Effects and mechanisms of growth differentiation factor 9 on activin A-regulated inhibin B and progesterone production in human granulosa cells

Activin A (homodimer of inhibin βA-subunit) is known to increase inhibin βB-subunit and inhibin B (heterodimer of inhibin α- and βB-subunit) levels and decrease progesterone accumulation in human granulosa cells. Growth differentiation factor 9 (GDF9) is a potent paracrine regulator of ovarian function, but its overall effects, particularly relating to activin A actions, are unknown. We examined the potential crosstalk between activin A and GDF9 in primary cultures of human granulosa-lutein (hGL) cells. Pretreatment of hGL cells with GDF9 for 24 h resulted in an increased expression of activin receptors and Smad2/3, and decreased inhibitory Smad7 activity. These effects were attenuated by BMP type II receptor ectodomain (BMPR2 ECD), a GDF9 antagonist. These GDF9-induced changes, in turn, increased the cellular response to activin A stimulation and resulted in significantly greater production of βB-subunit mRNA and inhibin B compared to activin A treatment alone. Interestingly, endogenous GDF9 mRNA and protein were detected in hGL cells. Reduction of endogenous GDF9 by GDF9 siRNA resulted in decreased levels of activin receptors and Smad2/3/4, but increased expression of Smad7. Consequently, GDF9 siRNA treatment significantly attenuated the stimulation of activin A on βB-subunit mRNA and inhibin B levels. Additionally, GDF9 suppressed the expression of follistatin (FST) and follistatin-like 3 (FSTL3), which are extracellular inhibitors of activin A. These effects were attenuated by BMPR2 ECD and GDF9 siRNA. Treatment with FST or FSTL3 siRNA augmented activin A-induced βB-subunit mRNA levels. Conversely, GDF9 enhancement of activin A-induced βB-subunit mRNA was attenuated by FST. Activin A decreased expression of StAR but not P450scc and 3βHSD, this effect lead to reduced basal and FSH-induced progesterone accumulation. GDF9 reversed these effects of activin A on StAR and progesterone; these GDF9 effects were attenuated by inhibin α-subunit siRNA. Together, these findings support a novel hypothesis that GDF9 exerts both paracrine and autocrine control of key components in the activin receptor-signaling pathway and the extracellular inhibition of activin A in hGL cells. As a result, GDF9 may serve to enhance activin A-induced accumulation of inhibin B, which in turn acts to reverse activin A suppression of progesterone accumulation during granulosa cell luteinization.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat