The role of ovarian proteases and their inhibitors in ovulation

THE ROLE OF OVARIAN PROTEASES AND THEIR INHIBITORS IN OVULATION Thomas E. Curry, .Jr.,a David D. Dean,b Sheryl L. Sanders,c Nancy G. Pedigo,a and Phrllrp B.C. Jonesd aDepartment of Obstetrics & Gynecolog CDepartment of Anatomy and Neurobiology, and dDepartment of Bioc K9 emistry, University of Kentucky, Lexington, KY 40536; bDepartment of Medicine, University of Miami, Miami, FL 33101, USA Corresponding

author:

Thomas E. Curry, Jr., Ph.D.

ABSTRACT To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later b 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after z uman chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 f 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 + 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 + 7.9 and 71.6 f 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization pro ram. Metalloproteinase inhibitor activity was evaluated in follicular flui 1 as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p
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INTRODUCTION

Ovulation is a dynamic, orchestrated process which culminates in a weakening of the follicular wall and extrusion of the oocyte. It is well documented in mammalian species that a surge of LH initiates a series of biochemical events which ultimately result in rupture of the follicle (1,2). The interaction of LH with a surface membrane receptor turns on the intracellular adenylate cyclase/cAMP pathway resulting in stimulation of protein kinase A. However, the exact mechanism by which LH leads to follicular rupture remains to be elucidated. For ovulation to occur, there must be a dissociation of the follicular wall and overlying connective tissue layers (3). Morphologically, there is a decrease in the apical collagenous matrix of the preovulatory follicle which occurs, in part, by the action of proteolytic enz mes (4). The activities of enz mes, such as the plasminogen activator/p rasmin system and the meta rloproteinase collagenase, increase during the preovulatory period in the rat and have been postulated to play a key role in ovulation (l-3). Although proteolytic enzyme activity increases prior to follicular rupture, little is known about the regulation of enzyme activity in the ovarian extracellular space. In other tissues, the regulation of plasminogen activator (PA) and collagenase activity occurs by the action of serumborne or tissue-derived inhibitors. Serum inhibitors, such as azmacroglobulin, account for the majority of inhibitor activity (5,6). Due to the large molecular size of az-macroglobulin (MW = 720,000), its role in regulating proteolysis at the tissue level has been questioned on the basis of its ability to cross endothelial cell junctions and enter the extracellular matrix. Along with az-macroglobulin, other plasma proteinase inhibitors are known to interact with lasminogen activators. For example, urokinase is inhibited by antit R rombin III and a-1-proteinase inhibitor, while tissue type PA (tPA) is inhibited by a-2-antiplasmin and a-lantitrypsin (7). In addition to serum-borne inhibitors, a second group of specific tissuederived PA or metalloproteinase inhibitors exists. For PA enzymes, there are two distinct inhibitors. Plasminogen activator inhibitor (PAI) type 1, or PAI-1, is present in endothelial cells, thrombocytes, and plasma. A second inhibitor, PAI t pe 2 (PAI-2), was originally purified from placental extracts but is also re reased by cultured monocyte-macrophages (8). A functional distinction between these plasminogen activator inhibitors is that PAI- inhibits both forms of PA, tPA, and urokinase, while PAI- is primarily an inhibitor of urokinase. For collagenase, a specific, tissue-derived inhibitor of metalloproteinases (TIMP) has been observed in numerous tissues (9-11). The inhibitor is a glycoprotein of approximately 27,000-29,000 MW that is stable to extremes of temperature and pH but is inactivated by reduction and alkylation (12). The purpose of the present study was to determine whether the ovary contains inhibitor activity, identify the inhibitors, and examine the changes in inhibitor activity during the periovulatory period in both rats and humans.

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MATERIALS AND METHODS Animals Immature female Sprague-Dawley rats (27 days of age) were injected SC with 20 IU of pregnant mare serum gonadotropin (PMSG) at 0900 h. The immature PMSG-primed model was utilized because it provides, by virtue of the development of a large number of follicles (30-40/ovary), an adequate amount of tissue necessary for measurement of ovarian collagenase and inhibitor activity, and allows ease of synchronization of the time of ovulation. Fifty-two h after PMSG administration, animals were injected SCwith vehicle (phosphate-buffered saline, PBS)or 10 IU of human chorionic gonadotropin (hCG) and killed at 0, 4, 8, 12, or 20 h later. These time points were chosen to examine enzyme and inhibitor activity before hCG stimulation (0 h), prior to ovulation (4 and 8 h), at the time of follicular rupture (12 h), and after ovulation (20 h). The ovaries were removed at the various time points and frozen at -20 C in 100 PI of PBS (pH 7.4) until assayed for collagenase or inhibitor activity (no longer than 4 weeks). Human follicular fluid and aranulosa cells Follicular fluid was collected from 10 women (N = 25) under oing ovarian superstimulation for either in vitro fertilization (IVF a or gamete intrafallopian tube transfer (GIFT) as described previously (13). Ovarian superstimulation for multiple follicular growth was induced with either human menopausal gonadotropin (Pergonal) or clomiphene citrate (Clomid) alone, or in combination, followed by hCG (10,000 IU) administration. Oocyte recovery was performed by laparoscopy 32 to 34 h later (i.e., 2-4 h prior to ovulation). Immediate1 after oocyte recovery, ranulosa cells and cellular debris were removed r rom the follicular fluid 1 y centrifugation at 1500 x g for 5 min. The supernatant was collected and frozen for protein, steroid, and inhibitor measurement or inhibitor purification. Granulosa cells were resuspended in guanidinium thiocyanate, vortexed, and total RNA was isolated using standard procedures (14,15). Only follicular fluids which were clear and not contaminated with red blood cells were utilized in this study. Metalloproteinase

inhibitor and collaaenase extraction in rat ovaries

To optimize extraction of the inhibitor, activity was measured in rat ovaries which were homo enized in detergent, extracted in a high calcium buffer, and heate 8 . Briefly, ovaries were homogenized in 10 volumes of 10 mM CaClz with 0.25Oh Triton X-100. The homogenate was centrifuged at 9000 x g for 30 minutes and the supernatant (Triton supernatant) saved for determination of inhibitor activity. Activity was also measured in ovarian tissue extracted by homogenizing the Triton pellet in a high calcium Tris buffer (50 mM Tris-HCI, 100 mM CaCl?, 0.15 M NaCI, pH 7.5). To further test the extraction of the inhibitor in this pellet, a portion of the resuspended Triton pellet was not heated while the remaining aliquot was heated to 60 C for 6 min. Both aliquots were

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centrifuged at 27,000 x g for 30 min and the supernatants (no heat and heat supernatants, respectively) saved. A portion of the Triton supernatant and both the no heat and heat supernatants were treated to determine whether the inhibitor activity could be abolished by a procedure reported to inactivate protease inhibitors (12) and concomitantly examine the changes in collagenase activity. A portion of each supernatant was reduced with 2 mM dithiothreitol for 30 min at 37 C followed by alkylation with 5 mM iodoacetamide for 30 min at 37 C, a treatment reported to destroy TIMP (12). All of these supernatants were dialyzed overnight against assay buffer (50 mM Tris-HCI, 10 mM CaCl2, 0.15 M NaCI, 0.05% Brij 35, 0.02Oh NaN3, pH 7.5) before measuring inhibitor and collagenase activity. Routinely, the heat supernatant was utilized for determination of inhibitor activity in extracts of ovaries collected at various times after hCG administration. Metalloproteinase The inhibitor assay is based on the inhibition of uterine neutral metalloproteinase activity usin a calorimetric substrate, Azocoll (Calbiochem, La Jolla, CA) as descri%ed previously (16). Metalloproteinase in sufficient volume (approximately 10-l 5 pl) to yield a change of optical density (OD) of 0.2 was mixed with either ovarian extract (O-300 pl), human follicular fluid (0 to 50 ul, diluted 1 :lOO), or column fractions (O100 I, discussed below) and preincubated for 1 h at 37 C. The activity of the Pully active metalloproteinase and its inhibition by aliquots of ovarian samples were quantitated by measuring the absorbance at 520 nm. Inhibitor activity was expressed as the percent inhibition of the total added uterine metalloproteinase activity. Total ovarian inhibitor activity was expressed as inhibitor units per ovary. An inhibitor unit takes into account the differences in percent inhibition between different assays and standardizes the percent inhibition against the enzyme activity (change in optical density). One inhibitor unit is the amount of ovarian sample resulting in a decrease in optical density of 0.05. Metalloproteinase

inhibitor purification

To determine whether follicular fluid contained metalloproteinase inhibitors, individual follicular fluids (2 ml) were chromatographed by gel filtration on a Sepharose 68 column (1.6 x 90 cm) equilibrated with assay buffer containing 1 M NaCl at 4 C. Fractions (3 ml) were dialyzed against assa buffer and inhibitor activity determined. A lar e molecular weight pea IT, approximately 700 kDa-protein, and a secon3 , smaller molecular weight peak, approximately 29kDa-protein, of inhibitor activity were observed. The small MW inhibitor was at-tially purified accordin to the protocol of Cawston and coworkers P12) to determine whet R er the inhibitor demonstrated the roperties of TIMP. Follicular fluid samples from an individual patient PN = 3) were pooled (8-12 ml), diluted 1:2 with assay buffer, applied to an Ultro el AcA-54 (LKB Instruments, Gaithersbur , MD) column (5 x 60 cm), an !I 15 ml fractions collected. Aliquots of eatYl

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fraction (50-100 PI) were assayed and fractions containin the small molecular weight inhibitor (second peak) pooled. The sma4I molecular weight inhibitor was further purified by lectin affinity chromatography. The fractions from the gel filtration column containing inhibitor activit were ap lied to a Concanavalin A-Sepharose (Pharmacra, Piscataway, NJ1 column P1.6 x 4 cm) and washed with assay buffer containing 1 mM MgClz and MnCl2 until the A280 of the eluent was equal to that of the column buffer. The inhibitor was eluted with a step gradient of column buffer containing 0.2 M I-0-methyl-a-D-glucop ranoside and 2-ml fractions collected. After dialysis against assay bu yrer, 100-200 PI aliquots were assa ed and the fractions with inhibitor activity were pooled. The inhi L itor was further purified by Heparin-Sepharose (Pharmacia) chromatography. Active fractions from the Concanavalin A-Sepharose column were dialyzed against 25 mM sodium cacodylate with 10 mM CaCl2 (pH 7.2) and applied to a 1 ml Heparin-Sepharose column. The column was washed with 25 ml of sodium cacodylate buffer and eluted with a linear salt gradient (O-l M NaCI). Inhibitor activity was measured in 150-200 ~1 aliquots of the dialyzed fractions. All columns were run at 4 C and all buffers contained the surfactant, 0.05% Brij 35, and NaN3 (0.02%) as a preservative. Sodium dodecvl sulfate sel electrophoresis The purity of the inhibitor fractions at each sta e of purification were monitored by sodium dodecyl sulfate (SDS) gel e sectrophoresis. Samples were denatured by heating to 100 C for 5 min in 65 mM Tris, 2% SDS, 25Oh lycerol, 1 mM EDTA, 4.75 mM dithiothreitol, and 0.05% bromophenol I!!lue (pH 6.8). Follicular proteins were separated by electrophoresis on 12% SDS-polyacrylamide gels (17) and counterstained with silver (18). Collaaenase assay Collagenase activity was determined by the degradation of 3H-labeled, telopeptide-free, type I collagen after incubation with the enzyme sample for 40-46 h at 30 C as described previously (19). The collagenolytic fragments were separated by precipitation in an equal volume of cold 20% trichloroacetic acid and pelleted by centrifugation. A 100 ul aliquot of the supernatant was placed in scintillation cocktail and counted. Since the ma’ority of collagenase present in ovarian extracts is in an inactive form ( 19). assays were performed in the presence of 0.5 mM aminophenylmercuric acetate (APMA), an activator of latent collagenase, to examine enzyme activity. Collagenase activit is reported as percent di estion which is calculated as [(digest - blan l!)/(total amount of 3H co9lagen-blank)] x 100 and where the blank represents 100 ul of assay buffer. A digestion of 2% equals 7.9 ng of collagen digested/h at 30C and represents the limits of detection of the assay. Northern analysis RNA samples from human granulosa cells (7 Pg) were denatured with glyoxyl, electrophoresed in 1.O”hagarose gels, and transferred onto nylon

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membranes (Bio-Rad, Richmond, CA) using standard procedures (15). The cDNA probes for tPA, urokinase, and PAI- were labeled b nicktranslation to a specific activity of 1.0-2.3 x 108 cpm/pg using [a-3 YP]dCTP Filters were hybridized with (New En land Nuclear, Boston, MA). radiolabe $ed cDNA probes according to the recommendations of the manufacturer (Bio-Rad). Autoradio raphs were analyzed with a LKB UltroScan XL laser densitometer to caBculate the relative content of mRNA in various samples. Radioimmunoassay of estradiol and proaesterone Follicular fluid levels of 17B-estradiol (E2) and progesterone were determined by radioimmunoassay with diagnostic commercial Coat-ACount kits (Diagnostic Products Corporation, Los Angeles, CA) as reported previously (13). Statistics An analysis of variance (ANOVA) was used to test differences in mean inhibitor activity. If si nificance was obtained with ANOVA, the StudentNewman-Keuls proce 3 ure was used to test for group comparisons (20). RESULTS Metalloproteinase

inhibitor and collaaenase extraction

The inhibitor activity in rat ovarian homogenates following Triton, no heat, or heat extraction is shown in Fig. 1A. The Triton supernatant had hi h levels of inhibitor activity which were unchanged following re1 uction and alkylation. Inhibitory activity was also present in the no heat and 60 C heat supernatants at similar levels. Treatment of the latter two extracts with reduction and alkylation, however, decreased (p
inhibitor activity after hCG treatment

Chan es in inhibitor activity in rat ovaries collected at 0,4,8, 12, and 20 h after 4 CC administration were measured in 100~pl aliquots of the heat

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supernatant with or without reduction and alkylation. The heat supernatant was examined to determine inhibitor activity in the same extract that had been previously utilized for the measurement of

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Fi ure 1A. Metalloproteinase inhibitor activity in extracts of rat ovaries collected 8 h a I?er hCG. Inhibition of uterine metalloproteinase activity is shown in lOO-pl aliquots of the Triton, no heat, or heat supernatants either untreated or reduced (2 mM dithiothreito!, 30 min at 37 C) and alkylated (5 mM iodoacetamide, 30 min at 37 C) to destroy inhibitor activity. All values represent the mean f SEM, N = 3.

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Figure 1B. Collagenase Collagenase activrty in treated with or without mM APMA is shown. All

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activity in extracts of rat ovaries collected 8 h after hCG. lOO-pl aliquots of the Triton, no heat, or heat supernatants reduction and alkylation and subsequently activated with 0.5 values represent the mean f SEM, N = 3.

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collagenase activity (19). Four h after hCG administration, there was a 1.7-fold increase (p
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HOURS Fi ure 2. Changes in inhibitor activity in extracts of rat ovaries collected at various times a R er hCG admmistration. Inhibitor activity is expressed as percent inhibition of the uterine metalloproteinase activity by a lOO- pl aliquot of the ovarian heat supernatant or as the total inhibitor units (defined in Materials and Methods) in the heat supernatant per ovary. All values represent the mean f SEM. N = 5. WA represents reduction and alkylation described in Materials and Methods. Inhibitor

activitv in human follicular fluid

Metalloproteinase inhibitor activity was observed to be present in all of the follrcular fluids (N = 25). The average inhibition was 0.74 + 0.05 inhibitor units per 0.5 pl aliquot and 51.6 f 9.0 units per individual

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follicle. Inhibitor activity ran ed from 0.34 to 1.57 units per 0.5 pl aliquot and 5.4 to 230.1 units per 3ollicle. Reduction and alkylation of the follicular fluid to destroy the inhibitor resulted in an average 75% decrease (p
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Figure 3. Correlation between inhibitor activity (expressed as inhibitor units defined in Materials and Methods) and estradiol concentration per follicle in human follicular fluid. Flurds were either untreated or reduced with dithiothreitol followed by alkylation with iodoacetamide to destroy inhibitor activity. Follicular fluids were diluted 1: 100 and a 50 I aliquot was mixed with uterine metalloproteinase (25 pl) in a total volume of 400 pl ! or 1 h before initiating the calorimetric inhibitor assay described in Materials and Methods. Regression lines for untreated fluids (dashed line), reduced and alkylated flurds (dotted line), and inhibitor activity susceptible to reduction and alkylation (i.e., untreated: reduced and alkylated, solid line) are shown. Individual values (solid circles), correlation coefficient, and p value are given for inhibitory activity susceptible to reduction and alkylation (solid line).

Inhibitor purification An initial separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitor activity; the large molecular wei ht inhibitor had an approximate MW of 700 kDa while the sma ?ler inhibitor was approximately 29 kDa in size (data not shown). A partial purification of the small MW inhibitor was performed to determine whether the inhibitor demonstrated the properties of TIMP. Pooled follicular fluid

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Figure 4. Gel filtration of pooled follicular fluid. Pooled follicular fluid (8-12 ml) from an individual patient was chromatographed on an Ultro el AcA54 column (5 x 60 cm). The sample was eluted at a flow rate of 25-28 ml/h an 8 15-ml fractions collected. 100 ~1 aliquots were assayed for inhibitor activity (filled circles). Eluted protein is shown by the change in absorbance at 280 nm (Azeo, open circles). CA = carbonic anhydrase, MW = 29,000; Cyto C = cytochrome C, MW = 12,500. 100

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Figure 5. Concanavalin A-Sepharose chromatography of follicular fluid. Pooled fractions from the Ultrogel AcA54 column (42-50 ml from fractions 32-38) were loaded on to a Concanavalin A-Sepharose column (1.6 x 4 cm) at 20 ml/h. Adsorbed protein was removed by washing with column buffer containin 8 0.2 M l-O-methyl o-Dglucopyranoside (indicated by the arrow). 125~pl aliquots rom the 2-ml fractions were assayed for inhibitor.

applied to an Ult;ogel AcA-54 column eluted a small molecular weight inhibitor in a broad peak that was relatively free of protein (Fig. 4). The fractions containing inhibitor activity were pooled and applied to a Concanavalin A-Sepharore column (Fig. 5). Fractions with inhibitor activity were combined, applied to a Heparin-Sepharose column, and eluted with a linear salt gradient (O-l .O M NaCI) as illustrated in Fig. 6.

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Fraction f%nber Figure 6. Heparin-Sepharose chromate raphy of follicular fluid. Pooled fractions from the Concanavalin A-Sepharose column P10-l 5 ml from fractions 25-30) were loaded on to a BioRad Econocolumn (1.6 x 4 cm). The column was washed with 25 ml of sodium cacodylate buffer at 20-22 ml/h and adsorbed proteins were eluted with a 0 to 1 M Nat1 gradient. Arrow indicates the beginning of the NaCl gradient. Inhibitor activity was measured in 150-200-pl aliquots of the 2-ml fractions.

Sodium dodecvl sulfate ael electrophoresis The purification of the inhibitor was monitored by SDS-gel electrophoresis (Fig. 7). The Heparin-Sepharose stage resulted in significant purification as demonstrated by a major protein with an approximate molecular weight of 28,500 (lane 4). RNA analysis In addition to collagenase and its associated inhibitors, we determined whether human preovulatory granulosa cells contain plasmino en activator mRNA. Total granulosa cells RNA from pooled follicles of 8 ive patients following exogenous gonadotropin stimulation was obtained as described previously (21). The RNA samples were hybridized with a radiolabeled human tPA cDNA probe (21) and analyzed by autoradiography. As shown in Fig. 8 (left panel), the presence of a band was detected after prolonged exposure in sample F. This signal represents the strongest signal observed in the granulosa cell samples. Since the lack of detectable granulosa tPA mRNA mi ht be attributed to a dilution effect resulting from either poolin ranu 9osa cells of different follicles or including mRNA from immature ?? 01 icles, we examined the tPA mRNA from 8 individual mature follicles. Tissue-type plasminogen activator mRNA was

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Figure 7. SDS-gel electrophoresis of aliquots from representative column fractions during artial purification of the small molecular wei ht inhibitor. Follicular fluid was applre dp to an AcA 54 gel filtration column (descrr% ed in Materials andMethods). Fractions with inhibitor activity (lane 2) were further separated on Concanavalin ASepharose (lane 3) and Heparin-Sepharose (lane 4). The major protein from the HeparinSepharose column has an approximate molecular weight of 28,500 (lane 4). Molecular weight standards (lane 1) are as follows: 8A = bovine albumin, MW = 66,000; EA = egg albumin, MW = 45,000; CD = glyceraldehyde-3-phos hate, MW = 36,000; CA= carbonic anhydrase, MW = 29,000; T = trypsinogen, MW = P4,000; and TI = trypsin inhibitor, MW = 20,100.

not detectable in individual follicles even though these follicles contained mature oocytes (data not shown). As a positive control, we anal zed the tPA mRNA content from a human breast cell line, HBL-100, w Kich was electrophoresed with the granulosa cell RNA samples. A stronger signal (about 4-fold) was detected from untreated HBL-100 cells (Fig. 8, right panel) than from granulosa cells. The size of the species detected with the human tPA cDNA probe is approximately 2700 nucleotides which is consistent with the size of human tPA mRNA (22). Dexamethasone treatment stimulated the accumulation of tPA mRNA in HBL-100 ceils by about 2-3-fold as previously reported (23). To examine the possibility that human granulosa cells contain the other form of plasminogen activator, urokinase, the RNA samples were hybridized with a human urokinase cDNA probe (24). As shown by Busso and coworkers (23), urokinase mRNA was detectable in HBL-100 RNA but it was not detectable in granulosa cell RNA (data not shown).

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c

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HBL-100

DEX Figure 8: Filter hybridization analysis of tPA mRNA in human granulosa cells and in human mammary epithelial cells (HBL-100). Northern analysis of total RNA was performed as described in Materials and Methods. The nylon membrane was exposed to Kodak XAR-5 film at -70 C for 14 h, using an intensifying screen. Size of RNA markers are indicated in kilobases. Left Panel: Human granulosa cell RNA from 2 of the 6 patient samples (samples E and F). Right Panel: Human mammary epithelial cell RNA, obtained from cells incubated in the absence or presence of 0.1 pM dexamethasone (DEX).

ABCDEF

Figure 9. Filter hybridization analysis of PAI- mRNA in human granulosa cells. Total RNA was prepared and analyzed as described in Materials and Methods. The nylon membrane was hybridized to a radiolabeled human PAI- cDNA probe and exposed to Kodak XAR-5 film at -70 C for 8 h, using an intensifying screen. Size of RNA markers are indicated in kilobases.

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To determine whether human granulosa cells have PAI-I mRNA, a human PAI-I cDNA probe (25) was used to examine granulosa cell expression of PAI- mRNA. As shown in Figure 9 (top panel), the PAI- EDNA probe detected a 3400 nucleotide species in all samples (A-F). Sample B contains a second species (about 2400 nucleotides) which is detectable in the other samples after prolonged exposure. Thus, onadotropin-stimulated human granulosa cells from preovulatory folBicles contain little or no detectable PA mRNA, whereas PAI- mRNA is present in total RNA from granulosa cells obtained from either pooled follicles or in single follicles. DISCUSSION The preovulatory LH surge results in an increase in the activity of both PA and collagenase (reviewed in l-3). The increase in proteolysis has been proposed to play a crucial role in the degradation of the apical connective tissue matrix and facilitate release of the oocyte (3,19,26). Although the stimulation of proteolysis by LH is well documented, subsequent control of follicular collagenolysis is unknown. Paradigms from other tissues, such as cartilage (16), skin (10,27), and bone (12,28), sug est that colla enase activit in the extracellular space is regu ?ated by metal 9oproteinase in z ibitors. We have previously observed a marked increase in collagenase activity from ovarian extracts following a procedure reported to destroy collagenase inhibitors, however, inhibrtor activity was not determined (19). These results suggested the presence of an ovarian metalloproteinase inhibitor which may act as an important modulator of enzyme activity. The present findings demonstrate that metalloproteinase inhibitors are present in both rat and human ovaries. In human follicular fluid, there is a correlation between increasing inhibitor activity and both estradiol and progesterone concentrations and thus, the stage of follicular maturation. In the rat, inhibitor activity increases during the preovulatory period. The inhibitor levels in rat ovarian extracts In the present study parallel previous findings of a preovulatory peak in collagenase activity at 8 h after hCG before declining at 20 h post-hCG treatment (19). An increase in inhibitor activity during the preovulatory period when collagenase is maximal is not unprecedented. In fibroblasts (1 l), endothelial cells (29). and osteoblast-like cells (28), the cell is capable of synthesizing both the enzyme and inhibitor simultaneously. Concomitant increases in collagenase and colla enase inhibitor activity have been roposed to maintain proteolytic Yl omeostasis and provide localize 8 control of extracellular degradation (9,11,28). In the ovary, a similar paradigm may For example, rat granulosa cell culture regulate collagen degradation. media contains colla enase-like activity (30,31). Following an LH stimulation of isolate 8 rat granulosa cells, there is a two-fold increase in inhibitor activity (32). Thus, the ovary may s nthesize both collagenase and a collagenase inhibitor during the preovu ratory period. The extracellular regulation of metalloproteinases in other tissues occurs by both serum-borne and tissue-derived inhibitors (5). Evidence from the gel filtration experiments indicates that both classes of inhibitor may be

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present in human follicular fluid. The large molecular weight inhibitor has many of the physiochemical characteristics of the serum-borne inhibitor, a2-macroglobulin (13). Although az-macroglobulin accounts for 95% of the collagenase inhibitory activity in serum, the role of this inhibitor in regulating metalloproteinase activity in the extracellular space has been questioned on the basis of its large size. However, such inhibitors may have a physiological role in modulating collagenolysis both prior to and following ovulation. As ovulation approaches, there is an increase in ovarian blood flow which would result in elevated levels of serum inhibitors in the ovary. Additionally, the permeability of the follicular capillary bed and the ranulosa cell basement membranes increases. The elevated blood 7 low in conjunction with increased permeability of capillary and basement membranes would permit extravasation of inhibitors into the extracellular matrix, diffusion into the follicular fluid, and thereby govern ovarian collagenase activity. Tissue-derived inhibitors may also regulate ovarian metalloproteinases. The small molecular weight inhibitor in human follicular fluid is a glycoprotein with approximate molecular size of 28,000 and similar in the physiochemical properties to TIMP, a tissue-derived inhibitor (12). Although the present studies do not attempt to elucidate the cellular origin of the inhibitor, granulosa cells may be one source of the follicular fluid TIMP-like inhibitor. Rat granulosa cells produce a metalloproteinase inhibitor in vitro, although the nature of the inhibitor has not been identified (32). In the present study, inhibitor activity in follicular fluid is at least IO-SO-fold higher than in serum from the same patient ~unpublish~ observation). The high levels of inhibitor activity ma reflect either synthesis of the inhibitor by an ovarian compartment suehy as granulosa cells or perhaps differences in clearance between follicular fluid and serum. We would propose, however, that ranulosa cells produce TIMP based upon both previous (32) and present 7indings as well as our recent observation that human granulosa cells express TlMP mRNA (unpublished data). Evidence from the present study suggests that the ovarian metalloproteinase inhibitor acts on collagenase. Treatment which inactivates the inhibitor increases collagenase activity in rat ovarian extracts. Also, human follicular fluid aliquots containing inhibitor activity inhibit both a partially purified active uterine collagenase and an active rat ovarian collagenase (13). These observations strengthen the concept that follicular fluid metalloproteinase inhibitors act on collagenase as well as other metalloproteinases, such as gelatinase or proteoglycanase, which may be present in the follicle. In concert, both endogenous (tissue-derived) and exogenous (serumborne) metalloproteinase inhibitors may modulate ovarian collagen degradation. Although the preovulatory increase in inhibitor activity parallels the collagenolytic changes, follicular de radation may occur due to an excess of the enzyme in relation to the inhi% itor only at the apex of the follicle. If cotta enase is being produced at the follicular apex as previously suggeste % (3,331, extravasation of serum-borne inhibitors

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would bind any free active enzyme not attached to apical collagen and thus regulate the site of collagenolysis. Enzyme activity may proceed until the follicular wall is degraded, at which time inhibitors being produced in a se arate compartment, such as the granulosa cell layer, would be ex osedpor mixed with apical collagenase. Additionally, there is rupture oft c:e follicular capillaries during ovulation resulting in further extravasation of serum-borne inhibitors (34). Thus, serum-borne and tissue-derived inhibitors may act to regulate the location and rate of follicular collagenolysis. Extracellular apical degradation may also involve the PA/plasmin system. In the rat, both ovarian PA activity and PA mRNA levels increase durin the preovulatory period (35-37). Gonadotropins appear to stimulate bot i! tPA and urokinase enzyme activities in rat ovarian cells (35,38). In granulosa cells, tPA enzyme activity is elevated by increasin the cellular concentration of tPA mRNA (37,39) as well as by decreasing tR e activity of a specific PA inhibitor (36). In the thecal cell compartment, urokinase is the predominant PA produced (38). Although the majority of the literature supports a role for the plasminogen activator/plasmin system in follicular rupture in the rat, the precise action of PA is unclear. Both in vivo (35) and in vitro (40) studies indicate that the role of the PA/plasmin system may be to activate colla enase. Palotie and coworkers (40) observed that PA enzyme activity in cuatured rat ovarian follicles increased at 2 h after hCG treatment and decreased by 4 h (possibl due to inhibition by PAI), while colla enase activity increased by 6 h. J vulation will take place at 12-14 h after ‘F,CC in this model. The injection of e-aminocaproic acid (an inhibitor of serine proteases such as PA and plasmin) to gonadotropin-treated rats prevented collagenolysis and ovulation when the drug was administered up to 2 h after hCG treatment (35). Similarly, administration of an inhibitor of PA, trans-4-(aminomethyl)cyclohexanecarbox lit acid, to perfused rat ovaries effectively inhibited ovulation O-3 z after hCG stimulation, but did not prevent ovulation when added at 5 or 7-9 h after hCG (41). In contrast, the administration of cysteine, an inhibitor of metalloproteinases such as collagenase, inhibited collagenolysis and ovulation 7 h after hCG treatment (26). Collectively, these observations suggest that PA is activated soon after the ovulatory stimulus and that this activation of the PA/plasmin system is followed in part by the activation of collagenase. Nevertheless, the inhibition of ovulation with indomethacin or colchicine does not block the preovulatory increase in ovarian PA activity, suggesting that PA does not represent the critical proteolytic enzyme required for follicular rupture (42). There are apparent species differences in PA/plasmin system. In contrast to the rat, Cani ari and coworkers (43) have demonstrated that mouse granulosa cells Prom preovulatory follicles contain urokinase mRNA and produce urokinase, rather than tPA, in response to gonadotropins. The results from the present study indrcate that the human granulosa cell PA/plasmin system differs from both the reported rat and mouse systems. Human granulosa cells from pooled preovulatory follicles contain almost

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levels of mRNA for tPA or urokinase. Furthermore, human cells contain abundant mRNA for PAI-1, which specifically and

undetectable

granulosa

rapidly inhibits both tPA and urokinase by the formation of a 1: 1 complex of the inhibitor and PA (44). Based upon the levels of mRNA for PA and PAI observed in the present study, very little active PA would be anticipated in human follicular fluid 2 to 4 h prior to ovulation. This concept is supported by our recent finding of a lack of measurable PA activity in human folhcular fluid from preovulatory follicles due to presence of an endogenous inhibitor (45). Thus, the differences in the ovarian PA/plasmin system in human and rat preovulatoty follicles may be related to differences in the period between LH stimulation and follicular rupture. In the human, ovulation takes place approximately 36 h after hCG administration, whereasfollicular rupture occurs 12-14 h after hCG in the rat. It is possible that in the human, PA activity increases after the LH surge, activates other proteases (such as collagenase), and declines prior to ovulation. In summary, the periovulatory ovary contains inhibitors for both collagenase and PA. Metalloproteinase inhibitor activity is elevated during the periovulatory period and this activity is apparently composed of both serum- and tissue-derived inhibitors. For PA, there is an abundance of PA inhibitor mRNA, whereas tPA and urokinase mRNA are almost undetectable. Thus, ovarian protease inhibitors may regulate connective tissue remodeling associated with follicular rupture. ACKNOWLEDGMENTS This manuscript represents both original, unpublished data and previously published information presented at the William J. LeMaire Reproductive Symposium, December 2,1988, Miami, Florida. The authors would like to thank Williams and Wilkins for the release of copyright information which has been included in this article. The didactic criticism of Dr. 1. Frederick Woessner throughout our studies on ovulation is kindly appreciated. The authors would like to thank Dr. Michael Vernon for performin the steroid RIA analysis. The expertise of Dr. Kathy Sharpe, Dr. Colom %o Giovanna, and Ms. Gail Sievert in rotein separation by SDSel electrophoresis is ratefully acknowledge I! . The authors would also Bike to ratefully than & Mr. Scott Estes, Ms. Marie Selzer, and Ms. Carol n Taplin 3or their technical assistance and Ms. Darleen Chamberlain fort Ke preparation of this manuscript. This work was supported by NIH grants HD-08747 and HD-06773 and by BRSGRR05374. Also, a note of tribute to Dr. David Puett for undertaking this compilation of scientific information which has been influenced by Dr. LeMaire. In closin a very heartfelt thank you to “Wim” for stimulating my personal an c3‘scientific growth (TEC).

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