Cortisol/glucocorticoid receptor: a critical mediator of the ovulatory process and luteinization in human periovulatory follicles.

STUDY QUESTION: Do cortisol/glucocorticoid receptors play an active role in the human ovary during ovulation and early luteinization? SUMMARY ANSWER: The ovulatory hCG stimulation-induced glucocorticoid receptor signaling plays a crucial role in regulating steroidogenesis and ovulatory cascade in human periovulatory follicles. WHAT IS KNOWN ALREADY: Previous studies reported an increase in cortisol levels in the human follicular fluid after the LH surge or ovulatory hCG administration. However, little is known about the role of cortisol/glucocorticoid receptors in the ovulatory process and luteinization in humans. STUDY DESIGN, SIZE, DURATION: This study was an experimental prospective clinical and laboratory-based study. An in vivo experimental study was accomplished utilizing the dominant ovarian follicles from 38 premenopausal women undergoing laparoscopic sterilization. An in vitro experimental study was completed using the primary human granulosa/lutein cells (hGLC) from 26 premenopausal women undergoing IVF. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study was conducted in a private fertility clinic and academic medical centers. Dominant ovarian follicles were collected before the LH surge and at defined times after hCG administration from women undergoing laparoscopic sterilization. Primary hGLC were collected from women undergoing IVF. hGLC were treated without or with hCG in the absence or presence of RU486 (20 µM; dual antagonist for progesterone receptor and glucocorticoid receptor) or CORT125281 (50 µM; selective glucocorticoid receptor antagonist) for 12 or 36 h. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and ovulatory cascade was studied with RT-quantitative PCR and western blotting. The production of cortisol, corticosterone, and progesterone was assessed by hormone assay kits. MAIN RESULTS AND THE ROLE OF CHANCE: hCG administration upregulated the expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), nuclear receptor subfamily 3 group C member 1 (NR3C1), FKBP prolyl isomerase 5 (FKBP5), and FKBP prolyl isomerase 4 (FKBP4) in human ovulatory follicles and in hGLC (P < 0.05). RU486 and CORT125281 reduced hCG-induced increases in progesterone and cortisol production in hGLC. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and the key ovulatory process was reduced by RU486 and/or CORT125281 in hGLC. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The role of cortisol/glucocorticoid receptors demonstrated using the hGLC model may not fully reflect their physiological roles in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Successful ovulation and luteinization are essential for female fertility. Women with dysregulated cortisol levels often suffer from anovulatory infertility. Deciphering the functional role of glucocorticoid receptor signaling in human periovulatory follicles enhances our knowledge of basic ovarian physiology and may provide therapeutic insights into treating infertility in women. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by P01HD71875 (to M.J., T.E.C., and M.B.) and R01HD096077 (to M.J.) from the Foundation for the National Institutes of Health and the BTPSRF of the University of Kentucky Markey Cancer Center (P30CA177558). The authors report no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Molecular changes in the equine follicle in relation to variations in antral follicle count and anti-Müllerian hormone concentrations.

REASONS FOR PERFORMING STUDY: The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. OBJECTIVES: To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. STUDY DESIGN: Observational study. METHODS: Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. RESULTS: Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. CONCLUSIONS: This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

Expression of basigin, an inducer of matrix metalloproteinases, in the rat ovary.

The extensive tissue remodeling that occurs during follicular development, ovulatory rupture, and the formation and regression of the corpus luteum (CL) requires local degradation of the extracellular environment by matrix metalloproteinases (MMPs). This report characterizes the expression pattern of basigin (Bsg), a putative regulator of MMP induction, in the rat ovary. An induced superovulation model (eCG/hCG) was used in immature rats to evaluate Bsg expression profiles in ovaries collected during the follicular phase, the preovulatory period, and the luteal lifespan. Levels of Bsg mRNA were unchanged through follicular growth (0-48 h post-eCG) and increased during postovulatory luteinization (24 and 48 h post-hCG; P < 0.01). Bsg expression persisted into pseudopregnancy (4-8 days post-hCG) and after functional luteal regression (12 days post-hCG). The profile of Bsg expression during regression of the CL was examined using a model of induced luteolysis. Both functional and structural regression was associated with a decline in Bsg expression levels. Bsg mRNA and protein localized to the theca of preovulatory follicles (12 h post-hCG) and formative and functional CL (24 h-8 days post-hCG). Bsg expression profiles in the induced ovulation and CL regression models were similar to observations made in naturally cycling mature rats. In the cycling ovary, Bsg signaling localized to newly forming CL, the theca of preovulatory follicles, and appeared to be lower in CL from previous estrous cycles. A putative regulatory mechanism of Bsg expression was identified using an in vitro model; treatment of cultured granulosa cells with hCG significantly augmented Bsg mRNA expression levels. The processes of ovulation and luteogenesis may be facilitated by Bsg expression and its induction or regulation of the MMPs.

Collagenase and gelatinase messenger ribonucleic acid expression and activity during follicular development in the rat ovary.

Metalloproteinases are members of a family of proteinases that remodel the extracellular matrix throughout the body. To test the hypothesis that metalloproteinases are regulated by gonadotropin-induced changes during follicular growth, rats were injected with eCG (20 IU, s.c.), and ovaries and serum were collected at the time of eCG administration (0 h) and at 6, 12, 24, 36, or 48 h later for analysis of metalloproteinase mRNA expression, metalloproteinase activity, and steroidogenesis. Serum estradiol levels increased from 18.9 pg/ml at 0 h to 503.8 pg/ml at 48 h. Analysis of mRNA expression was performed for collagenase-3, 72-kDa gelatinase, and 92-kDa gelatinase (n = 3-4). For collagenase-3, eCG stimulated a 32-fold increase in collagenase-3 mRNA at 48 h after eCG injection as compared to that in ovaries collected at the time of eCG administration (i.e., 0-h control). The mRNA levels for 72-kDa gelatinase were 2.8-fold compared to 0 h at 36 h after eCG treatment and returned to control levels by 48 h after gonadotropin treatment. Levels of the 92-kDa mRNA expression peaked at 24 h (4. 2-fold compared to 0 h) and returned to control levels by 36 h. Gel zymography revealed 3 gelatinolytic bands corresponding to the gelatinases of approximately 72 kDa, 92 kDa, and 105 kDa. Analysis of metalloproteinase activity as the degradation of collagen or gelatin per ovary showed an increase in gelatinolytic and collagenolytic activity between 12 and 48 h after eCG treatment. In summary, these findings demonstrate that the gonadotropin induction of folliculogenesis results in changes in the metalloproteinases that may be responsible for extracellular matrix remodeling associated with follicular growth.

Gonadotropin-releasing hormone agonist (GnRH-a) therapy alters activity of plasminogen activators, matrix metalloproteinases, and their inhibitors in rat models for adhesion formation and endometriosis: potential GnRH-a-regulated mechanisms reducing adhesion formation.

OBJECTIVE: To evaluate the effects of a gonadotropin-releasing hormone agonist (GnRH-a) on plasminogen activator (PA), matrix metalloproteinase (MMP), plasminogen activator inhibitor (PAI) and matrix metalloproteinase inhibitor (MMPI) activities in peritoneal fluid relative to GnRH-a-induced reduction of adhesion formation. DESIGN: Continuation of prospective randomized study using surgical models for adhesion formation. SETTING: Department of Obstetrics and Gynecology research laboratory at the University of Missouri School of Medicine. PATIENT(S): Forty reproductively cycling female Sprague-Dawley rats. INTERVENTION(S): Female rats were injected with depot GnRH-a or diluent and randomly assigned to adhesion and endometriosis surgeries. Peritoneal fluid was collected prior to (time 1) and 7 weeks from (time 2) initial surgery. MAIN OUTCOME MEASURE(S): Peritoneal fluid was analyzed for PA, PAI, MMP, and MMPI activities. RESULT(S): At time 1, MMP and MMPI activities were similar in all rats; however, PA and PAI activities were less in rats pretreated with GnRH-a than with diluent. Between time 1 and time 2, GnRH-a-treated rats showed an increase in PAI and MMPI activities without significant changes in PA or MMP activities, whereas rats receiving diluent showed a significant increase in PAI and MMP activities but no significant changes in PA or MMPI activities. At time 2, rats receiving GnRH-a had less PA and MMP activities than those receiving diluent. Adhesion scores showed a positive correlation with MMP activity. CONCLUSION(S): In the absence of GnRH-a therapy, surgical tissue manipulation increased peritoneal fluid MMP and PAI activity. Gonadotropin-releasing hormone agonist therapy decreased PA and MMP activities and also increased PAI and MMPI activities. This GnRH-a-induced shift to a less invasive phenotype may alter fibrinolysis and extracellular matrix remodeling and thereby play a role in the mechanism of GnRH-a-induced reduction in adhesion formation.

Effect of A23187 or angiotensin II on ovarian metalloproteinase inhibitors and steroidogenesis in rats.

The present study examined the effect of the calcium ionophore A23187 or angiotensin II (AII) on the expression of ovarian metalloproteinase inhibitor and activity in rat granulosa cells and intact ovaries. Granulosa cells were collected from rats primed with pregnant mares' serum gonadotrophin (PMSG) and cultured for 24 h with A23187, AII, or the AII receptor antagonist, saralasin, in the presence or absence of LH. Metalloproteinase inhibitor activity and progesterone concentrations were determined in the media. In the A23187 experiment, addition of A23187 to granulosa cells, cultured without LH, decreased inhibitor activity, especially at the concentrations of 10 and 100 mumol l-1 (decrease to 33 +/- 7% and 31 +/- 5% of control culture values, respectively). Addition of LH to the media increased inhibitor activity 3.04 +/- 0.39 times compared with the control; however, A23187 (10 and 100 mumol l-1), in the presence of LH, decreased inhibitor activity by approximately 67%. The ionophore had disparate effects on progesterone production. Without LH, A23187 increased progesterone production by 2.96 +/- 0.47 times at 10 mumol l-1 and by 5.53 +/- 0.65 times at 100 mumol l-1. However, in LH-stimulated cells, progesterone was inhibited by A23187 at 1 and 10 mumol l-1 but was unchanged at 100 mumol l-1. In the angiotensin experiment, addition of AII (0-10,000 nmol l-1) or saralasin (1 mumol l-1) did not affect inhibitor activity or progesterone concentrations compared with control values in the absence or presence of LH. For the angiotensin experiment in vivo, PMSG-primed rats were injected with hCG followed by saralasin (10 mmol l-1) 1 or 3 h later and killed at 4, 8, or 12 h after hCG. Expression of the ovarian tissue inhibitor of metalloproteinase-1 (TIMP-1) increased by 1.7 times at 4 h, 3.3 times at 8 h, and 3.0 times at 12 h after hCG compared with values in ovaries collected at the time of hCG injection. Administration of saralasin at 1 or 3 h after hCG had no effect on expression of TIMP-1 or on serum concentrations of progesterone or oestradiol. In summary, A23187 decreased granulosa cell-derived inhibitor activity, whereas All had no effect. We propose that calcium may play a role in modulating proteolysis associated with ovulation, while AII does not appear to regulate ovarian metalloproteinase inhibitor activity.

Assessment of the role of tissue inhibitor of metalloproteinase-1 (TIMP-1) during the periovulatory period in female mice lacking a functional TIMP-1 gene.

Tissue inhibitor of metalloproteinase (TIMP)-1 is a multifunctional peptide that has been implicated in the ovulatory process. To assess the function of TIMP-1 during the periovulatory period in vivo, mice incapable of expressing the TIMP-1 gene product were utilized. Twenty-three-day-old TIMP-1-deficient (n = 59) and wild-type (n = 61) female mice were injected with 5 IU eCG, followed 48 h later by an ovulation-inducing dose of hCG (5 IU). Animals were killed at the time of hCG injection (0-h hCG), at 12 h (12-h hCG), or at 24 h post-hCG (24-h hCG) administration. Serum was collected for the assessment of estradiol-17beta (0-h hCG groups) or progesterone content (12- and 24-h hCG groups), while ovaries were removed for either histological preparation or Northern analysis of TIMP-1, TIMP-2, and TIMP-3. The number of healthy and atretic follicles was determined in the 0-h hCG groups, as was the number of oocytes released in the 24-h hCG group. TIMP-1-deficient females in the 0-h hCG group showed reduced levels of ovarian TIMP-2 (0.29-fold decrease, p < 0.05) and TIMP-3 (3.0-fold decrease, p < 0.05) expression compared to wild-type counterparts. No significant difference was detected between genotypes in the 0-h hCG group for number of healthy or atretic follicles or for serum estradiol-17beta concentrations. Additionally, no significant differences were detected between genotypes in the 12- and 24-h hCG groups for serum progesterone concentrations, ovarian TIMP-2 and TIMP-3 expression, or number of oocytes released (24-h hCG group). To assess the effect of TIMP-1 on steroidogenesis in vitro, granulosa cells were obtained from 23-day-old, eCG-primed TIMP-1-deficient and wild-type females. Addition of recombinant human TIMP-1 significantly increased conditioned media estradiol-17beta concentrations in cell cultures from both mutant (1.32-fold over controls; p = 0.02; n = 4) and wild-type females (1.16-fold over controls; p = 0.04; n = 3). It is concluded from this study that TIMP-1 may modulate ovarian TIMP-2 and TIMP-3 mRNA expression during folliculogenesis. In addition, TIMP-1 exhibits steroidogenic activity in vitro, but no evidence was found for regulation of steroidogenesis in vivo.

Divergent effects of interleukin-1 beta on steroidogenesis and matrix metalloproteinase inhibitor expression and activity in cultured rat granulosa cells.

The periovulatory increase in ovarian matrix metalloproteinase inhibitor (MMPI) expression is regulated both in vitro and in vivo by LH, but the intermediary steps in this process are uncertain. The purpose of this experiment was to determine whether interleukin-1 beta (IL-1 beta), a known modulator of MMPI expression in other systems and one that is induced by LH in the ovary, is capable of regulating granulosa matrix metalloproteinase inhibitor expression and activity. Using an in vitro rat granulosa cell model, these parameters were assessed in response to IL-1 beta or LH administration alone or in combination. Granulosa cells were obtained from 24-day-old immature female rats primed with 20 IU PMSG at 22 days of age. Cells were cultured under serum-free conditions for 24 h at 37 C in the presence of medium (control), LH (100 ng/ml), IL-1 beta (10 ng/ml), or LH plus IL-1 beta. MMPI activity in the conditioned medium was assessed using a colorimetric assay (n = 6), whereas progesterone and estrogen concentrations in the conditioned medium were determined by RIA (n = 6). RNA was isolated from the granulosa cells and assessed for Northern analysis of tissue inhibitor of metalloproteinase-1 (TIMP-1; n = 4), TIMP-2 (n = 3), and TIMP-3 (n = 3) expression. When added to granulosa cells, IL-1 beta and LH each significantly (P < 0.05) increased MMPI activity in granulosa cell-conditioned medium above control values (40.9 +/- 3.0% inhibition for IL-1 beta and 67.1 +/- 5.6% inhibition for LH vs. 31.4 +/- 2.4% inhibition for controls). When added in combination, IL-1 beta had no effect on LH-stimulated inhibitor activity (67.1 +/- 5.6% inhibition vs. 69.9 +/- 5.1% inhibition for LH and LH plus IL-1 beta, respectively). Methylamine hydrochloride treatment revealed that the majority of inhibitor activity in all treatment groups was derived from TIMPs. The patterns of TIMP-1, TIMP-2, and TIMP-3 messenger RNA expression among the treatment groups paralleled the TIMP-derived inhibitor activity, in that both IL-1 beta and LH alone stimulated transcript expression of all three TIMPs. In addition, an increase in progesterone production was associated with IL-1 beta-stimulated (1.22-fold over control values; P = 0.0006) and LH-stimulated (9.6-fold over control values; P = 0.007) MMPI expression and activity. Lastly, IL-1 beta and LH significantly (P < 0.05) decreased estrogen production by approximately 33% compared to that in cultures with LH only. It is concluded from the current study that IL-1 beta is a mediator of MMPI expression as well as granulosa cell steroidogenesis, and that this cytokine has divergent actions in the presence and absence of LH.

Mifepristone and ovarian function.

In summary, RU 486 has been a powerful instrument in delineating progesterone action on the ovary. However, early experiments using RU 486 must be interpreted with the understanding that systemic administration of the antiprogestin may have had extraovarian sites of action, such as at the hypothalamic-pituitary axis or at the adrenal, that in turn led to indirect ovarian responses. Treatment with progesterone, agonist, or antagonist at periods during which the ovary lacks progesterone receptors would further suggest extraovarian sites of action or nongenomic mechanisms of action. Furthermore, the dose of ligand or antagonist administered and the hormonal milieu at the time of administration may dictate the ovarian response (Espey L, personal communication). For example, low doses of exogenous progesterone may elicit a biologic response, whereas high doses are without effect or may inhibit the biologic effect observed at lower doses. Although RU 486 is classically described as an antiprogestin, agonist actions have been observed in addition to its the well documented antiglucocorticoid effects. All of these variables may contribute to the confounding observations of progesterone and RU 486 action on the ovary. Regardless of these caveats, experimental paradigms have demonstrated that RU 486, either indirectly or directly, regulates ovarian folliculogenesis, stimulates and/or inhibits steroidogenesis depending on the species and time of RU 486 administration, inhibits ovulation, and modulates luteal function. These findings supports a progesterone-dependent mechanism in these varied aspects of ovarian function.

Galanin: a novel intraovarian regulatory peptide.

Galanin is a 29-amino acid peptide that acts as a neuropeptide in many tissues. To date, galanin action and the hormonal regulation of galanin gene expression have not been described in the ovary of any species. To study possible ovarian expression and regulation of galanin, immature gonadotropin-primed rats were given hCG (10 IU), and their ovaries were collected 0, 4, 8, 12, and 20 h after hCG treatment for determination of galanin messenger RNA (mRNA) concentration by solution hybridization. Galanin mRNA levels progressively increased after hCG administration, peaking at 12 h (2.4-fold increase vs. 0 h), with a subsequent return to 0 h levels at 20 h. To determine a possible ovarian role for galanin, rats were killed 48 h after gonadotropin administration, their ovaries were removed, and granulosa cells were harvested. These cells and the ovarian tissue remaining after granulosa cell collection (i.e. "shells") were each cultured for 24 h with increasing concentrations of galanin (0, 10, 100, and 1000 nM) in the presence or absence of LH. The medium was examined for steroid production and metalloproteinase inhibitor activity. In granulosa cell cultures, galanin increased the levels of estradiol by 26% and had no effect on progesterone, but decreased metalloproteinase inhibitor activity by 61% in the conditioned medium. In the shell cultures, galanin increased estradiol, progesterone, and androstenedione in the medium, suggesting that galanin acts on cells other than granulosa cells or that galanin action requires a paracrine interaction between granulosa and thecal cells. Our data demonstrate that galanin message is increased by hCG, and that galanin acts to amplify ovarian steroidogenesis while decreasing metalloproteinase inhibitor activity. These findings establish that ovarian galanin mRNA is hormonally stimulated and that galanin acts as an intraovarian regulatory peptide.

Antiprogesterone (RU486) effects on metalloproteinase inhibitor activity in human and rat granulosa cells.

OBJECTIVE: To determine if granulosa cells are a source of metalloproteinase inhibitor activity and whether P regulates this activity. DESIGN: Inhibitor activity was measured in media from human and rat granulosa cells cultured with the antiprogesterone mifepristone (RU486). Human granulosa cells were obtained at the time of oocyte retrieval from gonadotropin-stimulated patients after hCG administration. Rat cells were collected from gonadotropin-primed animals before the LH surge. Human and rat cells were cultured for 24 hours in the absence or presence of LH (100 ng/mL or 3.3 nmol/L) and/or RU486 (5 microM or 50 microM). Inhibitor activity and P were measured in the media. SETTING: Reproductive Endocrinology Laboratories, University of Kentucky. RESULTS: Media from human granulosa cells contained metalloproteinase inhibitor activity and the addition of LH did not change this activity. RU486 at 50 microM decreased inhibitor activity in cells cultured in the absence or presence of LH (0.59 +/- 0.12- and 0.24 +/- 0.18-fold change, respectively, versus control cultures; mean +/- SEM). In rat granulosa cells, inhibitor activity increased with LH treatment (1.97 +/- 0.12-fold change). RU486 decreased the activity present in cells cultured in the presence of LH. Progesterone production was stimulated by LH in both human and rat granulosa cells (3.71 +/- 0.90- and 7.18 +/- 0.24-fold change, respectively). In the human cells, RU486 inhibited P production whereas RU486 stimulated P production in the rat cells. CONCLUSIONS: These findings demonstrate for the first time that human granulosa cells are a source of metalloproteinase inhibitor activity. The decrease in granulosa cell-derived inhibitor activity by RU486 suggests that P stimulates inhibitor activity. Thus, P may regulate proteolysis associated with follicular rupture via its ability to stimulate granulosa cell production of metalloproteinase inhibitors. Differences in P production between the human and rat cells may be due to differences in hormonal stimulation regimens (i.e., hCG exposure).

Immunomodulation of rat endometriotic implant growth and protein production.

PROBLEM: The immune system has been implicated in the pathophysiology of endometriosis. To determine if modulation of the immune system influences endometriotic implant growth and protein production, the following experiment was conducted. METHOD: Female rats with surgically induced endometriosis were treated with either the immunosuppressive agent pentoxifylline (Pent; 5 mg/kg BW; N = 16) or a vehicle (N = 16) for 7 consecutive days, then killed. Twenty-four hours before death, 8 animals from each group were injected intraperitoneally with the immunostimulatory agent lipopolysaccharide (LPS; 200 micrograms/kg BW). At death, endometriotic implants were measured and protein production assessed by two-dimensional polyacrylamide gel electrophoresis. RESULT: Pentoxifylline significantly (P < 0.001) reduced endometriotic implant size (2.15 +/- 0.65 mm2 vs. 17.13 +/- 1.98 mm2) whereas LPS was without effect (18.32 +/- 2.57 mm2 vs. 17.13 +/- 1.98 mm2). Pentoxifylline also suppressed production of a portion of the proteins that comprise the implant specific group of proteins, ENDO-2, whereas LPS induced the production of two additional ENDO-2 proteins. CONCLUSION: Immunomodulatory agents can modulate rat endometriotic implant growth and production of implant-specific proteins.

The presence of luteinized unruptured follicle syndrome and altered folliculogenesis in rats with surgically induced endometriosis.

OBJECTIVE: Our purpose was to determine in the rat model whether endometriosis could influence ovarian function by altering oocyte release or folliculogenesis. STUDY DESIGN: We histologically examined the ovaries of reproductively cycling rats with (n = 16) and without (n = 10) surgically induced endometriosis. The rats in these two groups were further subdivided into unilaterally ovariectomized or ovarian-intact groups. Serial sections of ovaries were examined, and follicular development and frequency of luteinized unruptured follicles were determined. RESULTS: A significant tenfold increase in the number of luteinized unruptured follicles was observed in the ovaries from rats with endometriosis (2.7 per rat) compared with unoperated and sham-operated control groups (overall mean 0.26 per rat, p < 0.05). Additionally, ovaries from unilateral ovariectomized animals with endometriosis contained four times as many luteinized unruptured follicles (four per rat) as did the ovaries from bilaterally ovarian-intact rats with endometriosis (1.40 per rat, p < 0.01). Fewer follicles were present in rats with endometriosis (180 follicles per ovary) than in control rats (231 follicles per ovary, p < 0.05). CONCLUSION: In the rat model the presence of ectopic endometrium is associated with an increased frequency of luteinized unruptured follicles and altered follicular development.

Role of protein synthesis, prostaglandins, and estrogen in rat ovarian metalloproteinase inhibitor production.

Matrix metalloproteinases and their inhibitors regulate follicular connective tissue remodeling associated with ovulation. We examined the control of metalloproteinase inhibitor activity and gene expression by various treatments in cultured rat granulosa cells and intact whole ovaries. Cells were isolated from preovulatory follicles of immature eCG-primed rats and cultured with various treatments for 24 h. Metalloproteinase inhibitor activity was measured in the media. The addition of LH or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased inhibitor activity 2.5- and 2.6-fold above that in control cultures, respectively. Cycloheximide treatment blocked basal and LH- and TPA-stimulated inhibitor activity, suggesting that the increase in granulosa cell inhibitor activity resulted from de novo protein synthesis. Indomethacin, a prostaglandin synthase inhibitor, had no effect on basal or LH-induced granulosa cell inhibitor activity. Addition of the antiestrogen tamoxifen citrate or an aromatase inhibitor, 10-propargylestr-4-ene-3,17-dione, did not affect basal or LH-stimulated inhibitor activity, implying that estrogen is not involved in the signal transduction pathway leading to increased inhibitor activity. Northern analysis demonstrated the presence of mRNA for a tissue-derived metalloproteinase inhibitor, TIMP-1, which increased with LH stimulation, in rat granulosa cells. Similarly, an hCG stimulus increased TIMP-1 mRNA in periovulatory ovaries to the highest levels prior to ovulation. Neither cycloheximide nor indomethacin altered hCG-stimulated TIMP mRNA levels in periovulatory ovaries. The present study demonstrates that the LH- and TPA-induced increase in inhibitor activity resulted from de novo protein synthesis; however, de novo protein synthesis does not appear necessary for the increase in TIMP mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity.

Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.

Cellular localization of ovarian prostaglandin endoperoxide synthase during pseudopregnancy in the rat.

The specific cellular localization of prostaglandin endoperoxide (PGH) synthase, the enzyme responsible for initiating the conversion of arachidonic acid to prostaglandins, was studied throughout pseudopregnancy in the rat. Pseudopregnancy was induced by administration of eCG (20 IU) to immature, 27-day-old rats followed by hCG injection (10 IU) on Day 29. Animals were necropsied on Days 1 (Day 1 = 1 day post hCG), 5, 9, and 13 of pseudopregnancy. Ovaries were removed and processed for cellular identification of PGH synthase by immunohistochemistry. Immunoreactive PGH synthase was distributed throughout the CL at each of the 4 different days of pseudopregnancy, with the majority of the luteal cells exhibiting varying degrees of staining. The connective tissue centrum of the CL, however, lacked PGH synthase immunoreactivity. Quantitative assessment of the immunostaining distribution was accomplished with a computer-based image analysis program (Kontron IBAS). Results are expressed as the percentage of a digitized luteal area that contained intense immunoreactivity between Day 1 (0.6 +/- 0.2% immunoreactive area) and Day 5 (16.8 +/- 2.7%) of pseudopregnancy. The area of luteal immunostaining was similar on Day 5 and Day 9 (16.8 +/- 2.7% and 14.7 +/- 2.0%, respectively) followed by a decrease (p less than 0.05) in immunoreactivity on Day 13 (9.1 +/- 2.2%). The region of the CL adjacent to the germinal epithelium had an increase (p less than 0.01) in PG synthase staining distribution compared to the region of the CL adjacent to the ovarian medulla on all days of pseudopregnancy except Day 1. These findings demonstrate that PGH synthase is present in the rat CL throughout pseudopregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of matrix metalloproteinase inhibitors in rat granulosa cells and ovaries.

Metalloproteinases, such as collagenase or gelatinase, and their associated inhibitors appear to control connective tissue remodeling during follicular rupture. We examined the regulation of metalloproteinase inhibitor activity by various treatments in cultured rat granulosa cells. Granulosa cells were harvested from immature PMSG-primed rats and cultured with LH, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), cAMP, or forskolin. Inhibitor activity was measured in the medium. Increasing concentrations of either LH (0.1-1000 ng/ml) or TPA (2.5-100 nM) resulted in a dose-dependent increase in metalloproteinase inhibitor activity (2.9- and 2.4-fold increases above control, respectively). There was also a time-dependent induction of inhibitor activity in cells incubated in the presence of LH (100 ng/ml) for 6, 12, 18, or 24 h. Forskolin (0.1 mM) or cAMP (1 mM) treatment increased inhibitor activity 2.8- and 1.6-fold above that in control cultures. LH and TPA treatment in combination resulted in an additive increase in inhibitor activity compared to LH or TPA treatment alone. This finding suggested that the granulosa cell inhibitor activity might be induced through separate intracellular pathways. The inhibitor present in conditioned medium was isolated by chromatographic separation on a Sepharose 6B mol wt exclusion column. The inhibitor present was approximately 28,000 mol wt, which is consistent with the size of tissue inhibitor of metalloproteinase (TIMP). In addition to the granulosa cell experiments, changes in ovarian mRNA levels for TIMP were determined. There was a preovulatory increase in TIMP mRNA from whole rat ovaries, with the highest levels detected 12 h after hCG administration. The present study establishes that metalloproteinase inhibitor activity from rat granulosa cells is induced through separate pathways: a LH-cAMP-dependent protein kinase-A pathway and a cAMP-independent protein kinase-C pathway. Furthermore, a TIMP-like protein is observed in granulosa cell-conditioned medium, while TIMP mRNA is present in rat ovaries and increases before ovulation, suggesting that the granulosa cell metalloproteinase inhibitor is TIMP. We propose that TIMP acts in part to control the site and extent of follicular connective tissue remodeling associated with ovulation.

Alpha 2-macroglobulin and tissue inhibitor of metalloproteinases: collagenase inhibitors in human preovulatory ovaries.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

Identification and characterization of metalloproteinase inhibitor activity in human ovarian follicular fluid.

Metalloproteinase inhibitors regulate collagenase activity in the extracellular matrix. To assess the role of metalloproteinase inhibitors in the ovulatory process, inhibitor activity was examined in human follicular fluid collected 2-4 h before ovulation. The relationship between inhibitor activity and steroid content was determined, and the inhibitors were partially purified and characterized. Inhibitory activity in follicular fluid (n = 25) correlated with both follicular estradiol (P less than 0.001) and progesterone (P less than 0.02) concentrations per follicle. Chromatographic separation of the follicular fluid on Sepharose 6B isolated two peaks of inhibitory activity. The inhibitor from the small mol wt (Mr) peak shared many of the properties of tissue inhibitors of metalloproteinase. It was stable in response to heat (60 C) and methylamine (200 mM), and was destroyed by reduction and alkylation, a procedure reported to destroy previously characterized inhibitors. Partial purification by affinity and ion exchange chromatography demonstrated the inhibitor to be a glycoprotein with an approximate Mr of 28-29K. The large Mr inhibitor had an approximate size of 700K and exhibited many of the characteristics of alpha 2-macroglobulin, a serum-derived metalloproteinase inhibitor. It was sensitive to heat, methylamine, and reduction and alkylation. Thus, follicular fluid contains metalloproteinase inhibitor activity that is steroid related and may be hormonally regulated. Ovarian metalloproteinase inhibitors may act to regulate connective tissue remodeling during follicular rupture.

Ovarian prostaglandin synthase: immunohistochemical localization in the rat.

Ovarian prostaglandin synthase is stimulated by the luteinizing hormone surge resulting in the preovulatory increase in prostaglandins. In the present study, the ovarian cellular localization of prostaglandin synthase was identified by immunohistochemistry. Ovaries were collected from 27-day-old rats at the time of stimulation with pregnant mare serum gonadotropin (8 IU) (zero hours), 48 hours later, or 8 hours after administration of human chorionic gonadotropin (5 IU). At zero hours prostaglandin synthase immunostaining was present in the theca of larger follicles and interstitial regions. The number and intensity of immunostained cells in the theca increased between zero and 48 hours. The granulosa cell layer adjacent to the basement membrane in large antral follicles exhibited immunostaining. A similar staining pattern was observed 8 hours after human chorionic gonadotropin. These observations indicate that in the rat, the theca, interstitium, and granulosa contain prostaglandin synthase immunoreactivity and may contribute prostaglandins during follicular development and ovulation.