Fig 2: MIS12 localizes to kinetochores in mouse oocytes. (A) Exogenous MIS12 robustly localizes to kinetochores during both meiosis I (n = 13) and meiosis II (n = 12). Oocytes were immunostained with anti‐MYC and anti‐ACA antibodies, DNA is counterstained with DAPI. Three independent experiments were conducted. (B) Endogenous MIS12 clearly localizes to kinetochores in meiosis I (n = 15) and meiosis II (n = 13), as validated using antibody from Abcam. Three independent experiments were conducted. (C) Endogenous MIS12 clearly localizes to kinetochores in meiosis I (n = 11) and meiosis II (n = 14), as validated using antibody from Bioss. Three independent experiments were conducted. (D) MIS12 signal is absent from kinetochores in Mis12‐deleted oocytes. Oocytes after spreading were immunostained with anti‐MIS12 (Abcam) and anti‐ACA antibodies. DNA is counterstained with DAPI (Applies to A‐D). Scale bars: 10 µm (overview), 5 µm (enlarged view) (Applies to A–D). Three independent experiments were conducted. Three independent experiments were conducted. (E) Quantification of the MIS12/ACA ratio from (D) in control (n = 145) and Mis12‐deleted (n = 148) oocytes. Data are presented as mean ± SEM; *** p < 0.0001. The number of kinetochores analyzed (n) is indicated.

Fig 3: Chromosome‐anchoring of MIS12 rescues K‐MT attachment in Mis12‐deleted oocytes. (A) The H2B‐MIS12 fusion protein successfully localizes to chromosomes in mouse oocytes. Microtubules (magenta) and chromosomes (green) were visualized using MAP4‐mCherry and H2B‐MIS12‐Venus in live oocytes, respectively. Scale bars: 20 µm (overview), 5 µm (enlarged view). (B) Representative time‐lapse images of oocytes expressing EGFP‐MAP4 (microtubules) and H2B‐mCherry (chromosomes), comparing Mis12‐deleted oocytes with or without supplementation of H2B‐Mis12 fusion mRNA. Images were captured over 13 h with a frame interval of 10 min. Scale bar: 20 µm. Three independent experiments were conducted. (C) Chromosome alignment in meiosis I is restored by expressing the H2B‐MIS12 fusion protein in Mis12‐deleted oocytes (Control, n = 16; Mis12‐cKO, n = 18; Mis12‐cKO + H2B‐Mis12, n = 17). Data are presented as mean ± SEM; *** p < 0.0001. (D) Chromosome alignment in meiosis II is restored by expressing the H2B‐MIS12 fusion protein in Mis12‐deleted oocytes (Control, n = 15; Mis12‐cKO, n = 13; Mis12‐cKO + H2B‐Mis12, n = 16). Data are presented as mean ± SEM; *** p < 0.0001. (E) K‐MT attachment in meiosis II is rescued by H2B‐MIS12 (Mis12‐cKO, n = 12; Mis12‐cKO + H2B‐Mis12, n = 13). Oocytes were immunostained with anti‐α‐tubulin and anti‐ACA antibodies. DNA was counterstained with DAPI. Scale bars: 20 µm (overview), 5 µm (enlarged view).

Fig 4: NDC80‐anchoring MIS12 rescues K‐MT attachment in Mis12‐deleted oocytes. (A) Representative time‐lapse images of oocytes expressing EGFP‐MAP4 (microtubules) and H2B‐mCherry (chromosomes), comparing Mis12‐deleted oocytes with or without supplementation of Mis12 mRNA or Ndc80‐Mis12 fusion mRNA. Images were captured over 13 h with a frame interval of 10 min. Scale bar: 20 µm. Three independent experiments were conducted. (B) Chromosome alignment in meiosis I is restored by expressing either MIS12 or the NDC80‐MIS12 fusion protein in Mis12‐deleted oocytes (Control, n = 18; Mis12‐cKO, n = 19; Mis12‐cKO + Mis12, n = 19; Mis12‐cKO + Ndc80‐Mis12, n = 21). Data are presented as mean ± SEM; *** p<0.0001. (C) Chromosome alignment in meiosis II is restored by expressing either MIS12 or the NDC80‐MIS12 fusion protein in Mis12‐deleted oocytes (Control, n = 17; Mis12‐cKO, n = 18; Mis12‐cKO + Mis12, n = 16; Mis12‐cKO + Ndc80‐Mis12, n = 19). Data are presented as mean ± SEM; *** p<0.0001. (D) K‐MT attachment in meiosis II is successfully rescued by MIS12 or NDC80‐MIS12 (Mis12‐cKO, n = 19; Mis12‐cKO + Mis12, n = 16; Mis12‐cKO + Ndc80‐Mis12, n = 17). Oocytes were immunostained with anti‐α‐tubulin and anti‐ACA antibodies, DNA is counterstained with DAPI. Scale bars: 20 µm (overview), 5 µm (enlarged view).

Fig 5: MIS12 is required for K‐MT attachment in mouse oocytes. (A) Representative time‐lapse images of control and Mis12‐deleted oocytes expressing EGFP‐MAP4 (microtubules, green) and H2B‐mCherry (chromosomes, magenta), captured over 15 h with a frame interval of 10 min. Scale bar: 20 µm. Three independent experiments were conducted. (B) MIS12 deletion disrupts chromosome alignment. Quantification of the maximal equatorial chromosome width in meiosis I shows significant widening in mutant oocytes (n = 23) compared with control oocytes (n = 30). Data are presented as mean ± SEM; *** p < 0.0001. (C) Analysis of spindle bipolarity at metaphase I in control (n = 47) and Mis12‐cKO (n = 63) mouse oocytes. Data are presented as mean ± SEM; ns, not significant. (D) Analysis of spindle length at metaphase I in control (n = 39) and Mis12‐cKO (n = 52) mouse oocytes. Data are presented as mean ± SEM; *** p < 0.0001. (E) Analysis of spindle width at metaphase I in control (n = 39) and Mis12‐cKO (n = 52) mouse oocytes. Data are presented as mean ± SEM; ns, not significant. (F) Analysis of the spindle length/width ratio at metaphase I in control (n = 39) and Mis12‐cKO (n = 52) mouse oocytes. Data are presented as mean ± SEM; *** p < 0.0001. (G) Mis12 deletion accelerates meiosis I progression (control, n = 138; Mis12‐cKO, n = 152). Polar body extrusion (PBE) rates were recorded hourly from 4 to 15 h. Data are presented as mean ± SEM; ***p < 0.0001, ** p < 0.001, * p < 0.01. (H) MIS12 is essential for stable K‐MT attachment in meiosis I. Cold‐stable assay reveals increased microtubule detachment in Mis12‐deleted oocytes (n = 16) compared with control oocytes (n = 13). Oocytes were immunostained for α‐tubulin (white), ACA (kinetochores, blue), and DNA (magenta). Scale bars: 5 µm (overview), 1 µm (enlarged view). Three independent experiments were conducted. (I) Quantification of chromosome attachment errors from (H). The graph depicts the percentage of oocyte bivalents exhibiting normal bipolar attachment, unipolar (single‐side) attachment, or complete (double‐side) detachment. Data are presented as mean ± SEM; *** p < 0.0001.