Figure 2.

Effects of Smad4 cKO on Follicle Growth, Follicle Atresia, and FSH Target Genes Expression. A, Histology of WT and Smad4^gc−/− ovaries at PD23, with or without PMSG treatment. B, Immunohistochemistry results for pHistone H3 (Ser10) indicating GC proliferation in WT and Smad4^gc−/− ovaries, with or without PMSG treatment. C and D, Immunohistochemistry results for cleaved caspase 3 (C) and TUNEL assay (D) show an increase in GC apoptosis and follicle atresia in Smad4^gc−/− ovaries. E and F, Quantification results of phospho-histone H3 (pHH3) positive cells (E) and TUNEL-positive atretic follicles (F) in WT and Smad4^gc−/− ovaries at PD23 with PMSG treatment (44 h). G, Serum estrogen (E2) and P4 concentrations of WT and Smad4^gc−/− mice before and after PMSG injections. H, Quantitative RT-PCR results using GCs isolated from WT and Smad4^gc−/− ovaries (PMSG 44 h and PMSG 44 h+hCG 4 h) showing the expressions of the FSH target genes Cyp11a1, Fshr, and Lhcgr. I, Quantitative RT-PCR results for the effect of Smad4 depletion on Nppc/Npr2 expression. In WT ovaries before hCG treatment, Nppc was highly expressed in mural GCs and Npr2 was highly expressed in cumulus cells. After hCG treatment, Nppc and Npr2 mRNA levels were down-regulated in mural GCs and cumulus cells, respectively. However, this intricate regulation of Nppc/Npr2 expression was abolished in Smad4^gc−/− ovaries. J, Hematoxylin and eosin staining results showing precocious oocyte meiotic resumption in Smad4^gc−/− ovaries. K, Immunohistochemistry results for 3β-HSD expression patterns. This enzyme was predominantly expressed by mural GCs of antral follicles in WT ovaries, but was expressed in all GCs and cumulus cells in Smad4^gc−/− ovaries.