14-3-3ε蛋白在小鼠卵母细胞减数分裂恢复过程中对Cdc25B定位的影响

目的：探讨14-3-3ε蛋白在小鼠卵母细胞减数分裂恢复过程中对Cdc25B定位的影响，为阐明14-3-3ε蛋白对小鼠卵母细胞发育的调控机制奠定基础。方法：采用超排卵方法获得3周龄昆明系雌性小白鼠生发泡（GV）期卵母细胞分为未注射组、control siRNA注射组和14-3-3εsiRNA注射组。构建pmax-FP-Red-HA-14-3-3ε表达载体。间接免疫荧光技术检测14-3-3ε蛋白和Cdc25B在小鼠卵母细胞中的定位；直接免疫荧光技术观测14-3-3ε蛋白和Cdc25B蛋白在小鼠卵母细胞中的亚细胞定位；利用显微注射方法将14-3-3εsiRNA注射入GV期卵母细胞中，相差显微镜下观测卵母细胞的形态表现，计算卵母细胞的生发泡破裂（GVBD）发生率；Western blotting法检测卵母细胞中14-3-3ε蛋白的表达和Cdc2-pTyr15蛋白的相对表达水平；放射自显影检测卵母细胞中成熟促进因子（MPF）的活性。结果：间接和直接免疫荧光实验，野生型Cdc25B能与14-3-3ε蛋白共定位于细胞质；在GVBD前期，Cdc25B由胞浆穿梭进入细胞核。当Cdc25B蛋白的第321位丝氨酸突变为丙氨酸时，14-3-3ε蛋白的表达水平降低，Cdc25B的胞浆定位被取消。未注射组、control siRNA注射组卵母细胞在显微注射24 h后均未发生GVBD，GVBD发生率组间比较差异无统计学意义（P>0.05）；14-3-3εsiRNA注射组在显微注射后22和24 h卵母细胞的GVBD发生率均高于未注射组和control siRNA注射组（P<0.01），显微注射后24 h卵母细胞到达第2次减数分裂中期（MⅡ）的比例高于未注射组和control siRNA注射组（P<0.01）。结论：在小鼠卵母细胞减数分裂恢复过程中，Cdc25B的Ser321位点可能是14-3-3ε蛋白调控Cdc25B细胞内定位的具体作用位点。

Effect of 14-3-3ε protein on localization of Cdc25B during meiotic resumption of mouse oocytes

Objective:To explore the effect of 14-3-3εprotein on the localization of Cdc25B protein during the meiotic resumption of mouse oocytes,and to pay foundation for the further study on the molecular mechanism of 14-3-3εprotein in regulating the development of mouse oocytes.Methods:The Kunming genealology female mice aged 3 weeks were used to obtain the germinal vesicle (GV)-stage oocytes after superovulation.The GV-stage oocytes were divided into non-injection group,control siRNA injection group and 14-3-3εsiRNA injection group. The pmax-FP-Red-HA-14-3-3εexpression vector was constructed.Indirect immunofluorescence was used to observe the colocalization of 14-3-3εprotein and Cdc25B protein in the mouse oocytes;direct immunofluorescence was used to observe the subcellular localization of 14-3-3εprotein and Cdc25B protein in the mouse oocytes;14-3-3εsiRNA was microinj ected into the GV-stage oocytes;the morphology was observed under phase-contrast microscope;the germinal vesicle breakdown (GVDB)rates of the mouse oocytes were calculated;the expression level of 14-3-3εprotein and the relative expression level of Cdc2-pTyr15 protein were observed by Western blotting method;the matuation-promoting factor (MPF)activity in the oocytes was measured by autoradiography.Results:The indirect immunofluorescence and direct immunofluorescence results showed that the 14-3-3εprotein and wild Cdc25B protein were co-localized in the cytoplasm;Cdc25B was translocated from the cytoplasm to the nucleus shortly before GVBD.When the Ser321 of Cdc25B protein turned into Ala,the expression level of 14-3-3εprotein was decreased. None of the oocytes in non-injection group and control siRNA injection group were able to undergo GVBD until at least 24 h after injection,there was no significant differences in the rate of GVBD between non-injection group and control siRNA injection group (P>0.05);the GVBD rates of oocytes in 14-3-3εsiRNA injection group at 22 and 24 h after injection were significantly higher than those in non-injection group and control siRNA injection group (P<0.01);the rate of oocytes progressed to metaphaseⅡ (MII)in 14-3-3εsiRNA injection group at 24 h after injection was significantly higher than those in non-injection group and control siRNA injection group (P<0.01). Conclusion:Ser321 might be involved in the process of regulating the subcellular localization of Cdc25B by 14-3-3εprotein in the meiotic resumption of mouse oocytes.