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The Journal of Clinical Endocrinology and Metabolism logoLink to The Journal of Clinical Endocrinology and Metabolism
. 2018 Dec 11;104(5):1667–1676. doi: 10.1210/jc.2018-01705

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

Elie Hobeika 1, Marah Armouti 2, Hamsini Kala 2, Michele A Fierro 1, Nicola J Winston 1, Bert Scoccia 1, Alberuni M Zamah 1, Carlos Stocco 2,✉
PMCID: PMC6441017  PMID: 30541132

Abstract

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.

Oocyte-secreted factors synergize with FSH to promote CYP19A1 mRNA and protein expression and estradiol production in primary human granulosa cells via SMAD2/3, SMAD3, and IGF1R signaling.

Infertility is estimated to affect 15% of couples in the United States (1). Its prevalence is increasing in both developed and underdeveloped countries (2, 3). Infertility is commonly associated with poor follicle development and anovulation. Follicle development or folliculogenesis, the primum movens of in vitro fertilization (IVF), involves proliferation and differentiation of granulosa cells (GCs) and the maturation of the oocyte. Optimal development of preovulatory follicles requires FSH as well as local factors such as oocyte-secreted factors (OSFs) and IGFs. How these factors coordinate oocyte maturation with GC differentiation and follicular growth in humans remains unknown.

Under the effect of gonadotropins, primarily FSH, GCs acquire the capacity to produce high levels of estradiol by expressing aromatase (CYP19A1) and to respond to luteinizing hormone by expression of the luteinizing hormone receptor, which is required for ovulation and the formation and maintenance of the corpus luteum. During this process, preantral GCs differentiate into the mural and cumulus GCs. The cumulus cells are in direct contact with the oocyte (4); in fact, the oocyte is an active player in the GC differentiation process and actively suppresses mural-specific transcripts (5). Therefore, the current paradigm is that FSH and the oocyte establish opposing gradients of influence in the antral follicle, where FSH stimulates GC differentiation, whereas the oocyte inhibits FSH actions. However, whether this is the case in humans remains to be determined.

The oocyte participates in this bidirectional communication through OSFs, mainly growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). GDF9 and BMP15 cooperate to regulate GC proliferation and inhibit gonadotropin-induced differentiation in various animal models (6–8). In mice and sheep, GDF9 is essential not only for the stimulation of early follicular growth but also for cumulus expansion, ovulation, and oocyte competency (9–11). Similarly, BMP15 promotes mouse GC proliferation and inhibits FSH-induced progesterone synthesis (12). However, BMP15-knockout female mice exhibit normal folliculogenesis with slightly dysfunctional ovulation resulting only in subfertility and minimal ovarian histopathological defects (13).

Evidence suggests that BMP15 has species-specific functions, being more critical in mono-ovulatory species (sheep and humans) but superfluous in polyovulatory species, such as mice (14–18). For instance, in contrast to rodents, mutations in the BMP15 gene cause ovarian failure in the Inverdale sheep due to impaired follicle growth beyond the primary stage of development (19). More importantly, numerous reports have demonstrated that BMP15 mutations have been found in women with hypergonadotropic ovarian failure, premature ovarian insufficiency, primary or secondary amenorrhea, and polycystic ovary syndrome (PCOS) (20–26). Like BMP15, several studies on different human populations revealed that GDF9 mutations are also involved in diminished ovarian reserve, premature ovarian failure, and PCOS (27–30). Moreover, in patients with normal ovarian function undergoing IVF, BMP15 levels in the follicular fluid correlate positively with estradiol levels, higher fertilization rate, and better embryo development (31). Thus, GDF9 and BMP15 are crucial for follicle growth, oocyte quality, and embryo development in humans.

Our laboratory has validated the use and relevance of cumulus cells obtained from cumulus-oocyte complexes as an experimental approach to study FSH actions in humans (32, 33). This report examines the effect of GDF9 and BMP15 on aromatase expression and estradiol production, two important downstream effects of FSH signaling in GCs. In contrast to prior findings in rodents, our present work demonstrates that OSFs potentiate the expression of genes involved in estradiol production in primary human cumulus cells.

Materials and Methods

Human cumulus cell culture

Human cumulus cells were collected from patients undergoing IVF treatments at the University of Illinois infertility clinic under an Institutional Review Board–exempt protocol. No patient information was collected for reporting. After controlled ovarian stimulation, follicles were aspirated and cumulus oocyte complexes identified. Cumulus cells were separated from the oocyte manually. For each patient, the cumulus cells from all aspirated follicles were pooled, centrifuged at 2000g for 2 minutes, resuspended in phenol red–free DMEM F/12 medium (Sigma-Aldrich), and broken up to produce a single-cell suspension by gentle pipetting. Cells were then cultured on plates precoated with BD Matrigel (BD Biosciences) at a density of 6 × 104/mL in serum-free and phenol red–free DMEM/F12 supplemented with penicillin (50 IU/mL), streptomycin (50 g/mL), sodium bicarbonate (1.2 g/L; Sigma-Aldrich), and BSA (0.25 w/v; Sigma-Aldrich). 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&D Systems), BMP15 (R&D Systems), inhibitors of SMAD2/3 [SB431542 (SB); Tocris Bioscience], SMAD3 [SIS3 (SIS); Cayman Chemical Company], or SMAD1/5/8 [LDN-193189 (LDN); Selleck Chemicals], IGF1R inhibitor NVP-AEW451 (AEW; Calbiochem), and dibutyryl cAMP (dbcAMP; Sigma-Aldrich).

Total RNA isolation and gene expression quantification

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 Moloney murine leukemia virus reverse transcription (Invitrogen) at 37°C for 1 hour. The resulting cDNA was diluted to a final concentration of 10 ng/µL. Quantitative real-time PCR (qPCR) was performed using intron-spanning primers specific for the detection of ribosomal protein L19 (RPL19), CYP19A1, FSH receptor, and IGF2 as previously described (32, 33). All determinations were performed in duplicate, and the number of mRNA copies for each gene was calculated against a standard curve. Primer sequences are available upon request. Target gene expression was adjusted to the expression of RPL19, an internal control, for each sample.

Western blotting

Cultured cumulus cells were harvested in ice-cold radioimmunoprecipitation assay lysis buffer, and whole-cell lysates were used in Western blotting as previously described (32, 34). The primary antibodies used were CYP19A1 (1:1000; Abcam), SMAD3 (1:2000; Lifspan Biosciences, Inc.), phospho-SMAD3 (1:1000; Lifespan Biosciences, Inc.), and β-actin (1:1000; Proteintech) as a loading control. Image Laboratory software (Bio-Rad Laboratories) was used to quantify band intensities, which were adjusted relative to β-actin.

Promoter activity assay

The CYP19-Luc reporter was previously described by our group (35). Lentivirus containing this construct was generated as previously described (34). Empty plasmids were used as controls. Cells were infected with lentivirus and, after overnight incubation, treated with FSH, GDF9, BMP15, or their combination in the presence or absence of SMAD inhibitors LDN, SB, and SIS. At 48 hours, a luciferase activity was determined in 50 μL of lysates as previously described (34).

Statistical analysis

The number of patients used in each experiment is indicated in the corresponding figure legends. Data for continuous variables are presented as mean values ± SEM. Statistical comparisons of mean values between groups were performed with paired t tests, and multiple comparisons were performed with one-way ANOVA with repeated measures followed by Bonferroni adjustment where appropriate. Differences were considered to be statistically significant if the P values were <0.05.

Results

The combination of GDF9 and BMP15 potentiates FSH stimulation of CYP19A1

To determine whether GDF9 and BMP15 modulate the induction of aromatase by FSH, we performed pilot experiments in which human GCs were treated with GDF9 (G), BMP15 (B), or their combination (GB) in the presence or absence of FSH. GDF9 and BMP15 were used at a concentration of 5 ng/mL each, whereas FSH was used at a concentration of 50 ng/mL; these concentrations have been shown to have maximal effects in primary human GCs (32, 35). Primary cumulus cells were cultured in serum-free media in the presence of hormones for 48 hours before quantification of CYP19A1 mRNA transcript abundance. As previously shown, FSH significantly increased CYP19A1 mRNA transcripts when compared with nontreated cells (P < 0.05) (Fig. 1A). This stimulatory effect of FSH on CYP19A1 was significantly augmented by the addition of GB to the media (P < 0.01). In contrast, cotreatment with FSH in the presence of either GDF9 or BMP15 did not result in a statistically significant increase in CYP19A1 mRNA expression when compared with cells treated with FSH alone. Moreover, in the absence of FSH, GB treatment had no effect on CYP19A1 mRNA levels. These results suggest a positive interaction between GDF9 and BMP15 on the stimulation of aromatase by FSH in primary human cumulus cells.

Figure 1.

Figure 1.

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The combination of FSH, GDF9, and BMP15 potentiates FSH-induced Cyp19a1 expression, Cyp19a1 promoter activity, and estradiol production. Primary human cumulus cells were treated for 48 h with vehicle (C), GDF9 (G; 5 ng/mL), BMP15 (B; 5 ng/mL), and GB (5 ng/mL) in the presence or absence of FSH (F; 50 ng/mL). (A) Cyp19a1 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 with repeated measures and Bonferroni (a and b, P < 0.01). Control and FSH groups were compared by t test; *P < 0.05, n = 5. (B) Cyp19a1 expression was examined as in A but using a larger cohort of patients. Columns represent the mean ± SEM; columns with different letters differ significantly by one-way ANOVA with repeated measures and Bonferroni correction (a and b, P < 0.05; a and c, P < 0.0001; n = 24). (C) Cells were infected with lentivirus carrying the CYP19a1-Luc reporter and, after overnight incubation, treated for 48 h with vehicle (C), G, B, or GB in the presence or absence of FSH. Columns represent the mean ± SEM; columns with different letters differ significantly by one-way ANOVA with repeated measures and Bonferroni correction (a and b, P < 0.05; a and c, P < 0.0001; n = 5). (D) CYP19A1 protein levels were evaluated using Western blot. One-way ANOVA with repeated measures and Bonferroni correction indicated a global P value of 0.02. Columns represent the mean ± SEM; columns with different letters differ significantly. One-way paired t test was used to measure the difference between F and F+GB (FGB); *P < 0.05; n = 4. (E) Estradiol levels were quantified using ELISA. Columns represent the mean ± SEM; columns with different letters differ significantly by one-way ANOVA with Bonferroni correction (a and b, P < 0.01; b and c, a and c, P < 0.0001; n = 10).

To further support this conclusion, a larger cohort of patients (n = 24) was used to study the effect of the GB combination on the stimulation of aromatase by FSH. In this larger experiment, the combination of FSH with GDF9 and BMP15 significantly increased CYP19A1 mRNA transcripts by 9.2-fold when compared with nontreated cells (P < 0.0001) and by 3.4-fold compared with cells treated with FSH only (P < 0.0001) (Fig. 1B). Treatment with GB in the absence of FSH had no remarkable effects on CYP19A1 mRNA transcript levels. These findings confirm our previous publication demonstrating that the combination of GDF9 and BMP15 profoundly affects gene expression in primary cumulus cells and shows that these OSFs potentiate FSH actions on CYP19A1 gene expression.

CYP19a1 gene expression in the gonads is mainly controlled by the proximal promoter, which is also known as promoter II or the ovarian promoter (36). Because human GCs are difficult to transfect, we cloned −245 bp of the proximal CYP19a1 promoter into a lentiviral reporter plasmid (CYP19a1ov-LUC). To determine whether FSH plus GB regulated the proximal promoter II, cumulus cells were plated in 48-well plates and immediately infected with the lentivira for 24 hours. At this time, cells were treated with GDF9, BMP15, or GB in the presence of FSH. Luciferase activity was quantified 48 hours after the addition of FSH. FSH treatment alone increased the activity of the CYP19a1ov-LUC construct compared with the control group (P = 0.03) (Fig. 1C). In concordance with the regulation of Cyp19A1 mRNA and protein, FSH plus GB increased the activity of the CYP19a1ov-LUC construct to levels that were significantly higher than those observed with FSH alone or nontreated controls (P = 0.0006). Cotreatment with FSH in the presence of either GDF9 or BMP15 did not result in a statistically significant increase in CYP19a1ov-LUC activity when compared with cells treated with FSH alone. Moreover, in the absence of FSH, GB treatment had no effect on CYP19a1ov-LUC activity. These results suggest that the stimulatory effect of GB on the expression of CYP19a1 mRNA levels correlates with an increase on the activity of the proximal promoter of the CYP19a1 gene.

Next, we examined if the effect of GB on CYP19A1 mRNA levels translates into an increase in aromatase protein expression and estradiol production. In concordance with our findings at the mRNA levels, cotreatment with FSH and GB potentiated the stimulatory effect of FSH on aromatase protein expression (Fig. 1D). Protein quantification revealed that FSH and GB cotreatment increased CYP19A1 protein expression by 10-fold compared with the control group (P < 0.05) and fourfold compared with the group treated with FSH alone (P = 0.027). The changes in estradiol concentration in the media were consistent with the effects observed at the mRNA and protein levels of CYP19A1 (Fig. 1E). FSH stimulated estradiol production when compared with the GB and control groups (P < 0.05), whereas FSH plus GB increased estradiol production significantly when compared with FSH-treated cells. Taken together, these findings indicate a robust positive effect of the GDF9 and BMP15 combination on the stimulatory effect of FSH on estradiol production.

GDF9 and BMP15 potentiation of CYP19A1 expression occurs downstream of the FSH receptor

To further characterize the effect of GB on FSH actions, we first investigated whether the GB combination affected the expression of the FSH receptor. As shown in Fig. 2A, treatment with FSH and GB had no effect on FSH receptor expression. Next, we examined whether treatment with GB potentiates the stimulatory effect of cAMP, which is the primary second messenger activated by the FSH receptor. For this purpose, we treated GCs with dbcAMP, a cell-permeable analog of cAMP. As shown in Fig. 2B, treatment with dbcAMP alone stimulated Cyp19a1 mRNA levels when compared with the control group (P < 0.05). Cyp19a1 mRNA expression was significantly higher in cells treated with GB and dbcAMP when compared with dbcAMP alone (P < 0.05). Thus, as in the case of FSH, the stimulatory effect of dbcAMP was potentiated by the presence of GB in the media and suggest that the cross talk between FSH and GB occurs downstream of the FSH receptor.

Figure 2.

Figure 2.

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The cross talk between FSH and OSFs occurs downstream of the FSH receptor. Primary human cumulus cells were treated for 48 h with vehicle (C) or GB in the presence or absence of FSH (F) or dbcAMP (cAMP; 2 mM). mRNA levels were determined by qPCR and expressed relative to Rpl19. (A) Two-way paired t test showed no difference in Fshr mRNA expression. Columns represent the mean ± SEM; n = 5. (B) Columns represent the mean ± SEM; columns with different letters differ significantly (one-way ANOVA with repeated measures and Bonferroni correction; a and b, P < 0.05; a and c, P < 0.0001; b and c, P < 0.01; n = 5).

GDF9 and BMP15 regulate CYP19A1 via SMAD signaling

It has been shown that GDF9 signals through SMAD2/3, whereas BMP15 activates the SMAD1/5/8 signaling pathway (37–39). To investigate the mechanism used by GB to regulate Cyp19a1 in human GCs, we used inhibitors of SMAD2/3 (SB), SMAD3 (SIS), or SMAD1/5/8 (LDN). The concentration of SMAD inhibitors used was based on previous publicationa in mouse and humans GCs (40, 41). 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 Cyp19A1 mRNA determination. The results show that inhibition of SMAD1/5/8 had no marked effects on the induction of CYP19A1 levels by FSH plus GB (Fig. 3A). However, a strong inhibition was observed in cells treated with SB or SIS (Fig. 3A; P = 0.0002 and P = 0.005, respectively). This inhibition profile of SMAD3 with SIS3 was observed consistently in three additional patients at the CYP19A1 protein level (Fig. 3B). Treatment with SIS resulted in a fourfold decrease in CYP19A1 protein compared with treatment with FSH and GB (P = 0.02). These results suggest that SMAD3 plays a key role in the potentiating effect of GB on estradiol production in human GCs.

Figure 3.

Figure 3.

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GDF9 and BMP15 regulate CYP19A1 via SMAD signaling. Primary human cumulus cells were treated for 48 h with vehicle (C) or GB in the presence or absence of FSH (F), SMAD2/3 inhibitor SB (1 µM), SMAD3 inhibitor SIS (5 µM), or SMAD1/5/8 inhibitor LDN (100 nM). (A) mRNA levels were determined by qPCR and expressed relative to Rpl19. Two-tailed paired t test was used to detect difference in mRNA expression levels between F+GB and F+GB plus each SMAD inhibitor. *P < 0.05; **P < 0.001. Columns represent the mean ± SEM; n = 9. (B) Primary human cumulus cells from three different patients were treated similarly to A and CYP19A1 protein levels evaluated using Western blot. (C) Primary human cumulus cells were treated for 1 h with vehicle (C), F, GB, or F+GB. Western blots for phospho-SMAD3 (pSMAD3) and total SMAD3 (tSMAD3) were performed. A representative blot of four different patients is shown with protein quantification; n = 4. CTL, control.

To further investigate the role of SMAD3 on the regulation of cumulus cells by GB, we performed Western blot analysis for phosphorylated and total SMAD3 in cells treated with FSH and/or GB. As shown in Fig. 3C, SMAD3 is expressed in human cumulus cells but is not phosphorylated in the presence of vehicle (C) or FSH. In contrast, treatment with GB either in the presence or absence of FSH strongly stimulated SMAD3 phosphorylation (Fig. 3C).

Similarly, as shown for CYP19a1 mRNA and protein levels, the stimulation CYP19a1ov-LUC by FSH plus GB was blunted by the addition of the SMAD3 inhibitors SB (P < 0.015) and SIS (P < 0.036) (Fig. 4), suggesting that SMAD3 mediates GB contribution to the activation of the CYP19a1 proximal promoter. The addition of the SMAD1/5/8 inhibitor, LDN, also reduced promoter activity, but this effect was not considerable when compared with cells treated with F+GB without inhibitors.

Figure 4.

Figure 4.

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SMAD signaling mediates the stimulatory effect of GDF9 and BMP15 on the activity of the CYP19a1 ovarian promoter. Cells were infected with lentivirus carrying the CYP19-Luc reporter and, after overnight incubation, treated for 48 h with vehicle (C), FSH, and FSH+GB in the presence of different inhibitors: SMAD2/3 (SB; 1 µM), SMAD3 (SIS; 5 µM), or SMAD1/5/8 (LDN; 100 nM). Luciferase activity was quantified and expressed relative to the control. Columns represent the mean ± SEM. A one-tailed paired t test was used to evaluate the effect of each SMAD inhibitor on the stimulation of aromatase by F+GB. *P < 0.05; n = 4.

The combination of GDF9 and BMP15 potentiates FSH stimulation of IGF2 mRNA

IGF2 is essential for FSH-induced GC differentiation and for the induction of CYP19a1 (32, 42). Consequently, we sought to investigate whether the induction of IGF2 by FSH is also potentiated by FSH and GB cotreatment. To test this idea, we treated GCs with FSH, FSH plus GB, or GB alone and then quantified IGF2 mRNA transcript abundance 48 hours after the initiation of treatment. As expected, FSH stimulated IGF2 expression in primary human cumulus cells (Fig. 5). Like aromatase, the addition of GB to cells treated with FSH caused a significant increase in the expression of IGF2 when compared with cells treated with FSH alone (P < 0.0001). However, GB treatment in the absence of FSH had no effect on IGF2 expression.

Figure 5.

Figure 5.

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The combination of FSH, GDF9, and BMP15 potentiates Igf2 expression. Primary human cumulus cells were treated for 48 h with vehicle (C) or GB in the presence or absence of FSH (F). mRNA levels were determined by qPCR and expressed relative to Rpl19. One-way ANOVA with Bonferroni correction indicated a global P value < 0.0001. Columns represent the mean ± SEM; columns with different letters differ significantly. FSH stimulation of Igf2 mRNA was not significant using ANOVA, but the observed effect was confirmed using a two-way paired t test. **P = 0.01; n = 26.

IGF1R activation plays a major role on the stimulation of CYP19a1 by GDF9 and BMP15

To determine if the strong stimulation of IGF2 expression by GB and FSH plays any role on the stimulation of aromatase, human cumulus GCs were treated with AEW, a highly specific inhibitor of IGF1R activity (43). In previous publications, we have demonstrated that AEW abolished IGF1R activity and prevented AKT phosphorylation by IGF1 and IGF2; however, this inhibitor has no effect on insulin activation of AKT (32, 34, 42).

Granulosa cells were treated with AEW at a concentration of 1 μM (32, 34, 42) for 1 hour before the addition of FSH, GB, or both to the media. CYP19a1 mRNA levels were quantified 48 hours after the addition of FSH. The finding confirmed our previous report demonstrating that treatment with AEW decreases the basal expression of CYP19a1 mRNA to almost undetectable levels and that FSH is not able to stimulate CYP19a1 in the presence of AEW (32, 34, 42). The results also demonstrated that the potentiation of FSH plus GB on CYP19A1 mRNA was significantly decreased by the presence of AEW in the media when compared with cells treated with FSH plus GB alone (P = 0.04) (Fig. 6). However, the combination of FSH plus GB was still able to potentiate the effect of FSH on CYP19A1 mRNA levels in the presence of AEW (P = 0.001, ANOVA).

Figure 6.

Figure 6.

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IGF1R activity contributes to the stimulation of aromatase by FSH and GB. Primary human cumulus cells were treated for 48 h with vehicle (C) and F+GB (FGB) or with vehicle, FSH, F+GB, or GB in the presence of the IGF1R inhibitor AEW (1 μM). mRNA levels were determined by qPCR and expressed relative to Rpl19. One-way ANOVA with repeated measures and Bonferroni correction indicated a global P value of 0.001. Columns represent the mean ± SEM; columns with different letters differ significantly. A one-tailed paired t test revealed that the addition of AEW inhibits GDF9 and BMP15 potentiation of Cyp19a1 mRNA. *P = 0.04; n = 3.

Discussion

Our findings show that the oocyte have the capacity to regulate the expression of aromatase via the secretion GDF9 and BMP15. Only the combination of GDF9 and BMP15 was observed to potently stimulate the effect of FSH on CYP19a1 promoter activity, CYP19A1 mRNA and protein expression, and estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway.

In this study, we used human cumulus GCs collected from cumulus oocyte complexes isolated from women underdoing IVF as an experimental approach to study FSH actions. Our group has characterized this model and demonstrated that cumulus cells, unlike mural cells, do not luteinize and respond to gonadotropins and IGFs similarly to cells from preantral follicles (33). Our group has also used this model to investigate the interactions among FSH, IGFs, and OSFs in the regulation of anti-Müllerian hormone (32, 35, 42). Treatment of cumulus cells with GDF9 or BMP15, with or without FSH, does not lead to substantial changes in CYP19A1 and IGF2 mRNA production. Indeed, we recently showed that only cotreatment with GDF9 and BMP15 is effective in stimulating anti-Müllerian hormone in human primary cumulus cells (35). This strong synergism between GDF9 and BMP15 on GC function has been proposed to be mediated by a GDF9:BMP15 heterodimer (40, 41). However, GDF9:BMP15 heterodimers have not been detected in either the mouse ovary or human follicular fluid (40); therefore, whether these factors form heterodimers in vivo remains to be determined. In fact, a recent report suggested that monomeric forms of BMP15 and GDF9 might interact with their cell-surface receptors to initiate synergistic actions without the need to form heterodimers in solution (44).

Previous studies using bovine GCs showed that GDF9 and BMP15 have synergistic effects on the activation of the Smad signaling pathway and mural GC proliferation (39). Our data show a similar synergistic effect of these OSFs in human cumulus cells, acting through the SMAD2/3 and SMAD3 pathways and not through SMAD1/5/8. In addition, our findings demonstrate that treatment with GB strongly stimulates SMAD3 activity in human cumulus cells. Furthermore, our findings demonstrate that the cross talk between SMAD3 and the FSH-activated signaling occurs downstream of the FSH receptor as the GB combination does not affect the expression of this receptor. Finally, this cross talk leads to the stimulation of the proximal promoter of the CYP19A1 (aromatase) gene, although the molecular mechanisms involved in the control of this promoter by cAMP and SMAD3 remain to be investigated.

IGF2 is essential for FSH-induced GC differentiation and aromatase production (42). When in combination, FSH and IGF2 stimulate aromatase above the levels observed with each factor alone. This effect is prevented by the presence of a highly specific inhibitor of IGF1R activity (AEW), suggesting a key role for the IGF1R. In previous publications, we have demonstrated that AEW abolished IGF1R activity and prevents AKT phosphorylation by IGF1 and IGF2; however, this inhibitor has no effect on AKT activation by insulin (32, 34, 42). We have shown that cotreatment of GCs with FSH plus GB also potentiates the stimulation of IGF2 expression. However, although the addition of the IGF1R inhibitor resulted in a marked decrease in CYP19A1 mRNA levels, FSH plus GB was still able to increase CYP19A1 expression when compared with FSH alone. This suggests that GDF9 and BMP15 stimulate CYP19A1 via other pathways in addition to IGF2 stimulation. The elucidation of these pathways will contribute to a better understanding of the role of OSFs in the regulation of GC differentiation.

Cumulus cells were obtained from patients undergoing IVF. They were not screened or selected for the different etiologies of infertility for these studies. This could imply that the described results are basic intracellular signaling mechanisms that are not altered by the clinical characteristics of the patients. Nevertheless, there is an inherent bias in the methodology: the patients used in these experiments had to have a minimum of 100,000 cumulus GCs. Patients with diminished ovarian reserve or poor responders to controlled ovarian hyperstimulation tend to have fewer GCs and samples representative of these patients could not be used in our analysis. The possibility of overrepresentation of patients with PCOS in our population could affect the results because other groups have found differences in the relative expression of IGF1 in ovarian samples of women with PCOS (45). However, although we cannot discard the possibility that GCs from patients with different etiologies may respond differently to treatments with FSH plus GB, our findings demonstrate, nonetheless, a strong and very reproducible effect of GB treatment across a broad spectrum of patients, suggesting a crucial role for the mechanisms activated by OSFs on the regulation of human GCs.

Previous reports have used highly sensitive mouse GCs isolated from preantral follicles to study the effects of GDF9 and BMP15 (40, 41). Our data show that the combination of GDF9 and BMP15 behaves similarly in primary human cumulus cells recovered from mature follicles, which is highly remarkable and provides further support for the use of cumulus cells to study the molecular mechanism by which OSFs regulate human GCs. Based on these mouse studies, the current paradigm is that FSH and the oocyte establish opposing gradients of influence in the antral follicle where FSH stimulates GC differentiation, whereas the oocyte inhibits FSH actions. Our in vitro findings suggest that in humans, FSH and OSFs can cooperatively regulate critical aspects of human GC physiology, such as estradiol production.

In conclusion, the connection between GDF9 and BMP15 mutations and diminished ovarian reserve, premature ovarian failure, and PCOS points to a considerable pathogenic role for these factors in ovarian dysfunction. However, the biological mechanisms disrupted by GDF9 and BMP15 mutations and how this disruption would be linked to ovarian defects and early menopause remain unexplored in humans. Even in rodents, few genes are known to be regulated by GDF9 and BMP15 in GCs. Thus, our results show a unique and intriguing partnership between OSFs and FSH in the regulation of human GCs, highlighting the role of a healthy oocyte in follicular growth in humans. The evidence we present in this study suggests that only a combined treatment with FSH, GDF9, and BMP15 leads to maximal stimulation of aromatase and estradiol. Our observations also suggest that the combination of GDF9 and BMP15 activate a unique gene expression pattern that is significantly different from the one activated by GDF9 or BMP15 alone. As human oocytes produce both GDF9 and BMP15, our finding may represent the physiological effects of OSFs as compared with experiments using only GDF9 or BMP15. These findings could be used to develop new strategies to improve in vitro maturation of follicles.

Acknowledgments

Financial Support: This work was supported by National Institutes of Health Grant R56HD086054 (to C.S.).

Disclosure Summary: The authors have nothing to disclose.

Glossary

Abbreviations:

AEW

NVP-AEW451

BMP15

bone morphogenetic protein 15

dbcAMP

dibutyryl cAMP

GB

growth differentiation factor 9 and bone morphogenetic protein 15 combined

GC

granulosa cell

GDF9

growth differentiation factor 9

IVF

in vitro fertilization

LDN

LDN-193189

OSF

oocyte-secreted factor

PCOS

polycystic ovary syndrome

qPCR

quantitative real-time PCR

SB

SB431542

SIS

SIS3

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