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.

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.

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.

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.

IGF1R Expression in Ovarian Granulosa Cells Is Essential for Steroidogenesis, Follicle Survival, and Fertility in Female Mice.

Folliculogenesis is a lengthy process that requires the proliferation and differentiation of granulosa cells (GCs) for preovulatory follicle formation. The most crucial endocrine factor involved in this process is follicle-stimulating hormone (FSH). Interestingly, previous in vitro studies indicated that FSH does not stimulate GC proliferation in the absence of the insulinlike growth factor 1 receptor (IGF1R). To determine the role of the IGF1R in vivo, female mice with a conditional knockdown of the IGF1R in the GCs were produced and had undetectable levels of IGF1R mRNA and protein in the GCs. These animals were sterile, and their ovaries were smaller than those of control animals and contained no antral follicles even after gonadotropin stimulation. The lack of antral follicles correlated with a 90% decrease in serum estradiol levels. In addition, under a superovulation protocol no oocytes were found in the oviducts of these animals. Accordingly, the GCs of the mutant females expressed significantly lower levels of preovulatory markers including aromatase, luteinizing hormone receptor, and inhibin α. In contrast, no alterations in FSH receptor expression were observed in GCs lacking IGF1R. Immunohistochemistry studies demonstrated that ovaries lacking IGF1R had higher levels of apoptosis in follicles from the primary to the large secondary stages. Finally, molecular studies determined that protein kinase B activation was significantly impaired in mutant females when compared with controls. These in vivo findings demonstrate that IGF1R has a crucial role in GC function and, consequently, in female fertility.

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.