The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors involved in varied and diverse processes such as steroidogenesis, angiogenesis, tissue remodeling, cell cycle, apoptosis, and lipid metabolism. These processes are critical for normal ovarian function, and all three PPAR family members -alpha, delta, and gamma, are expressed in the ovary. Most notably, the expression of PPARgamma is limited primarily to granulosa cells in developing follicles, and is regulated by luteinizing hormone (LH). Although much has been learned about the PPARs since their initial discovery, very little is known regarding their function in ovarian tissue. This review highlights what is known about the roles of PPARs in ovarian cells, and discusses potential mechanisms by which PPARs could influence ovarian function. Because PPARs are activated by drugs currently in clinical use (fibrates and thiazolidinediones), it is important to understand their role in the ovary, and how manipulation of their activity may impact ovarian physiology as well as ovarian pathology.

PPARs are a family of nuclear hormone receptors involved in various processes that could influence ovarian function. We investigated the cellular localization and expression of PPARs during follicular development in ovarian tissue collected from rats 0, 6, 12, 24, and 48 h post-PMSG. A second group of animals received human CG (hCG) 48 h post-PMSG. Their ovaries were removed 0, 4, 8, 12, and 24 h post-hCG to study the periovulatory period. mRNAs corresponding to the PPAR isotypes (alpha, delta, and gamma) were localized by in situ hybridization. Changes in the levels of mRNA for the PPARs were determined by ribonuclease protection assays. PPAR gamma mRNA was localized primarily to granulosa cells, and levels of expression did not change during follicular development. Four hours post-hCG, levels of mRNA for PPAR gamma decreased (P < 0.05) but not uniformly in all follicles. At 24 h post-hCG, levels of PPAR gamma mRNA were reduced 64%, but some follicles maintained high expression. In contrast, mRNAs for PPAR alpha and delta were located primarily in theca and stroma, and their levels did not change during the intervals studied. To investigate the physiologic significance of PPAR gamma in the ovary, granulosa cells from PMSG-primed rats were cultured for 48 h with prostaglandin J(2) (PGJ(2)) and ciglitazone, PPAR gamma activators. Both compounds increased progesterone and E2 secretion (P < 0.05). These data suggest that PPAR gamma is involved in follicular development, has a negative influence on the luteinization of granulosa cells, and/or regulates the periovulatory shift in steroid production. The more general and steady expression of PPARs alpha and delta indicate that they may play a role in basal ovarian function.

PPARs are a family of nuclear hormone receptors involved in various processes that could influence ovarian function. We investigated the cellular localization and expression of PPARs during follicular development in ovarian tissue collected from rats 0, 6, 12, 24, and 48 h post-PMSG. A second group of animals received human CG (hCG) 48 h post-PMSG. Their ovaries were removed 0, 4, 8, 12, and 24 h post-hCG to study the periovulatory period. mRNAs corresponding to the PPAR isotypes (alpha, delta, and gamma) were localized by in situ hybridization. Changes in the levels of mRNA for the PPARs were determined by ribonuclease protection assays. PPAR gamma mRNA was localized primarily to granulosa cells, and levels of expression did not change during follicular development. Four hours post-hCG, levels of mRNA for PPAR gamma decreased (P < 0.05) but not uniformly in all follicles. At 24 h post-hCG, levels of PPAR gamma mRNA were reduced 64%, but some follicles maintained high expression. In contrast, mRNAs for PPAR alpha and delta were located primarily in theca and stroma, and their levels did not change during the intervals studied. To investigate the physiologic significance of PPAR gamma in the ovary, granulosa cells from PMSG-primed rats were cultured for 48 h with prostaglandin J(2) (PGJ(2)) and ciglitazone, PPAR gamma activators. Both compounds increased progesterone and E2 secretion (P < 0.05). These data suggest that PPAR gamma is involved in follicular development, has a negative influence on the luteinization of granulosa cells, and/or regulates the periovulatory shift in steroid production. The more general and steady expression of PPARs alpha and delta indicate that they may play a role in basal ovarian function.

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.

The preovulatory surge of gonadotropins induces meiotic maturation of the oocyte, the follicular/luteal phase shift in hormone production, and ovulation. This complex and rapid series of developmental changes is difficult to study in large mammals, such as primates and ruminants, because variability in the length of individual reproductive cycles makes it virtually impossible to predict the time of the LH surge. We have validated an experimental model for inducing the LH surge and ovulation in cattle and used it to study the sequence of changes in hormone secretion and some of the mechanisms of these changes. Luteolysis and a follicular phase were induced by injection of prostaglandin F(2alpha); injection of a GnRH analogue 36 h later induced an LH surge and ovulation. The LH surge peaked 2 h after GnRH and ovulation followed 22-31 h after the surge, consistent with the periovulatory interval in natural cycles. The ensuing luteal phase was normal, both in length and in concentrations of circulating progesterone. In experiment I, the uteroovarian effluent was collected, via cannulation of the vena cava, at frequent intervals relative to GnRH injection. Circulating estradiol declined progressively after GnRH, reaching a nadir by 8-10 h before ovulation, whereas concentrations of androstenedione and testosterone remained constant. In experiment II, preovulatory follicles were obtained at 0, 3.5, 6, 12, 18, or 24 h after GNRH: Concentrations of androgens and estradiol were measured in follicular fluid and medium from cultures of follicle wall (theca + granulosa cells); steady-state levels of mRNA for 17alpha-hydroxylase (17alphaOH) and P450 aromatase were measured in follicular tissue. Shortly after the LH surge (3.5 h post-GnRH) there was an acute increase in the capacity of follicular tissue to secrete androstenedione, but not estradiol, in vitro. Thereafter, both androgens and estradiol declined, both in follicular fluid and in medium collected from cultures of follicle wall. Levels of mRNA for 17alphaOH and aromatase in follicle wall decreased significantly by 6 h after GnRH, suggesting that declining levels of these enzymes underlie the decreases in steroid production by follicular cells. These results show that in cattle the preovulatory decrease in follicular estradiol production is mediated by redundant mechanisms, because androgen production and the capacity of granulosa cells to convert androgens to estradiol decline coordinately, in concert with decreases in mRNA for 17alphaOH and P450 aromatase.

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.

Follicular production of prostaglandins (PGs) is essential for ovulation, but the factors mediating gonadotropin-induced secretion of PGE and PGF(2alpha) remain largely unknown. We tested the hypothesis that gonadotropin-induced changes in progesterone and its receptor (PR) mediate the increase in periovulatory PGs. Heifers were treated with PGF(2alpha) and GnRH to induce luteolysis and the LH/FSH surge (ovulation occurs approximately 30 h after GnRH). Because there are two increases in intrafollicular progesterone/PR mRNA during the bovine periovulatory period, we first examined the temporal pattern of PG production by follicles collected at 0, 3.5, 6, 12, 18, and 24 h after GnRH. Although PGs did not increase in the follicular fluid until 24 h after GnRH, acute secretion of PGs by follicle wall (theca + granulosa cells) was initiated by 18 h and had increased manyfold by 24 h after GnRH. In vitro, FSH and LH induced dramatic transient increases in PG production by follicle wall and granulosa, but not theca, cells isolated from preovulatory follicles (0 h after GnRH). PG accumulation peaked on d 2 of culture, mimicking the secretion pattern after a gonadotropin surge in vivo. In cultures of follicle wall and granulosa cells, the PR antagonist mifepristone (MIFE, 1 microm) inhibited LH-induced PG secretion and the progestin medroxyprogesterone acetate (1 or 10 microm), but not the glucocorticoid dexamethasone (1 or 10 microm), overcame the effect of MIFE on PGs. Semiquantitative RT-PCR revealed that MIFE inhibited LH-induced expression of cyclooxygenase-2 mRNA in granulosa cells in vitro. Again, treatment with medroxyprogesterone acetate overcame the effect of MIFE. Together these results provide strong evidence that periovulatory increases in cyclooxygenase-2 mRNA, PGE, and PGF(2alpha) are mediated by gonadotropin-induced increases in progesterone/PR, indicating that in some species there is an important functional relationship between these pathways in the ovulatory cascade.

In cattle the development of large antral follicles occurs in two or three successive waves during the estrous cycle, with one follicle per wave selected for dominance. To test the hypothesis that negative feedback effects of steroids secreted by the dominant follicle are critical to the regulation of follicular waves, we examined temporal relationships among ovarian follicular growth, steroid secretion, and gonadotropin secretion. Follicular growth was monitored by ultrasonography. In the first experiment, blood was collected from 5 Holstein heifers every 8 h between Days 0 and 14 of the estrous cycle from both a jugular vein and the vena cava (to collect ovarian blood). Jugular samples were also collected every 12 min for 8 h during three periods (Days 3 or 4, 7 or 8, and 11, 12, or 13; n = 6) to characterize the pulsatile pattern of LH secretion. Both estradiol and testosterone concentrations in the vena cava increased as pre-wave elevations in FSH concentrations decreased (p < 0.05) between Days 1 and 4 (first follicular wave) and between Days 9 and 12 (second follicular wave). LH pulse amplitude was greater during the second period of frequent blood collection (Day 7 or 8, end of the growth phase of the first dominant follicle) compared to the other two periods (p < 0.05), suggesting that increased LH pulse amplitude may be important for the later stages of dominant follicle growth. In the second experiment, to determine whether ovarian steroids are secreted primarily by dominant follicles, blood samples were collected from the utero-ovarian veins draining ovaries with (n = 4) and without (n = 4) a dominant follicle during the first follicular wave. Testosterone, androstenedione, and estradiol concentrations in the utero-ovarian veins fluctuated in relation to the pattern of follicular growth (p < 0.05), and secretion was much greater from ovaries with a dominant follicle. In blood collected both from the vena cava and from the utero-ovarian veins, estradiol secretion reached a peak and started to decline before androgen concentrations peaked (p < 0.05), suggesting that the initial decrease in estradiol secretion from the dominant follicle is not due to a lack of androgen precursors. The results suggest that 1) a transient increase in LH pulse amplitude during the early-midluteal phase may be important for supporting the final stages of dominant follicle growth; 2) ovarian androgens, as well as estradiol, may play a critical role in the control of FSH secretion during waves of follicular development; and 3) the dominant follicle is responsible for fluctuations in circulating estradiol and androgens during follicular waves.