Oocyte-Derived Factors (GDF9 and BMP15) and FSH Regulate AMH Expression Via Modulation of H3K27AC in Granulosa Cells Free

Abstract

Anti–Müllerian hormone (AMH) produced by ovarian granulosa cells (GCs) plays a crucial role in ovarian function. It is used as a diagnostic and/or prognostic marker of fertility as well as for pathophysiological conditions in women. In this study, we investigated the underlying mechanism for regulation of AMH expression in GCs using primary mouse GCs and a human GC tumor–derived KGN cell line. We find that growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15) together (GDF9 + BMP15), but not when tested separately, significantly induce AMH expression in vitro and in vivo (serum AMH). Our results show that GDF9 + BMP15 through the PI3K/Akt and Smad2/3 pathways synergistically recruit the coactivator p300 on the AMH promoter region that promotes acetylation of histone 3 lysine 27 (H3K27ac), facilitating AMH/Amh expression. Intriguingly, we also find that FSH inhibits GDF9 + BMP15–induced increase of AMH/Amh expression. This inhibition occurs through FSH-induced protein kinase A/SF1-mediated expression of gonadotropin inducible ovarian transcription factor 1, a transcriptional repressor, that recruits histone deacetylase 2 to deacetylate H3K27ac, resulting in the suppression of AMH/Amh expression. Furthermore, we report that ovarian Amh mRNA levels are significantly higher in Fshβ-null mice (Fshβ^−/−) compared with those in wild-type (WT) mice. In addition, ovarian Amh mRNA levels are restored in Fshβ-null mice expressing a human WT FSHβ transgene (FSHβ^−/−hFSHβ^WT). Our study provides a mechanistic insight into the regulation of AMH expression that has many implications in female reproduction/fertility.

Anti–Müllerian hormone (AMH) or Müllerian inhibiting substance is a member of the TGF-β superfamily with well-established roles in reproductive organ differentiation and ovarian follicular development (1). In females, AMH is produced by the granulosa cells (GCs) of small growing follicles, and AMH levels subsequently decrease as these follicles grow to the antral stage. AMH levels are tightly regulated throughout folliculogenesis (2, 3) as AMH acts as a natural gatekeeper of follicle growth and maintains the follicle pool throughout reproductive life (4–7). Amh knockout mice develop premature ovarian insufficiency, and AMH directly or indirectly prevents or inhibits primordial follicles from entering the pool of growing follicles (6, 8). In women, AMH levels decrease with age, and a premature decrease in AMH causes pathophysiological conditions such as diminished ovarian reserve/premature ovarian insufficiency (9, 10). Similarly, AMH levels are significantly high in women with polycystic ovary syndrome, where follicles fail to grow beyond the small antral stage, form cysts, and do not regularly progress to ovulate (11, 12). In clinical practice, AMH level is associated with ovarian reserve and used as a diagnostic and/or prognostic marker for polycystic ovary syndrome and diminished ovarian reserve/premature ovarian insufficiency, as well as a predictor for ovarian response to fertility treatments (13, 14). Despite the critical role of AMH in follicular development and the widespread use of AMH as a clinical marker, the underlying mechanism of AMH actions or the regulation of AMH expression is poorly understood.

Recently, we reported that AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which target activin-receptor 2A and adenylate cyclase 9 (15). Moreover, we showed that AMH pretreatment of mice prior to superovulation improves oocyte yield; thus, AMH can, indeed, be a potential therapeutic option for women with low functional ovarian reserve. However, lack of knowledge of how AMH expression is regulated is a major limitation toward using AMH as a potential therapeutic option/target.

Studies across different species show that oocyte-derived factors, specifically growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15), induce AMH expression (16–19). GDF9 and BMP15 play key roles in GC development and fertility in most mammalian species (20–22). Moreover, studies show that mouse and human GDF9/BMP15 heterodimers are the most biopotent regulators of GC functions (23). A recent study (19) in human cumulus cells reported that GDF9 and BMP15 together are much more effective in inducing AMH expression compared with BMP15 or GDF9 alone. GDF9 and BMP15 belong to the TGF-β superfamily that signals through the ALK4/ALK6/BMPR2 receptor complex and activates the Smad pathway (24–27). Interestingly, the same study showed that FSH inhibits GDF9 + BMP15–induced AMH expression. Intriguingly, studies (28, 29) in women undergoing controlled ovarian stimulation also reported a decrease in plasma AMH levels with FSH treatment. However, the intracellular mechanism of GDF9 + BMP15–induced AMH expression or how FSH attenuates GDF9 + BMP15–induced AMH expression remains unexplored. Using primary mouse GC (mGC) culture and a human GC tumor cell line (KGN cells), in this study, we provide a mechanistic understanding of the regulation of AMH expression by GDF9 + BMP15 and FSH.

Regulation of gene expression is controlled at a number of different levels, one of which is modification of histones. The link between histone modifications and transcription has been extensively studied. It is now well established that individual histone modifications such as acetylation and methylation can be associated with transcriptional activation or repression (30). Acetylation of specific lysine residues on core histones facilitates DNA unwinding and increases accessibility to transcription factor binding (31). For example, acetylation of lysine 27 on histone 3 (H3K27ac) is considered a positive mark for transcription and associated with enhanced promoter activity (32). Histone acetylases and deacetylases play a critical role in altering chromatin structure and gene expression. In testis, SRY-related protein Sox9 regulates the transcription of AMH (33), and interestingly, Sox9 binding is associated with active regulatory regions, particularly H3K27ac (34). Here we hypothesize that in GCs, AMH expression is regulated in part through modulation of H3K27 acetylation. These data provide new insights into the regulation of AMH expression in GCs and may offer potential targets and/or options for therapeutic applications.

Material and Methods

Animals and cell culture

Mouse studies were performed in accordance with the guidelines for the care and use of laboratory animals and were approved by the University Committee on Animal Resources at the University of Rochester and Michigan State University. Unless otherwise mentioned, mouse experiments were performed in 8- to 9-week-old C57BL/6J mice (The Jackson Laboratory). Estrous cycle was determined by daily vaginal smears as described previously (15, 35), and on the day of estrus, mGCs were isolated by needle puncture under the microscope specifically from preantral and small antral follicles (puncturing of large antral follicles was avoided). GCs were then cultured for 48 hours prior to serum starvation and treatment, as described previously (15, 35–38). KGN cells (39), a human granulosa tumor cell line, were cultured for 24 hours in DMEM/F-12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Both mGCs and KGN cells were serum starved for 18 hours followed by stimulation with different concentrations of human recombinant GDF9 (R&D Systems), BMP15 (R&D Systems), and/or recombinant human FSH (Serono) for 24 hours. For experiments using inhibitors, cells were pretreated with the following inhibitors: PI3K inhibitor LY294002 (10 μM), MAPK inhibitor U0126 (10 μM), Smad 2/3 inhibitor SB431542 (10 μM), Smad 1/5/8 inhibitor LDN-193189 (100nM), protein kinase A (PKA) inhibitor H89 (10 μM), broad-spectrum histone deacetylase (HDAC) inhibitor trichostatin A (100 nM), or SF1 inhibitor SID7969543 (1 μM) (Tocris, Minneapolis, MN) for 30 minutes prior to stimulation. For all experiments with primary mGC cultures and KGN cells, cell viability was determined by trypan blue at the time of harvesting, and all experiments were repeated three times.

For in vivo studies, 8-week-old mice were injected intraperitoneally with different concentrations of recombinant human GDF9 (R&D Systems) and/or BMP15 (R&D Systems) once daily for 4 weeks (n = 5 mice per treatment). For GDF9 + BMP15 injections, GDF9 and BMP15 were mixed prior to injection. Thereafter, blood was collected on the day of estrus by cardiac puncture of mice under anesthesia and later used for determination of AMH levels.

Fshβ-null mice (Fshβ^−/−), Fshβ-null mice expressing a human FSHβ transgene (Fshβ^−/−hFSHβ^WT), and wild-type (WT) mice were originally developed and extensively characterized by T.R.K.’s laboratory as described (5, 40, 41). Ovaries (n = 3 per genotype) from these animals were used to isolate RNA and determine Amh mRNA levels by real-time PCR.

RNA extraction and real-time PCR

RNA from cells was isolated using the Omega E.Z.N.A. total RNA Kit I (Omega Bio-Tek, GA) according to the manufacturer’s instructions, and levels of specific mRNA expression were analyzed by the ∆∆CT method using OneStep Taqman gene expression assay primers (assay ID Mm00431795_g1-AMH, Hs00174915_m1-AMH, Hs00914223_m1-p300, Mm00625535_m1-p300, Rn01408381_m1-GIOT1, Hs00708538_s1-GIOT1, Mm03302249_g1-GAPDH, Hs03929097_g1-GAPDH, Hs02621185_s1-Hdac1, Hs00231032_m1-Hdac2, Hs00187320_m1-Hdac3, Hs01041648_m1-Hdac4, Hs00608351_m1-Hdac5, Hs00997427_m1-Hdac6, Hs00954353_g1-Hdac8, Hs00368899_m1-Hdac10, Mm00515108_m1-Hdac2; Applied Biosystems) and the Applied Biosystems StepOnePlus RT-PCR system. Then, 1 μg RNA was used for all of the RT-PCR reactions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.

Western blot analysis

Western blots were performed as described previously (15, 35, 42). Primary antibodies used were rabbit polyclonal Akt (43), phosphorylated Akt (S473) (44), GAPDH (45), phosphorylated Samd2/3 (Smad2-S465/467; Smad3-S423/425) (46), Smad2/3 (47) and (Cell Signaling Technology); AMH (48) and phosphorylated (S1834)–p300 (49) (Thermo Fisher Scientific); gonadotropin inducible transcription repressor 1 (50) (Abcam); and KAT3B/p300 (51), HDAC2 (52), and histone 3 acetyl K27 (Abcam) (53). Western blot data were quantified by densitometric analysis as described previously (35, 38). Briefly, protein levels were determined using computer-aided densitometry and expressed as relative increase in AMH/GAPDH or phosphorylated/total protein vs media or control.

Small interfering RNA knockdown experiments

Small interfering RNA (siRNA)–mediated knockdown experiments were performed as described previously (35, 36, 38, 42, 54). mGCs and KGN cells were treated with nontargeting siRNA pool and siRNA ON-TARGET Plus SMARTpool siRNAs (Dharmacon) for human or mouse according to the manufacturer’s instructions for 72 hours prior to 4 hours of serum starvation followed by 24 hours of stimulation. The SMART pool ON-TARGETplus siRNAs consist of a mixture of four siRNAs in a single reagent. The siRNAs used were EP300 siRNA (L-003486-00-00005), HDAC1 siRNA (L-003493-00-0005), HDAC2 siRNA (L-003495-02-0005), HDAC3 siRNA (L-003496-00-0005), HDAC4 siRNA (L-003497-00-0005), HDAC5 siRNA (L-003498-00-0005), HDAC6 siRNA (L-003499-00-0005), HDAC8 siRNA (L-003500-00-0005), HDAC10 siRNA (L-00472-00-0005), and ZNF461/GIOT1 siRNA (L-016102-00-0010). The mouse siRNAs used were EP300 siRNA (L-065607-00-0005) and HDAC2 siRNA (L-046158-00-0005). The siRNA for mouse gonadotropin inducible ovarian transcription factor 1 (GIOT1) was from the Thermo Fisher assay (ID 193349-GIOT1).

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) was performed as described previously (36, 38, 42) with the MAGnify Chromatin Immunoprecipitation System (Invitrogen), according to the manufacturer’s instructions. Chromatin fragments were immunoprecipitated with Dynabeads coupled with rabbit polyclonal anti-p300 (51), anti-H3K27ac (53), or anti-HDAC2 (52) ChIP grade antibody (Abcam) and IgG as nonspecific control. Quantitative PCR was performed using EXPRESS SYBR GreenER qPCR SuperMixes (Invitrogen) with primers (55) designed for three regions of the human AMH promoter (P1, P2, and P3).

Hormone assay

Serum AMH levels were measured by the Center for Research in Reproduction, Ligand Assay and Analysis Core at the University of Virginia. AMH levels were determined by AMH mouse and rat ELISA kit (Ansh Laboratories, Webster, TX). The reportable range for the assay was 3.36 to 215.0 ng/mL, and intra-assay coefficient of variation was 0.9%.

Nuclear and cytoplasmic fractionation

Separation of nuclear and cytoplasmic fractions was performed using the NE-PER Nuclear and Cytoplasmic extraction kit (Thermo Fisher Scientific) according to manufacturer’s protocol as described previously (36). Expression of GAPDH and lamin were used as cytoplasmic and nuclear controls, respectively.

Immunoprecipitation

Immunoprecipitation (IP) experiments (36) were done with nuclear fraction (nuclear and cytoplasmic fractionation) using Protein G-Agarose beads (Millipore) according to the manufacturer’s instructions. Antibodies used for IP were anti-rabbit Smad2/3, IgG (control) antibodies (Cell Signaling Technology), and anti-rabbit KAT3B/p300 (Abcam).

Statistical analysis

Each experiment for both mGCs and KGN cells was repeated at least three times, and each treatment was done in duplicate per experiment. Data are presented as mean ± SEM. For the in vivo study (n = 5 mice per treatment), data were log transformed. Statistical analysis was performed using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA). Statistical comparisons were made by two-way ANOVA followed by Tukey-Kramer analysis for multiple comparisons, and results with P ≤ 0.05 were considered significant.

Results

GDF9 and BMP15 together, but not separately, induce AMH expression in mGCs and KGN cells and increase serum AMH levels in vivo

Our studies in human KGN cells and mGCs show that GDF9 and BMP15 together significantly increase AMH/Amh mRNA expression in vitro. We find that GDF9 alone does not have any effect on AMH/Amh mRNA expression (Fig. 1A and 1E), whereas BMP15 treatment triggers a slight increase (1.5- to 1.8-fold) in AMH/Amh mRNA levels (Fig. 1B and 1E) in both KGN cells and mGCs. In contrast, GDF9 and BMP15 together, in a concentration-dependent manner, significantly induce AMH mRNA (Fig. 1C; 2.5- to 10.5-fold increase) and protein (Fig. 1D) levels in KGN cells. Similar results are also seen in mGCs [Amh mRNA (Fig. 1E); 10.6- to 16.1-fold increase and AMH protein (Fig. 1F)]. Quantitative analysis of AMH protein levels in KGN and mGCs is shown in the supplemental data (55).

For in vivo studies, results show that GDF9 or BMP15 injections did not have any effect on serum AMH levels (Fig. 1G). In contrast, GDF9 + BMP15 together significantly increased serum AMH levels (112.0 vs 169.6 ng/mL, vehicle vs GDF9 + BMP15). These results corroborate that GDF9 and BMP15 together are potent regulators of AMH expression in GCs.

GDF9 and BMP15 induce AMH expression through Smad2/3 and PI3K/Akt signaling

To identify the underlying mechanism involved in GDF9 + BMP15–induced AMH expression, we used inhibitors of different signaling pathways in KGN cells (Fig. 2) and mGCs (Fig. 3) as described in the Methods section. We find that inhibiting the Smad2/3 (SB431542) and PI3K/Akt pathway (LY294002) abrogates the GDF9 + BMP15–induced AMH/Amh mRNA (Fig. 2A and 3A) and AMH protein (Fig. 2B and 3B) expression in KGN and mGCs, respectively. Moreover, results show that GDF9 + BMP15 treatment can directly induce activation of Smad2/3 (Fig. 2C and 3C) and Akt (Fig. 2D and 3D) in both KGN and mGCs, respectively. This demonstrates that the GDF9 + BMP15–induced AMH expression is mediated through the Smad2/3 and PI3K/Akt pathway. Intriguingly, our studies shown in the supplemental data (55) demonstrate that GDF9 alone, but not BMP15, can induce Akt activation in KGN cells, whereas previous studies have shown that GDF9 can activate the PI3K/Akt pathway in murine GCs (56). Thus, it is likely that the observed activation of Akt by GDF9 + BMP15 treatment is primarily in response to GDF9. Moreover, inhibiting Smad2/3 (by SB431542) has no effect on GDF9 + BMP15–induced Akt activation (Fig. 2E), further demonstrating that GDF9 + BMP15–induced Akt activation is not mediated through the Smad2/3 pathway. Treatment of KGN cells and mGCs with the inhibitors alone (in the absence of GDF9 + BMP15) did not have any effect on basal AMH/Amh mRNA expression (55). Quantitative analysis of the protein levels in KGN cells and mGCs is shown in the supplemental data (55).

Interestingly, activation of the PI3K/Akt pathway alone is not sufficient to induce AMH expression. Treatment of KGN cells with insulin, which activates the PI3K/Akt pathway but not Smad2/3 (Fig. 2F), has no effect on AMH expression (Fig. 2G), indicating that both Smad2/3 and PI3K/Akt pathways are necessary for GDF9 + BMP15–induced AMH expression.

GDF9 + BMP15–induced Smad2/3 and PI3K/Akt signaling synergistically increase p300 recruitment to the AMH promoter, resulting in H3K27ac

Next, we tested how Smad2/3 and PI3K/Akt signaling induce AMH expression. The PI3K/Akt pathway plays a critical role in the regulation of gene expression induced by numerous stimuli. p300, a histone acetyltransferase (HAT) and transcriptional coactivator, acts in concert with transcription factors to facilitate gene expression. Phosphorylation of p300 at Ser-1834 by Akt is essential for its HAT and transcriptional activity (57). Interestingly, the 5′-promoter region of AMH has a number of p300 binding sites. We find that GDF9 + BMP15 treatment can induce phosphorylation of p300 at Ser-1834 that is blocked by the PI3K inhibitor, LY294002, in both KGN cells (Fig. 4A) and mGCs (Fig. 4D). Quantitative analysis of the protein levels in KGN cells and mGCs is shown in the supplemental data (55). TGF-β–induced Smad3/4 can directly activate the PKA pathway (58), and the latter has been reported (59) to phosphorylate p300. Thus, we used H89 (PKA inhibitor) as a negative control to demonstrate that the phosphorylation of p300 is mediated through the PI3K/Akt and not any other pathways. Moreover, siRNA-mediated knockdown of p300 in KGN cells (Fig. 4B) and mGCs (Fig. 4E) prevents the GDF9 + BMP15–induced increase in AMH/Amh mRNA levels (Fig. 4C and 4F). These data suggest that GDF9 + BMP15 through the PI3K/Akt pathway phosphorylate p300, which is involved in AMH expression.

Active enhancers usually can be identified by p300 binding (60). Thus, ChIP assay was done in KGN cells on two regions (P1 and P2) of the AMH promoter with an antibody against p300. Results [Fig. 5A and supplemental data (55)] show that GDF9 + BMP15 treatment significantly increased the recruitment of p300 on the AMH promoter region that was blocked by LY294002 (PI3K inhibitor) and SB431542 (Smad2/3 inhibitor). Moreover, given that p300 causes acetylation of H3K27, a positive marker for gene expression, we further performed ChIP studies with H3K27ac antibody. In accordance to p300 binding, there was a significant increase in H3K27ac mark on the AMH promoter with GDF9 + BMP15 treatment, and inhibition of the Smad2/3 (SB431542) and PI3K/Akt (LY294002) pathways blocked this effect [Fig. 5A and supplemental data (55)]. These studies demonstrate that in addition to the PI3K/Akt pathway, the Smad2/3 is also involved in the recruitment of p300 and in the acetylation of H3K27.

To determine whether Smad2/3 directly interacts with p300, we performed co-IP assays in nuclear extracts isolated from KGN cells (Fig. 5B) and mGCs (Fig. 5C) treated with GDF9 + BMP15. Upon GDF9 + BMP15 treatment, pSmad2/3 and p300 coprecipitated regardless of the precipitating antibody, confirming that they indeed form a complex in the nucleus, and this interaction is blocked by LY294002 and SB431542.

FSH inhibits GDF9 + BMP15–induced AMH/Amh expression through HDAC2-mediated deacetylation of H3K27ac

Intriguingly, we also found that FSH treatment inhibited GDF9 + BMP15–induced AMH/Amh expression. Results show that FSH inhibited GDF9 + BMP15–induced AMH/Amh mRNA (Fig. 6A and 6C) and AMH protein (Fig. 6A and 6B, insets) expression in primary KGN cells and mGCs. Although all tested doses of FSH (1, 5, 10, and 50 ng/mL) inhibited GDF9 + BMP15–induced AMH mRNA in both cell types, the dose response showed an inverted bell curve. However, this dose-dependent effect of FSH was seen on only the AMH/Amh mRNA but not AMH protein levels. Quantitative analysis of the AMH protein levels in KGN cells and mGCs following FSH treatment is shown in the supplemental data (55). The physiological actions of FSH are mediated through the cAMP-PKA-SF1 signaling pathway (61). We found that inhibition of FSH signaling with PKA (H89) and SF1 (SID7969543) specific inhibitors blocked the FSH-induced inhibition of GDF9 + BMP15–induced AMH/Amh expression in both KGN cells (Fig. 6B) and mGCs (Fig. 6D). This shows that the inhibitory effects of FSH are mediated directly through FSH signaling.

Because our studies show that GDF9 + BMP15–induced AMH expression is mediated through acetylation of H3K27, we wanted to test if FSH inhibits this acetylation process. Histone acetylation is primarily regulated by two groups of enzymes: the acetyltransferases, such as p300, which are involved in putting the acetyl mark on various histone residues, and deacetylases, which are involved in removing acetyl groups from lysine amino acids on a histone (62–65). Fig. 7A shows that trichostatin A treatment, a broad-spectrum HDAC inhibitor, significantly blocked the inhibitory effects of FSH on GDF9 + BMP15–induced AMH expression in KGN cells. This suggests that HDACs are involved in the inhibitory actions of FSH on AMH expression. To identify the specific HDAC(s) involved in the inhibitory effects of FSH, we determined the effect of siRNA-mediated knockdown of specific HDACs (55) on the inhibition of GDF9 + BMP15–induced AMH expression by FSH (Fig. 7B). Results show that siRNA-mediated knockdown of HDAC2 (Fig. 7B, inset) significantly reversed the inhibitor effects of FSH on GDF9 + BMP15–induced AMH expression (Fig. 7B). Similar results were also seen in mGCs (Fig. 7C). Intriguingly, however, we did not find any effect of FSH on HDAC2 mRNA or protein expression (data not shown). This indicates that although the inhibitory effects of FSH on GDF9 + BMP15–induced AMH/Amh expression are mediated through the FSH-induced PKA-SF1 pathway and involve HDAC2, it is not through regulation of HDAC2 expression.

Moreover, ChIP assay in KGN cells with antibodies against H3K27ac revealed that FSH treatment significantly lowered H3K27ac mark on the AMH promoter [Fig. 8 and supplemental data (55)]. To demonstrate that this decrease in H3K27ac levels on the AMH promoter is through FSH-induced modulation of HDAC2, we performed a corresponding ChIP showing increased association of HDAC2 on the AMH promoter following FSH treatment [Fig. 8 and supplemental data (55)].

FSH induces zinc finger protein GIOT1 gene expression, and GIOT1 acts as a repressor of AMH/Amh expression via recruitment of HDAC2

In our efforts to elucidate how FSH modulates HDAC2 actions to inhibit GDF9 + BMP15–induced AMH/Amh expression, we found that FSH induces the expression of GIOT1. GIOT1 is a novel transcriptional repressor, and its inhibitory effects are partly mediated through recruitment of HDAC2. Results show that although FSH stimulation increases GIOT1/Giot1 mRNA levels, inhibiting the FSH signaling pathway with H89 (PKA inhibitor) or SID7969543 (SF1 inhibitor) blocks this effect in both KGN cells and mGCs (Fig. 9A and 9D). Moreover, siRNA-mediated knockdown of GIOT1/Giot1 (Fig. 9B and 9E) reverses the inhibitory effects of FSH on GDF9 + BMP15–induced AMH expression (Fig. 9C and 9F).

The transcriptional repressor activities of GIOT1 are mediated, in part, through HDAC2 recruitment (66). Therefore, to determine if HDAC2 is recruited on the AMH promoter through GIOT1 upon FSH treatment, we first knocked down GIOT1 with siRNA in KGN cells. Thereafter, we cotreated the cells with FSH and GDF9 + BMP15 and performed ChIP assays on two regions (P1 and P2) of the AMH promoter with antibodies against H3K27ac and HDAC2. P1 is shown in Fig. 8 and P2 is shown in the supplemental data (55). Results show that knockdown of GIOT1 rescues the GDF9 + BMP15–induced H3K27ac mark on the AMH promoter. Moreover, knockdown of GIOT1 decreases the association of HDAC2 on the AMH promoter.

FSH inhibits AMH expression in vivo

Finally, given that FSH treatment inhibits AMH expression, we compared Amh mRNA levels in ovaries isolated from Fshβ-null mice (Fshβ^−/−) with those in WT and Fshβ-null mice expressing a human WT FSHβ transgene (Fshβ^−/−hFSHβ^WT). Results (Fig. 10A) show that Amh mRNA levels are significantly higher in Fshβ^−/− compared with ovaries from WT animals. Expression of human WT FSH on Fshβ-null genetic background (Fshβ^−/−hFSHβ^WT) significantly lowers the Amh mRNA levels, confirming FSH directly regulates AMH expression in mouse ovaries.

Discussion

Our study provides a mechanistic understanding of the regulation of AMH expression in GCs (Fig. 10B). We provide direct experimental evidence that oocyte-derived factors, such as GDF9 and BMP15, together through a synergistic effect between Smad2/3 and PI3K/Akt signaling, promote p300-mediated histone acetylation that induces the expression of AMH in GCs. We further show that FSH treatment inhibits GDF9 + BMP15–induced AMH expression through cAMP/PKA/SF1-mediated expression of GIOT1, a transcriptional repressor, and the latter recruits HDAC2 to deacetylate GDF9 + BMP15–induced p300-mediated H3K27ac, resulting in the suppression of AMH/Amh (Fig. 10B).

It has long been known that there exists an “oocyte-GC regulatory loop” involving complementary signaling pathways that are critical for follicle development (67). Our results, as well as studies by others (18, 19), now clearly establish that oocyte-derived factors, GDF9 and BMP15 together but not individually, induce AMH expression in GCs. It is likely that the effects of GDF9 and BMP15 cotreatment are mediated through the formation of the GDF9/BMP15 heterodimer termed “cumulin.” Previously (68), it has been shown that coaddition of separately expressed GDF9 and BMP15 results in formation of the heterodimer cumulin, which produces a strong synergistic response in GCs. Moreover, cumulin has enhanced signaling potency and can activate both Smad2/3 as well as Smad1/5/8 signaling pathways. Our studies show that GDF9 + BMP15–induced AMH expression is synergistically regulated by Smad (Smad2/3)–dependent and Smad-independent (PI3K/Akt) signaling pathways.

Interestingly, in human glomerular mesangial cells, the PI3K/Akt and Smad pathways crosstalk in response to TGF-β (69). In addition to our studies in KGN cells, studies in rat GCs from preantral follicles have reported that GDF9 causes activation of the PI3K/Akt pathway (56). Thus, it can be speculated that activation of the PI3K/Akt pathway by GDF9 + BMP15 is primarily mediated through GDF9 actions. Conversely, GDF9 + BMP15 have been reported (70) to activate the mechanistic target of rapamycin (mTOR) pathway in GCs through suppressing the expression of an mTOR inhibitor, Ddit4l (or REDD1), which inhibits the G protein–coupled receptor–activated mTOR pathway (71). This might be another possible mechanism by which GDF9 + BMP15 may stimulate the PI3K/Akt pathway. Therefore, the precise mechanism of GDF9 + BMP15–induced activation of Akt needs further investigation.

A recent study (19) in human cumulus cells reported that GDF9 + BMP15 induce AMH mRNA and protein levels. However, based on the AMH promoter activity assay using different lengths (2.2 kb) of AMH promoter, it was suggested that the regulation of GDF9 + BMP15–induced AMH expression occurs through a transcription-independent mechanism. Conversely, this study also proposed the possibility of additional elements beyond the 2.2-kb promoter region to be involved in regulation of AMH expression. We find that GDF9 + BMP15–induced AMH expression occurs through transcription activation of the AMH promoter mediated by Smad2/3-p300–induced H3K27 acetylation. In fact, there are p300 binding sites beyond 2.2 kb of the AMH promoter region, through which the observed transcriptional activities are likely being mediated. p300 is a HAT that can also acetylate nonhistone proteins related to transcription, as well as act as a coactivator in many processes, including growth factor–signaled activation. Our studies show that p300 gets phosphorylated by the PI3K/Akt pathway at Ser1834. This phosphorylation of p300 has been reported to be essential for transactivation of p300 by stimulating its HAT activity, assembling transcription factors, and recruiting basal transcriptional machinery (57, 72). Nevertheless, we find that phosphorylation of p300 at Ser1834 is not sufficient, and Smad2/3 signaling is also required for GDF9 + BMP15–induced AMH expression. Smad and p300 are transcriptional regulators of histone (73). It has been shown that SMAD2 induces changes in the chromatin landscape to regulate transcription, and SMAD2 binding correlates with histone acetylation (74–76). Moreover, previous studies (77, 78) have reported that TGF-β receptor phosphorylation of Smad3 promotes its interaction with p300 and synergistically augments transcriptional activation. In accordance with these studies, we also show that upon GDF9 + BMP15 treatment, Smad2/3 interacts with p300, and this corresponds to increased p300 binding and H3K27 acetylation of the AMH promoter. H3K27ac and p300 binding profile are widely used as a measure of enhancer activity and associated with higher levels of DNA accessibility (79). As SMAD2 cannot bind DNA itself, it requires additional transcriptional factors for recruitment to chromatin (76). Therefore, it is likely that Smad2/3-p300 recruits other transcriptional factors, forming a transcriptional complex to induce AMH expression. Further studies are needed to identify other transcriptional factors that may be involved in AMH expression.

The role of FSH in regulating AMH expression is controversial. Although some studies show that FSH can induce AMH expression (80), others have shown an inhibitory effect of FSH on AMH expression (19, 81). This difference has been attributed to cell and species specificity. Studies in human and mouse luteal GCs have primarily reported FSH to be stimulatory (80), whereas studies in human cumulus cells (19) and rat GCs (81), as well as our studies in primary mGCs and KGN cells, show that FSH inhibits GDF9 + BMP15–induced AMH/Amh expression. Also, clinical studies have reported that exogenous FSH administration decreases serum AMH levels in both normal-cycling women (28, 29) and patients with polycystic ovary syndrome (82). Despite these studies, the underlying mechanism of how FSH inhibits AMH expression remains unclear. Studies in human luteal GCs as well as in KK1 cells, a mouse luteal cell line, show that estradiol, through estrogen receptor–β, inhibits AMH expression (83). Our study demonstrates that FSH, through the regulation of GIOT1 expression, directly inhibits the GDF9 + BMP15–induced AMH/Amh expression. Gonadotropin-inducible ovarian transcription factors are a group of the C2H2 zinc finger protein family that are widely expressed in eukaryotic organisms (84). GIOT1 is predominantly expressed in steroidogenic, Leydig, granulosa, and theca cells; adrenal gland; and the pituitary (84). Previous studies in rat GCs show that FSH, through PKA, cAMP response element binding protein, and SF-1, induces GIOT1 (85). GIOT1 contains a Krüppel-associated box A domain in the N-terminal that is responsible for the transcriptional repressor activity. Moreover, GIOT1 has been shown to recruit HDAC2 to repress transcription (66). Our results are in accordance with these previous studies. Although we show that GIOT1 increases HDAC2 binding and decrease in H3K27ac mark on the AMH promoter, it is also possible that GIOT1, through the Krüppel-associated box A domain, physically interacts with Smad2/3 and/or p300 and interferes with the recruitment of p300 or directly binds to the AMH promoter region and represses its activation.

The fact that ovarian AMH mRNA levels are significantly higher in Fshβ-null (Fshβ^−/−) mice and restoration of FSH in these mice (Fshβ^−/−hFSHβ^WT) lowers the AMH expression serves as an in vivo proof for our in vitro studies. Fshb-null mice are infertile (40). These mice lack antral but have normal preantral follicles compared with those in ovaries of WT mice (5, 41). Therefore, the increase in Amh mRNA levels observed in the Fshβ-null ovaries is due to lack of inhibitory effects of FSH on Amh expression and not due to increased follicle number. Intriguingly, we and others have previously shown (5, 15) that AMH inhibits FSH action. In both mice (7) and humans (86), AMH is expressed by small growing follicles until the antral stage, after which AMH expression significantly decreases in large antral/preovulatory follicles. Taken together, our studies suggest the existence of a feedback loop during follicular development where AMH inhibits FSH actions in the preantral stage, whereas the latter in turn blocks AMH expression in antral follicles. In conclusion, these results demonstrate the underlying mechanism of AMH expression and its regulation in GCs. Given that AMH plays a major role in folliculogenesis and is widely used as a clinical biomarker for various pathophysiological conditions in women, results of the current study address a significant knowledge gap regarding the regulation of AMH expression during follicular development that has been a severe limitation in developing AMH as a potential therapeutic target and/or option for female fertility.

Abbreviations:

Abbreviations:
 
• AMH

  anti–Müllerian hormone

 
• BMP15

  bone morphogenetic factor 15

 
• ChIP

  chromatin immunoprecipitation

 
• GAPDH

  glyceraldehyde 3-phosphate dehydrogenase

 
• GC

  granulosa cell

 
• GDF9

  growth differentiation factor 9

 
• GIOT1

  gonadotropin inducible ovarian transcription factor 1

 
• H3K27ac

  acetylation of lysine 27 on histone 3

 
• HAT

  histone acetyltransferase

 
• HDAC

  histone deacetylase

 
• IP

  immunoprecipitation

 
• mGC

  mouse granulosa cell

 
• mTOR

  mechanistic target of rapamycin

 
• PKA

  protein kinase A

 
• siRNA

  small interfering RNA

 
• WT

  wild-type

Acknowledgments

Financial Support: This work was supported by grants from Ferring Pharmaceuticals (to A.S.), Michigan State University AgBioResearch (to A.S.), The Makowski Foundation (to T.R.K.), and the National Institutes of Health (RO1HD086062-01A1 to A.S. and RO1HD081162 and AG029531 to T.R.K.).

Disclosure Summary: A.S., N.G., and V.A.K. are listed as co-owners of pending US patents related to therapeutic use of AMH for regulation of fertility. N.G. is a shareholder in Fertility Nutraceuticals, LLC, and owner of the Center for Human Reproduction. N.G. receives patent royalties from Fertility Nutraceuticals, LLC. The remaining authors have nothing to disclose.

References