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

吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (02): 215-220.doi: 10.13481/j.1671-587x.20160205

14-3-3ε和Cdc25B在小鼠卵母细胞生发泡期阻滞中的作用

孟峻, 侯艳军, 张永梅, 呼格吉乐, 韩艳秋

内蒙古医科大学附属医院检验科, 内蒙古呼和浩特 010050

收稿日期:2015-07-10 发布日期:2016-03-31
通讯作者: 孟峻,主任检验师,硕士研究生导师(Tel:0471-6637610,E-mail:nmfrank@163.com) E-mail:nmfrank@163.com
作者简介:孟峻(1968-),男,内蒙古自治区乌兰察布市人,主任检验师,医学博士,主要从事生殖分子生物学方面的研究。
基金资助:
国家自然科学基金资助课题(81360109);内蒙古自然科学基金资助课题(2013MS1163)

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

MENG Jun, HOU Yanjun, ZHANG Yongmei, HU Gejile, HAN Yanqiu

Department of Clinical Laboratory, Affiliated Hospital, Inner Mongolia Medical University, Hohhot 010050, China

Received:2015-07-10 Published:2016-03-31

摘要/Abstract

摘要：

目的: 研究14-3-3ε和Cdc25B对小鼠卵母细胞生发泡(GV)期阻滞作用的影响,为阐明蛋白激酶A/Cdc25B/14-3-3ε通路在小鼠卵母细胞GV期阻滞中发育调控机制奠定基础。方法: 在体外将表达载体pcDNA3.1-ZEO-HA-14-3-3ε、pcDNA3.1-MYC-Cdc25B-WT、pcDNA3.1-MYC-Cdc25B-S321A和pcDNA3.1-MYC-Cdc25B-S321D转录成mRNA。超排卵方法获得小鼠GV期卵母细胞,实验分为未注射组、TE缓冲液注射组、14-3-3εmRNA单独注射组、14-3-3εmRNA+Cdc25B-WT(野生型)mRNA共注射组、14-3-3εmRNA+Cdc25B-S321D(模拟磷酸化型) mRNA共注射组、14-3-3εmRNA+Cdc25B-S321A(突变型)mRNA共注射组,收集各组母卵细胞后采用Western blotting法检测外源性14-3-3ε和Cdc25B蛋白表达及Cdc2-Tyr15的磷酸化状态;相差显微镜观察卵母细胞的形态学变化并计数生发泡破裂(GVBD)率。结果: 未注射组、TE缓冲液注射组、14-3-3εmRNA单独注射组、14-3-3εmRNA+Cdc25B-WT(野生型)mRNA共注射组、14-3-3εmRNA+Cdc25B-S321D(模拟磷酸化型) mRNA共注射组在显微注射后20h均未发生GVBD(P>0.05); 14-3-3εmRNA+Cdc25B-S321A(突变型)mRNA共注射组在显微注射后1、2和3h卵母细胞的GVBD率分别为(5.00±0.68)%、(62.00±3.56)%和(100.00±0.00)%,显微注射后20h到达第2次减数分裂中期(MII)的比例为(79.00±2.80)%,与未注射组和TE缓冲液注射组比较,GVBD率和到达MII的比例均显著升高(P<0.01)。结论: 14-3-3ε通过Cdc25B的321位丝氨酸磷酸化和脱磷酸化调控卵母细胞由GV期向GVBD的过渡。

关键词: 14-3-3&epsilon, Cdc25B, 卵母细胞, 生发泡, 生发泡破裂, 显微注射

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

中图分类号:

Q954.43

孟峻, 侯艳军, 张永梅, 呼格吉乐, 韩艳秋. 14-3-3ε和Cdc25B在小鼠卵母细胞生发泡期阻滞中的作用[J]. 吉林大学学报(医学版), 2016, 42(02): 215-220.

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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14-3-3ε和Cdc25B在小鼠卵母细胞生发泡期阻滞中的作用

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

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