Anti-Mullerian Hormone (AMH) Knockdown Increases Steroidogenesis in Ovine Granulosa Cells from Small Antral Follicles In Vitro.

In most species AMH is highly expressed in the granulosa cells (GCs) of gonadotropin-responsive preantral and small antral follicles and is widely regarded as an inhibitor of FSH-induced somatic cell differentiation. Recent data from our laboratory has indicated, however, that the differentiative response of ovine GC to supplementation with rhAMH, is bimodal, being stimulatory at low doses and inhibitory at high doses. The aim of this study was to evaluate the effect of knocking down endogenous AMH-bioactivity on FSH-induced GC differentiation utilizing a specific monoclonal antibody raised against the N-terminal region of ovine AMH (mAb) or by using short interfering RNA sequences (siRNA) to block AMH gene expression. Follicles <3mm in diameter were dissected from ovaries obtained at a local abattoir with GCs collected and seeded at appropriate cell density. For mAb experiments, GCs were incubated in GC serum-free media without (control) or with decreasing concentrations (1:2 to 1:2048 dilution) of mAb and media in which mAb were isolated (CM). For siRNA experiments, GCs were incubated in serum-free media (with or without FSH) before addition of siRNA (siRNA+FSH and siRNA-FSH) or media (NT+FSH and NT-FSH). Spent media from culture plates was collected every 24h (siRNA) or 48h (mAb) and stored at -20°C for estradiol (E2) and progesterone (P4) determination. Data was log-transformed and analyzed using ANOVA with experiment replicate (n=3) as blocking variable. Knockdown of AMH with mAb increased E2 (P<0.01) and decreased P4 (P<0.001) production compared to CM at higher concentrations of mAb (1:2 to 1:64 dilutions) between 96h and 192h of culture. At these concentrations, mAb cells showed higher (P<0.001) E2 and P4 production than control cells. Moreover, AMH RNA knockdown increased E2 production (P<0.01) at 72 hours post-transfection in siRNA with or without FSH supplementation compared to NT. In conclusion, knockdown of endogenous AMH bioactivity in vitro augmented FSH-induced aromatase activity in ovine GCs, thus supporting an inhibitory role for AMH in the modulation of gonadotropin-induced somatic cell differentiation.