Ovulation: a multi-gene, multi-step process

The luteinizing hormone (LH) surge initiates a cascade of proteolytic events that control ovulation. One of the genes induced by LH is the progesterone receptor (PR). Because mice with a mutant PR gene (PRKO) fail to ovulate and are infertile, we have used them as a model in which to determine PR target genes that might mediate the ovulatory process. The matrix metalloproteinases (MMPs: MMP2, MMP9, and MMP13) appear to be expressed in ovaries of PRKO mice in a manner similar to that in their wild-type littermates. However, the expression of two other types of proteases, cathepsin L (a member of the papain family) and ADAMTS-1 (A Disintegrin And Metalloproteinase with Thrombospondin-like motifs), are selectively induced in granulosa cells of preovulatory follicles by the LH surge. Maximal levels of these proteases are observed at 12-16 h after an LH surge, the time of ovulation. Furthermore, mRNAs encoding cathepsin L and ADAMTS-1 are reduced in the PRKO mice compared to their wild-type littermates. These novel observations indicate that these two proteases regulate some key step(s) controlling ovulation.     

Confirmation of ovulation and characterization of luteinizing hormone and progesterone secretory patterns in cycling,isosexually housed Bolivian squirrel monkeys (Saimiri boliviensis boliviensis)

In the female Bolivian squirrel monkey a much greater elevation of serum estradiol (E₂) was measured after mating than that observed in similary cycling monkeys that did not mate. This raised the possibility that cycling squirrel monkeys may not ovulate during nonmated cycles To test this hypothesis, we performed laparoscopies on nine isosexually housed, cycling monkeys to observe the ovaries after the luteinizing hormone (LH) surge, which was measured by mouse interstitial cell bioassay using LER 1909-2 as the standard. Single ovulatory stigmas were identified as well demarcated, red, punctate depressions at the center of dome-shaped elevations on the ovarian surface in eight monkeys, when laparoscopically examined 9-56 hr after the LH peak. One monkey examined laparoscopically prior to the LH surge had a large translucent cystic follicle, confirming the morphology of the mature prevulatory follicle. Mean progesterone (P) concentrations fell to a nadir 1 day prior to the LH surge and then began to rise on the LH surge. Peak P levels were found 2 days after the LH surge. In the ovulating animals in which periovulatory E₂ levels were measured, no value was greater than 800 pg/ml, indicating that the presence of follicular rupture was not sufficient to account for the elevated E₂ levels observed after mating. These data confirm ovulation and follicular rupture in the absence of mating and delineate the relationship between periovulatory LH, P, and E₂ secretory patterns in cycling squirrel monkeys.     

Gonadotropin surge induces two separate increases in messenger RNA for progesterone receptor in bovine preovulatory follicles

In mice deficient in progesterone receptor (PR), follicles of ovulatory size develop but fail to ovulate, providing evidence for an essential role for progesterone and PR in ovulation in mice. However, little is known about the expression and regulation of PR mRNA in preovulatory follicles of ruminant species. One objective of this study was to determine whether and when PR mRNA is expressed in bovine follicular cells during the periovulatory period. Luteolysis and the LH/FSH surge were induced with prostaglandin F(2alpha) and a GnRH analogue, respectively, and the preovulatory follicle was obtained at 0, 3.5, 6, 12, 18, or 24 h after GnRH treatment. RNase protection assays revealed a transient increase in levels of PR mRNA, which peaked at 6 h after GnRH and declined to the time 0 value by 12 h and a second increase at 24 h. The second objective was to investigate the mechanisms that regulate PR mRNA expression through in vitro studies on follicular cells of preovulatory follicles obtained before the LH/FSH surge. Theca and granulosa cells were isolated and cultured with or without a luteinizing dose of LH or FSH, progesterone, LH + progesterone, or LH + antiprogestin (RU486). Levels of PR mRNA increased in a time-dependent manner in granulosa cells cultured with LH or FSH and in theca cells cultured with LH, peaking at 10 h of culture. In contrast, progesterone (200 ng/ml) did not upregulate mRNA for its own receptor, and neither progesterone nor RU486 affected LH-stimulated PR mRNA accumulation. Furthermore, RU486 completely blocked LH-stimulated expression of oxytocin mRNA, indicating that PR induced by LH in vitro is functional. These results show that the gonadotropin surge induces a rapid and transient increase in expression of PR mRNA in both theca and granulosa cells of bovine periovulatory follicles followed by a second rise close to the time of ovulation and that the first increase in PR mRNA can be mimicked in vitro by gonadotropins but not by progesterone. These results suggest multiple and time-dependent roles for progesterone and PR in the regulation of periovulatory events in cattle.     

Gonadotropin and steroid control of granulosa cell proliferation during the periovulatory interval in rhesus monkeys

Progesterone produced in response to the midcycle gonadotropin surge is essential for ovulation and luteinization of the primate follicle. Because cell-cycle arrest is associated with the initiation of luteinization, this study was designed to determine the dynamics and regulation of granulosa cell proliferation by gonadotropin and progesterone during the periovulatory interval in the primate follicle. Granulosa cells or ovaries were obtained from macaques undergoing controlled ovarian stimulation either before (0 h) or as long as 36 h following the administration of an ovulatory hCG bolus with or without a 3beta-hydroxysteroid dehydrogenase inhibitor with or without a nonmetabolizable progestin. The percentage of cells staining positive for Ki-67, a nuclear marker for cell proliferation, decreased (P < 0.05) within 12 h of hCG administration in a steroid-independent manner. Levels of cyclin D2 and E mRNA did not decline during the periovulatory interval; however, cyclin B1 mRNA was reduced significantly by 12 h. Steroid depletion increased (P < 0.05) cyclin B1 mRNA at both 12 and 36 h post-hCG and was reversible by progestin replacement at 36 h. The cyclin-dependent kinase inhibitor p21(Cip1) was transiently increased 12 h post-hCG, whereas p27(Kip1) mRNA levels increased at 36 h in a steroid-independent fashion. These data suggest that a gonadotropin bolus inhibits mitosis in granulosa cells early (12 h) in the periovulatory interval, whereas progesterone may play a later, antiproliferative role in luteinized cells of primates.     

Mineralocorticoid synthesis during the periovulatory interval in macaques

Ovulation and luteal formation in primates are associated with the sustained synthesis of progesterone. The observed high intrafollicular concentrations of progesterone during the periovulatory interval raise the possibility that this steroid serves as a precursor for mineralocorticoids. The aim of this study was to determine if mineralocorticoids are synthesized by the luteinizing macaque follicle during controlled ovarian stimulation cycles in which follicular fluid and granulosa cell aspirates were obtained before or after an ovulatory hCG bolus. Follicular fluid concentrations of progesterone and 17alpha-hydroxyprogesterone increased within 3 h of an ovulatory hCG bolus. Their respective metabolites, 11-deoxycorticosterone (DOC) and 11-deoxycortisol, were not detectable before an ovulatory stimulus and increased starting at 6 h after hCG, while corticosterone and aldosterone were undetectable. Cortisol was present before and after hCG administration and had increased 2-fold at 24 h after an ovulatory stimulus. The expression of 21-hydroxylase (CYP21A2) mRNA increased within 3 h of hCG administration, while 11beta-hydroxylase-1 (CYP11B1) and 11beta-hydroxylase-2 (CYP11B2) mRNAs were not detectable. 11beta-Hydroxysteroid dehydrogenase-1 (HSD11B1) mRNA had increased at 12 h after hCG administration, and 11beta-hydroxysteroid dehydrogenase-2 (HSD11B2) had decreased by 3 h after hCG administration. Mineralocorticoid receptor mRNA levels did not change following hCG administration, while glucocorticoid receptor mRNA levels increased in response to an ovulatory stimulus.Treatment of granulosa cells with the mineralocorticoid receptor antagonist spironolactone blocked hCG-induced progesterone synthesis in vitro. These data indicate that macaque granulosa cells can synthesize mineralocorticoids in response to an ovulatory stimulus and that the mineralocorticoid receptor plays a key role in steroid synthesis associated with luteinization of macaque granulosa cells.     

Effects of high and low preovulatory concentrations of progesterone on ovulation from the isolated perfused rabbit ovary

Rabbit ovaries were isolated surgically before the ovulatory gonadotrophin stimulation and perfused in vitro. Untreated, control ovaries never ovulated. Ovaries treated in vitro with ovine LH ovulated 10-14 h later and the oocytes had undergone germinal vesicle breakdown (GVB). LH induced increases in progesterone secretion from the treated ovaries. A 3 beta-hydroxysteroid dehydrogenase blocker ('Compound A') effectively reduced progesterone secretion into the perfusate and follicular fluid to very low levels but had no effect on ovulation rate or on oocyte maturation. Excessively high progesterone levels were obtained artificially in perfusates by addition of exogenous steroid; the number of ovaries ovulating was markedly reduced but there was no effect on oocyte maturation. It is concluded that the rise in progesterone that normally occurs immediately after the LH surge is not a prerequisite for ovulation in the rabbit. However, progesterone may have a modifying effect on LH-induced follicle rupture when at a pharmacologically high level.     

Novel role of ADAMTS-5 protein in proteoglycan turnover and lipoprotein retention in atherosclerosis

Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE(-/-)) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE(441)) and aggrecan ((374)ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE(441) versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.     

Cellular localization of gelatinases and tissue inhibitors of metalloproteinases during follicular growth, ovulation, and early luteal formation in the rat

The matrix metalloproteinase (MMP) system consists of a proteolytic component, the metalloproteinases, and an associated class of tissue inhibitors of metalloproteinases (TIMPs). We investigated the cellular localization of the TIMPs and the gelatinase family of MMPs throughout the latter stages of follicular growth and during the periovulatory period. Immature female rats were injected with eCG, and ovaries were collected at the time of eCG administration (0 h) and at 6, 12, 24, or 36 h after eCG injection (i.e., follicular development group). A second group of animals (periovulatory) was injected with eCG followed by hCG 48 h later, and ovaries were collected at 0, 12, and 24 h after hCG. Ovaries were processed for the cellular localization of gelatinase or TIMP mRNA or gelatinolytic activity. Gelatinase mRNA (MMP-2 and MMP-9) was localized to the theca of developing follicles and to the stroma. Following a hCG stimulus, MMP-2 mRNA increased as the granulosa cells of preovulatory follicles underwent luteinization during formation of the corpus luteum (CL). MMP-9 mRNA remained predominantly in the theca during this period. In situ zymography for gelatinolytic activity demonstrated a pattern of activity that corresponded with the localization of MMP-2 and MMP-9 mRNA around developing follicles. Gelatinolytic activity was observed at the apex of preovulatory follicles and throughout the forming CL. The mRNA for TIMP-1, -2, and -3 was localized to the stroma and theca of developing follicles. TIMP-3 mRNA was present in the granulosa cells of certain follicles but was absent in granulosa cells of adjacent follicles. At 12 h after hCG, luteinizing granulosa cells expressed TIMP-1 and TIMP-3 mRNA, but TIMP-2 mRNA was at levels equivalent to the background. In the newly forming CL at 24 h after hCG administration, the luteal cells expressed TIMP-1, -2, and -3 mRNA, although the pattern of cellular expression was unique for each of the TIMPs. These findings demonstrate that the MMPs and TIMPs are in the cellular compartments appropriate for impacting the remodeling of the extracellular matrix as the follicle grows, ovulates, and forms the CL.     

Intraovarian thrombin and activated protein C signaling system regulates steroidogenesis during the periovulatory period

In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After treatment of gonadotropin-primed immature mice with an ovulatory dose of human chorionic gonadotropin (hCG) (a LH surrogate), major increases in the expression of THBD, EPCR, PAR1, and PAR4 were detected in granulosa and cumulus cells of preovulatory follicles. Immunoassay analyses demonstrated sustained increases in ovarian prothrombin and APC levels after hCG stimulation. We obtained luteinizing granulosa cells from mice treated sequentially with equine CG and hCG. Treatment of these cells with thrombin or agonists for PAR1 or PAR4 decreased basal and forskolin-induced cAMP biosynthesis and suppressed hCG-stimulated progesterone production. In cultured preovulatory follicles, treatment with hirudin (a thrombin antagonist) and SCH79797 (a PAR1 antagonist) augmented hCG-stimulated progesterone biosynthesis, suggesting a suppressive role of endogenous thrombin in steroidogenesis. Furthermore, intrabursal injection with hirudin or SCH79797 led to ipsilateral increases in ovarian progesterone content. Our findings demonstrated increased ovarian expression of key components of the thrombin-APC-PAR1/4 signaling system after LH/hCG stimulation, and this signaling pathway may allow optimal luteinization of preovulatory follicles. In addition to assessing the role of thrombin and associated genes in progesterone production by the periovulatory ovary, these findings provide a model with which to study molecular mechanisms underlying thrombin-APC-PAR1/4 signaling.     

Primate granulosa cell response via prostaglandin E2 receptors increases late in the periovulatory interval

Successful ovulation requires elevated follicular prostaglandin E2 (PGE2) levels. To determine which PGE2 receptors are available to mediate periovulatory events in follicles, granulosa cells and whole ovaries were collected from monkeys before (0 h) and after administration of an ovulatory dose of hCG to span the 40-h periovulatory interval. All PGE2 receptor mRNAs were present in monkey granulosa cells. As assessed by immunofluorescence, PTGER1 (EP1) protein was low/nondetectable in granulosa cells 0, 12, and 24 h after hCG but was abundant 36 h after hCG administration. PTGER2 (EP2) and PTGER3 (EP3) proteins were detected by immunofluorescence in granulosa cells throughout the periovulatory interval, and Western blotting showed an increase in PTGER2 and PTGER3 levels between 0 h and 36 h after hCG. In contrast, PTGER4 (EP4) protein was not detected in monkey granulosa cells. Granulosa cell response to PGE2 receptor agonists was examined 24 h and 36 h after hCG administration, when elevated PGE2 levels present in periovulatory follicles initiate ovulatory events. PGE2 acts via PTGER1 to increase intracellular calcium. PGE2 increased intracellular calcium in granulosa cells obtained 36 h, but not 24 h, after hCG; this effect of PGE2 was blocked by a PTGER1 antagonist. A PTGER2-specific agonist and a PTGER3-specific agonist each elevated cAMP in granulosa cells obtained 36 h, but not 24 h, after hCG. Therefore, the granulosa cells of primate periovulatory follicles express multiple receptors for PGE2. Granulosa cells respond to agonist stimulation of each of these receptors 36 h, but not 24 h, after hCG, supporting the hypothesis that granulosa cells are most sensitive to PGE2 as follicular PGE2 levels peak, leading to maximal PGE2-mediated periovulatory effects just before ovulation.     

Control of in vitro maturation of bovine cumulus-oocyte complex by preovulatory granulosa cells

The present study was designed to investigate (1) the influence of the secretions of follicular cells on the in vitro maturation of bovine cumulus-oocyte complexes (COCs) and (2) the origin of the factors controlling the metabolic function of cumulus cells during the preovulatory period. Preovulatory granulosa cells were collected from synchronized heifers either before or 7–9 hr after the luteinizing hormone (LH) surge, and their secretions were recovered after a 3 hr incubation. Follicular fluids (FFs) originating from the same follicles and sera from the same animals were also collected. The effects of FFs, sera, and secretions of granulosa cells on COC metabolism were compared during 24 hr of culture. FF stimulated cumulus expansion, progesterone secretion, and overall protein synthesis by COCs but decreased the amount of a major protein of 28 kDa. The time at which FF was collected influenced both cumulus expansion and protein synthesis by COCs. The effects of FF on COC metabolism were detected at the lowest protein concentration studied (0.073 mg/ml) and could be mimicked with serum, but only at a protein concentration 100-fold higher. The inhibitory effect of FF and serum on the amount of the 28 kDa protein was reproduced with the secretions of granulosa cells, acting at protein concentrations five- and 500-fold lower, respectively. However, the secretions of granulosa cells enhanced slightly cumulus expansion and had no effect on progesterone secretion and overall protein synthesis by COCs. These results suggest that COC metabolism is influenced both by endocrine and by local factors secreted by granulosa cells in response to gonadotropins. The paracrine control of COC metabolism by preovulatory granulosa cells could be exerted not only via intercellular contacts but also via substances secreted in FF. © 1993 Wiley-Liss, Inc.     

Mammalian oocytes are targets for prostaglandin E2 (PGE2) action

Background  

The ovulatory gonadotropin surge increases synthesis of prostaglandin E2 (PGE2) by the periovulatory follicle. PGE2 actions on granulosa cells are essential for successful ovulation. The aim of the present study is to determine if PGE2 also acts directly at the oocyte to regulate periovulatory events.     

Ovarian FAM110C (family with sequence similarity 110C): induction during the periovulatory period and regulation of granulosa cell cycle kinetics in rats

FAM110C belongs to a family of proteins that regulates cell proliferation. In the present study, the spatiotemporal expression pattern of FAM110C and its potential role were examined during the periovulatory period. Immature female rats were injected with equine chorionic gonadotropin (eCG) followed by human chorionic gonadotropin (hCG) and ovaries or granulosa cells were collected at various times after hCG administration (n = 3/time point). Expression levels of Fam110c mRNA and protein were highly induced both in intact ovaries and granulosa cells at 8 to 12 h after hCG treatment. In situ hybridization analysis demonstrated Fam110c mRNA expression was induced in theca and granulosa cells at 4 h after hCG, primarily localized to granulosa cells at 8 h and 12 h, and decreased at 24 h after hCG. There was negligible Fam110c mRNA detected in newly forming corpora lutea. In rat granulosa cell cultures, hCG induced expression of Fam110c mRNA was inhibited by RU486, whereas NS398 and AG1478 had no effect, suggesting that Fam110c expression is regulated in part by the progesterone receptor pathway. Promoter activity analysis revealed that an Sp1 site was important for the induction of Fam110c expression by hCG. Overexpression of FAM110C promoted granulosa cells to arrest at the G(1) phase of the cell cycle but did not change progesterone levels. In summary, hCG induces Fam110c mRNA expression in granulosa cells by activation of an Sp1-binding site and the actions of progesterone. Our findings suggest that FAM110C may control granulosa cell differentiation into luteal cells by arresting cell cycle progression.     

Lipoprotein receptor expression during luteinization of the ovarian follicle

Ovarian follicles luteinize after ovulation, requiring structural and molecular remodeling along with exponential increases in steroidogenesis. Cholesterol substrates for luteal steroidogenesis are imported via scavenger receptor-BI (SR-BI) and the low-density lipoprotein (LDL) receptor from circulating high-density lipoproteins and LDL. SR-BI mRNA is expressed in pig ovaries at all stages of folliculogenesis and in the corpus luteum (CL). An 82-kDa form of SR-BI predominates throughout, is weakly present in granulosa cells, and is robustly expressed in the CL, along with the less abundant 57-kDa form. Digestion of N-linked carbohydrates substantially reduced the SR-BI mass in luteal cells, indicating that differences between forms is attributable to glycosylation. Immunohistochemistry revealed SR-BI to be concentrated in the cytoplasm of follicular granulosa cells, although found mostly at the periphery of luteal cells. To examine receptor dynamics during gonadotropin-induced luteinization, pigs were treated with an ovulatory stimulus, and ovaries were collected at intervals to ovulation. SR-BI in granulosa cell cytoplasm increased through the periovulatory period, with migration to the cell periphery as the CL matured. In vitro culture of follicles with human chorionic gonadotropin induced time-dependent upregulation of 82-kDa SR-BI in granulosa cells. SR-BI and LDL receptor were reciprocally expressed, with the latter highest in follicular granulosa cells, declining precipitously with CL formation. We conclude that luteinization causes upregulation of SR-BI expression, its posttranslational maturation by glycosylation, and insertion into luteal cell membranes. Expression of the LDL receptor is extinguished during luteinization, indicating dynamic regulation of cholesterol importation to maintain elevated steroid output by the CL.