Fig. 1

Inhibition of enzyme activation or gene knockout of Ezh1/2impairs the oocyte development in fetal mouse ovaries. (A) Protein levels of EZH1, EZH2, and H3K27me3 in whole fetal ovaries cultured with Ezh1/2 inhibitor UNC1999 checked by western blotting (WB). DMSO is a negative control of the inhibitor treatment (vehicle group). β-actin was used as an internal control. (B, C) Representative images of immunofluorescence (IF) staining of DDX4 and H3K27me3 in fetal ovaries treated with EZH1/2 inhibitor UNC1999 or GSK343 (green color). DDX4 (red color) is an oocyte marker. The nucleus was stained with DAPI (blue). Scale bar:50 μm; (D) Protein levels of EZH1, EZH2, and H3K27me3 in wildtype (WT) or Ezh1/2 double knockout (dKO) embryonic ovaries checked by WB. β-actin was used as an internal control. (E) The levels of H3K27me3 and DDX4 stained by IF in fetal mouse ovaries after knockout Ezh1/2. The red color indicates H3K27me3 and the green color is DDX4. The nucleus was stained with DAPI (blue). Scale bar: 50 μm; (F-K) Quantification of H3K27me3 fluorescence intensity or DDX4 positive cells in B, C, and E. (L-N) TUNEL assays on fetal ovaries with treatment of EZH1/2 inhibitors or dKO ovaries. Green is a positive color. The nucleus was stained with DAPI (blue). Scale bar: 50 μm; (O-R) Quantitative analysis of TUNEL positive cells in L-N. (R, S) The protein level of active caspase3 and caspase3 checked by WB in fetal ovaries treated with UNC1999 or deleted Ezh1/2 gene. GAPDH was used as an internal control. Vehicle: an equivalent volume of DMSO. WT: wild type; dKO: double knockout of both Ezh1 and Ezh2 in germ cells. Each group had 20 ovaries. 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. The asterisk (*) denotes a statistically significant difference between the vehicle (or WT) and treatment groups. NS: no significant, *P < 0.05, **P < 0.01 (t-test)