Figure 4.

Effects of Smad4 cKO on Signaling Pathways Downstream of EGF-Like Factors and PGE2 A, Immunohistochemistry results for PTGS2 expression, which was significantly induced by hCG in GCs of preovulatory follicles, but was blocked in Smad4^gc−/− ovaries. B, In vitro cumulus expansion assay results. The COC expansion defect of Smad4^gc−/− ovaries could not be rescued by exogenous AREG (100 ng/mL) or PGE2 (500 nM). Images were acquired 18–20 hours after culture. C, Quantitative RT-PCR results. SB431542 (10 μM) did not block the PGE2-induced expression of Areg but did prevent the induction of PGE2 downstream genes related to cumulus expansion (Ptgs2, Has2, Tnfaip6, and Ptx3). D, Western blot results. Gonadotropin-induced ERK1/2 phosphorylation was reduced in Smad4^gc−/− ovaries, especially at 2 hours after hCG injection. E, Immunofluorescence results. Smad4 depletion did not affect PGE2-induced phosphorylation of cAMP responsive element binding protein (CREB) in cultured GCs. Primary GCs were isolated from the antral follicles of PMSG-primed WT and Smad4^gc−/− mice and treated with PGE2 (500 nM) for 30 minutes. F, Western blot results. SB431542 blocked EGF-stimulated ERK1/2 phosphorylation in cultured GCs. Primary GCs were treated with EGF (20 ng/mL) or EGF plus SB431542 (10 μM) for 10 minutes. G, Quantitative RT-PCR results. EGF receptor (Egfr) mRNA was markedly down-regulated in GCs of PMSG-primed Smad4^gc−/− ovaries. CREB, CRE-binding protein; DAPI, 4′,6-diamidino-2-phenylindole.