Centromere damage occurs in PCH. (A) Representative images showing γH2AX staining in oocytes 6 h after NEBD, treated with 40 µM Cen-ASO or 5MM-ASO. Damaged centromeres indicated by red boxes. Scale bar: 5 µm. (B) Percentage of centromeres containing DSBs, as assessed from γH2AX staining data in A (P < 0.0001, χ2 test; three independent experiments). (C) Representative image showing nonaligned bivalents with (red) and without (yellow) damaged centromeres, as assessed by the loss of MajSAT signal in one of the two sister chromatid pairs. Scale bar: 5 µm. (D) Line intensity plots of the MajSAT-mClover (green), tubulin (gray), and chromatin (blue) of bivalents indicated in C. Scale bar: 2 µm. (E) Bivalents with damaged (n = 28) or intact centromeres (n = 138) were analyzed individually and evaluated for three different measurements of alignment (166 bivalents from 10 oocytes). IKT distance, inter-kinetochore distance; deg, degree of bivalent angle. (F–H) The inter-kinetochore distance (F), alignment distance (G), and angle (H) were compared between bivalents with (red) and without (blue) damaged centromeres (****, P < 0.0001, t test). (I) Representative image to show kinetochore and Mad2 immunofluorescence in an oocyte injected with Cen-ASO. The damaged centromeres with Mad2 were shown in red boxes (enlarged version at the bottom). ACA, anticentromere antibody. Scale bars: 5 µm. (J) The percentage of kinetochores containing Mad2 in I, compared between centromeres that were either intact or damaged (****, P < 0.0001, χ2 test). Data from three independent experiments. Error bars indicate SD.
