Effect Of Ezrin On In Vitro Maturation Of Ooctye In Mouse

Ezrin is a member of the ERM protein family,acts as a linker between the cotex cytoskeleton and plasma membrane of cells,participates in a variety of cellular functions,such as cell morphology,cell adhesion,cell migration and signal transduction.References have shown ERM proteins involved in mouse oocyte cortex of tension control and positioning of the spindle when second meiotic division.In the previous studies,we found that Ezrin and its phosphorylated protein in the mouse oocytes from GV-stage to MII,but its role in mouse oocyte maturation process actually is unclear.Here,four experiments were designed and carried out to investigate effect of ezrin on mouse denuded oocytes(DOs)maturation:1.Ezrin level in mouse GV-stage DOs microinjected Ezrin-siRNAMouse GV-stage DOs microinjected Ezrin-siRNA or NC-siRNA(negative control)were blocked in M2 medium containning 2.5 mM milrinone for 0 h,6 h,12 h,18 h,24 h,30 h and 36 h,respectively,then stained with ezrin-specific antibody and FITC-labeled secondary antibody.The average fluorescence value analysis was conducted with Image J software.The results showed that: during the period of 18 ~ 30 h after microinjection,experimental group Ezrin expression quantity was significantly lower than control group(P < 0.05).2.Effect of milrinone-blocking duration on the efficency of in vitro maturation of mouse GV-stage DOsAfter blocked in M2 mediumn containning 2.5 mM milrinone for 0 h(control group),6 h,12 h,18 h,24 h,30 h and 36 h,respectively,Mouse GV-stage DOs were transferred into M2 medium without milrinone to continue in vitro maturation,and maturation rate of each group Dos was compared.Results: there’s no significant difference in maturation rate between groups bolcked 18 h or less and control group(P > 0.05);however,the oocyte maturation rate of groups blocked 24 or even longer was significantly lower than control group(P < 0.05).3.Effects on in vitro maturation of mouse oocytes after knockdown EzrinMicroinjection Ezrin-siRNA or NC-siRNA(negative control)in DOs of GV of mouse,after block 18 h in milrinone continue to mature and develop,it is found that after knockdown Ezrin,regular mouse oocyte in vitro maturation rate was significantly lower than control group(P < 0.05);Cortical granule(cortical granule,CGs)anomaly distribution rate and MII spindle position abnormal rate is significantly higher than control group(P < 0.05);Surface microvilli length decreased significantly(P < 0.05),the density significantly lower(P < 0.01).4.Effects of Ezrin-knockdown on efficiency of mouse oocyte IVFExperimental group(GV injection of Ezrin-siRNA)and control group(GV injection of NC-siRNA)mouse MII oocytes were gained by the in vitro fertilization(IVF),the results showed that: compared with control group,GV of knockdown Ezrin mouse mature oocytes fertilization rate has dropped,but no significant difference(51.34±4.94%)(P > 0.05),but the experimental blastocyst rate significantly lower than that of control group(P < 0.01).