Fig. 3

Inhibition of GSK-3β impeded meiotic progression and caused DSB repair deficiency in fetal mouse ovaries. a, b Oocytes at the diplotene stage were reduced following GSK-3β inhibition. Before the examination, ovaries at 14.5 dpc were cultured in vitro with DMSO or BIO for 4 days. a MSY2 (red) marked oocytes entering the diplotene stage, and DDX4 (green) marked germ cells. The nucleus was stained by Hoechst (blue). Oocytes lacking MSY2 staining are indicated by the dashed line. Scale bars, 200 μm. b Statistical analysis showed that the percentage of MSY2⁺ oocytes (number of cells both MSY2⁺ and DDX4⁺/number of cells DDX4⁺) per section decreased significantly following GSK-3β inhibition (Additional file 8: Individual data values). c, d Inhibition of GSK-3β caused meiotic DSB repair deficiency. Before the examination, ovaries at 14.5 dpc were cultured in vitro with DMSO or BIO for 3 days. c Representative images of the meiotic spread of the pachytene-stage oocytes with repaired or unrepaired DSBs. γ-H2AX (green) indicates unrepaired DSB sites. SYCP3 (red) demonstrates lateral elements. Scale bars, 10 μm. d Statistical analysis showed that the percentage of oocytes with unrepaired DSBs in the pachytene stage on chromosomes per slide increased significantly following GSK-3β inhibition (Additional file 8: Individual data values). e, f Inhibition of GSK-3β resulted in ectopic RAD51 expression. Before the examination, ovaries at 14.5 dpc were cultured in vitro with DMSO or BIO for 3 days. e Representative images of the meiotic spread of the pachytene-stage oocytes with normal or ectopic RAD51 foci. Oocyte chromosomes were co-stained with RAD51 (green) and SYCP3 (red). Scale bars, 10 μm. f Statistical analysis showed that the percentage of oocytes with ectopic RAD51 foci on chromosomes in the pachytene stage per slide increased significantly following GSK-3β inhibition (Additional file 8: Individual data values). The data are presented as mean ± s.d. The asterisk (*) denotes a statistically significant difference between the control and treatment groups. *P < 0.05, **P < 0.01, and ***P < 0.001 (t test)