SGK regulates pH increase and cyclin B-Cdk1 activation to resume meiosis in starfish ovarian oocytes

Tight regulation of intracellular pH (pHi) is essential for biological processes. Fully-grown oocytes, having a large nucleus called the germinal vesicle, arrest at meiotic prophase-I. Upon hormonal stimulus, oocytes resume meiosis to acquire fertilizability. At this time, pHi increases through Na+/H+ exchanger activity. However, regulation and function of this change remains obscure. Here we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated by the PI3K/TORC2/PDK1 signaling after hormonal stimulus, and is required for the pHi increase and cyclin B–Cdk1 activation. Furthermore, when we clamped pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulus normally induced cyclin B–Cdk1 activation; thereafter, oocytes initiated germinal vesicle breakdown (GVBD), but failed to complete it. Thus, SGK-dependent pHi increase is likely prerequisite for completion of GVBD in ovarian oocytes. We propose a model that SGK drives meiotic resumption through concomitant regulation of pHi and the cell-cycle machinery.

Introduction successful fertilization 22 . Starfish oocytes have been used as a model to study oocyte maturation for 48 more than half a century 23 . Previously, we reported that pH i of ProI-arrested ovarian oocytes is low 49 (~6.7) due to the relatively high CO 2 and low O 2 concentrations in the body cavity of female 50 starfish 17 . Soon after stimulation by the hormone 1-methyladenine (1-MA) 24 , pH i increases to ~6.9 17 , 51 after which the oocytes undergo GVBD and ultimately arrest at MI 16,17 . To maintain the MI arrest, 52 pH i is maintained at ~6.9 until spawning 16,17,25-28 . The increase in pH i following 1-MA stimulus is 53 known to require starfish NHE3 (sfNHE3) 16,17,25 . However, the upstream signaling and function of 54 this pH i increase remain elusive. 55 For analysis of pH i regulation, unstimulated starfish oocytes are isolated from ovaries, 56 placed in artificial seawater (ASW), and used for experiments because various techniques, such as 57 oocytes 17 , enabling us to examine the effects of altered pH i . In this study, to elucidate the regulation 83 and function of the pH i increase in oocytes, we examined the involvement of SGK in the pH i 84 increase following 1-MA stimulus and the effects of pH i alteration on meiotic resumption. We found 85 that SGK activation is prerequisite for two mutually independent events: pH i increase and cyclin 86 B-Cdk1 activation. Moreover, by clamping pH i at various values, we showed that oocytes exhibit 87 defective GVBD at reduced pH i values. Based on these findings, we propose a model for 88 SGK-dependent meiotic resumption in starfish ovarian oocytes. 89

Results 90
Starfish SGK is a homolog of human SGK3 91 To investigate the possible involvement of SGK in the pH i increase of oocytes during hormonal 92 stimulation with 1-MA in the starfish A. pectinifera, we cloned the cDNA of starfish SGK (sfSGK). 93 The open reading frame (ORF) of the cDNA encodes a polypeptide of 489 amino acids with a 94 predicted molecular mass of 56 kDa. At the amino acid sequence level, sfSGK is 52% identical to the 95 human SGK3 PX domain (Fig. 1a, blue open box) and 77% identical to the human SGK3 catalytic 96 domain (Fig. 1a, orange open box). Two phosphorylation sites, which are required for activation of 97 mammalian SGK, are conserved in sfSGK: Thr312 in the A-loop (corresponding to human SGK3 98 Thr320 which is phosphorylated by PDK1), and Thr479 in the HM (human SGK3 Ser486 which is 99 phosphorylated by mTORC2) (Fig. 1a). The transcriptome database contains no other SGK isoforms 100 for A. pectinifera, suggesting that SGK3 is the only member of this protein family in starfish. 101 For this study, we generated two types of antibodies: one against a recombinant fragment 102 of sfSGK lacking the N-terminal 50 amino acids (anti-sfSGK-ΔN50), and the other against a 103 C-terminal peptide (17 amino acids) containing the HM of sfSGK (anti-sfSGK-HM). We evaluated 104 the reactivity of these antibodies by immunoblotting starfish oocytes. Both antibodies detected a 105 protein of 56 kDa, corresponding the predicted molecular mass of sfSGK in unstimulated oocytes 106 ( Fig. 1b, left and middle panels), suggesting that these antibodies recognized a sfSGK protein. The 107 sfSGK band underwent a mobility shift after 1-MA treatment (Fig. 1b, left and middle panels), 108 suggesting that the protein is phosphorylated after 1-MA stimulation. 109 Full activation of mammalian SGK is achieved by PDK1-dependent phosphorylation of 110 A-loop 35,41 . The amino acid sequence of the A-loop in mammalian SGK is quite similar to that of 111 sfSGK (Fig. 1a). Hence, to determine whether sfSGK is phosphorylated in the A-loop in stimulated 112 starfish oocytes, we used a commercial polyclonal anti-human phospho-SGK antibody that 113 recognizes a phosphorylated amino acid in the A-loop of mammalian SGK. The antibody detected a 114 ~59 kDa protein in stimulated oocytes, but not in unstimulated oocytes (Fig. 1b, right panel). In 115 addition, this band ran at the same position as the shifted bands recognized by the anti-sfSGK-ΔN50 116 and anti-sfSGK-HM antibodies (Fig. 1b). To confirm that the protein detected by the anti-human 117 phospho-SGK antibody was sfSGK, we immunoprecipitated sfSGK using the anti-sfSGK-HM 118 antibody and immunoblotted with the anti-human phospho-SGK antibody. As expected, we detected 119 protein in the bead fraction ( Fig.1 c,  Next, we investigated the dynamics of phosphorylation of these proteins in ovarian 138 oocytes in the body cavity, where MI arrest occurs after GVBD 16,17,27 . To this end, we injected 1-MA 139 into the body cavities of female starfish, and then at each time point, isolated two pieces of ovary: 140 one for counting GVBD, and the other for immunoblotting analysis. We found that 95% of ovarian 141 oocytes underwent GVBD within 25 min after 1-MA injection; spawning from the female started at 142 30 min. To prepare ovarian oocytes for immunoblotting, the piece of ovary was placed directly into 143 sample buffer immediately after isolation. Although these samples contained not only oocytes but 144 also other somatic cells derived from ovarian germinal epithelium, we ignored the involvement of 145 the germinal epithelium because the sfSGK, Cdc25 and Cdk1 proteins were not detectable in the 146 germinal epithelium (Fig, 2b). Analysis of oocyte-derived sfSGK, Cdc25, and Cdk1 proteins 147 revealed that both sfSGK and Cdc25 in ovarian oocytes were phosphorylated within 5 min, and that 148 Tyr15 of Cdk1 was dephosphorylated within 25 min (Fig. 2c). These results indicate that sfSGK, 149 Cdc25, and cyclin B-Cdk1 in ovarian oocytes in the body cavity were activated similarly to those in 150 isolated oocytes. 151 It should be noted that the shifted bands of sfSGK (Fig. 2a,  sfSGK should depend on TORC2. As expected, A-loop phosphorylation was blocked by pp242, a 166 specific inhibitor of TOR, the catalytic subunit of TORC2 (Fig. 2d, lane 6). Notably, the mobility 167 shift of sfSGK was reduced by pp242, but was not affected by BX795 (Fig. 2d, lanes 6 and 10), 168 suggesting that the shift is caused by HM phosphorylation, which is dependent on TORC2 but not 169

PDK1. 170
It should be noted, however, that TOR forms two types of complex: TOR complex 1 171 (TORC1) and TORC2 48 , both of which are inhibited by pp242. To confirm that the inhibition of 172 sfSGK phosphorylation by pp242 was due to inhibition of TORC2 rather than TORC1, we used the 173 TORC1 inhibitor rapamycin at a concentration previously shown to be sufficient for inhibition of 174 inhibitor BX795 and the TORC1/2 inhibitor pp242 (Fig. 2f, the top band, lane 5 and 6), but was 190 unchanged by the TORC1 inhibitor rapamycin. In addition, we found that the intensity of the middle 191 band was reduced by pp242, but not by BX795 or rapamycin (Fig. 2f,  Next, we investigated whether sfSGK is involved in the pH i increase after 1-MA stimulus. Given that 200 the anti-sfSGK-HM antibody immunoprecipitated sfSGK protein (Fig. 1c), the binding of the 201 antibody to the HM may be strong enough to prevent the interaction of TORC2 with the HM of 202 sfSGK and thereby block phosphorylation of the HM. Indeed, the mobility shift of sfSGK after 203 1-MA stimulus was blocked in oocytes injected with the antibody (Fig. 3a). More importantly, 204 phosphorylation of the A-loop was also blocked (Fig. 3a), as observed in oocytes treated with pp242 205 ( Fig. 2d), indicating that the anti-sfSGK-HM antibody inhibited sfSGK activation. This inhibition 206 was specific to sfSGK, because the HM of Akt was phosphorylated even in oocytes pre-injected with 207 the anti-sfSGK-HM antibody (Fig. 3a); accordingly, hereafter we refer to this antibody as an 208 sfSGK-neutralizing antibody. To determine whether sfSGK activation is required for the pH i increase 209 after 1-MA treatment, we injected a pH-sensitive fluorescent dye, BCECF-dextran, into unstimulated 210 oocytes along with the sfSGK-neutralizing antibody, and monitored the dynamics of pH i by 211 measuring fluorescence every 30 seconds before and after 1-MA addition. The sfSGK-neutralizing 212 antibody blocked the pH i increase, whereas the pH i of oocytes injected with control IgG increased 213 soon after 1-MA treatment (Fig. 3b). These results indicate that the pH i increase after 1-MA 214 treatment depends on activation of sfSGK. 215 216

Activation of sfSGK, but not pH i increase, is required for cyclin B-Cdk1 activation 217
Previously, we showed that GVBD is initiated by 1-MA in isolated oocytes even when the pH i 218 increase is blocked 16 . Nonetheless, to our surprise, GVBD was blocked by injection of the 219 sfSGK-neutralizing antibody, whereas control IgG had no inhibitory effects on GVBD (Fig. 4a, b). 220 When we checked the phosphorylation status of Cdc25 and Cdk1 of the oocytes injected with the 221 sfSGK-neutralizing antibody, we found that neither hyperphosphorylation of Cdc25 nor 222 dephosphorylation of Tyr15 of Cdk1 after 1-MA stimulus occurred (Fig, 4c), indicating that this 223 antibody blocked 1-MA-dependent signal transduction leading to cyclin B-Cdk1 activation. 224 To verify that the inhibitory effects of the antibody were caused by specific inhibition of 225 sfSGK activation, we next performed a rescue experiment. Specifically, we replaced Thr479 of 226 sfSGK with Glu (T479E), which was expected to mimic the negatively charged phosphate group, 227 thereby causing PDK1-dependent A-loop phosphorylation even in the presence of the neutralizing 228 antibody. We co-injected mRNA encoding the mutant sfSGK-T479E and the sfSGK-neutralizing 229 antibody into unstimulated oocytes. After a 17-h incubation, the mutant was expressed and partially 230 phosphorylated on its A-loop even in the absence of 1-MA (Fig. 4d). This partial activation did not 231 induce GVBD. 1-MA stimulation induced enhancement of A-loop phosphorylation on the mutant 232 sfSGK (Fig. 4d), as well as GVBD (Fig. 4e), whereas the A-loop phosphorylation and mobility shift 233 of endogenous sfSGK were blocked in these oocytes (Fig. 4d). On the basis of these findings, we 234 concluded that the antibody-injected oocytes are rescued by the additional expression of the mutant 235 sfSGK-T479E, and that the inhibitory effect of the antibody is highly specific. 236 Interestingly, oocytes expressing the T479E mutant underwent GVBD several minutes 237 faster than the control intact oocytes (Fig. 4e), suggesting that partial activation of the mutant in 238 unstimulated oocytes likely shortened the time to GVBD following 1-MA treatment, further 239 supporting the idea that sfSGK participates in 1-MA signaling leading to GVBD. We also found that 240 the band of the T479E mutant phosphorylated on the A-loop underwent an upward mobility shift 241 after 1-MA stimulation, indicating that the mutant was phosphorylated on a site outside the A-loop 242 and the HM; however, the function of this phosphorylation event remains unclear. Taken together, 243 these results suggest that activation of sfSGK is required for cyclin B-Cdk1 activation. 244 Although the 1-MA-induced pH i increase and cyclin B-Cdk1 activation were 245 simultaneously blocked by the sfSGK-neutralizing antibody, they were independent of each other. 246 We base this claim on two lines of evidence: 1) The increase in pH i occurred earlier than cyclin B-247 Cdk1 activation (Fig. 2a, 3b), and was not affected by treatment with the Cdk1 inhibitor, roscovitine 248 (Supplementary Fig. 1), indicating that the pH i increase is independent of cyclin B-Cdk1 activation. 249 2) GVBD in isolated oocytes, in which initial pH i before 1-MA stimulation is ~7.0, is initiated even 250 when the pH i increase was blocked by an NHE inhibitor or by treatment with sodium-free ASW 16 , 251 indicating that the cyclin B-Cdk1 activation leading to GVBD is independent of the pH i increase 252 (see also below, Fig. 5a). Thus, the pH i increase and cyclin B-Cdk1 activation are mutually 253 independent events. 254 255

B-Cdk1 activation 257
To elucidate the role of the pH i increase, we investigated effect of altered pH i on 1-MA-induced 258 meiotic resumption in starfish oocytes. To clamp pH i , oocytes were incubated in sodium-free ASW 259 containing CH 3 COONH 4 (modified ASW), in which the pH i was adjusted to the desired value with 260 CH 3 COONH 4 but did not increase after 1-MA stimulation because of the absence of sodium ion 17 261 (see also Materials & Methods). We clamped pH i at 6.7, corresponding to the value in unstimulated 262 ovarian oocytes 17 ; as well as 7.0 and 7.2, similar to the values in isolated oocytes before and after 263 1-MA stimulus, respectively 17 . First, we examined effects of pH i clamping on sfSGK and cyclin B-264 Cdk1 activation after 1-MA stimulus. Immunoblotting analysis showed that the phosphorylation 265 status of sfSGK, Cdc25, and Cdk1 in oocytes at all clamped pH i values was basically same as in 266 oocytes in ASW (Fig. 5a). This observation suggests that 1-MA-induced signaling leading to cyclin 267 B-Cdk1 activation does not depend on pH i , and further supports our conclusion that the pH i increase 268 is not required for cyclin B-Cdk1 activation. 269 After cyclin B-Cdk1 activation, the onset of GVBD was delayed for several minutes at a 270 clamped pHi of 6.7 (Fig. 5b) Supplementary Fig.  285 3). These observations suggest that progression of granule invasion is sensitive to pH i ; in particular, 286 completion of GVBD is drastically impaired at a pH i of 6.7. Given that the pH i of unstimulated 287 ovarian oocytes is around 6.7 (ref. 17 ), the pH i increase mediated by sfSGK is likely required for 288 completion of GVBD in ovarian oocytes. 289

Discussion 290
The results of this study show that sfSGK is activated by TORC2 and PDK1 in a PI3K-dependent 291 manner after 1-MA stimulus in starfish oocytes. We found that sfSGK is required for regulation of 292 two mutually independent pathways leading to pH i increase and cyclin B-Cdk1 activation. 293 Furthermore, we identified the presence of a pH i -sensitive process in meiotic resumption: although 294 1-MA signaling leading to cyclin B-Cdk1 activation is pH i -independent, the subsequent onset time 295 and completion of GVBD are pH i -dependent. 296 On the basis of previous studies and the present findings, we propose a model for meiotic 297 resumption in ovarian oocytes (Fig. 6). In this model, sfSGK exerts two essential functions: cyclin 298 B-Cdk1 activation, which induces meiotic resumption, and pH i increase, which promotes completion 299 of GVBD (Fig. 6). In living starfish, ovarian oocytes reside in coelomic fluid in the body cavity. 300 Previously, we reported that the coelomic fluid has relatively high CO 2 and low O 2 concentrations 301 relative to ASW 17 . Under these gas conditions, the pH i of unstimulated ovarian oocytes is around 6.7, 302 and increases after 1-MA stimulation 17 . As shown in Fig. 2c, sfSGK is activated in ovarian oocytes 303 after 1-MA stimulus in the body cavity. Subsequently, sfSGK likely induces the pH i increase and 304 cyclin B-Cdk1 activation in ovarian oocytes (Fig. 6), as demonstrated in isolated oocytes (Figs. 3  305 and 4). Thereafter, cyclin B-Cdk1 causes GVBD. However, if pH i in ovarian oocytes was not 306 increased from 6.7 soon after 1-MA stimulus, GVBD would initiate after a delay and fail to complete, 307 as we observed when pH i was clamped at 6.7 in isolated oocytes (Fig. 5). Thus, in ovarian oocytes, 308 the sfSGK-dependent pH i increase would determine the time of GVBD onset after cyclin B-Cdk1 309 activation and be a prerequisite for completion of GVBD (Fig. 6). 310 In the ovaries, starfish oocytes arrest at MI after GVBD 16,17,27 . This arrest is released upon 311 spawning, at which time the oocytes are ready to be fertilized 16,17,27 . Fertilization during the period 312 between release from the MI arrest and the first polar body formation is important for monospermy, 313 because insemination before GVBD or after polar body formation tends to result in polyspermy 27,50 . 314 Thus, the sfSGK-dependent pH i increase soon after 1-MA may ensure that ovarian oocytes proceed 315 to MI arrest before spawning, helping to promote monospermy. In addition, to ensure fertilization 316 before polar body formation, MI arrest must be maintained until spawning 27 . To maintain MI arrest, 317 sfSGK-dependent pH i increase should not exceed 7.0, because release from MI arrest occurs at 318 pH i >7.0 (ref. 16,17,26,27 ). In this study, A-loop phosphorylation of sfSGK became undetectable around 319 the time of the end of pH i increase (Fig. 2a, c). Therefore, sfSGK inactivation may be important to 320 stop the pH i increase for maintaining MI arrest. Interestingly, the A-loop was dephosphorylated even 321 in the presence of phosphorylated HM (Fig. 2a, c), implying the presence of regulatory mechanism 322 of A-loop dephosphorylation that is not associated with dephosphorylation of the HM, such as 323 up-regulation of an A-loop phosphatase. To our knowledge, regulation of SGK inactivation has never 324 been investigated in any animal, and this issue should be examined in a future study. 325 Although mammalian SGKs are involved in cell proliferation 51,52 , their function in 326 M-phase remains unclear. Here, we showed that sfSGK is required for cyclin B-Cdk1 activation in 327 starfish oocytes (Fig. 4). In parallel with the present study, Hiraoka et al. demonstrated that sfSGK 328 directly phosphorylates Cdc25 and Myt1 to activate cyclin B-Cdk1, and that sfSGK, rather than Akt, 329 is the major kinase responsible for induction of cyclin B-Cdk1 activation 53 . These are the first 330 reports to demonstrate the involvement of SGK in cyclin B-Cdk1 activation. 331 In addition, we showed that sfSGK is required for the pH i increase (Fig. 3b). Previously, 332 we reported that this increase depends on sfNHE3 16,17,25 . In human cultured cells, NHE-dependent 333 pH i increase is upregulated by phosphorylation of C-terminal region of NHE that leads to NHE 334 activation 7,9 , and/or by accumulation of NHE protein on the plasma membrane through trafficking of 335 endosomal NHE to the plasma membrane 6 . Although it is not known whether these mechanisms are 336 functional in starfish oocytes, it is clear from our previous work that the C-terminus of sfNHE3 is 337 phosphorylated by human SGK1 in vitro 25 . Therefore, we speculate that sfSGK upregulates sfNHE3 338 by direct phosphorylation of its C-terminus. 339 In the process of GVBD in starfish oocytes, the nuclear pore complex on nuclear envelopes 340 (NEs) of the GV are first disassembled 54-56 , and then the NEs are rapidly broken into membrane 341 fragments, followed by complete mixing of cytoplasm and nucleoplasm 54,55 . GVBD onset, as judged 342 by differential interference contrast (DIC) microscopy in this study, likely represents the time of 343 membrane fragmentation, as a clear boundary between cytoplasm and GV was still observed by DIC 344 during nuclear pore disassembly 55 . One possible interpretation of the remarkable blockage of GVBD 345 completion at a clamped pH i of 6.7 (Fig. 5b, c) is that incomplete NE fragmentation occurred and 346 disrupted invasion of cytoplasmic granules. Given that NE fragmentation depends on F-actin shell 347 formed on the inner surface of the GV 56 , our present observations imply that the formation or 348 function of this shell is blocked at pH i 6.7. Although this possibility is yet to be tested, F-actin 349 architecture is known to be disrupted by lower pH i , at least in mouse mammary tissue 57 . 350 pH i increase after release from ProI arrest has also been reported in frog 13 , urodele 58 , and 351 surf clam 12 . In Xenopus oocytes, NHE-dependent pH i increase after hormonal stimulation 13,14,59 has 352 been suggested to accelerate cyclin B-Cdk1 activation by promoting accumulation of Mos protein, 353 which participates in cyclin B-Cdk1 activation 60 . By contrast, we found that cyclin B-Cdk1 354 activation was independent of the pH i increase. One potential reason for this difference could be 355 cyclin B-Cdk1 activation in starfish oocytes does not require synthesis of new protein 61 , including 356 Mos 62 . In addition, pH i increase in Xenopus oocytes plays a role in migration of GV to the animal 357 pole before GVBD 63 . Such migration does not occur in starfish oocytes because the GV already 358 resides at the animal pole even in ProI-arrested oocytes. Instead, we found that completion of GVBD 359 depends on pHi. Thus, starfish and Xenopus oocytes provide suitable models for understanding 360 different aspects of pH i -dependent regulation of the meiotic cell cycles. 361 In summary, we showed that sfSGK is required for pH i increase and cyclin B-Cdk1 362 activation in starfish oocytes, and that completion of GVBD is a pH i -sensitive process. These 363 findings reveal a novel role for SGK in control of meiotic cell cycles and emphasize the importance 364 of tight regulation of pH i in oocyte meiosis. 365

Materials and methods 366
Oocyte preparation. Starfish (Asterina pectinifera) were collected on Pacific coast of Japan, and 367 kept in laboratory aquaria supplied with circulating seawater at 14°C. Fully-grown ProI-arrested 368 oocytes were isolated from ovaries and treated with calcium-free artificial seawater (calcium-free Because 3' and 5' rapid amplification of cDNA ends (RACE) with specific primers designed against 393 fragment 1 were unsuccessful, we obtained another fragment (fragment 2) that partially overlapped 394 with fragment 1, as follows: a forward specific primer (5'-CCCGACAAGCTCTAC-3') was 395 designed against fragment 1, and a reverse degenerate primer (GLPPFY, 396 5'-CCNRANGGNGGNAARAT-3') was designed against a conserved amino acid sequence in the 397 SGK alignment that is located in a region approximately 85 amino acids C-terminal to the region of 398 the first reverse degenerate primer. The 360-bp fragment 2 was obtained by RT-PCR using these 399 primers. Next, to identify the 3' end, another first-strand cDNA library was generated from the total 400 mRNA using RNA PCR Kit (AMV) Ver. 2.1 and Prime Script Reverse Transcriptase (Both, Takara 401 Bio; Shiga, JP) using the oligo dT-Adaptor primer from the kit. Then, RT-PCR was performed on the 402 cDNA library using a specific forward primer designed against the 5' region of fragment 2 403 (5'-CCCCAGAAGTCTTGAAGAA-3') and the adaptor primer (5'-GTTTTCCCAGTCACGAC-3'). 404 Using the RT-PCR product as a template, nested PCR was performed with another forward specific 405 primer (5'-GGAGTATGATCGTAGTGTAG-3') and the adaptor primer, resulting in amplification of 406 a ~600 bp 3' RACE product. Next, the 5' ends were identified by RACE using 5'-Full RACE Core 407 Set (Takara Bio; Shiga, JP) with specific primers (reverse for generating first-strand cDNA: CaCl 2 ), the pH i of oocytes was invariably ~0.2 units higher than pH o 17 . Hence, to clamp pH i of 513 oocytes at 6.7, 7.0, and 7.2, unstimulated oocytes were incubated with modified ASW with a pH of 514 6.5, 6.8, and 7.0, respectively, for 20 min. These oocytes were then treated with 1-MA, followed by 515 observation of GVBD or immunoblotting analysis. It should be noted that because sodium ion is not 516 present in modified ASW, the sfNHE3-dependent pH i increase mediated by Na + /H + exchange does 517 not occur, so pHi is maintained at the clamped value even after 1-MA treatment. As a control, 518 isolated oocytes were incubated in ASW (pH o 8.2), in which the pHi of unstimulated oocytes was 519 ~7.0. Because ASW contains sodium ions, a sfNHE3-dependent pH i increase to 7.2-7.3 occurs after 520 1-MA treatment 17 ; see also the control IgG-injected oocytes in Fig. 3b.  BCECF-dextran and either anti-sfSGK-HM antibody or control IgG were co-injected into 757 unstimulated oocytes. After a 1-h incubation, 1-MA was added, and the fluorescence intensity ratio 758 was measured every 30 seconds before and after 1-MA addition. pH i was calculated from the 759 fluorescence intensity ratio and plotted. Data represent means ± SE of three independent 760 experiments. 761 activation. In a manner that depends on PI3K, sfSGK is activated via phosphorylation of the A-loop 799 by PDK1 after prior phosphorylation of the HM by TORC2. Activation of sfSGK is essential for 800 cyclin B-Cdk1 activation and sfNHE3-dependent pH i increase. These SGK-dependent pathways are 801 pH i -independent (blue box). On the other hand, time to GVBD onset after cyclin B-Cdk1 activation 802 and completion of GVBD are pH i -dependent (yellow box), and both are defective at pH i of 6.7. 803 Because pH i of ovarian oocytes before 1-MA stimulation in body cavity is ~6.7, the 804 sfSGK-dependent pH i increase after 1-MA stimulus accelerates GVBD onset and is required for 805 completion of GVBD in ovarian oocytes. By contrast, the pH i of isolated oocytes before 1-MA 806 stimulation in ASW is ~7.0, which is already permissive for GVBD. Therefore, a pH i increase is not 807 essential for GVBD in isolated oocytes. 808