Carolyn M. Komar

Peroxisome proliferator-activated receptors (PPARs) and ovarian function – implications for regulating steroidogenesis, differentiation, and tissue remodeling

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.

Decline in Circulating Estradiol During the Periovulatory Period Is Correlated with Decreases in Estradiol and Androgen, and in Messenger RNA for P450 Aromatase and P450 17α-Hydroxylase, in Bovine Preovulatory Follicles

Abstract 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 F2α; 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 17α-hydroxylase (17αOH) 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 17αOH 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 17αOH and P450 aromatase.

Gonadotropin Surge Induces Two Separate Increases in Messenger RNA for Progesterone Receptor in Bovine Preovulatory Follicles

Abstract 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 F2α 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.

Expression of Messenger RNA for ADAMTS Subtypes Changes in the Periovulatory Follicle after the Gonadotropin Surge and During Luteal Development and Regression in Cattle

Abstract Extensive remodeling of the extracellular matrix occurs in the ovary during the periovulatory period. Matrix metalloproteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases, are believed to play integral roles in this highly regulated series of cellular events, but their specific roles remain unclear. Recent cloning studies have identified a novel family of metalloproteinases, the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family. The regulated expression of distinct ADAMTS subtypes has been shown to be required for tissue morphogenesis during embryonic development and for maintaining the integrity of tissues in the adult. In the present studies, we have determined that multiple ADAMTS subtypes are present in the bovine ovary using a reverse transcription-polymerase chain reaction strategy. In particular, ADAMTS-1, -2, -3, -4, -5 (also known as ADAMTS-11), -7, -8, and -9, but not ADAMTS-6, -10, or -12, mRNA transcripts were detected in granulosa cells of nonatretic ovarian follicles and corpora lutea. The levels of mRNA for these ovarian ADAMTS were up- or down-regulated or remained unchanged in the granulosa and/or theca cells of the dominant follicle following the preovulatory surge of gonadotropins, depending on the subtype and/or the cell compartment, and in the corpus luteum during the luteal phase of the estrous cycle. The complex expression patterns observed for the distinct ADAMTS subtypes in the granulosa and theca cells of the periovulatory follicle and in the luteal tissues of the bovine ovary suggest that these novel proteases mediate, at least in part, the remodeling events underlying folliculogenesis and ovulation and the formation, maintenance, and regression of the corpus luteum.

Localization and Expression of Messenger RNAs for the Peroxisome Proliferator-Activated Receptors in Ovarian Tissue from Naturally Cycling and Pseudopregnant Rats

Abstract 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 (α, δ, and γ) 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 PPARγ 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 PPARγ mRNA. The PPARα mRNA was localized mainly in the theca and stroma, and PPARδ mRNA was expressed throughout the ovary. Levels of mRNA for PPARγ decreased between Days 1 and 4 of pseudopregnancy, and PPARα mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARδ mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARγ 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 PPARδ mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARα may play a role in lipid metabolism in the theca and stroma.

Inverse Relationship Between the Expression of Messenger Ribonucleic Acid for Peroxisome Proliferator-Activated Receptor γ and P450 Side Chain Cleavage in the Rat Ovary

Abstract Messenger RNA for peroxisome proliferator-activated receptor γ (PPARγ) has been found in granulosa cells, and its expression decreases after the LH surge. We determined which developmental stage of ovarian follicle expresses mRNA for PPARγ and evaluated the impact of PPARγ agonists on steroidogenesis. Ovaries were collected from immature eCG/hCG-treated rats at 0 (no eCG), 24, and 48 h post-eCG and 4 and 24 h post-hCG. Ovarian tissue was serially sectioned and processed for in situ hybridization to localize mRNA corresponding to PPARγ, aromatase, and the LH receptor, and P450 side chain cleavage (P450SCC) and to determine whether apoptotic cells were present. During follicular development, there was no correlation between the expression of mRNAs for PPARγ and aromatase or the presence of apoptotic cells, but a general inverse correlation was observed between the expression of PPARγ mRNA and LH receptor mRNA. At 4 h post-hCG, follicles expressing P450SCC mRNA had lost expression of PPARγ mRNA. This inverse pattern of expression between PPARγ and P450SCC mRNAs was also observed 24 h post-hCG, with developing luteal tissue expressing high levels of P450SCC mRNA but little or no PPARγ mRNA. To determine the impact of PPARγ on steroidogenesis, granulosa cells were collected from ovaries 24 h post-eCG and cultured alone, with FSH alone, or with FSH in combination with the PPARγ agonists ciglitazone or 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2). Treatment of granulosa cells with PGJ2 stimulated basal progesterone secretion, whereas ciglitazone or PGJ2 had no significant effect on FSH-stimulated steroid production. These findings suggest that 1) PPARγ may regulate genes involved with follicular differentiation and 2) the decline in PPARγ in response to LH is important for ovulation and/or luteinization.

Localization and Expression of Tissue Inhibitor of Metalloproteinase-4 in the Immature Gonadotropin-Stimulated and Adult Rat Ovary

Abstract The tissue inhibitors of metalloproteinases (TIMPs) are important regulators of the matrix metalloproteinases (MMPs), proteolytic enzymes essential for controlling the coordinated tissue remodeling that takes place in the ovary. In the present study, we characterized the ovarian expression pattern of TIMP-4. The localization of TIMP-4 mRNA was determined by in situ hybridization in adult cycling rats. TIMP-4 mRNA on the day of estrus was expressed in a punctate pattern in stroma and in corpora lutea (CL) from previous cycles but not in newly formed CL or follicles. At metestrus, TIMP-4 mRNA was present in certain CL from the current and previous cycles and continued to exhibit a punctate pattern of expression in the stroma. By diestrus, TIMP-4 mRNA was detected in the thecal layer surrounding follicles, and a relatively high level of expression was observed in a punctate pattern within new and previous CL and in the stroma. TIMP-4 mRNA was also observed in the thecal layer at proestrus, but the punctate pattern within CL and stroma was absent. To correlate the changes in cellular localization with changes in overall TIMP-4 levels, ovarian mRNA and protein levels were examined in adult cycling rats and in gonadotropin-stimulated immature rats. In cycling rats, there was no change in mRNA or protein levels across the cycle, although there was a trend towards higher levels during estrus (P = 0.08). In gonadotropin-treated rats, there was an increase in TIMP-4 mRNA 48 h after eCG administration with a corresponding doubling of TIMP-4 protein. Although TIMP-4 mRNA and protein tended to decline after hCG treatment, this trend was not significant (P = 0.08). These findings indicate that TIMP-4 could play an important role in regulating MMPs in a localized manner in follicles and CL throughout the cycle.

Periovulatory Changes in Ovarian Metalloproteinases and Tissue Inhibitors of Metalloproteinases (TIMPS) Following Indomethacin Treatment

The periovulatory luteinizing hormone (LH) surge sets in motion a series of biochemical and biophysical changes culminating in follicular rupture and release of the oocyte. Among these biochemical events is a stimulation of prostaglandin production in conjunction with a stimulation of proteolytic enzyme activity (reviewed in Refs. 1, 2). The LH-stimulated increase in prostaglandins (PGs) has been suggested to be of pivotal importance in the ovulatory process (1, 2). Such a postulate is supported by reports that PGF2α (3, 4) and prostacyclin (5) are able to induce follicular rupture in the absence of gonadotropins. Further evidence for the role of PGs in ovulation is the observation that blockade of PG production by various PG synthase inhibitors (e. g., indomethacin) results in an inhibition of ovulation in the rat, rabbit, sheep, pig, primate, and human (reviewed in Refs. 1, 2). The mechanism(s) by which PGs may regulate follicular rupture, however, is unknown. It is hypothesized that local PG production may impact ovarian blood flow, smooth muscle contractility, oxygen-free radical formation, and action, as well as proteolysis associated with apical connective tissue degradation and oocyte extrusion (1, 2).

Potential role for peroxisome proliferator-activated receptor γ in regulating luteal lifespan in the rat

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to stimulate progesterone production by bovine luteal cells. We previously reported higher expression of PPARgamma in old compared with new luteal tissue in the rat. The following studies were conducted to determine the role of PPARgamma in rat corpora lutea (CL) and test the hypothesis that PPARgamma plays a role in the metabolism of progesterone and/or luteal lifespan. Ovaries were removed from naturally cycling rats throughout the estrous cycle, and pseudopregnant rats. mRNA for PPARgamma and P450 side-chain cleavage (SCC) was localized in luteal tissue by in situ hybridization, and protein corresponding to PPARgamma and macrophages identified by immunohistochemistry. Luteal tissue was cultured with agonists (ciglitazone, prostaglandin J2) or an antagonist (GW-9662) of PPARgamma. Progesterone was measured in media by RIA and levels of mRNA for 20alpha-hydroxysteriod dehydrogenase (HSD) and bcl-2 were measured in luteal tissue after culture by RT-PCR. An inverse relationship existed between the expression of mRNA for SCC and PPARgamma. There was no effect of PPARgamma agonists or the antagonist on luteal progesterone production in vitro, or levels of mRNA for 20alpha-HSD. PPARgamma protein was localized to the nuclei of luteal cells and did not correspond with the presence of macrophages. In new CL, ciglitazone decreased mRNA for bcl-2 on proestrus, estrus, and metestrus. Interestingly, GW-9662 also decreased mRNA for bcl-2 on proestrus and diestrus in old and new CL, and on metestrus in new CL. These data indicate that PPARgamma is not a major player in luteal progesterone production or metabolism but may be involved in regulating luteal lifespan.

Polarized Ovaries of the Long-Tongued Bat, Glossophaga soricina: A Novel Model for Studying Ovarian Development, Folliculogenesis, and Ovulation

Glossophaga soricina is a spontaneously ovulating, monovular, polyestrous bat with a simplex uterus, exhibiting true menstruation. Studies conducted on reproductively active, captive‐maintained animals established that G. soricina also has polarized ovaries, with the ovarian surface epithelium (OSE) restricted to the medial side of the ovary, and primordial follicles limited to an immediately adjacent zone. Follicles selected for further development are recruited from the medullary side of this zone, and ovulation is restricted to the portion of the ovary covered by the OSE. To further develop G. soricina as a model for studying ovarian development and physiology, ovaries were collected from fetal, neonatal, and adult females and processed for morphological and immunohistochemical analyses. The latter included staining for factor VIII‐related antigen (von Willebrand factor) to assess regional differences in ovarian vascularity. The ovarian structure in fetal and neonatal animals was very similar to that in other species that do not have polarized ovaries at comparable stages of development. This indicated that polarization of the ovary does not occur during fetal development, but rather sometime between the neonatal period and adulthood. Vascular elements were abundant in areas of the ovary surrounding early growing follicles, but sparse in the zone of the ovary containing primordial follicles. The polarized nature of the ovaries in G. soricina suggests that this species might be used as a model to investigate the formation, long‐term maintenance, and activation of primordial follicles, and the role of the OSE in ovulation and folliculogenesis. Anat Rec, 290:1439–1448, 2007.