Mouse oocyte-secreted factors and gdf-9 stimulate granulosa cell proliferation via bmpr-ii and activate the smad2/3 pathway

Abstract

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 uncharacterized. This study was conducted to identify the receptor/signalling system utilized 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-morphogenic 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 neutralize 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 antagonize TGF-b and partially antagonized activin-A bioactivity, demonstrating its specificity. The TGFbR-II ECD, ActR-II ECD and ActR-IIB ECD all failed to neutralize oocyte- or GDF-9-stimulated GC DNA synthesis, whereas they did antagonize the activity of their respective ligands. The BMPRII ECD also completely antagonized 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-b (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-b 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-b/activin intracellular pathway. Thus oocytes utilize an unusual interaction of the two TGF-b superfamily receptor/signalling systems that are generally considered distinct.