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 invitro.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-14-3-3εand 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 14-3-3εmRNA+ Cdc25B-Ser321A mRNA group at 20 h (79.00%±2.80%)after injection progressed to MII (P<0.01).Conclusion:14-3-3εcan regulate the transition from GV to GVBD of mouse oocytes by means of phosphorylation and dephosphorylation of S321-Cdc25B.%目的：研究14-3-3ε和Cdc25 B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／Cdc25 B／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-Cdc25 B-S321 D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＋Cdc25 B-WT （野生型） mRNA共注射组、14-3-3εmRNA ＋Cdc25 B-S321 D （模拟磷酸化型）mRNA共注射组、14-3-3εmRNA＋Cdc25 B-S321 A （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＋Cdc25 B-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20 h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3 h 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20 h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Cdc25 B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。
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