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morphogenetic protein 15 (BMP15) on AMH production in primary human cumulus cells. DESIGN: Prospective in vitro studies using human primary cumulus granulosa cell cultures. MATERIALS AND METHODS: Follicular aspirates of women undergoing in vitro fertilization (IVF) at a University IVF Center were used. Cumulus cells were mechanically separated from the oocyte, seeded on 24-well culture dishes pre-coated with extracellular matrix at a density of 3x10 4 cells/well and cultured for 24 hours before treatment. Cells were treated with a combination of GDF9, BMP15, recombinant FSH, and specific signaling inhibitors for 48 hours. Cells were harvested for protein and RNA isolation. AMH mRNA and protein levels were quantified by real-time PCR and Western blot, respectively. RESULTS: 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 and protein expression. Increasing concentration of G+B (0.6, 2.5, 5 and 10 ng/ml each) stimulated AMH in a dose-dependent manner showing 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 tended to decrease the stimulation of AMH expression by G+B but without reaching statistical significance. Treatment with G+B had a tendency to increase the activity of a reporter construct carrying a 2kb section of the AMH promoter. Finally, the effect of G+B on the expression of AMH was significantly decreased by inhibitors of the SMAD2/ 3 signaling pathway. CONCLUSIONS: These findings show, for the first time, that AMH production is regulated by oocyte-secreted growth factors in primary human granulosa cells. We demonstrated that only the combination of GDF9 and BMP15 potently stimulates AMH production supporting the idea that these factors form heterodimers. Inhibition of SMAD signaling blocks the stimulatory effect of G+B, suggesting that these heterodimers are likely to activate the BMPR2-ALK4-ALK6 receptor complex.

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