小鼠卵母细胞体内成熟过程中CDK1蛋白及其mRNA的动态变化

Dynamic changes of CDK1 protein and mRNA levels during oocyte maturation in vivo in mouse

目的 探讨小鼠卵母细胞体内成熟过程中细胞周期蛋白依赖性激酶-1(CDK1)蛋白和CDK1 mRNA表达的动态变化。方法 将6~8周龄SPF级ICR雌性小鼠68只随机分为4组,根据获取卵母细胞的时期分为GV组、GVBD组、MⅠ组和MⅡ组,每组17只。腹腔注射10 U孕马血清促性腺激素(PMSG)48 h后,颈椎脱臼处死法处死小鼠,获取GV期卵母细胞;注射PMSG 48 h后注射10 U人绒毛膜促性腺激素(HCG),分别于注射HCG后4 h、8 h和12 h后获取GVBD、MⅠ和MⅡ期卵母细胞。Western blot检测各阶段卵母细胞CDK1蛋白的表达,实时荧光定量PCR(qRT-PCR)检测各阶段卵母细胞中CDK1 mRNA的表达,并采用Spearman相关分析检验CDK1 mRNA和蛋白表达的相关性。结果 Western blot结果显示,CDK1蛋白在GV、GVBD、MⅠ和MⅡ期卵母细胞均有表达,且随着卵母细胞的体内成熟,CDK1蛋白表达量持续增加(P<0.05)。qRT-PCR结果显示,CDK1 mRNA在GV、GVBD、MⅠ和MⅡ期卵母细胞均有表达;与GV期比较,GVBD期和MⅠ期CDK1 mRNA表达水平均显著下调(P<0.05),而MⅡ期CDK1 mRNA表达水平显著上调(P<0.05)。Spearman相关分析显示,卵母细胞体内成熟过程中CDK1蛋白与CDK1 mRNA表达的相关性较差(R=0.200)。结论 卵母细胞体内成熟过程中CDK1蛋白的增加并不完全是由转录水平的变化引起的,其他一些机制可能在转录后水平上影响CDK1蛋白的翻译。本研究为进一步深入探讨卵母细胞成熟的分子机制奠定了一定基础。

Objective:To investigate dynamic changes of the levels of protein and mRNA of cyclin dependent kinase-1(CDK1)during oocyte maturation in vivo in mouse.Methods:Sixty-eight SPF grade ICR female mice aged 6-8 weeks were randomly divided into 4 groups.According to the period of oocyte retrieved,they were divided into GV group,GVBD group,MⅠgroup and MⅡgroup,with 17 mice in each group.The mice were killed by cervical dislocation method 48 hours after intraperitoneal injection of 10 U pregnant horse serum gonadotropin(PMSG)to obtain GV stage oocytes.The mice were injected 10 U human chorionic gonadotropin(HCG)48 hours after PMSG injection,GVBD,MⅠand MⅡoocytes were obtained respectively at 0,4,8 and 12 hours after HCG injection.Western blot was used to detect the expression of CDK1 protein in oocytes at each stage,and qRT-PCR was used to detect the expression of CDK1 mRNA in oocytes at each stage.Spearman was used for correlation analysis.Results:Western blot showed that CDK1 protein was expressed in oocytes at GV,GVBD,MⅠand MⅡstage,and CDK1 protein expression increased continuously with oocytes maturation in vivo(P<0.05).The results of qRT-PCR showed that CDK1 mRNA was expressed in oocytes at GV,GVBD,MⅠand MⅡstage,and compared with the GV stage,the mRNA expression was down-regulated significantly in oocytes at GVBD and MⅠstage(P<0.05),while the expression of CDK1 mRNA in MⅡstage was significantly up-regulated(P<0.05).Spearman correlation analysis indicated that CDK1 protein expression was poor correlated with CDK1 mRNA expression during oocytes maturation in vivo(R=0.200).Conclusions:The increase in CDK1 protein is not a consequence of change CDK1 transcription levels,and the other mechanisms might affect CDK1 protein expression at the post-transcriptional level.This study is helpful to further understand the molecular mechanism of oocyte maturation.