MI arrest is induced by K-Mt attachment defects. (A) Representative time-lapse images of oocytes expressing H2B-mCherry (gray) and MajSAT-mClover (green; see Video 1). Damaged centromeres were labeled by yellow arrows; time from NEBD. Scale bar: 5 µm. (B) Maturation rates and the mean time for anaphase were measured in A. (C) Evaluation of bivalent alignment levels at 6 h after NEBD. 200 bivalents (10 oocytes) were captured by time-lapse imaging for each group. Green: aligned bivalents; red: non-aligned bivalents. IKT distance, inter-kinetochore distance; deg, degree of bivalent angle. The detailed measurement methods are described in Fig. S2. (D) K-Mt attachments were detected by immunofluorescence after cold treatment. The attached and nonattached kinetochores are indicated by the blue and red boxes. ACA, anticentromere antibody Scale bar: 5 µm (white); 1 µm (yellow). (E) The percentage of nonattached centromeres, from D, was measured (****, P < 0.0001, χ2 test). (F) CENPC localization by immunofluorescence. Scale bar: 5 µm. (G) CENPC intensity, from F, with background subtraction. Data are normalized with respect to the mean intensity of 5MM-ASO (t test). All data from three independent experiments. Error bars indicate SD.
