Effects of Endogenous Growth Differentiation Factor 9 on Activin A-Induced Inhibin B Production in Human Granulosa-Lutein Cells

Background We recently reported on the effects of exogenous growth differentiation factor 9 (GDF9) in enhancing activin A-induced inhibin βB-subunit mRNA and inhibin B levels in human granulosa-lutein (hGL) cells by modulating key components of the activin signaling pathway. We undertook the following study to characterize the role of endogenous GDF9 in this regard. Methods We compared inhibin subunit (α, βA, and βB) mRNA and inhibin B levels and activation of activin receptors (ACVRs) and Smad signaling pathway in hGL cells obtained from women undergoing in vitro fertilization and cultured with and without activin A treatment after GDF9-targeting small interfering RNA transfection. GDF9, inhibin subunits, ACVR2B/1B and Smad2/3/4/7 mRNA and/or protein levels, Smad phosphorylation, and inhibin B were assessed with RT-PCR, immunoblotting, and ELISA. Data were analyzed by ANOVA followed by Tukey’s test. Results GDF9 was detected as mRNA and protein in hGL cells and protein in follicular fluid from all 11 patients tested. Reduced endogenous GDF9 expression after targeting small interfering RNA transfection was associated with decreased ACVR2B/1B and Smad2/3/4 but increased inhibitory Smad7 mRNA and protein levels and, consequently, reduced activin A-induced βB-subunit mRNA and inhibin B levels. Conclusions We report here for the first time autocrine roles for endogenous GDF9 in hGL cells in enhancing activin A-induced βB-subunit mRNA and inhibin B levels via key components of the activin signaling pathway. However, the relative contributions of GDF9 in granulosa cells vs. oocyte as autocrine/paracrine regulators of βB-subunit and inhibin B production in normal and abnormal human ovarian functions remain to be determined.