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

Abstract

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

Figure 1. The combination of GDF9 and BMP15 stimulate Amh mRNA expression in human cumulus cells

Cumulus cells were treated for 48 hours with vehicle (C), GDF9 (2.5, 10ng/ml), BMP15 (2.5, 10ng/ml), or the combination of GDF9 and BMP15 (0.1, 0.6, 2.5, 5, or 10ng/ml of each). Amh mRNA levels were determined by qPCR and expressed as relative to Rpl19. Columns represent the mean ± SEM, Columns with different letters differ significantly a–b P<0.05, a–c P<.001 vs controls, one-way ANOVA, Bonferroni test, n=15.

Figure 2. AMH protein levels in human cumulus cells treated with GDF9 and BMP15

A) Cumulus cells from three different patients (Patient 1, 2, and 3) were treated with 5 ng/ml of both GDF9 and BMP15 (GB) for 48 hours. AMH β-actin (ACTB) protein levels were determined by Western blotting. On the left, the average (± SEM) of the relative optical density units of AMH to ACTB is shown (**P< 0.001, t-test, n=3). B) Cumulus cells from Patient 4 were treated with 2.5, 5, or 20 ng/ml of both GDF9 and BMP15. AMH protein levels were determined as in A. Expression of ACTB is shown as a loading control.

Figure 3. FSH inhibits the stimulation of GDF9 and BMP15 on Amh mRNA expression

Cumulus cells were treated for 48 hours with GDF9 and BMP15 (G+B; 5 ng/ml of each) in the presence or absence of FSH (50 ng/ml). Amh mRNA levels were determined by qPCR and expressed relative to Rpl19. Columns with different letters differ significantly a–b P<0.001, a–c P<0.05, b–c P<0.01, one-way ANOVA, Bonferroni test, n=11.

Figure 4. Effect of cell signaling inhibition on GDF9 and BMP15 induced AmhmRNA levels

Cumulus cells were treated for 1 hour with the following specific SB431542 (SMAD2/3; 10 μM), or LDN-193189 (SMAD1/5/8; 100 nM) followed by a 48-hour co-treatment with GDF9 and BMP15 (5 ng/ml of each). Amh mRNA levels were determined 48 h after by qPCR and expressed relative to Rpl19. Columns with different letters differ significantly a–b P<0.001, one-way ANOVA, Bonferroni test, n=9.