Figure S1.
Dual spindle characteristics in bovine zygotes. (A–C and E) IF staining of bovine zygotes fixed at 27.5 h after IVF. Maximum intensity projections orthogonal to the spindle axis of confocal sections are shown for microtubules (α-tubulin, green), chromatin (Hoechst, blue), sperm flagellum (acetyl tubulin, cyan), and pericentrosome (Nedd1 or Cep192, magenta). Scale bars, 5 µm. (A) Arrowheads indicate spermatozoan flagellum adjacent to one spindle pole, confirming monospermic fertilization. For further details, see Materials and methods. (B) Distant dual spindles with distinct mitotic timing inside same cytoplasm. (C and D) Diverse centrosome positioning in proximate (fused) and distant dual spindles. (C) Arrowheads indicate the number and position of pericentrosomes. (D) Abundance (%) of centrosome positions/pole characteristics as illustrated in C. (E–H) Comparison of centrosome positioning in bicentrosomal (contralateral) metaphase spindles after IF of unperturbed (n = 16) or cold-treated (3 min cold shock on ice, n = 9) zygotes. d1, centrosome to spindle distance; d2, spindle half-length; d3, centrosome to chromosome distance. (E) Illustration of the measurements in exemplary metaphase spindle after cold treatment (see also Fig. 1 D); red line illustrates projected spindle axis orthogonal to chromosomes. (F) Distance in micrometers between centrosomes and spindle body (d1, see arrow in E). For assessment of spindle body, see Materials and methods. Error bars indicate SEM distance of unperturbed (3.9 µm) versus cold shock–treated (6.5 µm) zygotes (P = 0.01, significant). (G) Relative distance (%) between centrosome and spindle microtubules respective to spindle half-length (d1/d2, see arrows in E). Error bars indicate SEM distance in unperturbed (49.1%) versus cold shock–treated (87.3%) zygotes (P = 0.006, significant). (H) Distance in micrometers between centrosomes and the chromosome centroid (d3, see arrow in E). Error bars indicate SEM distance in unperturbed (11.7 µm) versus cold shock–treated (13.7 µm) zygotes (P = 0.15). (F–H) Average measurements for both centrosomes from same zygote are depicted. Statistical tests: two-tailed unpaired Student’s t test.

Dual spindle characteristics in bovine zygotes. (A–C and E) IF staining of bovine zygotes fixed at 27.5 h after IVF. Maximum intensity projections orthogonal to the spindle axis of confocal sections are shown for microtubules (α-tubulin, green), chromatin (Hoechst, blue), sperm flagellum (acetyl tubulin, cyan), and pericentrosome (Nedd1 or Cep192, magenta). Scale bars, 5 µm. (A) Arrowheads indicate spermatozoan flagellum adjacent to one spindle pole, confirming monospermic fertilization. For further details, see Materials and methods. (B) Distant dual spindles with distinct mitotic timing inside same cytoplasm. (C and D) Diverse centrosome positioning in proximate (fused) and distant dual spindles. (C) Arrowheads indicate the number and position of pericentrosomes. (D) Abundance (%) of centrosome positions/pole characteristics as illustrated in C. (E–H) Comparison of centrosome positioning in bicentrosomal (contralateral) metaphase spindles after IF of unperturbed (n = 16) or cold-treated (3 min cold shock on ice, n = 9) zygotes. d1, centrosome to spindle distance; d2, spindle half-length; d3, centrosome to chromosome distance. (E) Illustration of the measurements in exemplary metaphase spindle after cold treatment (see also Fig. 1 D); red line illustrates projected spindle axis orthogonal to chromosomes. (F) Distance in micrometers between centrosomes and spindle body (d1, see arrow in E). For assessment of spindle body, see Materials and methods. Error bars indicate SEM distance of unperturbed (3.9 µm) versus cold shock–treated (6.5 µm) zygotes (P = 0.01, significant). (G) Relative distance (%) between centrosome and spindle microtubules respective to spindle half-length (d1/d2, see arrows in E). Error bars indicate SEM distance in unperturbed (49.1%) versus cold shock–treated (87.3%) zygotes (P = 0.006, significant). (H) Distance in micrometers between centrosomes and the chromosome centroid (d3, see arrow in E). Error bars indicate SEM distance in unperturbed (11.7 µm) versus cold shock–treated (13.7 µm) zygotes (P = 0.15). (F–H) Average measurements for both centrosomes from same zygote are depicted. Statistical tests: two-tailed unpaired Student’s t test.

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