Chromosome isolation from oocytes.

(A) Oocytes after zona pellucida removal. Left panel: MI oocyte. Right panel: MII oocyte with visible polar body. Scale bar = 10 μm. (B) Spindle isolation process. Left panel: Spindle flowing out from the oocyte after oocyte lysis. Middle panel: a chromosome being isolated from the spindle-chromosome complex. Right panel: chromosome captured between two pipettes. Scale bar = 10 μm.

Chromosome stiffness measurement.

(A) Example images of chromosome isolation. Left: MII oocyte chromosome. Right: MI oocyte chromosome. Scale bar = 10 μm. (B) Chromosome stiffness comparison across different cell types: mitotic cells (n=8), WT spermatocytes at prophase I (n=8), MI oocytes (n=8) and MII oocytes (n=8). Young’s Modulus of MI oocyte chromosomes (3790 ± 700 Pa) is much higher than that of mitotic cells (370 ± 70 Pa, P=0.0002) and MII oocytes (670 ± 130 Pa, P=0.0006). Data are presented as mean ± SEM. All statistical analyses were performed via t-test.

Chromosome stiffness measurement in meiosis-specific cohesin mutants.

(A) Images of chromosome isolation from Stag3-/- spermatocytes. Scale bar = 10 μm. (B) Chromosome stiffness comparison across various cell types: WT spermatocytes at prophase I (n=8), Rec8-/- spermatocytes at prophase I (n=8), Stag3-/-spermatocytes at prophase I (n=9) and Rad21l-/- spermatocytes at prophase I (n=10). Young’s Modulus of WT spermatocyte chromosomes (2710 ± 610 Pa) is not significantly different from that of Rec8-/- spermatocyte chromosomes (2580 ± 620 Pa, P= 0.8884), Stag3-/- spermatocyte chromosomes (2240 ± 210 Pa, P= 0.4533) and Rad21l-/- spermatocyte chromosomes (2050 ± 370 Pa, P= 0.3514). Data are presented as mean ± SEM. All statistical analyses were conducted using t-test.

Chromosomes in aged oocytes are stiffer than those in younger oocytes.

(A) Images showing the isolation of chromosomes from an aged MI oocyte. Left panel: aged MI oocyte images. Middle panel: spindle isolated from aged MI oocyte. Right panel: MI chromosome isolated from the spindle-chromosome complex. Scale bars = 10 μm. (B) Chromosome stiffness comparison between MI oocytes from 3–4-week-old mice (n=8) and 48-week-old mice (n=5). Young’s Modulus of 3–4-week-old MI oocyte chromosomes (3790 ± 700 Pa) is significantly lower than that of 48-week-old MI oocyte chromosomes (8150 ± 1590 Pa, P=0.0150). Data are presented as mean ± SEM and statistical analysis was performed using t-test.

Etoposide treatment reduces chromosome stiffness.

(A) Images of chromosome isolation from MI oocyte treated with 50 μg/ml etoposide. Left panel: a spindle after cell lysis. Middle panel: a spindle captured with pipettes. Right panel: chromosome isolation. Scale bar = 10 μm. (B) DAPI staining of control and 50 μg/ml etoposide-treated MI oocytes. Scale bar = 10 μm. (C) Chromosome stiffness comparison between mitotic cells (n=8), control MI oocytes (n=8), 5 μg/ml etoposide-treated MI oocytes (n=8), 25 μg/ml etoposide-treated MI oocytes (n=8) and 50 μg/ml etoposide-treated MI oocyte (n=8). Young’s Modulus of control MI oocyte chromosomes (3790 ± 700 Pa) did not differ significantly from that of 5 μg/ml etoposide-treated MI oocyte chromosomes (3930 ± 400 Pa, P = 0.8624). However, it was significantly higher than that of 25 μg/ml etoposide-treated MI oocyte chromosomes (1640 ± 340 Pa, P = 0.015) and 50 μg/ml etoposide-treated MI oocyte chromosomes (1710 ± 430 Pa, P=0.0245). Data are presented as mean ± SEM, with statistical analysis conducted using t-test.