Reduced pHi perturbed chromosome transport and microtubule organization for spindle assembly. (A) Oocytes were treated and stained as in Fig. 6 at the indicated time points after GVBD. Typical images of GV region or spindles (max projection and/or single slices) from two independent experiments are shown. GVBD occurred around 17 min after 1-MA stimulation in ASW and around 13, 10, and 18 min at clamped pHi values of 7.2, 7.0, and 6.7, respectively. Bars, 10 µm. The stained samples were also used in B–D. (B) Oocytes with spindles were counted at the indicated times after GVBD. n.d., not determined. (C) Spindles assembled at clamped pHi values of 7.0 (45−50 min after GVBD) and 7.2 (20−30 min after GVBD) were checked for chromosome alignment (aligned or unaligned; typical images are shown). Arrowheads indicate unaligned chromosomes. Bars, 10 µm. (D) At the indicated time points after GVBD, oocytes at a clamped pHi of 6.7 were classified according to their chromosome distribution (typical images are shown): (i, cyan) scattered within the entire GV region; (ii, green) somewhat gathered toward the animal pole, but not in a compact group; (iii, magenta) gathered in a compact group near the animal pole. Dotted lines, boundary of the GV region. Bars, 10 µm. In B–D, results from two females (starfish 1 and 2) are shown. In B–D, 20 and 25 oocytes were observed at each time point in starfish 1 and 2, respectively.
