Fig 2: F-actin loss exacerbates reproductive age-related premature chromatid separation in mammalian eggs.(A) Unpaired chromatid identification pipeline from maximum intensity projected immunofluorescence images of CENP-A, topoisomerase II, and microtubules in young and aged metaphase II–arrested eggs. CENP-A spots detection and intercentromere distance measurements in Imaris were used to identify chromatids that did not contain two centromeres and assign them as single chromatids. Boxes mark regions that are magnified in insets. (B) Quantification of the number of single chromatids [identified as in (C)] in DMSO- or cytochalasin D–treated young and aged metaphase II–arrested eggs. Each filled black circle in graph represents a single egg, and red bars represent mean values. Data are from three independent experiments. (C) Representative maximum intensity projected immunofluorescence images of microtubules, centromeres, chromatid pairs, and single chromatids in DMSO- or cytochalasin D–treated young and aged metaphase II–arrested eggs. Boxes mark regions that are magnified in insets. (D) Representative maximum intensity projected immunofluorescence images of centromeres and chromatids in metaphase II chromosomal spreads of DMSO- or cytochalasin D–treated young eggs. Boxes mark regions that are magnified in insets. (E) Quantification of the number of single chromatids in metaphase II chromosomal spreads of DMSO- or cytochalasin D–treated young eggs. Each filled black circle in graph represents chromosomal spread from a single egg, and red bars represent mean values. Data are from three independent experiments. Statistical significance was evaluated using Fisher’s exact test (B and E) * P=0.0131. The number of analyzed oocytes is specified in italics. Cyto D, cytochalasin D.

Fig 3: Impaired DSB repair after CENP-A depletion. (A) Representative images of control and CENP-A Trim-away oocytes stained with p-MDC1 and ACA antibodies. Scale bar, 10 μm. (B) Quantification of ACA and p-MDC1 intensities. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.0001. (C) Representative images of control and CENP-A Trim-away oocytes stained with p-MDC1 and CIP2A antibodies after ETP treatment and recovery (ETP + R). Scale bar, 10 μm. (D, E) Ratio of p-MDC1 and CIP2A intensities at chromosomes over spindle poles. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.0001. ns, not significant. (F) Representative images of chromosome spreads showing TUNEL signals in control and CENP-A Trim-away oocytes after ETP treatment and recovery. Representative individual chromosomes are enlarged (bottom). Scale bar, 10 μm (uncropped); 5 μm (enlarged). (G, H) Quantification of TUNEL intensity on entire chromosomes and at centromeres. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.0001. ns, not significant. (I) Schematic diagram depicting the roles of CENP-A in CIP2A–MDC1–TOPBP1 chromosomal recruitment.

Fig 4: Centromeres(A) Spermatocyte chromosome spreads of Smc1β+/+, Smc1β−/−, and Smc1β−/−,1a mice, stained with anti-SYCP3 (red) for AEs/LEs and with anti-CENP-A (green) for centromere.(B and C) Spermatocyte chromosome spreads of Smc1β+/+, Smc1β−/−, and Smc1β−/−,1a mice, stained with anti-SYCP3 (green) for AEs/LEs, anti-CENPA (red), and anti-H1t (gray) showing H1t-positive and H1t-negative spermatocytes, respectively. AEs/LEs stained with anti-SYCP3 and centromere with anti-CENP-A. White arrows indicate abnormal centromere structure (scale bar, 5 μm).See also Figures S5 and S7.

Fig 5: Suv39h1/Suv39h2 deficiency generates small and compact mitotic chromosomes.a–f, Flow sorting and size measurements of chromosomes (Chr) 19 and X from WT or Suv39h dn mouse ESCs and MEFs. Flow karyotype of mitotic chromosomes is isolated from WT or Suv39h dn ESCs (a) and MEFs (d); gates used to isolate chromosomes 19 and X are indicated. Representative images (right) of mitotic chromosomes 19 (b and e) and X (c and f) from WT or Suv39h dn ESCs (b and c) and MEFs (e and f) are shown, where DAPI stain (gray) and Cenpa label (green) indicate the chromosome body and centromere, respectively. Scale bar, 5 μm. Boxplots (left of images) show area measurements of individual chromosomes and centromeres for WT and Suv39h dn cells. Minimum, lower quartile, median, upper quartile and maximum values are indicated (n = minimum 100 chromosomes analyzed for each cell line over 3 independent experiments). P values of statistically significant changes, measured by unpaired, two-tailed Student’s t-tests, are indicated. g,h, Representative images of WT or Suv39h dn ESC metaphase spreads stained with either chromosome 19 painting probe (green) (g) or chromosome X painting probe (green) (h), in addition to γSat probe (red) and DAPI (blue). Scale bars, 4 μm and 1 μm for the metaphase spread and zoom-in images, respectively. Chromosome and centromere sizes of chromosomes 19 and X were calculated on metaphase spreads of WT and Suv39h dn ESCs. Boxplots show chromosome and centromere area measurements for WT and Suv39h dn spreads. Minimum, lower quartile, median, upper quartile and maximum values are indicated (n = minimum 25 chromosomes analyzed on metaphase spreads for each line over 3 independent experiments). b,c,e–h, P values of statistically significant changes, measured by unpaired, two-tailed Student’s t-tests, are indicated. Source data, including the precise numbers of chromosomes analyzed, are provided.Source data