The kinase cyclin B–Cdk1 complex is a master regulator of M-phase in both mitosis and meiosis. At the G2/M transition, cyclin B–Cdk1 activation is initiated by a trigger that reverses the balance of activities between Cdc25 and Wee1/Myt1 and is further accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently proposed to be the cyclin A–Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M transition, in which hormonal stimuli induce cyclin B–Cdk1 activation, cyclin A–Cdk1 is nonessential and hence the trigger remains elusive. Here, we show that SGK directly phosphorylates Cdc25 and Myt1 to trigger cyclin B–Cdk1 activation in starfish oocytes. Upon hormonal stimulation of the meiotic G2/M transition, SGK is activated by cooperation between the Gβγ-PI3K pathway and an unidentified pathway downstream of Gβγ, called the atypical Gβγ pathway. These findings identify the trigger in oocyte meiosis and provide insights into the role and activation of SGK.

Hosoda et al. show that SGK is required for an increase in intracellular pH (pHi) and cyclin B–Cdk1 activation upon meiotic resumption in starfish oocytes. The pHi increase is a prerequisite for chromosome transport and spindle assembly in ovarian oocytes.

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Oocyte maturation is a process wherein an oocyte arrested at prophase of meiosis I resumes meiosis to become a fertilizable egg. In starfish ovaries, a hormone released from follicle cells activates the oocytes, resulting in an increase in their intracellular pH (pHi), which is required for spindle assembly.Herein, we describe a protocol for pHi measurement in living oocytes microinjected with the pH-sensitive dye BCECF. For in vivo BCECF calibration, we treated oocytes with artificial seawater containing CH3COONH4 to clamp pHi, injected pH-standard solutions, and converted the BCECF fluorescence intensity ratios to pHi values. Of note, if the actual pHi is higher or lower than the known pH of injected standard solutions, the BCECF fluorescence intensity ratio will decrease or increase, respectively. On the other hand, the pH of the injected solution displaying no change in fluorescence intensity should be considered the actual pHi. These methods for pHi calibration and clamping are simple and reproducible.

12The kinase cyclin B-Cdk1 complex is a master regulator of M-phase in both mitosis and 13 meiosis. At the G2/M transition, cyclin B-Cdk1 activation is initiated by a trigger that 14 reverses the balance of activities between Cdc25 and Wee1/Myt1, and is further 15 accelerated by autoregulatory loops. In somatic cell mitosis, this trigger was recently 16proposed to be the cyclin A-Cdk1/Plk1 axis. However, in the oocyte meiotic G2/M 17 transition, in which hormonal stimuli induce cyclin B-Cdk1 activation, cyclin A-Cdk1 18 is non-essential and hence the trigger remains elusive. Here, we show that SGK directly 19 phosphorylates Cdc25 and Myt1 to trigger cyclin B-Cdk1 activation in starfish oocytes. 20After hormonal stimulation of the meiotic G2/M transition, SGK is activated by 21 cooperation between the Gβγ-PI3K pathway and an unidentified pathway downstream 22 of Gβγ, called the atypical Gβγ pathway. These findings identify the trigger in oocyte 23 meiosis and provide insights into the role and activation of SGK. 24 B-Cdk1, which is activated downstream of extracellular hormonal stimuli 16 ; hence this 49 is known as the meiotic G2/M transition. However, this transition has not been reported 50 to require cyclin A 16-21 . Furthermore, Plk1 14,22,23 and Aurora 24-26 are non-essential in 51 some oocytes. Thus, mechanisms other than the cyclin A-Cdk1/Plk1 axis likely trigger 52 cyclin B-Cdk1 activation at the meiotic G2/M transition in oocytes. 53In vertebrate oocytes, meiotic G2 arrest requires cAMP-dependent protein kinase 54 A (PKA), and downregulation of this kinase leads to meiotic G2/M transition 16,20,27,28 . 55In mice, PKA appears to directly upregulate Wee1B and downregulate Cdc25; however, 56 it remains unclear how downregulation of PKA reverses the balance between Wee1B 57 and Cdc25 activities to trigger cyclin B-Cdk1 activation 29 . In Xenopus, the hormone 58 progesterone stimulates protein synthesis of Moloney sarcoma oncogene (Mos) and 59 cyclin B, which redundantly trigger cyclin B-Cdk1 activation 30 . This redundant process 60 is possibly mediated in two manners: activating phosphorylation of Cdc25 by 61 Mos/mitogen activated protein kinase (MAPK) cascade, and inhibitory phosphorylation 62 of Myt1 by newly synthesized cyclin B-associated Cdk1 23 . However, the molecular link 63 between downregulation of PKA and de novo protein synthesis of Mos and cyclin B 64 remains elusive 31,32 . 65In contrast with vertebrate oocytes, the mechanisms by which activation of cyclin 66 B-Cdk1 is triggered at the meiotic G2/M transition have been well studied in 67 invertebrate starfish oocytes since the existence of a trigger kinase was first 68 reported 16,21,33 . In starfish, cAMP and PKA are likely not involved in meiotic G2 69 arrest 34 . The physiological maturation-inducing hormone 1-methyladenine (1-MeAde) 70induces the meiotic G2/M transition 35 without a requirement for new protein synthesis 36 . 71Cyclin A, Wee1, and Mos are not present, and Aurora and Plk are not required for 72 1-MeAde stimulation only whe...