Extra-ovarian expression and activity of growth differentiation factor 9

Abstract

Growth differentiation factor 9 (GDF9) produced within the ovary plays an essential role during follicle maturation through actions on granulosa cells, but extra-ovarian expression, signalling and actions of GDF9 are less well characterised. The present studies confirm GDF9 expression in the mouse testis, pituitary gland and adrenocortical cancer (AC) cells, and establish its expression in LbT2 gonadotrophs, and in mouse adrenal glands, particularly foetal and neonatal cortical cells. AC, LbT2, TM3 Leydig and TM4 Sertoli cells express the requisite GDF9 binding signalling components, particularly activin receptor-like kinase (ALK) 5 and the bone morphogenetic protein (BMP)/GDF type II receptor, BMPRII (BMPR2). We therefore compared GDF9 activation of these potential extra-ovarian target cell types with its activation of granulosa cells. Recombinant mouse GDF9 stimulated expression of activin/transforming growth factorb-responsive reporters, pGRAS-luc or pAR3-lux, in TM4 and AC cells (IC50Z145 ng/ml in the latter case), and two granulosa cell lines, KGN and COV434. The ALK4/5/7 inhibitor, SB431542, blocked GDF9 activity in each case. By contrast, GDF9 lacked specific effects on TM3 cells and rat primary pituitary and mouse LbT2 gonadotrophs. Our findings show that GDF9 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 (TGFBR1) and R-SMADs 2 and 3 may not be sufficient for a cell to respond to GDF9.

Growth Differentiation Factor 9 (GDF9) Forms an Incoherent Feed-forward Loop Modulating Follicle-stimulating Hormone β-Subunit (FSHβ) Gene Expression

“…According to that knock-out model, ovarian GDF9 has a major influence on both LH and FSH serum levels, which does not preclude a distinct autocrine/ paracrine role of GDF9 in the pituitary. Overall, whereas the role of GDF9 in the ovary is clearly established, our work and the previous detection of GDF9 expression in rat primary pituitary cultures and L␤T2 gonadotropes (15) and in the human (41) (GEO accession GDS3834 (42)), (GEO accession GDS1096 (43)), and bovine pituitary (44) support the view that GDF9 has an important functional role in the pituitary.…”

“…The identification of novel secreted FSH␤ regulators might help elucidate the fundamental mechanisms underlying frequency sensitivity. Growth differentiation factor 9 (GDF9), a member of the transforming growth factor ␤ (TGF␤) superfamily that is expressed in primary pituitary cultures and gonadotrope cell lines (15), was one candidate selected for study. We found that GDF9 is a strong autocrine inducer of FSH␤ that is suppressed by GnRH and forms a network motif contributing to the GnRH frequency sensitivity of FSH␤ regulation.…”

Activation of Latent Human GDF9 by a Single Residue Change (Gly391Arg) in the Mature Domain

“…To confirm this, we assessed the ability of conditioned medium from mGDF9 or hGDF9 expressing cells to activate a phosphorylated mothers against decapentaplegic (Smad)-2/3-responsive luciferase reporter (pGRAS) in a murine adrenocortical cell line (18). Treatment of cells with mGDF9 resulted in a dose-dependent increase in luciferase activity (EC 50 28ng/ml) (Fig.…”

“…Wild-type and mutant hGDF9 were compared with mGDF9 for their ability to stimulate a luciferase response in a mouse adrenocortical cell line (AC) (18) or in a human granulosa cell line (KGN). Briefly, AC or KGN cells were plated in 48-well plates at 114,000 cells/well or 40,000 cells/well, respectively.…”

“…We also acknowledge the Victorian Government's Operational Infrastructure Support Program and the Montgomery Trust (Prince Henry's Institute data audit no. [11][12][13][14][15][16][17][18]. …”