Abstract
This study was conducted to characterize the receptor/signalling system utilized by the oocyte-secreted growth differentiation factor 9 (GDF9) to promote granulosa cell (GC) growth in the marmoset monkey. Seven adult female marmosets were primed with hFSH for 6 days, whole ovaries were removed on day 7, follicles manually excised, and GC collected from 3 size classes: periantral (PA; 0.42–0.66mm), small antral (SA; 0.66–1.5mm) and large antral (LA; >1.5mm). RNA was extracted from oocytes and GC and subjected to RT-PCR using human primers. In all follicle size classes oocytes expressed GDF9 mRNA and GC expressed mRNA for key GDF9 signalling molecules; bone morphogenetic protein receptor II, activin receptor-like kinase (ALK) 5 and Smad 3. To examine the intracellular response to GDF9, cultured GC from LA follicles were transfected with luciferase reporter constructs and treated with growth factors. CAGA-luciferase (Smad 3 pathway) in transfected GC was stimulated by TGFβ1 (18× above control), GDF9 and mouse oocytes (both ~5×), but not by BMP7. Conversely, neither TGFβ1, GDF9 nor oocytes activated BRE-luciferase (Smad 1/5/8 pathway), which was stimulated 30× by BMP7. 3H-thymidine incorporation was used to determine the effects of GDF9 on GC proliferation. Basal incorporation was highly dependent on follicle size, with PA follicles ~10× higher than SA and ~30× higher than LA follicles. GDF9 stimulated 3H-uptake in GC from all sized follicles, most potently in PA and SA cells. The mitogenic effect of GDF9 was amplified by IGF1; ~3× in SA GC and ~5× in LA GC. In contrast, in the presence of FSH or FSH+IGF1, GDF9 did not stimulate GC proliferation. Treatment of GC with an ALK4/5/7 kinase inhibitor, SB431542, antagonized both GDF9 and GDF9+IGF1 mitogenic effects, in a dose-dependent manner. Thus, GDF9 potently stimulates primate GC proliferation utilizing components of the TGFβ signalling system initially identified in rodents.
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