Extra-ovarian expression and activity of growth differentiation factor 9

GDF-9 produced within the ovary plays essential roles during follicle maturation through actions on granulosa cells, but extra-ovarian expression, signalling and actions of GDF-9 are less well characterized. The present studies confirm GDF-9 expression in the mouse testis, pituitary gland, and adrenocortical cancer (AC) cells, and establish its expression in LbetaT2 gonadotrophs, and in mouse fetal and neonatal, but not adult, adrenal glands. AC, LbetaT2, TM3 Leydig and TM4 Sertoli cells express the requisite GDF-9 binding/signalling components, particularly activin receptor-like kinase (ALK)5 and the bone morphogenetic protein (BMP)/GDF type II receptor, BMPRII. We therefore compared GDF-9 activation of these potential extra-ovarian target cell types with its activation of granulosa cells. Recombinant mouse GDF-9 stimulated expression of activin/TGF-beta-responsive reporters, pGRAS-luc or pAR3-lux, in TM4 and AC cells (IC50=145 ng/ml in the latter case), and two granulosa cell lines, KGN and COV434. The ALK4/5/7 inhibitor, SB431542, blocked GDF-9 activity in each case. In contrast, GDF-9 lacked specific effects on TM3 cells and rat primary pituitary and mouse LbetaT2 gonadotrophs. Our findings show that GDF-9 regulates the expression of R-Smad2/3-responsive reporter genes through ALK4, 5 or 7 in extra-ovarian (adrenocortical and Sertoli) cells with similar potency and signalling pathway to its actions on granulosa cells, but suggest that expression of BMPRII, ALK5 and R-Smads 2 and 3 may not be sufficient for a cell to respond to GDF-9.