Dynein is essential for maturation of MTOCs and spindle, whereas kinesin-5 is required for spindle bipolarization. (A and B) Live imaging of mouse zygotes during the first division under inhibition of dynein by P150-CC1 (A) or of kinesin-5 by monastrol (B) at prophase (left), early (middle), and late prometaphase (right), respectively. Z-projected images of confocal sections (3 µm thick) show microtubules (EGFP-MAP4; gray). Time indicates hours and minutes after NEBD. Note that the monopolar spindle is formed in the kinesin-5–inhibited zygote (asterisk in B; right). (C) Distance of each cytoplasmic MTOC from the nucleus measured by tracking MTOCs in control, dynein-, and kinesin-5–inhibited zygotes (n = 4, 1, and 3 embryos, respectively) and plotted against time after NEBD. (D) Speed of each MTOC movement toward the nucleus in relation to the initial distance of the MTOC from the nucleus in control, dynein-, and kinesin-5–inhibited embryos. Insets show representative tracks in zygotes. (E) Whisker box plot of speed of MTOC movement toward the nucleus in control (n = 48 tracks derived from four zygotes), dynein-inhibited (n = 38 tracks derived from three zygotes), and kinesin-5–inhibited (n = 32 tracks derived from three zygotes) embryos. MTOC movement is significantly slower in dynein-inhibited embryos and faster in kinesin-5–inhibited embryos than in controls (P < 0.05, Welch two-sample t test). The lines near the middle of the boxes represent the median (50th percentile). The bottom and top of the boxes are the 25th and 75th percentile, respectively. The whiskers extend to the most extreme data point, which is no more than 1.5 times the percentile range of the box. The dots represent the extreme data point extending out of the 1.5 times percentile range of the box. (F) Summary of the potential mechanism leading to acentrosomal spindle assembly in the mouse zygote before (left) and after (right) nuclear envelope breakdown (NEBD). Bars, 10 µm.