Molecular crosstalk between insulin-like growth factors and follicle-stimulating hormone in the regulation of granulosa cell function.

Background: The last phase of folliculogenesis is driven by follicle-stimulating hormone (FSH) and locally produced insulin-like growth factors (IGFs), both essential for forming preovulatory follicles. Methods: This review discusses the molecular crosstalk of the FSH and IGF signaling pathways in regulating follicular granulosa cells (GCs) during the antral-to-preovulatory phase. Main findings: IGFs were considered co-gonadotropins since they amplify FSH actions in GCs. However, this view is not compatible with data showing that FSH requires IGFs to stimulate GCs, that FSH renders GCs sensitive to IGFs, and that FSH signaling interacts with factors downstream of AKT to stimulate GCs. New evidence suggests that FSH and IGF signaling pathways intersect at several levels to regulate gene expression and GC function. Conclusion: FSH and locally produced IGFs form a positive feedback loop essential for preovulatory follicle formation in all species. Understanding the mechanisms by which FSH and IGFs interact to control GC function will help design new interventions to optimize follicle maturation, perfect treatment of ovulatory defects, improve in vitro fertilization, and develop new contraceptive approaches.

Oocyte-secreted factors strongly stimulate sFRP4 expression in human cumulus cells.

Secreted frizzled-related protein-4 (SFRP4) belongs to a family of soluble ovarian-expressed proteins that participate in female reproduction, particularly in rodents. In humans, SFRP4 is highly expressed in cumulus cells (CCs). However, the mechanisms that stimulate SFRP4 in CCs have not been examined. We hypothesise that oocyte-secreted factors such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are involved in the regulation of SFRP4. Human CCs were collected from patients undergoing fertility treatments and treated with GDF9 or BMP15 or their combination in the presence of FSH or vehicle. FSH treatment significantly decreased SFRP4 mRNA levels when compared with nontreated cells. However, SFRP4 mRNA levels were increased significantly by GDF9 plus BMP15 in a concentration-dependent manner in the presence or absence of FSH. The combination of GDF9 plus BMP15 also increased SFRP4 protein levels and decreased the activity of the ß-catenin/T cell factor-responsive promoter significantly. GDF9 plus BMP15 inhibited steroidogenic acute regulatory protein and LH/hCG receptor stimulation by FSH, while treatment with SFRP4 blocked the stimulatory effect of FSH on these genes. The evidence demonstrates that GDF9 and BMP15 act in coordination to stimulate SFRP4 expression and suggests that SFRP4 mediates the anti-luteinising effects of the oocyte in human CCs.

Genome-wide interactions between FSH and insulin-like growth factors in the regulation of human granulosa cell differentiation.

Study question: Is the genome-wide response of human cumulus cells to FSH and insulin-like growth factors (IGFs) comparable to the response observed in undifferentiated granulosa cells (GCs)? Summary answer: FSH actions in human cumulus cells mimic those observed in preantral undifferentiated GCs from laboratory animals, and approximately half of the regulated genes are dependent on the simultaneous activation of the IGF1 receptor (IGF1R). What is known already: Animal studies have shown that FSH and the IGFs system are required for follicle growth and maturation. In humans, IGF levels in the follicular fluid correlate with patients' responses to IVF protocols. The main targets of FSH and IGFs in the ovary are the GCs; however, the genomic mechanisms involved in the response of GCs to these hormones are unknown. Study design, size, duration: Human cumulus cells isolated from IVF patients were cultured for 48 h in serum-free media in the presence of vehicle, FSH, IGF1R inhibitor or their combination. Participants/materials, setting, methods: Discarded cumulus cells were donated to research by reproductive-aged women undergoing IVF due to non-ovarian etiologies of infertility at a university-affiliated clinic. The effect of FSH and/or IGF1R inhibition on cumulus cell function was evaluated using Affymetrix microarrays, quantitative PCR, western blot, promoter assays and hormone level measurements. Main results and the role of chance: The findings demonstrate that human cumulus cells from IVF patients respond to FSH with the expression of genes known to be markers of the preantral to preovulatory differentiation of GCs. These results also demonstrate that ~50% of FSH-regulated genes require IGF1R activity and suggest that several aspects of follicle growth are coordinately regulated by FSH and IGFs in humans. This novel approach will allow for future mechanistic and molecular studies on the regulation of human follicle maturation. Large scale data: Data set can be accessed at Gene Expression Omnibus number GSE86427. Limitations, reasons for caution: Experiments were performed using primary human cumulus cells. This may not represent the response of intact follicles. Wider implications of the findings: Understanding the mechanisms involved in the regulation of GC differentiation by FSH and IGF in humans will contribute to improving treatments for infertility. Study funding/competing interest(s): The project was financed by the National Instituted of Health grant number R56HD086054 and R01HD057110 (C.S.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. We have no competing interests to declare.

Regulation of AMH by oocyte-specific growth factors in human primary cumulus cells.

The regulation of AMH production by follicular cells is poorly understood. The purpose of this study was to determine the role of the oocyte-secreted factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on AMH production in primary human cumulus cells. Cumulus cells from IVF patients were cultured with a combination of GDF9, BMP15, recombinant FSH and specific signaling inhibitors. Stimulation with GDF9 or BMP15 separately had no significant effect on AMH mRNA levels. In contrast, simultaneous stimulation with GDF9 and BMP15 (G + B) resulted in a significant increase in AMH mRNA expression. Increasing concentration of G + B (0.6, 2.5, 5 and 10 ng/mL) stimulated AMH in a dose-dependent manner, showing a maximal effect at 5 ng/mL. Western blot analyses revealed an average 16-fold increase in AMH protein levels in cells treated with G + B when compared to controls. FSH co-treatment decreased the stimulation of AMH expression by G + B. The stimulatory effect of G + B on the expression of AMH was significantly decreased by inhibitors of the SMAD2/3 signaling pathway. These findings show for the first time that AMH production is regulated by oocyte-secreted factors in primary human cumulus cells. Moreover, our novel findings establish that the combination of GDF9 + BMP15 potently stimulates AMH expression.

GATA4 and GATA6 Knockdown During Luteinization Inhibits Progesterone Production and Gonadotropin Responsiveness in the Corpus Luteum of Female Mice.

The surge of luteinizing hormone triggers the genomic reprogramming, cell differentiation, and tissue remodeling of the ovulated follicle, leading to the formation of the corpus luteum. During this process, called luteinization, follicular granulosa cells begin expressing a new set of genes that allow the resulting luteal cells to survive in a vastly different hormonal environment and to produce the extremely high amounts of progesterone (P4) needed to sustain pregnancy. To better understand the molecular mechanisms involved in the regulation of luteal P4 production in vivo, the transcription factors GATA4 and GATA6 were knocked down in the corpus luteum by crossing mice carrying Gata4 and Gata6 floxed genes with mice carrying Cre recombinase fused to the progesterone receptor. This receptor is expressed exclusively in granulosa cells after the luteinizing hormone surge, leading to recombination of floxed genes during follicle luteinization. The findings demonstrated that GATA4 and GATA6 are essential for female fertility, whereas targeting either factor alone causes subfertility. When compared to control mice, serum P4 levels and luteal expression of key steroidogenic genes were significantly lower in conditional knockdown mice. The results also showed that GATA4 and GATA6 are required for the expression of the receptors for prolactin and luteinizing hormone, the main luteotropic hormones in mice. The findings demonstrate that GATA4 and GATA6 are crucial regulators of luteal steroidogenesis and are required for the normal response of luteal cells to luteotropins.

Abruption-induced preterm delivery is associated with thrombin-mediated functional progesterone withdrawal in decidual cells.

Plasma progesterone levels remain elevated throughout human pregnancy, suggesting that reduced reproductive-tract progesterone receptor (PR) initiates labor. Placental abruption and excess thrombin generation elicit preterm delivery (PTD). PR, glucocorticoid receptor (GR), and total and p-ERK1/2 in decidual cells (DCs) and interstitial trophoblasts (IT) were assessed via immunohistochemical staining in abruption-associated PTD versus gestational-age matched control placentas, and in cultured DCs incubated with estradiol (E2) ± medroxyprogesterone acetate (MPA) ± thrombin. Immunostaining for PR was lower in DC nuclei in abruption versus control decidua and was absent from ITs; GR was higher in IT than DCs, with no abruption-related changes in either cell type; p-ERK1/2 was higher in DCs in abruption than control decidua, with total ERK 1/2 unchanged. Immunoblotting of cultured DCs demonstrated strong E2, weak MPA, and intermediate E2+MPA mediated elevation of PR-A and PR-B levels, with constitutive GR expression. In cultured DCs, thrombin inhibited PR but not GR mRNA levels, reduced PR binding to DNA and [(3)H]progesterone binding to PR, and enhanced phosphorylated but not total ERK1/2 levels. Coincubation with a specific p-ERK1/2 inhibitor reversed thrombin-enhanced p-ERK1/2 and lowered PR levels. Thus, abruption-associated PTD is initiated by functional progesterone withdrawal, as indicated by significantly reduced DC nuclear expression of PR-A and PR-B. Functional withdrawal of progesterone results in increased p-ERK1/2, and is thus one pathway initiating abruption-associated PTD.

Insulin-like growth factor 1 enhances follicle-stimulating hormone-induced phosphorylation of GATA4 in rat granulosa cells.

Preovulatory granulosa cell (GC) differentiation is essential for the maturation and release of oocytes from the ovary. We have previously demonstrated that follicle-stimulating hormone (FSH) and insulin-like growth factors (IGFs) closely interact to control GC function. Similarly, we showed that GATA4 mediates FSH actions and it is required for preovulatory follicle formation. This report aimed to determine in vivo the effect of FSH on GATA4 phosphorylation and to investigate whether FSH and IGF1 interact to regulate GATA4 activity. In rat ovaries, treatment with equine chorionic gonadotropin (eCG) increased the phosphorylation of GATA4, which was confined to the nucleus of GCs. Using primary rat GCs, we observed that GATA4 phosphorylation at serine 105 increases the transcriptional activity of this transcription factor. Like FSH, IGF1 stimulated GATA4 phosphorylation at serine 105. Interestingly, GATA4 phosphorylation was significantly higher in cells cotreated with FSH and IGF1 when compared to FSH or IGF1 alone, suggesting that IGF1 augments the effects of FSH on GATA4. It was also found that the enhancing effect of IGF1 requires AKT activity and is mimicked by the inhibition of glycogen synthase kinase-3 ß (GSK3ß), suggesting that AKT inhibition of GSK3ß may play a role in the regulation of GATA4 phosphorylation. The data support an important role of the IGF1/AKT/GSK3ß signaling pathway in the regulation of GATA4 transcriptional activity and provide new insights into the mechanisms by which FSH and IGF1 regulate GC differentiation. Our findings suggest that GATA4 transcriptional activation may, at least partially, mediate AKT actions in GCs.

Salt-inducible kinases regulate androgen synthesis in theca cells by enhancing CREB signaling.

Ovulation is the pinnacle of folliculogenesis, a process that requires an interplay between the oocyte, the granulosa cells, and the theca cells (TCs). TCs are the only source of ovarian androgens, which play a vital role in female fertility. However, abnormally elevated androgen levels reduce fertility. Therefore, uncovering novel mechanisms regulating androgen synthesis in TCs is of great significance. We have shown that salt-inducible kinases (SIKs) regulate granulosa cell steroidogenesis. Here, we investigated whether SIKs regulate androgen production in TCs. SIK2 and SIK3 were detected in the TCs of mouse ovaries and isolated TCs. Next, TCs in culture were treated with luteinizing hormone (LH) in the presence or absence of a highly specific SIK inhibitor. SIK inhibition enhanced the stimulatory effect of LH on steroidogenic gene expression and androgen production in a concentration-dependent manner. SIK inhibition alone stimulated the expression of steroidogenic genes and increased androgen production. Activation of adenylyl cyclase with forskolin or emulation of increased intracellular cyclic AMP levels stimulated steroidogenesis, an effect that was enhanced by the inhibition of SIK activity. The stimulatory effect of downstream targets of cyclic AMP was also significantly augmented by SIK inhibition, suggesting that SIKs control targets downstream cyclic AMP. Finally, it is shown that SIK2 knockout mice have higher circulating testosterone than controls. This evidence shows that TCs express SIKs and reveal novel roles for SIKs in the regulation of TC function and androgen production. This information could contribute to uncovering therapeutic targets to treat hyperandrogenic diseases.

Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.

The molecular control of corpus luteum formation, function, and regression.

The corpus luteum (CL) is one of the few endocrine glands that forms from the remains of another organ and whose function and survival are limited in scope and time. The CL is the site of rapid remodeling, growth, differentiation, and death of cells originating from granulosa, theca, capillaries, and fibroblasts. The apparent raison d'etre of the CL is the production of progesterone, and all the structural and functional features of this gland are geared toward this end. Because of its unique importance for successful pregnancies, the mammals have evolved a complex series of checks and balances that maintains progesterone at appropriate levels throughout gestation. The formation, maintenance, regression, and steroidogenesis of the CL are among the most significant and closely regulated events in mammalian reproduction. During pregnancy, the fate of the CL depends on the interplay of ovarian, pituitary, and placental regulators. At the end of its life span, the CL undergoes a process of regression leading to its disappearance from the ovary and allowing the initiation of a new cycle. The generation of transgenic, knockout and knockin mice and the development of innovative technologies have revealed a novel role of several molecules in the reprogramming of granulosa cells into luteal cells and in the hormonal and molecular control of the function and demise of the CL. The current review highlights our knowledge on these key molecular events in rodents.

Salt-inducible Kinases Are Critical Determinants of Female Fertility.

Follicle development is the most crucial step toward female fertility and is controlled mainly by follicle-stimulating hormone (FSH). In ovarian granulosa cells (GCs), FSH activates protein kinase A by increasing 3',5'-cyclic adenosine 5'-monophosphate (cAMP). Since cAMP signaling is impinged in part by salt-inducible kinases (SIKs), we examined the role of SIKs on the regulation of FSH actions. Here, we report that SIKs are essential for normal ovarian function and female fertility. All SIK isoforms are expressed in human and rodent GCs at different levels (SIK3>SIK2>SIK1). Pharmacological inhibition of SIK activity potentiated the stimulatory effect of FSH on markers of GC differentiation in mouse, rat, and human GCs and estradiol production in rat GCs. In humans, SIK inhibition strongly enhanced FSH actions in GCs of patients with normal or abnormal ovarian function. The knockdown of SIK2, but not SIK1 or SIK3, synergized with FSH on the induction of markers of GC differentiation. SIK inhibition boosted gonadotropin-induced GC differentiation in vivo, while the genomic knockout of SIK2 led to a significant increase in the number of ovulated oocytes. Conversely, SIK3 knockout females were infertile, FSH insensitive, and had abnormal folliculogenesis. These findings reveal novel roles for SIKs in the regulation of GC differentiation and female fertility, and contribute to our understanding of the mechanisms regulated by FSH. Furthermore, these data suggest that specific pharmacological modulation of SIK2 activity could be of benefit to treat ovulatory defects in humans and to increase the propagation of endangered species and farm mammals.

Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells.

CONTEXT: Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown. OBJECTIVE: To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs. DESIGN: Primary human cumulus GCs in culture. SETTING: University infertility center. PATIENTS OR OTHER PARTICIPANTS: GCs of women undergoing in vitro fertilization. INTERVENTION(S): Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic homolog (SMAD) inhibitors. MAIN OUTCOME MEASURE(S): Quantification of mRNA, protein, promoter activity, and DNA methylation. RESULTS: FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P < 0.0001) in a concentration-dependent manner showing a maximal effect at 5 ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P < 0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P < 0.009). CONCLUSIONS: For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.

Prolactin signaling through the short isoform of the mouse prolactin receptor regulates DNA binding of specific transcription factors, often with opposite effects in different reproductive issues.

BACKGROUND: It has been well established that prolactin (PRL) signals through the long form of its receptor (PRL-RL) and activates the Jak/Stat pathway for transcription of PRL target genes. However, signaling pathways mediated through the short PRL-R isoform (PRL-RS) remains controversial. Our recent finding that PRL signaling through PRL-RS represses two transcription factors critical for follicular development lead us to examine other putative PRL/PRL-RS target transcription factors in the decidua and ovary, two well-known target tissues of PRL action in reproduction. METHODS: In this investigation we used mice expressing PRL-RS on a PRL-R knockout background and a combo protein/DNA array to study the transcription factors regulated by PRL through PRL-RS only. RESULTS: We show that PRL activation of the PRL-RS receptor either stimulates or inhibits the DNA binding activity of a substantial number of transcription factors in the decidua as well as ovary. We found few transcription factors to be similarly regulated in both tissues, while most transcription factors are oppositely regulated by PRL in the decidua and ovary. In addition, some transcription factors are regulated by PRL only in the ovary or only in the decidua. Several of these transcription factors are involved in physiological pathways known to be regulated by PRL while others are novel. CONCLUSION: Our results clearly indicate that PRL does signal through PRL-RS in the decidua as well as the ovary, independently of PRL-RL, and activates/represses transcription factors in a tissue specific manner. This is the first report showing PRL/PRL-RS regulation of specific transcription factors. Many of these transcription factors were not previously known to be PRL targets, suggesting novel physiological roles for this hormone.

Prolactin signaling through the short form of its receptor represses forkhead transcription factor FOXO3 and its target gene galt causing a severe ovarian defect.

Prolactin (PRL) is a hormone with over 300 biological activities. Although the signaling pathway downstream of the long form of its receptor (RL) has been well characterized, little is known about PRL actions upon activation of the short form (RS). Here, we show that mice expressing only RS exhibit an ovarian phenotype of accelerated follicular recruitment followed by massive follicular death leading to premature ovarian failure. Consequently, RS-expressing ovaries of young adults are depleted of functional follicles and formed mostly by interstitium. We also show that activation of RS represses the expression of the transcription factor Forkhead box O3 (FOXO3) and that of the enzyme galactose-1-phosphate uridyltransferase (Galt), two proteins known to be essential for normal follicular development. Our finding that FOXO3 regulates the expression of Galt and enhances its transcriptional activity indicates that it is the repression of FOXO3 by PRL acting through RS that prevents Galt expression in the ovary and causes follicular death. Coexpression of RL with RS prevents PRL inhibition of Galt, and the ovarian defect is no longer seen in RS transgenic mice that coexpress RL, suggesting that RL prevents RS-induced ovarian impairment. In summary, we show that PRL signals through RS and causes, in the absence of RL, a severe ovarian pathology by repressing the expression of FOXO3 and that of its target gene Galt. We also provide evidence of a link between the premature ovarian failure seen in mice expressing RS and in mice with FOXO3 gene deletion as well as in human with Galt mutation.

Sp1 regulates steroidogenic genes and LHCGR expression in primary human luteinized granulosa cells.

Luteinizing hormone and human chorionic gonadotropin (hCG) bind to the luteinizing hormone/chorionic gonadotropin receptor (LHCGR). LHCGR is required to maintain corpus luteum function but the mechanisms involved in the regulation of LHCGR in human luteal cells remain incompletely understood. This study aimed to characterize the expression of LHCGR mRNA in primary human luteinized granulosa cells (hLGCs) obtained from patients undergoing in vitro fertilization and to correlate LHCGR expression with the response of hLGCs to hCG by assessing the expression of genes known to be markers of hCG actions. The results show that LHCGR expression is low in freshly isolated cells but recovers rapidly in culture and that hCG maintains LHCGR expression, suggesting a positive feedback loop. The activity of a LHCGR-LUC reporter increased in cells treated with hCG but not with follicle-stimulating hormone. Treatment with hCG also stimulated the expression of genes involved in steroidogenesis in a time-dependent manner. LHCGR promoter expression was found to be regulated by SP1, which we show is highly expressed in hLGCs. Moreover, SP1 inhibition prevented the stimulation of steroidogenic genes and the increase in LHCGR-LUC reporter activity by hCG. Finally, we provide evidence that a complex formed by SP1 and GATA4 may play a role in the maintenance of LHCGR expression. This report reveals the mechanisms involved in the regulation of the LHCGR and provides experimental data demonstrating that the proximal region of the LHCGR promoter is sufficient to drive the expression of this gene in primary hLGCs.

Oocyte-Secreted Factors Synergize With FSH to Promote Aromatase Expression in Primary Human Cumulus Cells.

CONTEXT: The role of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) on aromatase regulation is poorly understood in humans. OBJECTIVE: Determine GDF9 and BMP15 effects on FSH stimulation of estradiol production in primary human cumulus granulosa cells (GCs). We hypothesized that the combination of GDF9 and BMP15 potentiates FSH-induced aromatase expression. DESIGN: Primary human cumulus GCs in culture. SETTING: University infertility center. PATIENTS OR OTHER PARTICIPANTS: GCs of 60 women undergoing in vitro fertilization were collected. INTERVENTIONS: Cells were treated with GDF9 and/or BMP15 (GB) in the presence or absence of FSH, dibutyryl cAMP, or SMAD inhibitors. MAIN OUTCOME MEASURES: Promoter activity, mRNA, protein, and estradiol levels were quantified. RESULTS: FSH and GB treatment increased CYP19A1 promoter activity, mRNA, and protein levels as well as estradiol when compared with cells treated with FSH only. GB treatment potentiated cAMP stimulation of aromatase and IGF2 stimulation by FSH. GB effects were inhibited by SMAD3 inhibitors and IGF1 receptor inhibitors. GB, but not FSH, stimulates SMAD3 phosphorylation. CONCLUSION: The combination of GDF9 and BMP15 potently stimulates the effect of FSH and cAMP on CYP19a1 promoter activity and mRNA/protein levels. These effects translate into an increase in estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway and involves, at least in part, the effect of the IGF system.

Aromatase expression in the ovary: hormonal and molecular regulation.

Estrogens are synthesized by the aromatase enzyme encoded by the Cyp19a1 gene, which contains an unusually large regulatory region. In most mammals, aromatase expression is under the control of two distinct promoters a gonad- and a brain-specific promoter. In humans, this gene contains 10 tissue-specific promoters that are alternatively used in various cell types and tumors. Each promoter is regulated by a distinct set of regulatory sequences and transcription factors that bind to these specific sequences. The cAMP/PKA/CREB pathway is considered to be the primary signaling cascade through which the gonad Cyp19 promoter is regulated. Very interestingly, in rat luteal cells, the proximal promoter is not controlled in a cAMP dependent manner. Strikingly, these cells express aromatase at high levels similar to those found in preovulatory follicles, suggesting that alternative and powerful mechanisms control aromatase expression in luteal cells and that the rat corpus luteum represents an important paradigm for understanding alternative controls of the aromatase gene. Here, the molecular and cellular mechanisms controlling the expression of the aromatase gene in granulosa and luteal cells are discussed.

Follicle-stimulating hormone-induced activation of Gata4 contributes in the up-regulation of Cyp19 expression in rat granulosa cells.

Several studies have suggested that the transcription factor GATA4 plays an important role in ovarian function. This study evaluated the effects of GATA4 on the regulation of the Cyp19 gene in primary rat granulosa cells under basal conditions and in response to stimulation by FSH. A significant increase in GATA4 mRNA, protein, and DNA binding activity was observed in rats treated with pregnant mare serum gonadotropin, a hormone that binds to the FSH receptors, and in granulosa cells incubated with FSH. Enrichment of the Cyp19 promoter was observed in granulosa cells treated with FSH after chromatin precipitation with an anti-GATA4 antibody. Mutation of the GATA binding site on the Cyp19 promoter and inhibition of GATA4 expression with specific small interfering RNA significantly reduced FSH-enhanced Cyp19 expression, whereas overexpression of GATA4 increased Cyp19 promoter activity. A synergistic effect observed between GATA4 overexpression and FSH treatment in Cyp19 expression was abolished by mutating Ser105 in the GATA4 protein or by pretreating granulosa cells with a protein kinase A inhibitor. Inhibition of phosphatidylinositol-dependent kinase (PI3-K)/casein kinase 2 or ERK1/2 attenuated GATA4/FSH synergism, whereas the simultaneous blockade of PI3-K/casein kinase 2 and ERK1/2 activity eliminated Cyp19 stimulation. Finally, we demonstrated that FSH increases GATA4 phosphorylation and that GATA4 activation requires the activation of multiple kinases, including ERK1/2, PI3-K, and protein kinase A. These findings demonstrate that GATA4 contributes in the regulation of Cyp19 expression in the rat ovary and provide the first evidence that FSH regulates GATA4 activity.

GATA Regulation and Function During the Ovarian Life Cycle.

GATA4 and GATA6 are the sole GATA factors expressed in the ovary during embryonic development and adulthood. Up today, GATA4 and GATA6 are the only transcription factors that have been conditionally deleted during ovarian development and at each major stage of follicle maturation. The evidence from these transgenic mice revealed that GATA4 and GATA6 are crucial for follicles assembly, granulosa cell differentiation, postnatal follicle growth, and luteinization. Thus, conditional knockdown of both factors in the granulosa cells at any stage of development leads to female infertility. GATA targets impacting female reproduction include genes involved in steroidogenesis, hormone signaling, ovarian hormones, extracellular matrix organization, and apoptosis/cell division.