Ezrin对小鼠卵母细胞体外成熟的影响

发布时间：2018-03-01 01:27

  本文关键词： 小鼠 Ezrin 卵母细胞 体外成熟　出处：《安徽农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：Ezrin是ERM蛋白家族一员,在多种细胞中桥接细胞骨架与质膜,参与细胞形态维持、细胞粘附、细胞迁移及信号转导等多种细胞功能。有研究表明该家族蛋白参与了小鼠卵母细胞皮层紧张度的调控及第二次成熟分裂时纺锤体的定位。我们前期的研究发现,小鼠GV期到MII期的卵母细胞均有Ezrin及其磷酸化修饰的活性形式存在,但其在小鼠卵母细胞成熟过程究竟发挥什么作用尚不清楚。本试验以小鼠GV期裸卵为材料,通过显微注射特异性siRNA敲低Ezrin的方法研究了该蛋白在小鼠卵母细胞发育成熟中的作用以及对受精后胚胎发育潜力的影响。研究内容与结果如下:1.显微注射Ezrin-siRNA后不同时间小鼠GV期裸卵内Ezrin的表达水平向小鼠GV期裸卵显微注射Ezrin-siRNA或NC-siRNA(阴性对照),在含Milrinone(抑制细胞核分裂)M2中分别抑制0 h、6 h、12 h、18 h、24 h、30h和36 h后,用Ezrin特异性抗体进行荧光染色并用Image J软件分析所获图片的平均荧光值。结果发现:在显微注射后18~30 h期间,试验组Ezrin表达量显著低于对照组(P0.05)。2.Milrinone阻滞不同时间对小鼠GV期裸卵体外成熟率的影响将小鼠GV期裸卵在Milrinone培养液中分别阻滞0 h(对照组)、6 h、12 h、18 h、24 h、30 h和36 h后,统计各组卵母细胞成熟率(以排出PBI计)。结果显示,阻滞时间≤18 h,各组卵母细胞成熟率与对照组无显著差异(P0.05);阻滞时间≥24 h,则卵母细胞成熟率显著低于对照组及阻滞时间≤18 h的各试验组(P0.05)。3.敲低Ezrin对小鼠卵母细胞细胞体外成熟的影响向小鼠GV期裸卵中显微注射Ezrin-siRNA或NC-siRNA(阴性对照)后,Milrinone阻滞18 h再继续成熟培养,结果发现:敲低Ezrin蛋白后,小鼠卵母细胞体外成熟率显著低于对照组(P0.05);皮层颗粒(cortical granule,CGs)异常分布率及MII期纺锤体位置异常率显著高于对照组(P0.05);表面微绒毛长度显著降低(P0.05),密度极显著降低(P0.01)。4.敲低Ezrin后对小鼠卵母细胞IVF的影响分别取试验组(GV期注射Ezrin-siRNA)和对照组(GV期注射NC-siRNA)所获得的小鼠MII期卵母细胞进行体外受精(IVF),结果发现:与对照组相比,GV期敲低Ezrin导致小鼠成熟卵母细胞受精率有下降(51.34±4.94%),不过差异不显著(P0.05),但试验组的囊胚率极显著低于对照组(P0.01)。
[Abstract]:Ezrin is a member of ERM protein family, bridging cytoskeleton and plasma membrane in a variety of cells, participating in cell morphology maintenance and cell adhesion. Several cell functions such as cell migration and signal transduction. Some studies have shown that the family proteins are involved in the regulation of mouse oocyte cortical tension and the localization of the spindle during the second maturation and division. The active forms of Ezrin and phosphorylation were present in mouse oocytes from GV phase to MII stage, but it was not clear what role they played in the maturation process of mouse oocytes. The role of the protein in mouse oocyte maturation and its effect on embryo development potential after fertilization were studied by microinjection of specific siRNA knockdown Ezrin. The contents and results of the study are as follows: 1. After microinjection of Ezrin-siRNA, the effect of the protein on the development and maturation of mouse oocytes was studied. At the same time, the expression level of Ezrin in the nude eggs of mice at GV stage was microinjected with Ezrin-siRNA or NC-siRNA (negative control group) into the nude eggs of GV phase, and was inhibited in Milrinone- containing (inhibiting nuclear mitotic M 2) for 0 h, 6 h, 12 h, 18 h, 18 h, 24 h, 30 h and 36 h, respectively. Ezrin specific antibody was used for fluorescence staining and Image J software was used to analyze the average fluorescence value of the images. The results showed that: during 1830 h after microinjection, The expression of Ezrin in the experimental group was significantly lower than that in the control group (P 0.05). 2. The effect of Milrinone on the maturation rate of mouse GV naked eggs at different time. The mice GV naked eggs were blocked in Milrinone medium for 0 h (control group 6 h, 12 h, 18 h, 18 h, 24 h) for 30 h and 36 h, respectively. The maturation rate of oocytes in each group was calculated by excretion of PBI. When the block time was less than 18 h, the maturation rate of oocytes in each group was not significantly different from that in the control group (P0.05), but the maturation rate of oocyte was significantly lower than that of the control group and the control group (鈮，

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