Oocyte/GDF9 Regulation of Granulosa/Cumulus Cell Functions Requires EGF Receptor/MAPK3/1 Signaling.

Oocyte-secreted growth differentiation factor 9 (GDF9) plays a critical role, in collaboration with FSH/EGF, during folliculogenesis through to ovulation. The molecular mechanism behind this dual requirement however remains unclear. One hypothesis on the relationship between these two pathways is that GDF9-activation of Smad2/3 enables FSH to exert its effects through MAPK signaling. Alternatively, this study investigated the need for MAPK3/1 signaling to allow oocyte/GDF9 to regulate key granulosa/cumulus cell functions. In the absence of FSH or EGF, 3H-thymidine incorporation and upregulation of matrix genes (Ptx3 and Has2) in granulosa cells stimulated by oocyte coculture or recombinant mouse GDF9 was, as expected, inhibited by SB431542 (5 µM), which prevents Smad2/3 activation. Surprisingly, this oocyte/GDF9 effect was inhibited as well by the EGF receptor inhibitor AG1478 (5 µM), despite the absence of FSH and EGF. This suggests that a minimal "basal" level of EGF receptor activity is required for oocyte/GDF9 bioactivity in granulosa cells. In addition to inhibition by SB431542 and AG1478, the oocyte- and GDF9-stimulated 3H-thymidine incorporation was inhibited by the MEK1 inhibitor U0126 (10 µM) and the Src inhibitor PP2 (10 µM), while the bone morphogenetic protein (BMP) type 1 receptor Alk2/3/6 inhibitor dorsomorphin (10 μM) was without effect. GDF9-stimulated 3H-thymidine incorporation was not affected by the NF-κB inhibitor SN50 (10 µg/ml) or the matrix metalloproteinase inhibitor GM6001 (10 µM), excluding the NF-κB pathway or shedding of membrane-bound EGF ligands as participating in GDF9 bioactivity. Interestingly, activin A (10 ng/ml)- and TGFβ1 (2 ng/ml)-stimulated 3H-thymidine incorporation had similar inhibitor sensitivity. When treated as cumulus-oocyte complexes, cumulus cell 3H-thymidine incorporation was also inhibited by the Alk4/5/7, EGF receptor and Src inhibitors. These results suggest that growth factors signaling through the Smad2/3 pathway not only need an active type 1 receptor, but also share a common requirement of EGF receptor-dependant and Src-dependant MAPK3/1 phosphorylation. Western blot analysis showed that granulosa cells have a basal (ligand-independant) level of MAPK3/1 phosphorylation that can be inhibited by using an EGF receptor or a Src inhibitor and increased by treating with EGF. EGF-stimulated MAPK3/1 phosphorylation was not affected by inhibition of Alk4/5/7 or by adding exogenous recombinant GDF9. Using a construct possessing a Smad3-activated promoter coupled with the luciferase gene transfected into mouse granulosa cells, GDF9-stimulated promoter activation was inhibited by Alk4/5/7, EGF receptor, MEK1 and Src inhibitors. Conversely, GDF9-stimulated Smad3-promoter activity was increased in a dose-dependent manner by EGF. These experiments suggest that MAPK3/1 activation amplifies Smad2/3-mediated growth factor effects on granulosa/cumulus cells, while MAPK3/1 inhibition abolishes their bioactivity. These results lead us to hypothesize that, Src/EGFR-dependant MAPK3/1 phosphorylation is required for oocyte/GDF9-stimulated functions of granulosa and cumulus cells. Hence, apart from its role in the transmission of the ovulatory LH signal, EGF-like molecules might serve as a critical modulators of GDF9 action on granulosa/cumulus cells. This research was supported by the NHMRC of Australia to RBG and DLR and a postdoctoral fellowship from NSERC of Canada to MS. (platform)

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