Reduced pHi delays GVBD and blocks cytoplasmic granule invasion into the GV region. (A–C) To clamp the pHi at ∼6.7, 7.0, and 7.2, unstimulated oocytes were incubated with modified ASW for 20 min (see also Fig. S1 B). As a control, unstimulated oocytes were incubated in ASW for 20 min, in which the basal pHi is ∼7.0 but increases to ∼7.2 after 1-MA stimulation (Moriwaki et al., 2013; see also Fig. S1 B). These oocytes were stimulated with 1-MA, followed by immunoblotting (A); monitoring GVBD (B; means ± SE of three independent experiments); and time-lapse DIC imaging (C; images captured every 10 s; selected images after GVBD are shown; see Videos 1–4 for complete image sequences). Asterisk in A, nonspecific bands. Closed and open arrowheads in A indicate the positions of the upper and lower sfSGK bands, respectively. Bar in C, 50 µm. GVBD morphology is shown in Fig. S3. Z-stacks acquired after the time-lapse imaging are shown in Fig. S4. The results in A and C are representative of two independent experiments.
