/04/$15.00/0 Endocrinology 145(4): Copyright 2004 by The Endocrine Society doi: /en

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1 /04/$15.00/0 Endocrinology 145(4): Printed in U.S.A. Copyright 2004 by The Endocrine Society doi: /en Tissue-Type Plasminogen Activator and Its Inhibitor Plasminogen Activator Inhibitor Type 1 Are Coordinately Expressed during Ovulation in the Rhesus Monkey YI-XUN LIU, KUI LIU, QIANG FENG, ZHAO-YUAN HU, HAI-ZHEN LIU, GUO-QING FU, YIN-CHUAN LI, RU-JIN ZOU, AND TOR NY State Key Laboratory of Reproductive Biology (Y.-X.L., Q.F., Z.-Y.H., H.-Z.L., G.-Q.F., Y.-C.L., R.-J.Z.), Institute of Zoology, Chinese Academy of Sciences, Beijing , China; and Department of Medical Biochemistry and Biophysics (K.L, T.N.), Umeå University, SE Umeå, Sweden OVULATION, TRIGGERED BY a surge of LH released from the pituitary and recurring every reproductive cycle in female mammals, is an essential prerequisite for fertilization and subsequent embryonic development (1 4). A mature follicle that is destined to ovulate usually protrudes markedly from the surface of the ovary, and for the ovum to escape from this structure, an extensive breakdown and remodeling of basement membranes and connective tissues that constitute the follicular wall is required (1 5). Factors leading to the occurrence of this very complex process have fascinated biologists for many years. Several lines of evidence have suggested that plasmin-mediated proteolysis plays a role in the breakdown of the follicle wall during ovulation in rodents (for reviews, see Refs. 1, 4, 6 9). The plasminogen activator (PA) system is a versatile, temporally controlled enzymatic system that comprises plasminogen, which is activated to the proteolytic enzyme plasmin by either of the two physiological PAs, tissue-type PA Abbreviations: FF, Follicular fluid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GC, granulosa cell; hcg, human chorionic gonadotropin; PA, plasminogen activator; PAI, PA inhibitor; PMSG, pregnant mare serum gonodotropin; SDS, sodium dodecyl sulfate; SSC, NaCl and Na citrate; TIC, theca-interstitial cell; tpa, tissue-type PA; upa, urokinase-type PA. Endocrinology is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community. Ovulation is a gonadotropin-controlled process that is essential for the propagation of all mammalian species. In the present study, we used a pregnant mare serum gonodotropin/ human chorionic gonadotropin (hcg)-induced, synchronized ovulation model in rhesus monkeys and systematically investigated the roles of the plasminogen activator (PA) system in the ovulatory process of the primate. At different follicular developmental stages throughout the periovulatory period, samples of ovaries, granulosa cells, and theca-interstitial cells as well as follicular fluid were collected, and levels of PA and PA inhibitor type-1 (PAI-1) were evaluated by fibrin overlay, reverse fibrin overlay, Northern blot analysis, and in situ hybridization, respectively. We showed that in response to an injection of ovulation-triggering hcg, which mimics the preovulatory surge of LH in the circulation, granulosa cellderived tissue-type PA (tpa) was substantially elevated in preovulatory follicles and reached its maximum level just before ovulation. Although theca-interstitial cell-derived PAI-1 was also stimulated by pregnant mare serum gonodotropin and hcg treatments, however, the maximum level of PAI-1 appeared 12 h earlier than that of tpa. When ovulation approached, accompanying the highest tpa level in the preovulatory follicles, the follicular PAI-1 level declined dramatically to its minimum value. Moreover, our data on the expression of follicular PA and PAI-1 over the periovulatory period were reinforced by results obtained at the mrna level. Our data suggest that the coordinated expression of tpa and PAI-1 may be of importance for the follicular rupture process during ovulation in the primate. (Endocrinology 145: , 2004) (tpa) and urokinase-type PA (upa). Activation of this system is initiated by release of tpa or upa by specific cells in response to external signals such as hormones, growth factors, or cytokines that lead to locally expressed extracellular proteolytic activities (1, 4, 9, 10). The PA system is also regulated by specific inhibitors directed against PAs and plasmin, including PA inhibitor (PAI) type-1, PAI-2, protease nexin 1, and 2 -antiplasmin (4, 9, 11). Many studies have suggested that the PA system plays a crucial role in the degradation of the follicular wall during ovulation in rodents. In rats, ovulation is preceded by a transient and cellspecific expression of tpa and PAI-1, which causes a proteolytic activity localized to the surface of the ovary just before ovulation (for reviews, see Refs. 1, 4, 8, 9). Studies with intrabursal injections of 2 -antiplasmin or antibodies against tpa partially block gonadotropin-induced ovulation in rats (12). Our previous results with gene-deficient mice have demonstrated that tpa and upa double-knockout mice have a 26% decreased ovulation rate (13). However, plasminogen knockout mice showed a normal ovulation rate, indicating that plasmin is not required for efficient follicular rupture or for activation of other proteases involved in the mouse ovulatory process. Alternatively, the role of plasmin may be effectively compensated for by other mechanisms in the absence of plasmin in the mouse ovary (14). Thus, the role of the PA system in ovulation is still not fully 1767

2 1768 Endocrinology, April 2004, 145(4): Liu et al. PA System in Monkey Ovulation understood. Little is known about whether the PA system participates in the ovulation process in the primate. Answering this question has been somewhat hindered by limited availability of primate ovarian materials that represent the entire periovulatory process. In this study, we used a gonadotropin-induced, synchronized ovulation model in rhesus monkeys and collected ovarian samples at different follicular developmental stages throughout the periovulatory process. The expression and functions of tpa, upa, and their physiological inhibitor PAI-1 were investigated in follicles during the ovulatory process. Materials and Methods Rhesus monkeys (Macaca mulatta) Fifteen prepubertal (2 2.5 yr old) and 13 adult (5 7 yr old) female rhesus monkeys were obtained from the Primate Research Center of Kunming Institute of Zoology, Chinese Academy of Sciences (Kunming, China). The use of monkeys for this study was approved by the Academic Committee of the Institute of Zoology, Chinese Academy of Sciences, and a World Health Organization Review Committee. As experimental animals, rhesus monkeys have long pregnancy periods and low birth rates, and their availability can be limited from time to time. Thus, in this study both adult and prepubertal monkeys were used. Our previous studies (15 17) have shown that follicles from both prepubertal and adult monkeys can grow and ovulate upon gonodotropin stimulation. The only difference is that adult monkeys have fewer, and comparatively smaller, developing follicles (4 10 follicles of 3 8 mm in diameter per ovary), compared with those of prepubertal monkeys (15 20 follicles of 5 8 mm in diameter per ovary). Ovaries from prepubertal monkeys can therefore generate more granulosa cells (GCs) and theca-interstitial cells (TICs). Also as a result of their availability, we used prepubertal monkeys for the protein level analyses shown in Figs. 1 5 and adult monkeys for mrna level analyses in Figs. 6 and 7. When adult female monkeys were used, they were treated with gonadotropins only during the reproductive season (from October to February in Kunming, China) when their ovaries were capable of responding to exogenous hormone treatment To induce ovulation, animals were treated with a pregnant mare serum gonodotropin (PMSG)/human chorionic gonadotropin (hcg) based on a regimen developed by the Wisconsin Regional Primate Research Center (15 18). After an initial im injection of 950 IU PMSG on d 1 (which was any day for the prepubertal monkeys and the second day of menstrual bleeding for the adult monkeys), 300 IU PMSG was administered on d 2, 4, and 6, followed by daily injections of 300 IU PMSG ond8 12. On d 13, a single im injection of 4000 IU hcg was administered. At various time points after hormone treatment, follicular sizes were monitored during surgery when they were collected for experiments. Typically, follicles before PMSG treatment were very small with diameters less than 0.8 mm in both prepubertal and adult monkeys. After the 12 d of treatment with PMSG, developing follicles normally reached 5 8 mm in diameter in prepubertal monkeys and 3 8 mmin adult monkeys. Further treatment with hcg did not alter the follicular sizes significantly. Typically, at a time point of 36 h after hcg injection, we observed large preovulatory follicles full of follicular fluid protruding from ovaries, which were very close to the start of ovulation. Also, we observed newly formed corpora lutea full of blood h after hcg injection. Thus, it was fairly obvious that ovulation in our gonadotropininduced monkeys took place h after hcg treatment. After 12 d of PMSG injection (representing the stage of developed follicles) and 12, 24, and 36 h of hcg injection (representing the preovulatory stage), the animals were maintained under ketamine hydrochloride-induced anesthesia, and the ovaries were removed by abdominal surgery in a standard operation room. Reagents and hormones McCoy s 5A medium (modified, without serum), penicillin, streptomycin, l-glutamine, HEPES, trypan blue, plasminogen, and thrombin were obtained from Sigma Chemical Co. (St. Louis, MO). Acrylamide, N,N-methylenebisacrylamide, sodium dodecyl sulfate (SDS), TEMED, ammonium persulfate, and Coomassie brilliant blue were purchased from Bio-Rad Laboratories (Richmond, CA). PMSG was obtained from Changchun Institute of Biological Products (Changchun, China), and hcg was obtained from the State Key Laboratory of Dr. Yushu Xu (Institute of Zoology, Chinese Academy of Sciences, Beijing, China). Purified human tpa, upa, goat antihuman tpa, and goat antihuman PAI-1 antibodies were obtained from Biopool (Umeå, Sweden). Goat polyclonal anti-upa antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fibrinogen was obtained from Calbiochem (La Jolla, CA). Preparation of GCs, TICs and follicular fluid Monkey ovaries at different stages were dissected by surgery and rinsed in McCoy s 5A medium containing penicillin and streptomycin. For preparation of GCs, developed or preovulatory follicles were punctured with 25-gauge hypodermic needles, and the released cells were collected by centrifugation. After removing GCs, the residual ovaries were washed thoroughly in McCoy s 5A medium to remove free cells, transferred into McCoy s 5A medium containing 0.08% collagenase, and incubated at 37 C for 10 min. The dissociated GCs were discarded and the residual ovarian fragments were further incubated in McCoy s 5A medium containing 0.4% collagenase at 37 C for approximately 1 h to yield TICs. The cells were washed twice with culture medium, and viability was determined by trypan blue exclusion. As revealed by microscopy, the GC and TI cell preparations were essentially pure, especially the GCs. To determine the purity of the prepared GCs and TICs more accurately, we cultured the cells in the presence of androgen, which is a substrate for estrogen synthesis by GCs. Because TICs do not express aromatase, they cannot synthesize estrogen from androgen. This is in contrast to GCs, which express aromatase and can therefore convert androgen to estrogen (19). Judging from the estrogen production by the cultured GC and TIC preparations, we estimated that the isolated TICs contain less than 5% GC contamination. Aliquots of each preparation of GCs or TICs containing cells or cells were frozen at 80 C for further analysis of PA and PAI-1 activities or mrna content, respectively. Follicular fluid (FF) was also collected from preovulatory follicles 12, 24, and 36 h after hcg treatment. Cell cultures GCs and TICs ( cells/well) were seeded in 24-well culture plates (Corning Glass Works, Corning, NY) containing 0.5 ml/well of serum-free McCoy s 5A medium supplemented with l-glutamine (2 mm), penicillin (100 U/ml), and streptomycin sulfate (100 g/ml). The cells were cultured in 95% air and 5% CO 2 at 37 C. After 24 h of incubation, the conditioned media were harvested, centrifuged, and stored at 20 C for measurement of tpa and PAI-1 activities. SDS-PAGE PA and PAI-1 antigens in cell culture medium and cell lysates were fractionated by SDS-PAGE. Electrophoresis was performed at 50 V overnight until the dye front reached the bottom of the gel. After electrophoresis, the gel was washed for 2 45 min in 2.5% (vol/vol) Triton X-100 solution to remove SDS in the gel and rinsed once with distilled water before being applied to the surface of a fibrin/agar indicator gel. Fibrin overlay and reversed fibrin overlay assays The fibrin-agar indicator gel for PA activity assay was prepared based on a method developed by Granelli-Piperno and Reich (20), with modifications. The fibrin-agar gel contained plasminogen (25 mg/ml), fibrinogen (2.4 mg/ml), and thrombin (0.5 U/ml) to allow the formation of fibrin as the substrate for PA-converted plasmin. For detection of PAI-1 activity in the gel, a reversed fibrin overlay assay was used (20). In addition to plasminogen, fibrinogen, and thrombin, the reversed fibrin-agar indicator gel contained 0.05 U/ml upa to allow autolysis of the gel. The emergence of opaque, lysis-resistant bands indicated the presence of PAI-1 activities in samples fractionated by the SDS-PAGE gel. The rinsed SDS gel was laid carefully onto the fibrin-agar gel and

3 Liu et al. PA System in Monkey Ovulation Endocrinology, April 2004, 145(4): incubated at 37 C in a humid chamber for about 16 h for PA activity assay and 4 5 h for PAI-1 activity assay. Western blot analysis of PAI-1 antigen For Western blot analysis, samples of follicular fluid or culture media were fractionated by SDS-PAGE. After electrophoresis, the proteins were transferred electrophoretically to nitrocellulose membranes and probed using a polyclonal goat antihuman PAI-1 antibody. Antibody binding was visualized using antigoat IgG antibodies conjugated with alkaline phosphatase. Synthesis of antisense RNA probes for detection of monkey tpa and PAI-1 mrna The riboprobes for rhesus monkey tpa and PAI-1 were prepared as previously described (16). Briefly, fragments of monkey tpa (346 bp) and PAI-1 (388 bp) cdna were obtained by RT-PCR and subcloned into pgem-3z vectors. Sequencing results revealed that the monkey tpa and PAI-1 cdna fragments had and 96.79% identity to the corresponding human cdna sequences, respectively. For Northern blot analysis, cdna templates were linearized so that the antisense crna probes could be labeled with 32 P-uridine 5-triphosphate with an in vitro transcription kit (Roche Diagnostics Corp., Indianapolis, IN). The specific activity of the probes ranged between and cpm/ g RNA. RNA preparation and analysis Total RNA from GCs or TICs was prepared by the Nonidet P-40 method. For hybridization analysis, total RNA was fractionated on 1% agarose gels in the presence of 2.2 m formaldehyde and transferred to nylon filters. After cross-linking under UV light, the filters were prehybridized at 62 C for 2hinasolution containing 50% formamide, 5 SSC (1 SSC 0.15 m NaCl, m Na citrate), 8 Denhardt s solution (1.6 mg/ml Ficol, 1.6 mg/ml polyvinylpyrrolidone, and 1.6 mg/ml BSA), 0.1% SDS, 10 mm EDTA, and 25 mm yeast trna. The hybridization was performed in the same solution with 32 P-uridine 5-triphosphate probes at 62 C overnight, followed by two to three washes in 0.1 SSC containing 0.1% SDS at 65 C for about 1 h. The hybridized filters were exposed to the screen of the PhosphorImager cassette (Molecular Dynamics, Sunnyvale, CA), and data were analyzed using PhosphorImager programs (Molecular Dynamics). The relative amount of mrna was determined by densitometric scanning of autoradiographs and normalized against levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the samples. In situ hybridization of tpa and PAI-1 mrnas Frozen sections (10 12 m) were prepared from adult monkey ovaries at various stages and in situ hybridization was performed as described previously (21). Slides to be used for comparison were prepared and hybridized at the same time. To monitor background levels and the specificity of the hybridization, the sense strands of the probes were included in each experiment. Photographs were taken with a camera (Carl Zeiss, New York, NY) attached to a Axioplan microscope (Zeiss) at a magnification 10. Data analysis All experiments were repeated at least three times. For each time point of a single experiment, ovarian materials from one rhesus monkey were used. Representative photography using dark-field illumination for PA and PAI-1 activities as well as mean sd of protein and mrna levels from three experiments were shown. Data were analyzed by ANOVA. Differences among groups were calculated by Tukey s multiple-comparison test (22), and a difference was considered to be significant with P Results Identification of two types of PA (tpa and upa) in the monkey ovary Ovarian lysate (50 g) obtained 12 h after hcg injection was immunoprecipitated with normal rabbit serum, goat antihuman tpa, or antimouse upa monoclonal antibodies. PA activities in the supernatant after immunoprecipitation were analyzed by SDS-PAGE/fibrin-overlay autography. As shown in Fig. 1, two types of PA with molecular weights equivalent to human tpa and upa (70 and 50 kda, respectively; data not shown) were observed in monkey ovarian lysate immunoprecipitated with normal rabbit serum. However, only upa was detected in ovarian lysate after immunoprecipitation with anti-tpa antibody, and only tpa was seen in ovarian lysate after immunoprecipitation with antiupa antibody. Gonadotropin regulation of tpa and upa activities in monkey GCs during the periovulatory period GCs obtained from follicles of prepubertal monkeys treated with PMSG/hCG were cultured for 24 h, and PA activities were analyzed in the conditioned media. As shown in Fig. 2A, conditioned medium from GCs after PMSG treatment contained a relatively low but measurable amount of tpa. Treatment with hcg dramatically increased tpa activity, which reached a high level just before ovulation (36 h after hcg injection). No upa activity was detected in GCs before ovulation (Fig. 2A, lanes PMSG, h after hcg injection). However, after ovulation (3 d after hcg injection), tpa activity in the luteinized GCs disappeared, whereas a dramatically increased upa activity was observed. Although tpa and upa are secreted proteins, we also measured the contents of tpa and upa in GC extracts. As shown in Fig. 2B, the tpa activities measured in GC lysate were similar to those secreted into the culture media. The activity of tpa was undetectable in GC lysate after PMSG treatment, whereas it increased to high levels before ovulation (12 36 h after hcg injection). Although upa was not detected in the conditioned medium of GC cultures before ovulation (Fig. 2A), hcg treatment stimulated the upa content of the GC and luteal cells, and the upa produced by luteal cells (3 d after hcg injection) was secreted into the culture medium (Fig. 2B). Gonadotropin regulation of upa and tpa activities in monkey TICs during the periovulatory period TICs obtained from prepubertal monkey follicles treated with PMSG/hCG were cultured for 24 h, and PA activities FIG. 1. Identification of tpa and upa activities in the monkey ovary. Ovarian lysate (50 g) obtained 12 h after hcg injection was immunoprecipitated with normal rabbit serum (S), goat antihuman tpa (Anti-tAb), or antimouse upa (Anti-uAb) monoclonal antibodies. PA activities in the supernatant after immunoprecipitation were analyzed by SDS-PAGE/fibrin overlay autography. The analysis was performed with a mixture of ovarian tissues from three PMSG/hCGtreated female monkeys.

4 1770 Endocrinology, April 2004, 145(4): Liu et al. PA System in Monkey Ovulation FIG. 2. Gonadotropin regulation of tpa and upa activities in monkey GCs during the periovulatory period. Prepubertal monkeys were treated with PMSG and/or hcg. The ovaries were removed at various times after gonadotropin treatment and GCs were prepared as described in Materials and Methods. GCs ( cells/time point) were either incubated at 37 C in 0.5 ml of McCoy s 5A medium for 24 h or directly frozen at 70 C for preparation of cell lysate. The PA activities in the conditioned media (A) or the cell lysates (B) were measured by fibrin overlay technique. PMSG: 12 d after PMSG treatment; 12 h, 24 h, 36 h, 3d: time points after hcg treatment. For each time point of an experiment, cells from the ovaries of one female monkey were used. Representative photograph from three independent experiments as well as average relative levels of tpa and upa activities (mean SD) of three experiments were shown. Small letters (a, b, c, and d) indicate significant differences (P 0.05). were measured in the culture medium. As shown in Fig. 3, A and B, TICs obtained from the monkey follicles treated with only PMSG produced undetectable quantities of tpa and upa. Only a small amount of tpa was produced by TICs just before ovulation (36 h after hcg injection). However, TIC secreted a considerable amount of upa activity after hcg induction, at a time point just before ovulation (36 h after hcg injection). FIG. 3. Gonadotropin regulation of upa and tpa activities in monkey TICs during the periovulatory period. Prepubertal monkeys were treated with PMSG and/or hcg. The ovaries were removed at various times after gonadotropin treatment, and TICs were prepared as described in Materials and Methods. TICs ( cells/time point) were incubated at 37 C in 0.5 ml McCoy s 5A medium for 24 h, and PA activities in the conditioned media were measured by fibrin overlay technique. PMSG: 12 d after PMSG treatment; 12 h, 24 h, 36 h, 3d: time points after hcg treatment. For each time point of an experiment, cells from the ovaries of one female monkey were used. A, A representative photograph from three independent experiments; B, Average relative levels of tpa and upa activities (mean SD) of three experiments. Small letters (a, b, and c) indicate significant differences (P 0.05). Gonadotropin regulation of PAI-1 secretion during the periovulatory period To study the secretion of PAI-1 by cultured GCs and TICs, cells obtained from prepubertal monkey ovaries at various times after gonadotropin treatment were cultured for 24 h and PAI-1 protein levels were measured in the culture medium. Cultured GCs did not secrete any detectable quantity of PAI-1 (data not shown). However, as shown in Fig. 4, A and B, the PAI-1 level in the TIC conditioned medium increased in a time-dependent manner after treatment with PMSG and hcg and reached a maximum level 24 h after hcg injection. Subsequently, as ovulation approached (36 h after hcg treatment), the levels of PAI-1 were remarkably reduced. The PAI-1 level increased again after ovulation in the corpus luteum (3 d after hcg treatment). PA and PAI-1 activities in FF during the periovulatory period PAs (tpa and upa) and PAI-1 are secreted proteins, and PAI-1 can interact with PAs to form PA-PAI-1 complex, which neutralizes PA activities and regulates the level of free active PAs (11). To study how the coordinated expression of PAs from GCs and PAI-1 from TICs is correlated with the

5 Liu et al. PA System in Monkey Ovulation Endocrinology, April 2004, 145(4): ovulatory process, we collected FF samples from preovulatory follicles (12, 24, and 36 h after hcg injection) of prepubertal monkeys treated with PMSG and hcg. Corresponding PA activity and PAI-1 activity/antigen levels in the FF were examined by fibrin overlay, reverse fibrin overlay, and Western blot, respectively. Although a considerable amount of tpa activity was produced and secreted by GCs from follicles h before ovulation (Fig. 2, A and B), very little free tpa activity was measured in FF of follicles at h after hcg treatment. Most of the tpa activity in the FF was found to be in complexes with PAI-1 12 h after hcg treatment (Fig. 5A, 12 h). As ovulation approached (24 h after hcg injection), the free tpa level was slightly increased, whereas PA activities bound in the PA-PAI-1 complexes also increased (Fig. 5A, 24 h), which is in agreement with our data in Figs. 2 and 4 that both tpa and PAI-1 levels had increased at this time point. Just before the onset of ovulation (Fig. 5A, 36 h after hcg injection), free tpa activities in the FF were dramatically elevated, whereas PA activities bound in PA-PAI-1 complexes became very low. Fluctuations in PAI-1 activity (Fig. 5B) and protein levels (Fig. 5C) were also evaluated in the FF of preovulatory follicles. A maximum level of PAI-1 was detected in FF 24 h after hcg treatment, which was 12 h earlier than the highest free tpa level in FF (Fig. 5A). However, just before ovulation (36 h after hcg treatment), the PAI-1 level decreased to an undetectable level (Fig. 5, B and C). FIG. 4. Gonadotropin regulation of PAI-1 secretion by monkey TICs during the periovulatory period. Prepubertal monkeys were treated with PMSG and/or hcg. The ovaries were removed at various times after gonadotropin treatment and TICs were prepared as described in Materials and Methods. TIC ( cells/time point) were incubated at 37 C in 0.5 ml McCoy s 5A medium for 24 h, and PAI-1 antigen levels in the conditioned media were measured by Western blot. PMSG: 12 d after PMSG treatment; 12 h, 24 h, 36 h, 3d: times after hcg treatment. For each time point of an experiment, cells from the ovaries of one female monkey were used. A, A representative photograph from three independent experiments. B, Average relative levels of tpa and upa activities (mean SD) of three experiments. Small letters (a, b, and d) indicate significant differences (P 0.05). Northern blot analysis of tpa mrna content in GCs of adult monkey ovaries At various time points after the hormone treatment, GCs were obtained by puncturing developed follicles of adult monkey ovaries. Total RNA from each group was prepared, fractionated by agarose gel electrophoresis and transferred onto nylon filters. The filters were hybridized with 32 P- labeled monkey tpa riboprobe. The relative levels of tpa mrna were calculated by densitometric scanning of autoradiographs and normalized against the corresponding GAPDH mrna levels. As shown in Fig. 6, GC from PMSG treated (12 d) adult monkey ovaries contained very low levels of tpa mrna. Treatment with hcg considerably induced tpa mrna synthesis in the GCs in a time-dependent manner, reaching its maximum level 36 h after hcg injection. FIG. 5. PA and PAI-1 levels in FF during the periovulatory period. Prepubertal monkeys were treated with PMSG and/or hcg. The ovaries were removed at various times after gonadotropin treatment. FF was collected from preovulatory follicles of prepubertal monkeys 12, 24, and 36 h after hcg injection, as described in Materials and Methods. PA (A) and PAI-1 (B) activities as well as PAI-1 antigen levels (C) were determined by fibrin overlay, reverse fibrin overlay, and Western blot analysis, respectively. For each time point of the experiment, FF from the ovaries of one female monkey was used. The result shown is a representative photograph from three independent experiments; 12, 24, and 36 h: time after hcg treatment.

6 1772 Endocrinology, April 2004, 145(4): Liu et al. PA System in Monkey Ovulation FIG. 6. Changes of tpa mrna levels in granulosa cells of adult monkeys treated with PMSG and hcg. Adult monkeys were treated with PMSG for 12 d and followed by injection of hcg as described in Materials and Methods. Total RNA from GCs of each group ( cells) was prepared by the Nonidet P-40 method and fractionated by agarose gel electrophoresis before being transferred onto nylon filters and hybridized with 32 P-labeled antisense monkey tpa riboprobe. For each time point of one experiment, cells from two ovaries of one monkey were used. The relative amounts of tpa mrna (mean SD) were estimated by densitometric scanning of the autoradiographs and normalized against the corresponding amounts of GAPDH mrna from three Northern blot analyses. Different letters indicate significant difference between the points (P 0.05). PMSG: monkeys treated with PMSG for 12 d; PH12, PH36, and D3: 12 h, 36 h, and 3 d after hcg injection. The quantity of tpa mrna then decreased to a significantly lower level after ovulation (3 d after hcg treatment). In situ hybridization analysis of tpa and PAI-1 mrnas in adult monkey follicles To confirm the coordinated expression of tpa and PAI-1 in monkey ovaries during the periovulatory period, we also measured the mrna levels of tpa and PAI-1 in adult monkey ovaries during the periovulatory period. The results were consistent with the changes in their mrna levels measured by Northern blot. Specifically, low levels of tpa (Fig. 7A) and PAI-1 (Fig. 7E) mrnas were expressed in the monkey follicles treated with only PMSG for 12 d. In the preovulatory follicles treated with PMSG and hcg, tpa mrna expression start to increase at 24 h after hcg treatment (Fig. 7B) in the GCs and was further increased in GCs at 36 h after hcg injection (Fig. 7C). PAI-1 mrna was elevated in follicles in the TIC 24 h after hcg treatment (Fig. 7F). However, in accordance with our results at the protein level, PAI-1 mrna was very low in TICs just before ovulation (36 h after hcg injection, Fig. 7G). The mrna levels of both tpa and PAI-1 decreased to undetectable levels in copora lutea at 5 d after hcg injection (Fig. 7, D and H). Discussion Ovulation is a complex physiological process that is controlled by multiple factors including hormones, growth factors, and cytokines (1 4). Over the last century, theories concerning elements that cause ovulation have been proposed (3). Data published over the last decade have suggested that proteolytic activities mediated by the PA system may play a role in the ovulatory process in rodents (1, 4, 6 9). To evaluate the roles of PA and PAI-1 in ovulation of the primate, we used a PMSG/hCG-induced, synchronized ovulation model in the rhesus monkey. We have demonstrated for the first time that the coordinated expression of tpa and its physiological inhibitor PAI-1 takes place during the ovulatory process of rhesus monkeys. In this study, we have shown that in monkey ovaries, GCs are the major source of PA production, whereas TICs are the major source of PAI-1 production. Expression of both tpa and PAI-1 was induced in monkey follicles by treatment with gonadotropins (PMSG and hcg), and maximum levels were reached before ovulation. However, the peak levels of tpa and PAI-1 appeared at different times before ovulation, with the peak of PAI-1 occurring about 12 h earlier than that of tpa. When tpa reached its highest level, the expression of PAI-1 had already decreased to its lowest level. Thus, our data indicate that tpa and PAI-1 expression is temporally coordinated during the ovulatory process of the monkey. Such coordinated expression of tpa and PAI-1 may generate high tpa activity in follicles just before ovulation, which may facilitate the breakdown of the follicular wall during ovulation. We also found that although monkey GCs can produce a considerable amount of upa, upa was not secreted from the GC cells before ovulation but was secreted by luteal cells after ovulation at the time when secreted tpa decreased to undetectable levels. Our results suggest that tpa produced by GCs is the major PA species taking part in the process of follicle rupture during ovulation in rhesus monkey ovaries. On the other hand, upa is the major PA species that participates in the active tissue remodeling and angiogenesis processes during corpus luteum formation and development (16, 17). Little is known about the roles of the PA system in human ovaries. Human GCs collected from preovulatory follicles were reported to contain little or no tpa or upa mrna (23). Nevertheless, in the FF of human preovulatory follicles, low levels of tpa were detected (24). Moreover, relatively abundant amounts of PAI-1 mrna were reported in human GCs or granulosa-luteal cells (23 25). The difference in the expression of tpa and PAI-1 in GCs and FF of preovulatory human follicles compared with the results we obtained from monkey ovaries may have several explanations. There might be species variations between humans and monkeys. However, we have also noticed that the expression of PA and PAI-1 during the periovulatory process in monkeys showed a temporal pattern that changed in a matter of hours. In our experiments, GC or FF samples obtained at different time points during the periovulatory period contained significantly different levels of PA and PAI-1. Therefore, it seems to us that what type of results can be obtained from human GCs and FF may also depend on the stage and condition of the clinical samples. The hypothesis that tpa and PAI-1 play a role in monkey ovulation is supported by previous studies in rodents. In the rat, GCs synthesize most of the follicular tpa activity (26), whereas the follicular wall (TICs) contributes most of the PAI-1 activity (27, 28). PAI-1 may therefore serve as a specific barrier to localize the tpa activity within the follicle (4, 9, 27, 28). As ovulation approaches, the level of PAI-1 in TICs and GCs dramatically decreases, whereas tpa activity in the follicle rises to its maximum level. The coordinated expression

7 Liu et al. PA System in Monkey Ovulation Endocrinology, April 2004, 145(4): FIG. 7. Localization of tpa and PAI-1 mrnas in preovulatory monkey follicles. Frozen sections (10 12 m) from ovaries of adult female monkeys 12 d after PMSG treatment (A, E), 24 h (B, F), 36 h (C, G), or 5 d (D, H) after treatment with hcg were hybridized with digoxigenin-labeled monkey tpa (A D) and PAI-1 (E H) probes as described in Materials and Methods. Slides for comparison were prepared at the same time. Photographs were taken at original magnification 10. Representative pictures from three individual experiments (n one monkey per time point for each experiment) are shown. CL, Corpus luteum. of tpa and PAI-1 may therefore lead to a short pulse of proteolytic activity that could play a role in rupture of the follicle (4, 9, 27, 28). However, an important challenge is to determine the roles of the PA system in primate ovulation, compared with the limited positive data from PA and plasminogen knockout mice (13, 14). It is well known that species differences exist in the expression of the PA system in ovaries. For example, both upa and tpa have been detected in the mouse ovary (29 31). In contrast to the rat, 70 80% of secreted PA activities by mouse GCs were from upa, and both tpa and upa were induced by gonadotropins and reached their maximum levels before ovulation (30, 31). In addition, no PAI-1 activity was detected in mouse GCs and FF, whereas the activity of a plasmin inhibitor, 2 -antiplasmin, was found in the FF (32). Using chicken and pig models, Politis et al. (33) and Tilly et al. (34) also demonstrated that tpa is the principal PA in the preovulatary follicles of these species. Despite the abovementioned results, we also examined the ovarian PA activities in other species, such as amphioxus, rabbit, cat, hamster, and giant panda. Ovaries of these species contained mainly tpa activities, which were regulated by gonadotropins (our unpublished data). Thus, we suggest that tpa may be the key PA type that plays roles in ovulation of various species. Studies of PA and plasminogen-deficient mouse strains have indicated that other families of proteases including the matrix metalloproteinases and their inhibitors may also be involved in follicle rupture during ovulation (14, 35 38).

8 1774 Endocrinology, April 2004, 145(4): Liu et al. PA System in Monkey Ovulation Ovulation is also modulated by endogenously produced local factors that can regulate the expression and localization of PAs and matrix metalloproteinases (for papers and reviews, see Refs. 1 5, 9, 39 42). In addition, the gonadotropininduced ovulatory process in mammals involves gross physiological events that are similar to acute inflammatory reactions regulated by various genes in the ovary (43 45). Thus, it seems to us that the next challenge is to identify how multiple paracrine and autocrine factors regulate other proteases and protease inhibitors during the periovulatory period in the primate. Acknowledgments The authors thank Dr. Ann Clark for proofreading the manuscript. 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