Abstract
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 become fertilizable. At this time, the pHi increases via Na+/H+ exchanger activity, although the regulation and function of this change remain obscure. Here, we show that in starfish oocytes, serum- and glucocorticoid-regulated kinase (SGK) is activated via PI3K/TORC2/PDK1 signaling after hormonal stimulus and that SGK is required for this pHi increase and cyclin B-Cdk1 activation. When we clamped the pHi at 6.7, corresponding to the pHi of unstimulated ovarian oocytes, hormonal stimulation induced cyclin B-Cdk1 activation; thereafter, oocytes failed in actin-dependent chromosome transport and spindle assembly after germinal vesicle breakdown. Thus, this SGK-dependent pHi increase is likely a prerequisite for these events in ovarian oocytes. We propose a model that SGK drives meiotic resumption via concomitant regulation of the pHi and cell cycle machinery.
Highlights
Intracellular pH is tightly regulated in living cells
Phosphoinositide-dependent kinase 1 (PDK1) interacts with the phosphorylated hydrophobic motif (HM) and phosphorylates the A-loop[41], resulting in full activation of SGK35,36,42,43. It is not known whether serum- and glucocorticoid-regulated kinase (SGK) is activated in starfish oocytes, we previously showed that the C-terminus of starfish NHE3 (sfNHE3) has a putative consensus sequence for phosphorylation by SGK, and can be phosphorylated by recombinant human SGK1 in vitro[25]
When we checked the phosphorylation status of Cdc[25] and Cdk[1] of the oocytes injected with the starfish SGK (sfSGK)-neutralizing antibody, we found that neither hyperphosphorylation of Cdc[25] nor dephosphorylation of Tyr[15] of Cdk[1] after 1-MA stimulus occurred (Fig, 4c), indicating that this antibody blocked 1-MA–dependent signal transduction leading to cyclin B–Cdk[1] activation
Summary
Intracellular pH (pHi) is tightly regulated in living cells. Increases in pHi are required for a variety of physiological and pathological processes, including early embryonic development[1,2,3] and cancer cell survival[4]. A key pHi regulator, the sodium–proton exchanger (NHE) is a 12-transmembrane protein that increases pHi by exporting intracellular H+ and importing extracellular Na+ Upon extracellular stimuli by maturation-inducing hormones, ovarian oocytes resume the meiotic cell cycle via activation of cyclin B–Cdk[1] and subsequently undergo germinal vesicle breakdown (GVBD)[19,20,21]. Oocytes undergo another arrest at the metaphase of meiosis I or II (MI or MII arrest) that enables successful fertilization[22]. The upstream signaling and function of this pHi increase remain elusive
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