Fig. 4

MEK1/2 signalling inhibition permitted STRA8 expression but failed to effectively induce meiosis in XY germ cells. Analysis of XY or XX E12.5 gonad-mesonephros tissue cultured with DMSO or 500 nM FGFRi or MEKi for 72 h (A–C) or 96 h (D–F). A Immunofluorescent images demonstrating STRA8 localisation. Top panel: DAPI (blue), MVH (green), STRA8 (red), SMA (cyan). Bottom panel: STRA8 (grey). B Percentage STRA8 positive germ cells determined by flow cytometry. C,D Immunofluorescent images demonstrating SYCP3 (C) and phospho-γH2AX (p-γH2AX) localisation. Left panel: DAPI (blue), MVH (green), SYCP3 (red; C) or SMA (red; D) and p-γH2AX (cyan). Middle panel: SCP3 (grey; C). Right panel: p-γH2AX (grey). E Percentage p-γH2AX-positive germ cells determined by flow cytometry. F Flow cytometric cell cycle analysis of G0/G1, S-phase and G2/M based on the incorporation of EdU (S-phase) and propidium iodide (DNA content). A, C, D scale bar: 50 μm. Replicates: A, C, D n = 3–4, B n = 8, E,F n = 8–10. Statistics: B Ordinary one-way ANOVA with Tukey’s multiple comparison, E Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons, F Repeated measures two-way ANOVA with Tukey’s multiple comparisons. Error bars: mean ± SEM. Significance between controls and treatment: *P < 0.05, **P < 0.01, ****P < 0.0001