 10% Ammoniumpersulfate 60 p1 (form GIBCO, BRL) 40% Acrylamine-bisacrylamide 1.3 ml ddH2Q 2.4 ml
Total volume is 5 ml, enough for one 0.75 mm gel.
Stacking buffer (fom GIBCO, BRL) 1.35 ml 1 0% Ammoniurnpersul fate 38 pl 40% Acrylamine-bisacrylamide 150 pl (form GIBCO, BRL)
Total volume is 1.54 ml, enough for one 0.75 mm gel.
Bring up to 500 ml with ddH20. Adjust pH to 7.2. Store at room temperature.
Tris Glycine 10% SDS
Bring up to 50 ml with ddH20. Stored at 4OC
Tris CaC12 10 mM ZnC12
Bring up to 50 mi with dW20. Adjust pH to 8. Stored at 4OC
Cciowsie blue stain
Methano 1 Coommasie blue Glacial acetic acid
Bring up to 200 ml with ddHzO.
Methanol Glacial acetic acid
Bring up to 500 ml with ddH20. Store at roorn temperature.
onre
Tris Glycerol SDS Bromophenol blue
Bring up to 50 ml with ddHH,O. Store at -20°C.
Chromogenic assay
BSA Tween 20 Sodium azide 10x PBS
Bring up to 100 ml with dW20. Store at 4OC.
gelatin Tween 20 10x PBS
Bring to 500 ml with ddH20. Autoclave for 90 min and Store at 4 ' ~ .
BrUig to 500 ml with ddH20. Adjust pH to 7.5. Store at room temperature.
5,S-dithiobis (2-nitrobenzoic acid) 43.6 mg Na2HP04 (50 mM) 5 ml
Store at -20°C.
Na-CBZ-L-lysine thiobenzyl ester 38.6 mg
Dissolved in 5 ml ddH20. Store at -20°C.
APPENDIX III STANDARD CURVE OF GELATIN ZYMOGRAPHY
The untreated EOSC conditioned medium containhg various amount of protein (0.1-0.5 pg/lane) was applied to a 7.5% acrylamide gelatin zyrnography. Mer incubation ovemight, the gel was stained with Coomassie Bnlliant Blue G-250. In the upper panel, lysis bands represent gelatinases as detected by gelatin zymography. Bands representing gelatinase A (72 D a ) were analyzed densitometrically as shown in the lower panel. There is a linear relationship between the amount of gelatinase A in the conditioned medium which was loaded ont0 the gel and the area of lysis zones.
1 2 3 4 5 (ulllane)
O 1 2 3 4 5 6 Volume of conditioned medium (ul)
APPENDIX N STANDARD CURVE OF REVERSE GELATIN ZYMOGWPHY
A series of volumes (0.5-8 pl) of p"fied mouse TIMPs was appiied to a 12.5% acrylarnide gel containhg gelatin (0.1%) and an MMP preparation. Afier incubation overnight, the gel was stained with Coomassie Brilliant Blue G-250 and destained ovemight. In the upper panel, lysis-resistant bands represent mouse TIMP-1,-2 and -3 as detected by reverse gelatin zymography. Bands representing TLMP-1 are analyzed densitometrically show in the lower panel. There is a linear relationship between the amount of TIMP-1 loaded ont0 the gel and the area of lysis-resistant zones.
O I 1 I I I 1
O 1 2 3 4 5 6
Volume of mouse TlMP (ul)
APPENDK V STANDARD CURVE OF FIBRIN AUTOGRAPHY
A series of various amounts of human kidney uPA (0.2-1.0 IUAane) was applied to a 10% acrylamide gel. After electrophoresis, indicator film containing fibnn and plasminogen was overlaid on top of the gel. The gel sandwich was incubated at 37°C overnight. In the upper panel, lysis bands represent human kidney uPA as detected by fibrin autography. Bands representing uPA were andyzed densitometrically as shown in the lower panel. There is a linear relationship between the amount of uPA loaded ont0 the gel and the area of lysis zone.
Concentration of human uPA (IUIlane)
APPENDIX VI STANDARD CURVE OF REVERSE FIBRIN AUTOGRAPHY
A series of various arnount of human recombinant PM-1 (1 .O-5.0 ng/lane) was applied to a 1 0% acrylamide gel. After electrophoresis, indicator film containing fi brin, human kidney uPA and plasminogen was overlaid on top of the gel. The gel sandwich was incubated at 37'C overnight. In the upper panel lysis-resistant bands represent hurnan recombinant PAL1 as detected by reverse fibrin autogram. Bands representing PM-1 are analyzed densitometrically as shown in the lower panel. There is a Iinear relationship between the amount of PA14 Ioaded ont0 the gel and the area of lysis-resistant zone.
O 1 2 3 4 5 6
Concentration of human PAI-1 (ngtlane)
IMAGE EVALUATION TEST TARGET (QA-3)
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