Fig. 4
Increased apoptosis and mitochondrial calcium overload in the GCs of 6-week-old Amh-cre; Foxj2tg/tg mice. A A Volcano diagram depicting differentially expressed genes. Red dots represent 2162 upregulated genes; green dots represent 2142 downregulated genes; blue dots represent 22,761 genes with no significant differences in expression. B, C Gene set enrichment analysis (GSEA) -based GO enrichment plots for representative gene sets: intrinsic apoptotic signaling pathway (B) and mitochondrial calcium ion homeostasis (C). D, E Transmission electron microscopy (TEM) images of ovaries from 6-week-old control mice and Amh-cre; Foxj2tg/tg mice. Abbreviations: O - oocyte; ZP - zona pellucida; M - mitochondria. Red dashed lines delineate the borders of the ZP. Red arrows indicate transport vesicles within the ZP. Yellow arrows point to the microvilli of granulosa cells (GCs) in the ZP. Scale bar: 5 μm (top panel of D), 2 μm (bottom panel of D and top panel of E), 500 nm (bottom panel of E). F, G Representative immunohistochemical staining (IHC) images (F) and statistical results (G) of Cl-caspase3 protein levels in ovaries of Amh-cre; Foxj2tg/tg and control mice. Data are presented as the mean ± SEM (n = 6). **P < 0.01. H, I Representative western blot images (H) and statistical results (I) of Cl-caspase3, bcl-2, bax, γH2AX protein levels in isolated ovarian granulosa cells from Amh-cre; Foxj2tg/tg and control mice. Data are presented as the mean ± SEM (n = 4). *P < 0.05, **P < 0.01. J, K, L Quantitative analysis of mean fluorescence intensity of Rhod-2AM (representing mitochondrial Ca2+ levels) (J), Rhod-5 N AM (representing of endoplasmic reticulum Ca2+ levels) (K), Cal-590 AM (representing cytoplasmic Ca2+ levels) (L) in GCs from Amh-cre; Foxj2tg/tg and control mice. Data are presented as the mean ± SEM (n = 3). *P < 0.05, ****P < 0.0001