Fig. 4

Determination of molecular mechanism in male determination and generation of eSCs. a A schematic diagram of derivation of eSCs in 10.5–12.5 dpc. b Growth curves of different groups in 12–20 days. Results were expressed as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of difference in the mean cell number calculated by one-way ANOVA method. (*P value < 0.05, **P value < 0.01, ***P value < 0.001). c Optical and IF micrographs were taken in groups (W.G) and (W.G.S1) at 14 days. DAPI staining showed blue in cell nucleus. WNT4⁺ cells showed red. SOX9⁺ cells showed green. Scale bar = 200 μm. d Optical and IF micrographs were taken in groups (W.G.S1) and (W.G.S1.S9) at 14 days. DAPI staining showed blue in cell nucleus. FGFR2⁺ cells showed green. Scale bar = 20 μm. e AMH⁺ cells were determined via FCM in different groups at 14 days. The control peak took the result of isotype antibody showing dark color. The positive result refers to the control result. f The transcriptional levels of Sry were determined in different groups. Results were expressed relative to the highest mean value as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of differences in the mean gene expression calculated by one-way ANOVA method. (*P value < 0.05, **P value < 0.01, ***P value < 0.001). g The transcriptional levels of Sf1 were determined in different groups. Results were expressed relative to the result of control group (group (Induced IM)) at 14 days as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of differences in the mean gene expression calculated by one-way ANOVA method. (*P value < 0.05, **P value < 0.01, ***P value < 0.001). h The transcriptional levels when Sox9 was overexpressed were determined by qPCR at 14 days. Results were expressed related to the mean value of control group (group (Induced IM)) as mean ± SD (n = 3 independent experiments). Nucleotide band of relevant genes was detected. The band of control group was on the left. The band of test group was on the right. i The transcriptional levels of Wnt4 were determined in different groups. Results were expressed relative to the highest mean value as mean ± SD (n = 3 independent experiments). Asterisks indicate statistical significance of differences in the mean gene expression calculated by one-way ANOVA method. (*P value < 0.05, **P value < 0.01, ***P value < 0.001)