14-3-3ε和Cdc25B在小鼠卵母细胞生发泡期阻滞中的作用

doi:10.13481/j.1671-587x.20160205

Abstract

Abstract:

Objective: To study the role of 14-3-3εand Cdc25B in germinal vesicle (GV)-stage arrest of mouse oocytes, and to pay foundation for further study on the molecular mechanism of PKA/Cdc25B/14-3-3εpathway in GV-stage arrest of mouse oocytes.Methods: The eukaryotic expression vectors of pcDNA3.1-ZEO-HA-14-3-3ε, pcDNA3.1-MYC-Cdc25B-WT, pcDNA3.1-MYC-Cdc25B-S321A, and pcDNA3.1-MYC-Cdc25B-S321D were transcribed into mRNA in vitro.The mouse GV-stage oocytes were collected after superovulation and divided into no injection group, TE buffer microinjection group, 14-3-3εmRNA injection group, 14-3-3εmRNAs+Cdc25B-WT mRNA injection group, and 14-3-3εmRNA+Cdc25B-S321A mRNA injection group, 14-3-3εmRNA+Cdc25B-S321D mRNA injection group.The protein expression levels of HA-14-3-3ε and MYC-Cdc25B and the phosphorylation status of Cdc2-pTyr15 were observed by Western blotting method.The morphological changes and germinal vesicle breakdown(GVDB) rates of mouse oocytes were observed under phase-contrast microscope.Results: None of the oocytes in no injection group, TE buffer microinjection group, 14-3-3εmRNA injection group, 14-3-3εmRNA+Cdc25B-WT mRNAs injection group and 14-3-3εmRNA+Cdc25B-S321D mRNA were able to undergo GVBD until at least 20 h after injection(P>0.05);the GVBD rates of oocytes in 14-3-3εmRNA+Cdc25B-S321A mRNA group at 1 h(5.00%±0.68%), 2 h (62.00%±3.56%) and 3 h(100.00%±0.00%) after injection were significantly higher than those in no injection group and TE buffer injection group(P<0.01);the oocytes in 14-3-3ε mRNA+Cdc25B-Ser321A mRNA group at 20 h(79.00%±2.80%) after injection progressed to MII(P<0.01).Conclusion: 14-3-3ε can regulate the transition from GV to GVBD of mouse oocytes by means of phosphorylation and dephosphorylation of S321-Cdc25B.

Key words: 14-3-3ε, Cdc25B, oocytes, germinal vesicle, germinal vesicle breakdown, microinjection

CLC Number:

Q954.43

MENG Jun, HOU Yanjun, ZHANG Yongmei, HU Gejile, HAN Yanqiu. Role of 14-3-3εand Cdc25B in GV-stage arrest of mouse oocytes[J].Journal of Jilin University Medicine Edition, 2016, 42(02): 215-220.

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Role of 14-3-3εand Cdc25B in GV-stage arrest of mouse oocytes

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