Growth differentiation factor-9 stimulates proliferation but suppresses the follicle-stimulating hormone-induced differentiation of cultured granulosa cells from small antral and preovulatory rat follicles

In addition to pituitary gonadotropins and paracrine factors, ovarian follicle development is also modulated by oocyte factors capable of stimulating granulosa cell proliferation but suppressing their differentiation. The nature of these oocyte factors is unclear. Because growth differentiation factor-9 (GDF-9) enhanced preantral follicle growth and was detected in the oocytes of early antral and preovulatory follicles, we hypothesized that this oocyte hormone could regulate the proliferation and differentiation of granulosa cells from these advanced follicles. Treatment with recombinant GDF-9, but not FSH, stimulated thymidine incorporation into cultured granulosa cells from both early antral and preovulatory follicles, accompanied by increases in granulosa cell number. Although GDF-9 treatment alone stimulated basal steroidogenesis in granulosa cells, cotreatment with GDF-9 suppressed FSH-stimulated progesterone and estradiol production. In addition, GDF-9 cotreatment attentuated FSH-induced LH receptor formation. The inhibitory effects of GDF-9 on FSH-induced granulosa cell differentiation were accompanied by decreases in the FSH-induced cAMP production. These data suggested that GDF-9 is a proliferation factor for granulosa cells from early antral and preovulatory follicles but suppresses FSH-induced differentiation of the same cells. Thus, oocyte-derived GDF-9 could account, at least partially, for the oocyte factor(s) previously reported to control cumulus and granulosa cell differentiation.     

Effects of methoxychlor on IGF-I signaling pathway in rat ovary

Follicular development and other ovarian functions are regulated by growth factors that can be affected by exogenous agents. Methoxychlor (MXC) is an organochloride pesticide that causes female infertility. We investigated how MXC affects the distribution of developing ovarian follicles in adult rats after treatment between embryonic day (E) 18 and postnatal day (PND) 7. We also measured insulin-like growth factor-I (IGF-I) and its receptor, IGF-IR, expressions in ovarian follicles and investigated whether MXC changed the levels of IGF-I and IGF-IR in the ovary. Using immunohistochemical (IHC) staining, we detected IGF-I expression in oocytes and granulosa cells of the follicles, luteal cells, interstitial cells, theca externa and theca interna, and the smooth muscle of ovarian vessels. IGF-IR was co-localized with IGF-I in the ovary except for the theca externa. IGF-I expression was decreased in granulosa cells of preantral and antral follicles after treatment with MXC compared to granulosa cells of preantral and antral follicles of the control group. We also observed that oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the MXC treated groups showed increased IGF-IR expression compared to oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the control group. We also detected more secondary and preantral follicles, and fewer primordial and antral follicles after MXC administration compared to controls. Therefore, the IGF signaling pathway may participate in MXC induced ovary dysfunction and female infertility.     

Mouse antral oocytes regulate preantral granulosa cell ability to stimulate oocyte growth in vitro

In this study we evaluated whether mouse oocytes derived from early antral or preovulatory follicles could affect the ability of preantral granulosa cells to sustain oocyte growth in vitro. We found that early antral oocytes with a diameter > or =75 microm did not grow any further during 3 days of culture on preantral granulosa cell monolayers in vitro, while most of the oocytes with a smaller diameter increased significantly in size. Similarly, about 65% of growing oocytes isolated from preantral follicles grew when cultured on preantral granulosa cells. By coculturing with growing oocytes fully grown early antral or preovulatory oocytes, a small proportion (about 10%) of growing oocytes increased in diameter, and changes in granulosa cell morphology were observed. Such effects occurred as a function of the fully grown oocyte number seeded and were not associated with a decrease in coupling index values. By avoiding physical contact between antral oocytes and granulosa cells, the proportion of growing oocytes undergoing a significant increase in diameter was about 36%. These results indicate that fully grown mouse oocytes can control preantral granulosa cell growth-promoting activity through the production of a soluble factor(s) and the maintenance of functional communications with surrounding granulosa cells.     

Androgen synthesis during follicular development: evidence that rat granulosa cell 17-ketosteroid reductase is independent of hormonal regulation

Although androgens have been implicated in follicular atresia, ovarian follicular androgen synthesis is required for preovulatory follicular growth. To localize the site(s) of androgen biosynthesis and to obtain a better understanding of the regulation of the androgenic pathway(s) in rat ovarian follicles we examined the relative abilities of developing follicles to accumulate specific androgens [testosterone (T) and dihydrotestosterone (DHT)] using both radioimmunoassay (RIA) and 3H-substrate metabolism techniques. Small antral and preovulatory follicles were obtained from control or human chorionic gonadotropin (hCG)-primed immature rats, respectively (Richards and Bogovich, 1982). Small antral follicles, theca and granulosa cells produced little immunoassayable androgen (T + DHT) when incubated with or without 8-bromo-cAMP. In contrast, preovulatory follicles and theca produced more androgen than small antral tissues and in a manner acutely stimulable by cAMP. Granulosa cells produced little androgen under these conditions. Inclusion of [3H] androstenedione in the incubates yielded increased accumulation of [3H] T and [3H] DHT for all small antral and preovulatory tissues. Indeed, granulosa cells from both small antral and preovulatory follicles possessed a remarkable ability to accumulate [3H] T. This ability was not altered by hypophysectomy or subsequent treatment with estradiol and/or follicle-stimulating hormone (FSH). These results suggest that 17-ketosteroid reductase may be a constitutive enzyme in granulosa cells.     

Expression of mRNA of lipoprotein receptor related protein 8, low density lipoprotein receptor, and very low density lipoprotein receptor in bovine ovarian cells during follicular development and corpus luteum formation and regression

Lipoproteins in the plasma are the major source of cholesterol obtained by the ovarian theca and granulosa cells for steroidogenesis. In this study, we have identified mRNA expression in bovine theca and granulosa cells of two lipoprotein receptors, low density lipoprotein receptor (LDLr) and very low density lipoprotein receptor (VLDLr) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. In the corpus luteum (CL) both these receptors were found in the developing and differentiating stages whereas only mRNA for VLDLr was detected in the regression stage. This study also described for the first time, the presence of lipoprotein receptor related protein (LRP8) in granulosa cells from small antral follicles through preovulatory follicles and in theca cells from large and medium sized antral follicles. This may indicate a role of LRP8 in cholesterol delivery to steriodogenic cells. LRP8 was not detected in any of the CL stages. The roles of the LDLr superfamily in lipid transport to ovarian cells and its participation in follicular and CL development and regression is discussed.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Expression and regulation of mRNAs for insulin-like growth factor (IGF-I), IGF-binding protein-2, and LH receptor in the process of follicular atresia

Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in pr     

mRNA expression pattern of gonadotropin receptors in bovine follicular cysts

Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis.     

CNP/NPR2 signaling maintains oocyte meiotic arrest in early antral follicles and is suppressed by EGFR‐mediated signaling in preovulatory follicles

Oocyte meiosis is arrested at prophase I by factors secreted from surrounding somatic cells after oocytes acquire meiotic competence at an early antral stage, and meiosis resumes in preovulatory follicles as a result of the luteinizing hormone (LH) surge. Recently, signaling by C‐type natriuretic peptide (CNP) through its receptor, natriuretic peptide receptor 2 (NPR2), was found to be essential for meiotic arrest at the late antral stage. Whether or not CNP/NPR2 signaling maintains oocyte meiotic arrest in earlier follicular stages and how it is associated with meiotic resumption induced by the LH surge is unclear. In this study, we examined the expression of Nppc and Npr2, respectively encoding CNP and NPR2, in the ovaries of immature mice. Nppc and Npr2 mRNA were specifically expressed in the outer and inner granulosa cell layers, respectively, in early antral follicles. Histological analysis of mice with a mutation in Npr2 revealed precocious resumption of oocyte meiosis in early antral follicles. Ovaries of mice treated with excess human chorionic gonadotropin (hCG) exhibited markedly decreased Nppc mRNA levels in granulosa cells of preovulatory follicles. Moreover, we found that amphiregulin, a mediator of LH/hCG activity through epidermal growth factor receptor (EGFR), suppressed Nppc mRNA levels in cultured granulosa cells. These results suggest that CNP/NPR2 signaling is essential for oocyte meiotic arrest in early antral follicles and that activated LH/amphiregulin/EGFR signaling pathway suppresses this signal by downregulating Nppc expression. Mol. Reprod. Dev. 79: 795–802, 2012. © 2012 Wiley Periodicals, Inc.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Anticoagulant heparan sulfate proteoglycans expression in the rat ovary peaks in preovulatory granulosa cells

Ovarian granulosa cells synthesize anticoagulant heparan sulfate proteoglycans (aHSPGs), which bind and activate antithrombin III. To determine if aHSPGs could contribute to the control of proteolytic activities involved in follicular development and ovulation, we studied the pattern of expression of these proteoglycans during the ovarian cycle. aHSPGs were localized on cells and tissues by (125)I-labeled antithrombin III binding followed by microscopic autoradiography. Localization of aHSPGs has shown that cultured granulosa cells, hormonally stimulated by gonadotropins to differentiate in vitro, up-regulate their synthesis and release of aHSPGS: In vivo, during gonadotropin-stimulated cycle, aHSPGs are present on granulosa cells of antral follicles and are strongly labeled in preovulatory follicles. These data demonstrate that aHSPG expression in the ovarian follicle is hormonally induced to culminate in preovulatory follicles. Moreover, we have shown that five heparan sulfate core proteins mRNA (perlecan; syndecan-1, -2, and -4; and glypican-1) are synthesized by granulosa cells, providing attachment for anticoagulant heparan sulfate chains on the cell surface and in the extracellular matrix. These core proteins are constantly expressed during the cycle, indicating that modulations of aHSPG levels observed in the ovary are likely controlled at the level of the biosynthesis of anticoagulant heparan sulfate glycosaminoglycan chains. This expression pattern enables aHSPGs to focus serine protease inhibitors in the developing follicle to control proteolysis and fibrin formation at ovulation.     

Localization of androgen receptor and cytochrome P450 aromatase in the follicle and corpus luteum of the porcine ovary

The following study was undertaken to localize androgen receptors (AR) and aromatase cytochrome P450 (P450arom) in porcine ovarian tissue because ovarian androgens may act locally to modulate follicular and luteal function in various species. Androgen receptor was detected immunohistochemically in granulosa and theca cells of preantral as well as in growing antral follicles. The most intensive staining was observed in the antral granulosa layer. Luteinizing granulosa cells of preovulatory follicles, and luteal cells from the early and midluteal phases stained weakly for the androgen receptor. Fully regressed corpora lutea in the early follicular phase of the next cycle did not stain for androgen receptor. In contrast, granulosa cells were very weakly stained for aromatase in early stages of follicular development. The P450arom was maximally expressed with the same intensity in mural and antral layers in large ovulatory follicles. Corpora lutea from the early luteal phase showed positive staining, whereas those from midluteal phase did not stain for aromatase, some cells of regressed corpora lutea unexpectedly exhibited aromatase staining.     

Spatiotemporal expression of epidermal growth factor receptor messenger RNA and protein in the hamster ovary: follicle stage-specific differential modulation by follicle-stimulating hormone,luteinizing hormone,estradiol, and progesterone

Spatiotemporal expression, endocrine regulation, and activation of epidermal growth factor receptor (EGFR) in the hamster ovary were evaluated by immunofluorescence and in situ hybridization localization. Whereas granulosa cells (GC) of primordial through large preantral (stage 6, 7-8 layers GC) follicles had low immunoreactivity, granulosa cells of antral follicles, theca, and interstitial cells had intense EGFR immunoreactivity. EGFR expression in GC of primordial and small preantral follicles increased progressively from estrous through proestrous, but a significant increase occurred in mural GC of antral follicles following the gonadotropin surge. Interstitial cells around small preantral follicles had strong immunofluorescence, and the intensity increased significantly in fully differentiated thecal cells. Distinct EGFR protein was localized in the nucleus of the oocytes and granulosa cells. FSH significantly stimulated EGFR expression in the GC, especially the mural GC, theca, and interstitial cells in hypophysectomized hamster. Estrogen stimulated EGFR expression in preantral GC as well as in interstitial cells. Progesterone and hCG effect was limited to theca and interstitial cells. EGFR expression correlated well with EGFR activation following endogenous or exogenous gonadotropin exposure. Receptor mRNA expression closely followed the protein expression, with increased mRNA expression in mural GC of antral follicles. These results suggest that low levels of EGF signal as a consequence of low levels of receptors in preantral GC may be critical for cell proliferation, but higher receptor density may evoke increased signal intensity due to activation of other intracellular signal pathways, which activate cellular processes related to granulosa, theca, and interstitial cell differentiation. The spatiotemporal cell type and follicle stage-specific expression of receptor mRNA and protein and EGFR activation is critically regulated by gonadotropins and ovarian steroids, primarily estradiol.     

Induction of apoptosis by 9,10-dimethyl-1,2-benzanthracene in cultured preovulatory rat follicles is preceded by a rise in reactive oxygen species and is prevented by glutathione

The polycyclic aromatic hydrocarbon (PAH) 9,10-dimethyl-1,2-benzanthracene (DMBA) destroys primordial, primary, and secondary ovarian follicles in rodents, but its effects on antral follicles have received limited attention. PAHs are metabolized to reactive species, some of which can undergo redox cycling to generate reactive oxygen species (ROS). We previously showed that ROS initiate apoptosis in preovulatory follicles cultured without gonadotropin support and that glutathione (GSH) depletion induces apoptosis in the presence of gonadotropins. In the present study, we tested the hypothesis that DMBA induces apoptosis in preovulatory follicles, which is mediated by ROS and prevented by GSH. Preovulatory follicles were isolated from ovaries of 25-day-old rats 48 h after the injection of 10 IU of eCG and were cultured with DMBA in the presence of FSH for 2 to 48 h. DMBA induced granulosa cell (GC) and theca cell (TC) apoptosis at 48 h, as judged by TUNEL and activated caspase-3 immunostaining. DMBA treatment also increased the numbers of GCs and TCs that immunostained for the proapoptotic protein BAX. Follicular ROS levels were significantly increased in DMBA-treated follicles at 12, 24, and 48 h. GSH supplementation protected against and GSH depletion enhanced the induction of apoptosis in GCs and TCs by DMBA. These findings suggest that GSH is a critical protective mechanism against DMBA-induced apoptosis in antral follicles and that ROS generation may mediate DMBA-induced GC apoptosis.