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The Journal of Clinical Endocrinology and Metabolism logoLink to The Journal of Clinical Endocrinology and Metabolism
. 2019 Oct 7;105(1):327–335. doi: 10.1210/clinem/dgz057

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

Elie Hobeika 1, Marah Armouti 2, Michele A Fierro 1, Nichola Winston 1, Humberto Scoccia 1, Alberuni M Zamah 3, Carlos Stocco 2,✉
PMCID: PMC6938692  PMID: 31588501

Abstract

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.

Folliculogenesis is a long process lasting several months in humans that transforms a primordial follicle into a dominant preovulatory follicle (1). The success of folliculogenesis depends on a close interaction between the two main components of the follicle, the granulosa cells (GCs) and the oocyte. The active role of the oocyte in folliculogenesis was described in the early 1970s, when it was observed that oocyte ablation leads to impaired folliculogenesis and follicle luteinization (2). Later, Nekola et al. (3) confirmed these findings and showed that GCs cultured near oocytes appeared to be less luteinized than those cultured without oocytes. It was not until two decades later that landmark studies identified two oocyte-specific growth factors (OSFs), growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) (4, 5). The influence of these factors on the process of folliculogenesis is now well accepted.

GDF9 and BMP15 have a high degree of homology in their sequence and structure. They have similar expression patterns and functions in the ovary (6–8). Moreover, recent evidence suggests that GDF9 and BMP15 form heterodimers, which mediate some of their actions (9, 10). However, the role of GDF9 and BMP15 in the control of folliculogenesis has been elucidated exclusively using animal models and cell lines. These reports showed that GDF9 is a critical player in the follicular development of mice and sheep, whereas BMP15 is not essential for fertility in mice but critical in sheep (4, 5, 8). However, GDF9 and BMP15 function in the human ovary and in human primary ovarian cells remains unexplored, mainly due to the lack of appropriate experimental approaches.

We previously validated the use of cumulus cells obtained from patients undergoing in vitro fertilization (IVF) as a proxy of undifferentiated GCs to study follicle-stimulating hormone (FSH) actions in humans (11, 12). Also, we examined the interaction between FSH, GDF9, and BMP15 on GC function and showed that, whereas FSH inhibits anti-Müllerian hormone (AMH) production in primary human GCs, the combination of GDF9 and BMP15 (GB) potentiates the production of AMH (13). The interaction between FSH and OSFs is not always antagonistic. For instance, our most recent report shows that GDF9 and BMP15 potentiate FSH stimulation of aromatase and estrogen production, two hallmarks of GC differentiation (14).

Previous studies demonstrated an essential role of the insulin-like growth factor (IGF) system on the induction of aromatase and estradiol synthesis in human GCs (11, 15, 16). Interactions between FSH and IGFs have also been shown to upregulate the production of estradiol and progesterone in several species such as rodent (17), porcine (18), and bovine (19), beyond that of either factor alone. However, the IGF system differs significantly between rodents and humans, making the use of animal models unsuitable for studies aiming to elucidate the regulation of the IGF system in humans. For instance, while IGF1 is mostly expressed in mouse GCs, IGF2 is the only IGF expressed in human GCs (11, 16, 20). We have also demonstrated that FSH inhibits IGF1 expression in rodent GCs, whereas FSH stimulates IGF2 expression in human GCs (11, 15, 16). These findings were confirmed in a recent report showing that IGF2 expression in human GCs increases approximately 64-fold as follicles progress from the small antral to the preovulatory stage, a process that is entirely regulated by FSH (21). However, the mechanisms controlling the expression of IGF2 in human GCs remains unexplored. Here, we examined the interaction between GDF9, BMP15, and FSH on the regulation of IGF2 in human primary cumulus GCs. We tested the hypothesis that GDF9 and BMP15 interact with FSH to stimulate the expression of IGF2 and that this effect mediates, at least in part, the enhancing effect of OSFs on aromatase and estradiol production.

Materials and Methods

Human cumulus cell culture

Human cumulus cells were collected from patients undergoing IVF at the University of Illinois IVF Center under an Institutional Review Board-exempt protocol. No patient information was collected for reporting. After controlled ovarian stimulation, mature follicles were aspirated from women undergoing IVF. The cumulus–oocyte complexes were then removed from the follicular aspirates, and the cumulus cells were separated from the oocyte manually. The cumulus cells from all follicles for a single patient were then pooled and transported immediately to the laboratory where they were dispersed by hyaluronidase digestion (80 IU/μL) and then centrifuged at 500 × g for 2 minutes. Cells were incubated at room temperature in red blood cell lysis buffer for 5 minutes to eliminate contaminating erythrocytes, centrifuged again at 500 × g for 2 minutes. Cells were suspended in 0.4 mL of serum-free and phenol red-free DMEM/F12-0.25% BSA (Sigma-Aldrich) media supplemented with penicillin (50 IU/mL), streptomycin (50 g/mL), and sodium bicarbonate (1.2 g/L; Sigma-Aldrich). Cells were then cultured on plates precoated with BD Matrigel (BD Biosciences) at a density of 6 × 104/mL. Cells were cultured for at least 48 hours, followed by treatment with various combinations of hormones and signaling inhibitors. Treatments included human recombinant FSH (Serono), GDF9 (R&B), BMP 15 (R&B), inhibitors of SMAD2/3 (SB431542, Tocris), SMAD 3 (SIS3; Cayman Chemical Company), or SMAD1/5/8 (LDN-193189, Selleck Chemicals), IGF1R inhibitor AEW (NVP-AEW451) (Calbiochem), and dbcAMP (Sigma). Cells of different individuals were not pooled together.

Polymerase chain reaction

Total RNA was isolated using TRIzol Reagent (Invitrogen) as recommended in the manufacturer’s protocol. Total RNA (1 µg) was reverse-transcribed using anchored oligo-dT primers (Integrated DNA Technologies), and reverse transcriptase from Moloney murine leukemia virus (Invitrogen) at 37°C for 2 hours. The resulting cDNA was diluted to a final concentration of 10 ng/µL. Quantitative real-time polymerase chain reaction (qPCR) was performed using intron-spanning primers specific for the detection of ribosomal protein L19 (RPL19) and promoter 3-specific IGF2 transcripts. The number of copies for each gene was calculated using a standard curve made with a serial dilution of the respective cDNA (22). Once the number of copies of the gene of interest was obtained, the relative expression is calculated as the ratio between the copy number of the gene of interest and the copy number of the housekeeping gene Rpl19. Primer sequences are available upon request.

Promoter activity assay

The IGF2p3–Luc reporter was cloned into a lentivirus reporter vector using the method described previously (14). Lentiviruses containing this construct were generated as described (14). Empty plasmids were used as controls. Cells were infected with lentivirus and after overnight incubation treated with FSH, GDF9, BMP15, or their combination. At 48 hours, luciferase activity was determined in 50 μL of lysates as previously described (14).

Promoter 3 methylation

We examined the methylation status of the 37 CpG sites found in the IGF2 promoter 3 using bisulfite sequencing. Cumulus cells were treated with vehicle or G+B in the presence or absence of FSH for 48 hours before genomic DNA isolation. An aliquot of DNA was treated with sodium bisulfite and purified using Wizard DNA clean-up system (Promega). To quantify CpG methylation, IGF2 promoter 3 was amplified using primers specific for bisulfite-treated DNA. Then, next-generation sequencing libraries were prepared from the original PCR amplicon using primers that introduce Illumina adaptors. Samples were then sequenced using Illumina NextSeq. A threshold of 98% conversion of no-CpG C to uracil was set for the sample to be included in the analysis. The sequences were aligned to the IGF2-P3 sequence and 5 mC percentage calculated.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) for IGF2 protein secretion was performed on the supernatant of human GCs treated with the different combinations of FSH, GDF9, and BMP15 using the Quantikine® ELISA kit for Human IGF2 (R&D Systems).

Statistical analysis

Each experiment was run at least in duplicate, and data for continuous variables are presented as mean values ± standard error of the mean (SEM). Statistical comparisons of mean values between groups were performed with paired t-tests, and multiple comparisons were performed with one-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni adjustment, Fisher’s least significant difference (LSD) test or Tukey’s multiple comparisons test, where appropriate. Differences were considered to be statistically significant if P < 0.05.

Results

The combination of GDF9 and BMP15 potentiates FSH induction of IGF2

We previously demonstrated that the combination of GDF9 and BMP15 potentiates the stimulatory effect of FSH on IGF2 expression (14). To further evaluate the impact of GDF9 and BMP15 on the expression of IGF2 mRNA, primary hGCs were treated with increasing concentrations of GDF9 and BMP15 in the presence of 50 ng/mL of FSH. The concentration of FSH was initially chosen based on that used in previous reports (13, 14). As previously shown, FSH significantly increased IGF2 mRNA transcripts when compared with nontreated cells using the t-test (P <0.05) (Fig. 1A). This stimulatory effect of FSH on IGF2 was significantly augmented by the addition of the combination GDF9 and BMP15 (G+B) to the media (P < 0.01). As shown in Fig. 1A, IGF2 mRNA levels were significantly increased in human GCs treated with G+B at concentrations of 2.5 and 10 ng/mL in the presence of FSH compared with other treatments and the untreated control cells. Thus, treatment with FSH and either GDF9 or BMP15 at concentrations of 10 ng/mL, or treatment with 10 ng/mL G+B in the absence of FSH did not stimulate IGF2 compared with FSH-only treated cells. To maintain consistency with previous reports, a dose of 5 ng/mL for each GDF9 and BMP15 was used in the remainder of the experiments (13, 14).

Figure 1.

Figure 1.

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The combination of FSH, GDF9, and BMP15 (GB) potentiate FSH-induced IGF2 expression in a dose-dependent manner. A. Primary human cumulus cells were treated for 48 hours with vehicle (C), GDF9 (G: 10 ng/mL), BMP15 (B: 10ng/mL), GDF9 and BMP15 combined (GB: 0.6, 2.5, and 10 ng/mL for each of G and B), in the presence or absence of follicle-stimulating hormone (FSH) (F: 50 ng/mL). IGF2 mRNA levels were determined by quantitative real-time polymerase chain reaction (qPCR) and expressed relative to Rpl19. Columns represent the mean ± SEM (standard error of the mean). Columns with different letters differ significantly by one-way analysis of variance (ANOVA) analysis with Fisher’s least significant difference (LSD) test, a–b P < 0.05, a–c P < 0.01, b–c P < 0.05, n = 9. Control and FSH groups were compared by t test; *P < 0.05, n = 9. B. Primary human cumulus cells were treated for 48 hours with vehicle (C), GDF9 (G: 5 ng/mL), BMP15 (B: 5 ng/mL), in the presence or absence of FSH (F: 5, 25, 100 ng/mL). IGF2 mRNA levels were determined by qPCR and expressed relative to Rpl19. Columns represent the mean ± SEM. Columns with different letters differ significantly by one-way ANOVA analysis with Fisher’s LSD test, a–b P < 0.05, a–c P < 0.01, b–c P < 0.05, n = 5.

Next, we examined whether G+B was able to enhance the effect of increasing concentrations of FSH. As shown in Fig. 1B, although G+B cotreatment enhanced the effect of FSH, increasing concentrations of FSH did not further increase IGF2 expression, suggesting that the stimulation of IGF2 appears to be maximal in the presence of FSH and G+B.

GDF9 and BMP15 potentiate FSH stimulation of IGF2 protein levels

Next, we examined whether the effect of G+B on IGF2 mRNA levels translates into an increase in IGF2 protein. IGF2 was measured in the supernatant of cultures treated as indicated above using ELISA. In concordance with findings at the mRNA levels, cotreatment of FSH with G+B potentiated the stimulatory effect of FSH on IGF2 protein expression (Fig. 2). IGF2 concentration in the supernatant of cells treated with FSH and G+B cotreatment was increased 1.9-fold compared with the control group (66.67 ± 5.5 vs 33.99 ± 0.6 pg/mL, P < 0.01) and 1.5-fold compared with the group treated with FSH alone (66.67 ± 5.5 vs 43.15 ± 0.4 pg/mL, P < 0.05). A paired t-test comparing the FSH-treated group with the untreated control group showed a statistically significant increase in IGF2 protein production (43.15 ± 0.4 vs 33.99 ± 0.6 pg/mL, P < 0.01). Taken together, these findings indicate a robust positive effect of the GDF9 and BMP15 combination on the stimulatory effect of FSH on IGF2 protein production.

Figure 2.

Figure 2.

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The combination of GDF9 and BMP15 potentiates follicle-stimulating hormone (FSH)-induced IGF2 expression and protein synthesis. IGF2 protein levels were quantified by enzyme-linked immunosorbent assay. Columns represent the mean ± standard error of the mean, columns with different letters differ significantly by one-way analysis of variance analysis with repeated measures and Bonferroni correction a–b P < 0.001. One-way paired t-test was used to measure the difference between C and F, (**) for P < 0.01, n = 3.

GDF9 and BMP15 potentiate cAMP stimulation of IGF2

Next, we determined whether G+B interacts with cAMP, the primary second messenger of the FSH receptor, to stimulate IGF2. As shown in Fig. 3, treatment with dbcAMP (a cAMP analog) alone stimulated IGF2 mRNA levels compared with the control group (P < 0.05). However, IGF2 mRNA expression was significantly higher in cells treated with GDF9, BMP15, and dbcAMP than in cells treated with dbcAMP alone (P < 0.05) or the control (C) group (P < 0.01). Thus, as in the case of FSH, the stimulatory effect of dbcAMP was potentiated by the presence of G+B in the media, suggesting that the crosstalk between FSH and G+B occurs downstream of the FSH receptor.

Figure 3.

Figure 3.

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The potentiation of FSH actions by GDF9 and BMP15 (GB) occurs downstream of cAMP. Primary human cumulus cells were treated for 48 hours with vehicle (C) or the combination of GDF9 and BMP15 (GB: 5 ng/mL) in the presence or absence of dbcAMP (cAMP: 2 mM) and the absence of FSH. mRNA levels were determined by quantitative real-time polymerase chain reaction and expressed relative to Rpl19. One-way ANOVA analysis with repeated measures and Bonferroni correction. Columns represent the mean ± SEM, columns with different letters differ significantly, a–b P < 0.05, a–c P < 0.01, b–c P < 0.05, n = 5.

GDF9 and BMP15 increase IGF2 transcripts derived from the promoter 3 but have no effect on promoter activity

IGF2 gene transcription is driven by four different promoters (23). The activity of each promoter generates IGF2 mRNA with promoter-specific untranslated exons upstream of the protein-coding exons 7 to 9 that are common to all transcripts. We have demonstrated that IGF2 expression in human GCs is controlled by promoters 3 and 4, of which only promoter 3 is regulated by FSH (16). For this reason, the results listed in Figs. 1 and 3 were performed using primers that amplify transcripts derived from promoter 3. To further examine the regulation of promoter 3 by G+B, we cloned 511 bp of the IGF2 promoter 3 into a lentiviral reporter plasmid (IGF2p3–LUC). To determine whether G+B regulates IGF2p3–LUC, cumulus cells were plated in 48-well plates and immediately infected with the lentivirus for 24 hours. At this time, cells were treated with vehicle, FSH, G+B, or FSH plus G+B. Luciferase activity was quantified 48 hours after the addition of FSH. FSH treatment alone increased the activity of the IGF2p3–LUC construct compared with the control group (P < 0.05) (Fig. 4A). In contrast to the findings observed on promoter 3 transcript levels, treatment with FSH plus G+B did not increase the activity of the IGF2p3–LUC construct above the levels observed with FSH alone. This lack of agreement between the observed regulation of promoter 3 transcripts and the promoter 3 luciferase reporter by G+B suggests alternative mechanisms by which GDF9 and BMP15 regulate IGF2 expression.

Figure 4.

Figure 4.

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Effect of GDF9 and BMP15 (GB) on IGF2 promoter 3 activity. A. Cells were infected with lentivirus carrying the IGF2p3–LUC reporter and after overnight incubation treated for 48 hours with vehicle (C), follicle-stimulating hormone (FSH), FSH+GB, or GB. Luciferase activity was quantified and expressed relative to the control. Columns represent the mean ± SEM, columns with different letters differ significantly, n = 4. B. H19 transcripts were measured in cumulus cells treated with increasing doses of G+B and FSH (50 ng/mL) for 48 hours. Expression of H19 was determined by quantitative real-time polymerase chain reaction and expressed relative to Rpl19. Columns represent the mean ± standard error of the mean. Columns with different letters differ significantly by one-way analysis of variance analysis followed by Tukey’s multiple comparisons test (n = 5).

IGF2 mRNA upregulation in cancer cells correlates with demethylation of CpG islands in the genomic region corresponding to promoter 3, which contains 37 CpG islands (24, 25). Therefore, we examined the methylation status of all CpG sites in this genomic region using bisulfite sequencing. Cumulus cells were treated with vehicle or G+B in the presence or absence of FSH for 48 hours before genomic DNA isolation and bisulfite sequencing. The results demonstrated that treatment with G+B in the presence or absence of FSH had no effect on the percentage of methylation of the IGF2 promoter 3 (data not shown), suggesting that promoter 3 methylation is unlikely to mediate the effects of G+B.

To examine other possible mechanisms for the regulation of IGF2 by G+B, we examined the expression of the H19 gene. H19 and IGF2 form a reciprocally imprinted cluster (IGF2/H19) located on human chromosome 11 (26). It is well known that IGF2 and H19 expression is inversely regulated via a differentially methylated region upstream of the H19 gene (27). Therefore, we quantified the mRNA levels of H19 in cells treated with vehicle or G+B in the presence or absence of FSH. The results demonstrated that treatment with G+B and FSH strongly stimulated the expression of H19 reaching levels that were significantly higher than those found in cells treated with FSH or GB alone (Fig. 4B).

GDF9 and BMP15 regulate IGF2 via SMAD signaling

GDF9 and BMP15 activate SMAD2/3 and SMAD1/5/8 signaling pathways, respectively (28–30). To investigate the mechanism of G+B-induced IGF2 expression in the presence of FSH, we used inhibitors of SMAD2/3 (SB431542), SMAD3 (SIS3), or SMAD1/5/8 (LDN-193189). The concentration of SMAD inhibitors used was based on previous publications in mouse and humans GCs (10, 30). The inhibitors were added to the media 1 hour before the addition of FSH or FSH plus GB. Forty-eight hours later, cells were harvested for IGF2 mRNA determination. The results show that inhibition of SMAD1/5/8 had no significant effects on the induction of IGF2 mRNA levels by FSH plus G+B (Fig. 5). However, the potentiation of IGF2 expression by G+B in the presence of FSH was inhibited by the addition of SB431542 and SIS3 (P = 0.018 and P = 0.007, respectively).

Figure 5.

Figure 5.

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GDF9 and BMP15 regulate IGF2 via SMAD signaling. Primary human cumulus cells were treated for 48 hours with vehicle (C) or the combination of GDF9 and BMP15 (GB: 5 ng/mL) in the presence or absence of FSH (50 ng/mL), SMAD2/3 inhibitor SB431542 (SB: 1 µM), SMAD3 inhibitor SIS3 (SIS: 5 µM), or SMAD1/5/8 inhibitor LDN-193189 (LDN: 100 nM). IGF2 mRNA levels were determined by qPCR and expressed relative to Rpl19. t-Test was used to evaluate the difference between mRNA expression in the F+GB with and without the different SMAD inhibitors. Columns represent the mean ± standard error of the mean, columns with different letters differ significantly, a–b P < 0.05, a–c P < 0.01, b–c P < 0.05, n = 8.

IGF1R activity is necessary for GDF9 and BMP15 potentiation of IGF2 expression

In human GCs, we previously reported that FSH stimulation of IGF2 requires IGF1R activity (16). To determine whether the stimulatory effect of G+B on IGF2 also requires the IGF1R, human GCs were treated with NVP-AEW451, a known inhibitor of IGF1R activity, at a concentration of 1 μM for one hour before the addition of FSH, G+B, or both to the media. IGF2 mRNA levels were quantified 48 hours after the addition of FSH. The potentiation of FSH plus G+B on IGF2 mRNA was significantly decreased by the presence of NVP-AEW451 in the media when compared with cells treated with FSH plus G+B alone (P = 0.02) (Fig. 6). However, G+B was still able to potentiate the effect of FSH on IGF2 mRNA levels in the presence of NVP-AEW451 (P = 0.008, ANOVA).

Figure 6.

Figure 6.

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The combination of GDF9 and BMP15 potentiates FSH stimulation of IGF2 mRNA partially through IGF1R signaling. Primary human cumulus cells were treated for 48 hours, as described in Fig. 2A in the presence or absence of the IGF1R inhibitor NVP-AEW451 (1 μM). mRNA levels were determined by qPCR and expressed relative to Rpl19. One-way ANOVA analysis with repeated measures and Bonferroni correction indicated a global P = 0.008 for the group. Columns represent the mean ± SEM, columns with different letters differ significantly. A t-test revealed that the addition of NVP-AEW451 inhibits GDF9 and BMP15 potentiation of IGF2 mRNA (**P = 0.02), n = 4.

Discussion

We have shown for the first time that GDF9 and BMP15, acting in coordination with FSH, maximally stimulate IGF2 in primary human GCs. The enhancing effect of GDF9 and BMP15 occurs downstream of the FSH receptor and is inhibited by SMAD2/3 and SMAD3 pathway inhibitors. Only the combination of GDF9 and BMP15 enhances IGF2 expression. These findings support the hypothesis that GDF9 and BMP15 form heterodimers (9, 10). However, GDF9:BMP15 heterodimers have not been detected in either animal or human models. On the other hand, a recent report suggested that monomers of BMP15 and GDF9 activate cell-surface receptors initiating a synergistic effect without the need for heterodimer formation (31). Therefore, additional experiments are needed to determine the mechanism of the collaborative effect between GDF9 and BMP15 on the regulation of GCs.

The use of human cumulus GCs collected from cumulus–oocyte complexes isolated from women undergoing IVF has been adopted by other groups and us to investigate human GC biology (11, 13–16, 32). We demonstrated that cumulus cells have distinct properties compared with mural cells such as lack of luteinization and the ability to respond to FSH and IGFs (12). The use of human cells is highly relevant because of the specific characteristics of the IGF system in the human ovary that are not conserved in other species (see earlier) (11, 16, 20).

IGF2 plays a central role in the differentiation of human GCs. Our group was the first to show that FSH enhances IGF2 mRNA expression in human GCs (16) and showed that IGF2 is necessary for FSH-induced upregulation of steroidogenic enzymes such as Cyp19a1, Cyp11a1, and Star (11). Together, these findings and the current report support the hypothesis that the IGF2 system is also involved in the selection of the dominant follicle. Thus, considering that IGF2 is vital for GC proliferation and that OSFs increase IGF2 expression, it is reasonable to propose that a healthy oocyte will secrete higher quantities of GDF9 and BMP15, hence sensitizing GCs to FSH. An oocyte producing high levels of GDF9 and BMP15 will enhance the survival of its surrounding GCs in the presence of low levels of FSH, leading to its selection from the cohort.

We proved previously and confirmed in this report that FSH stimulates IGF2 expression by increasing promoter 3-derived transcripts in a v-akt murine thymoma viral oncogene homolog 1 (AKT) activation dependent manner (16). However, the molecular mechanisms involved in the transcriptional activation of IGF2 in humans are unknown. We show here for the first time that FSH increases IGF2 transcription using a reporter vector in which luciferase activity is controlled by 511 bp of the genomic region of human P3 promoter. The mechanisms and factors participating in the stimulation of promoter 3 activity by FSH remain to be determined. Of note, in silico analysis of the promoter showed the presence of binding sites for specificity protein 1 (SP1), early growth response protein 1 (ERG1), activation protein 2 (AP2), and CCAAT enhancer binding protein beta (C/EBPb). However, this analysis revealed the absence of binding sites for cAMP/CREB, which mediates FSH actions, suggesting an atypical regulation of IGF2 expression by FSH in human GCs.

Inhibition of SMAD2/3 activation abolishes IGF2 stimulation by G+B, suggesting a major role of SMAD2/3 in IGF2 regulation. Upon phosphorylation by type 1 receptor kinase, Smads form heteromeric complexes with co-Smads and translocate into the nucleus to activate gene transcription. Additionally, a well-documented mechanism of R-Smad regulation is through phosphorylation of the linker region found between the MH1 and MH2 domains (33), which profoundly affects SMAD signaling. Among the kinases targeting this region is AKT. We have previously demonstrated that AKT is essential for the induction of IGF2 by FSH (16). Thus, in human GCs, the activation of SMAD by G+B could be further enhanced by FSH via activation of AKT. We speculate that this mechanism could play a central role in mediating the crosstalk between the signaling activated by oocyte-secreted factors and FSH. Future studies will be needed to test this hypothesis.

How GDF9 and BMP15 regulate the expression of P3 transcripts is also unclear. Our findings suggest that combined these factors enhance promoter 3 IGF2 transcript expression in the presence of FSH. Strikingly, our promoter studies showed that GDF9 and BMP15 do not enhance the stimulation of the P3-Luc reporter by FSH. Thus, GDF9 and BMP15 may regulate the steady-state levels of IGF2 by affecting gene transcription indirectly, or by regulating the stability of the IGF2 mRNA. For the first of these possibilities, we considered two possible mechanisms: (1) changes occur in the methylation status of promoter 3, or (2) methylation changes occur upstream of the H19 gene (27), which is known to upregulate IGF2 and reduce H19 expression. Our results suggest that the methylation of promoter 3 is not affected by GDF9+BMP15 treatment. Moreover, IGF2 and H19 are regulated similarly by GDF9 and BMP15, indicating that methylation on the imprinting-control region (ICR) region upstream of the H19 gene is most probably not affected by these factors. Therefore, the evidence points to the idea that GDF9 and BMP15 are not involved in the regulation of IGF2/H19 methylation. However, further studies will be required to evaluate this possibility fully.

Changes in mRNA stability may also play a role in the regulation of IGF2 mRNA by GDF9 and BMP15. For instance, it will be of great interest to determine if GDF9 and BMP15 regulate the expression of mRNA binding proteins known as IGF2 mRNA-binding proteins (IMPs). IMPs have been shown to participate in the post-transcriptional regulation of IGF2 (34). A recent report demonstrated that the expression of all IMP isoforms is relatively constant during human follicular development (21). This report also showed a 64-fold increase in IGF2 expression from small antral to preovulatory follicle maturation (21). Moreover, we observed previously that inhibition of IGF1 receptor activity abolishes IGF2 induction by FSH (16). However, the lack of IGF1R activity does not prevent the enhancement of IGF2 expression by GDF9 and BMP15. Taken together, our findings and previous reports suggest that IGF2 regulation in human GCs may involve several interdependent mechanisms, which agrees with our hypothesis that IGF2 plays a central role in follicle maturation and selection in humans.

A limitation of our study includes the under-representation of women with diminished ovarian reserve or low oocyte yield at the time of oocyte retrieval, which would yield a suboptimal number of GCs. Thus, the possibility of a variable response of GCs from patients with different etiologies of infertility cannot be ruled out. However, the strength of the study is that GCs from different patients were not pooled together, suggesting that the mechanisms activated by OSFs on the regulation of human GCs are conserved despite human subject variability.

In conclusion, GDF9 and BMP15 play a significant role in follicle development. In addition to their partnership with FSH in the regulation of aromatase, we present evidence of their synergism in the stimulation of IGF2. This in itself sensitizes the follicle to FSH-induced aromatase expression, which is of utmost importance in follicular growth in humans. This report adds to our understanding of the unique role that OSFs play in folliculogenesis. Furthermore, these findings can be used in efforts to develop new strategies to improve in vitro maturation of follicles.

Acknowledgments

Financial Support: NIH grant R56HD086054 (C.S.).

Glossary

Abbreviations

AMH

anti-Müllerian hormone

ANOVA

analysis of variance

BMP15

bone morphogenetic protein 15

ELISA

enzyme-linked immunosorbent assay

FSH

follicle-stimulating hormone

GDF9

growth differentiation factor 9

hGC

Human granulosa cell

IGF2

insulin-like growth factor 2

IVF

in vitro fertilization

LSD

least significant difference

OSF

oocyte-secreted factor

qPCR

quantitative real-time polymerase chain reaction

SEM

standard error of the mean

Additional Information

Disclosure Summary: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Data availability: All data generated or analyzed during this study are included in this published article or in the data repositories listed in References.

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