
%0 Journal Article
%A Gilchrist, R. B. 
%A Ritter, L. J. 
%A Myllymaa, S. 
%A Kaivo-Oja, N. 
%A Amato, F. 
%A Ritvos, O. 
%A Mottershead, D. G. 
%T 247. Molecular basis of oocyte&#8211;paracrine signalling that promotes mouse granulosa cell proliferation
%J Reproduction, Fertility and Development
%D 2004/08/25
%V 16
%N 9
%P 247-247
%K 
%R https://doi.org/10.1071/SRB04ABS247
%U https://www.publish.csiro.au/paper/SRB04ABS247 
%X Oocytes regulate follicle growth and development by secreting paracrine growth factors that act on granulosa cells (GC). We have recently determined that growth differentiation factor-9 (GDF-9) accounts for ~50% of the total mitogenic activity of oocytes, the remaining portion is as yet uncharacterised. This study was conducted to identify the receptor/signalling system utilised by oocytes to promote GC proliferation. We used an established oocyte-secreted mitogen bioassay, where denuded oocytes are co-cultured with primed-mouse mural GC. In this system, oocytes, GDF-9, TGF-b1 and activin-A all promoted GC DNA synthesis in a dose-dependent manner, but bone-morphogenetic protein-6 (BMP-6) and BMP-7 did not. The type-II receptor for GDF-9 is BMPRII and using real-time RT-PCR, cumulus cells (CC) and mural GC were found to express equivalent levels of BMPRII mRNA. We tested the capacity of the receptor ectodomain (ECD) to neutralise oocyte-stimulated mural GC proliferation. The BMPRII ECD antagonised both oocyte and GDF-9 bioactivity in a dose-dependent manner, completely abolishing activity of both mitogens at 1 ug/mL. The BMPRII ECD did not antagonise TGF-&#946; and partially antagonised activin-A bioactivity, demonstrating its specificity. The TGF&#946;R-II ECD, activin R-II ECD and activin R-IIB ECD all failed to neutralise oocyte- or GDF-9-stimulated GC DNA synthesis, whereas they did antagonise the activity of their respective ligands. The BMPRII ECD also completely antagonised oocyte-stimulated CC DNA synthesis. Using this oocyte-factor bioassay with mural GC transfected with Smad luciferase reporter constructs, we found that oocytes, GDF-9 and TGF-&#946; (but not BMP-6) activated the Smad2/3 pathway. Consistent with this, oocytes and GDF-9 led to phosphorylation of GC Smad2 molecules as detected by Western blot. Conversely the Smad1/5/8 pathway was activated by BMP-6, but not by GDF-9, TGF-&#946; nor surprisingly by oocytes. This study provides evidence that BMPRII is a key receptor for transmitting the paracrine actions of oocytes in GC. However, oocyte-secreted factors do not activate the BMP intracellular signalling pathway but rather the TGF-&#946;/activin intracellular pathway. 
