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	Figure 1. sfSGK protein and phosphorylation of its A-loop are detectable in starfish oocytes. (A) The amino acid sequence of sfSGK was aligned with that of human SGK3. Colored boxes indicate conserved domains: PX domain (blue), A-loop (magenta), HM (purple), and catalytic domain (orange). Magenta and purple asterisks indicate conserved residues phosphorylated by PDK1 and TORC2, respectively (see also Fig. 2). (B) Unstimulated oocytes were incubated with 1-MA for 4 min, followed by immunoblotting with the indicated antibodies. Asterisks, nonspecific bands. (C) Immunoprecipitation (IP) was performed with anti-sfSGK-HM antibody or control (Cont.) IgG using extracts of unstimulated or 1-MA−stimulated oocytes. Input extract (Input), flow-through (FT), and beads (Beads) samples were analyzed by immunoblotting (IB). Asterisk, nonspecific bands. The indicated results in B and C are representative of three and two independent experiments, respectively. Closed and open arrowheads in B and C indicate positions of the upper and lower bands of sfSGK, respectively.
	Figure 2. sfSGK is activated by PDK1 and TORC2 in a PI3K-dependent manner. (A) Unstimulated oocytes were treated with 1-MA and analyzed by immunoblotting with anti-human phospho-SGK (sfSGK-pT312 [A-loop]), anti-sfSGK-HM (sfSGK), anti-sfAkt phospho-Ser477 (Akt-pS477[HM]), anti-sfAkt C-terminal fragment (Akt), anti-sfCdc25 (Cdc25), anti-Cdk1 phospho-Tyr15 (Cdk1-pY15), and anti-PSTAIR (Cdk1) antibodies. GVBD occurred at 17 min. (B) A piece of ovary was separated into oocytes and germinal epithelium, and subjected to immunoblotting. (C) To stimulate ovarian oocytes, 1-MA was injected into the body cavity of female starfish. Pieces of ovaries were then recovered at the indicated times and analyzed via immunoblotting or monitored for GVBD timing. GVBD occurred within 25 min. Spawning started at 30 min. (D) Unstimulated oocytes were treated with 1-MA for 4 min in the presence of indicated inhibitors or DMSO, and then subjected to normal SDS-PAGE, followed by immunoblotting. Closed and open arrowheads in A–D indicate the positions of the upper and lower sfSGK bands, respectively. Asterisks in A–D, nonspecific bands. (E and F) Unstimulated oocytes were treated with 1-MA for 4 min (E) or treated as in D (F), and then subjected to Phos-tag SDS-PAGE, followed by immunoblotting (IB). The results shown are representative of three independent experiments in A, E, and F, and two independent experiments in B–D.
	Figure 3. Activation of sfSGK is required for rapid pHi increase after 1-MA stimulus. (A) Unstimulated oocytes were injected with an anti-sfSGK-HM antibody or control IgG, treated with 1-MA for 4 min, and then subjected to immunoblotting. Asterisk, nonspecific bands. Closed and open arrowheads indicate positions of the upper and lower band of sfSGK, respectively. The result shown is representative of three independent experiments. (B) BCECF-dextran and either anti-sfSGK-HM antibody or control IgG were coinjected into unstimulated oocytes. After a 1-h incubation, 1-MA was added, and the fluorescence intensity ratio was measured before and after 1-MA addition. pHi was calculated from the fluorescence intensity ratio and plotted (means ± standard error [SE] of three independent experiments).
	Figure 4. Activation of sfSGK is required for cyclin B–Cdk1 activation. (A–C) Unstimulated oocytes were injected with anti-sfSGK-HM antibody or control IgG and treated with 1-MA, followed by monitoring GVBD (A; means ± SE of three independent experiments); imaging by DIC microscopy (B); and immunoblotting (C) at the indicated times. An oil drop introduced along with the antibodies as a mark of the injection can be seen on the right of the GV in B. Bar in B, 50 µm. Asterisk in C, nonspecific bands. Closed and open arrowheads in C indicate positions of the upper and lower bands of sfSGK, respectively. (D and E) Unstimulated oocytes were injected with anti-sfSGK-HM antibody, incubated for 1 h, and further injected with mRNA encoding a mutant sfSGK (T479E or K183M/T479E), followed by additional incubation for 22 h. These oocytes were treated with 1-MA, followed by immunoblotting (D) and monitoring GVBD (E; means ± SE of three independent experiments). exo. and endo. in D indicate exogenous and endogenous sfSGK, respectively. Lanes 3 and 12 in D represent oocytes that were collected at the time of GVBD under each condition (∼16 and ∼14 min, respectively). The results shown in B–D are representative of three independent experiments.
	Figure 5. 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.
	Figure 6. F-actin shell, meshwork, and patches were formed at all clamped pHi values. (A and B) Oocytes stimulated with 1-MA in ASW or in modified ASW to clamp the pHi at ∼7.2, 7.0, or 6.7 were fixed upon GVBD or 5 min after GVBD and then triple-stained with phalloidin for F-actin (gray), anti-α-tubulin antibody for microtubules (green), and DAPI for chromosomes (magenta). Typical images of GV region (max projections and single slices) from two independent experiments are shown in A. Single slices including the actin patches (white boxes with enlarged images) observed 5 min after GVBD are shown in B. Bars in A and B, (main images) 10 µm, (insets) 5 µm.
	Figure 7. 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.
	Figure 8. Model for sfSGK-dependent meiotic resumption in ovarian oocytes. A model for 1-MA–induced meiotic resumption in ovarian oocytes was prepared on the basis of previous and current findings. 1-MA stimulus induces Gβγ-dependent PI3K activation. In a manner that depends on PI3K, PDK1 and TORC2 activate sfSGK. Activation of sfSGK is essential for cyclin B–Cdk1 activation and sfNHE3-dependent pHi increase. These SGK-dependent pathways prefer lower pHi (magenta box). Processes leading to GVBD after cyclin B–Cdk1 activation prefer higher pHi (green box). After GVBD, actin-dependent chromosome gathering and microtubule organization for spindle assembly as well as cytoplasmic granule invasion into the GV region require a pHi of ∼7.0 or higher (cyan box) although spindles contains unaligned chromosomes at pHi ∼7.0. Because the pHi of ovarian oocytes before 1-MA stimulation in the body cavity is ∼6.7, the sfSGK-dependent pHi increase accelerates GVBD and is required for the processes leading to meiotic spindle assembly in ovarian oocytes. By contrast, the pHi of isolated oocytes before 1-MA stimulation in ASW is ∼7.0, which is already permissive for the processes toward spindle assembly.

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