Fig. 6

Nuclear translocation and transcriptional activation of β-catenin were detrimental to fetal oocytes. a qRT-PCR analysis showed that mRNA levels of target genes of β-catenin (normalized to β-actin) significantly decreased following combined (BIO+ICG-001) treatment compared with single (BIO) treatment (Additional file 8: Individual data values). b–d Nuclear translocation and transcriptional activation of β-catenin resulted in premature upregulation of TAp63 in the fetal ovary. Before the examination, ovaries at 14.5 dpc were cultured in vitro with DMSO or single or combined inhibitors for 2 days. b qRT-PCR analysis of mRNA levels of TAp63 (normalized to β-actin) (Additional file 8: Individual data values). c Western blotting analysis of TAp63 protein levels. GAPDH was used as an internal control. d Immunofluorescence staining for TAp63 (green) and DDX4 (red). The nucleus was stained by Hoechst (blue). e Blockage of β-catenin transcriptional activity rescued DSB repair deficiency in oocytes following GSK-3β inhibition. Before the examination, ovaries at 14.5 dpc were cultured in vitro with DMSO or single or combined inhibitors for 3 days. Statistical analysis showed that the percentage of oocytes with persistent γ-H2AX signals in the pachytene stage on chromosomes per slide increased significantly following GSK-3β inhibition but reduced significantly following combined treatment (Additional file 8: Individual data values). f, g Blockage of β-catenin transcriptional activity rescued fetal oocyte loss following GSK-3β inhibition. Before the examination, ovaries at 14.5 dpc were cultured in vitro with DMSO or single or combined inhibitors for 4 days. f Oocytes were stained with DDX4 (green). The nucleus was stained by Hoechst (blue). g Statistical analysis showed that the total number of oocytes decreased significantly following a single treatment but could be rescued efficiently following combined treatment (Additional file 8: Individual data values). h, i Blockage of β-catenin transcriptional activity alleviated oocyte apoptosis following GSK-3β inhibition. Before the examination, ovaries at 14.5 dpc were cultured in vitro with DMSO single or combined inhibitors for 3 days. h Active Caspase-3 signals (green) corresponded to apoptotic cells. Oocytes were stained with DDX4 (red). The nucleus was stained by Hoechst (blue). i Statistical analysis showed that the number of apoptotic oocytes per section increased significantly following single treatment but decreased following combined treatment (Additional file 8: Individual data values). The data are presented as means ± s.d. Different letters (a–c) denote a statistically significant difference between the groups (ANOVA and Holm-Sidak test). Scale bars, 200 μm