Fig. 2

Defective Ezh1/2 function leads to failure of DNA break repair and disrupts oocyte meiosis progression in embryonic mouse ovaries. (A) Representative images of γ-H2AX with IF staining in fetal mouse ovaries treated with EZH1/2 inhibitor UNC1999. Red is a positive color. DDX4 is green. The nucleus was stained with DAPI (blue). Scale bar: 50 μm, enlarged scale bar: 10 μm; (C) Quantitative analysis of γ-H2AX fluorescence intensity (A). (B, E, F) Representative images γ-H2AX (red), SCP3 (red), and DAPI (blue) by IF staining in pachytene stage oocytes with chromosome spread. SCP3, the major cytoskeletal protein of the synapsis complex, is continuously expressed throughout premeiotic phase I and is a chromosomal linkage site. Here SCP3 staining is present in the displayed chromosome. Oocytes were isolated from fetal ovaries treated with EZH1/2 inhibitors (UNC1999 (B) and GSK343(E)) or dKO ovaries (F). Scale bar:2 μm; (D, G, H) Quantitative analysis of γ-H2AX foci in B, E, F. (I-L) Representative images of IF staining of MSY2 (a marker of diplotene phase of meiosis) in fetal ovaries with the treatment of EZH1/2 inhibitor (UNC1999 (I) or GSK343 (J)), negative control chemical RLS3 (K), and lentivirus (dKO, L). Green is a positive color. DDX4 staining indicated oocytes. H3K27me3 was stained as red color. The nucleus was stained with DAPI (blue). Scale bar: 50 μm, enlarged scale bar: 10 μm; (M-P) Quantitative analysis of MSY2 fluorescence intensity in I-L. (Q, R) Representative images of SCP1 IF staining in fetal ovaries treated with UNC1999 or dKO ovaries. SCP1 was stained as green color. DDX4 is red. The nucleus was stained with DAPI (blue). Scale bar: 50 μm, enlarged scale bar:10 μm; (S, T) Quantitative analysis of SCP1 fluorescence intensity (Q and R). The number of images assessed in a single experiment was at least 3. The experiments were repeated at least 3 times (N = 3). The data were presented as mean ± S.D. *P < 0.05, **P < 0.01 (t-test)