ABSTRACT
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.