关键词: 亚细胞定位, 14-3-3ε, Cdc25B, 卵母细胞

Abstract: Objective:To explore the effect of 14-3-3ε protein 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 14-3-3ε siRNA injection group.The pmax-FP-Red-HA-14-3-3ε expression vector was constructed. Indirect immunofluorescence was used to observe the colocalization of 14-3-3ε protein and Cdc25B protein in the mouse oocytes; direct immunofluorescence was used to observe the subcellular localization of 14-3-3ε protein and Cdc25B protein in the mouse oocytes;14-3-3ε siRNA was microinjected 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 14-3-3ε protein 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 14-3-3ε protein 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 14-3-3ε protein 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 14-3-3ε siRNA 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 14-3-3ε siRNA 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 14-3-3ε protein in the meiotic resumption of mouse oocytes.

Key words: 14-3-3ε, oocyte, subcellular localization, Cdc25B