Hippo信号通路核心蛋白YAP在小鼠卵母细胞减数分裂中的作用  

Role of Hippo signaling pathway core effector YAP in meiosis of mouse oocytes

作　　者：崔秋盈 马雪山 姚桂东[1] 路新乐 毕钡钡 胡琳莉[1] CUI Qiu-ying;MA Xue-shan;YAO Gui-dong;LU Xin-le;BI Bei-bei;HU Lin-li(Reproductive Medicine Center,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052)

机构地区：[1]郑州大学第一附属医院生殖医学中心,郑州450052

出　　处：《生殖医学杂志》2022年第8期1111-1118,共8页Journal of Reproductive Medicine

基　　金：国家重点基础研究发展计划(2019YFA0110903)。

摘　　要：目的 探究Hippo信号通路的核心蛋白YAP对小鼠卵母细胞减数分裂过程的影响及其可能的机制。方法 收集5周龄雌性小鼠的生发泡(GV)期卵母细胞并随机分为3组进行相应处理,分别为对照组(仅使用M2培养液培养)、抑制剂组(使用含有1μmol/L Verteporfin的M2培养液培养)和激动剂组(使用含有6μmol/L XMU-MP-1的M2培养液培养),20枚/组。于体外培养4 h和12 h后在体视镜下观察统计各组生发泡破裂(GVBD)率和第一极体排出率,并使用实时定量PCR技术检测各组卵母细胞中Hippo信号通路主要成分Yap1、Lats1和Tead1的mRNA表达水平,采用免疫荧光技术分析各组卵母细胞内YAP蛋白的分布情况和对纺锤体、微丝帽等结构的影响。结果 Yap1 mRNA在减数分裂的各个时期都有表达并且表达水平逐渐上升,Lats1 mRNA在GV、GVBD、MⅠ期表达量偏低,到了MⅡ期表达量显著增高(P<0.05),而Tead1 mRNA在GV期和GVBD期没有表达,到MⅠ、MⅡ期才逐渐开始表达。YAP蛋白在GV期定位于细胞质内,细胞核中没有YAP蛋白的分布。在随后的GVBD、MⅠ和MⅡ期中,YAP蛋白均匀地分布在卵母细胞胞质中;体外培养12 h后,抑制剂组与对照组相比第一极体排出率相近,而激动剂组与对照组相比第一极体排出率显著下降(P<0.05);并且随着XMU-MP-1浓度的增加,卵母细胞第一极体排出率逐渐下降。免疫荧光实验结果显示,激动剂组卵母细胞在第一次减数分裂中期染色体排列异常,纺锤体缩短变形,同时卵母细胞膜上未能形成正常的微丝帽结构。结论 YAP蛋白的活性增强会破坏小鼠卵母细胞的纺锤体结构和阻碍微丝帽的形成,进而导致其阻滞于第一次减数分裂中期。Objective:To investigate how YAP protein,the key component of Hippo signaling pathway,regulates the meiosis of mouse oocytes and its mechanism.Methods:The germinal vesicle(GV)oocytes of mice were collected and randomly divided into 3 groups:the control group(cultured with M2 medium only),the inhibitor group(cultured with M2 medium containing 1μmol/L Verteporfin)and the agonist group(cultured with M2 medium containing 6μmol/L XMU-MP-1),20 oocytes in each group.After oocytes were cultured in vitro for 4 hours and 12 hours,the germinal vesicle breakdown(GVBD)rate and first polar body extrusion(PBE)rate were observed under a stereoscope.RT-qPCR was used to detect the mRNA expressions of Yap1,Lats1 and Tead1 of the Hippo signaling pathway in oocytes.Immunofluorescence technique was used to analyze the location of YAP protein in oocytes and its effect on the structure of spindles and actin caps.Results:Yap1 mRNA was expressed throughout all phases of meiosis(GV,GVBD,MⅠand MⅡphases),and the expression level gradually increased.The expression of Lats1 mRNA was low at GV,GVBD and MⅠphases,and significantly increased at MⅡphase(P<0.05).Tead1 mRNA was not expressed in GV and GVBD phases,and gradually began to be expressed at MⅠand MⅡphases.YAP protein was localized in cytoplasm at GV phase,and there was no distribution of YAP protein in the nucleus.In the subsequent GVBD,MⅠand MⅡphases,YAP protein was evenly distributed in the cytoplasm of oocytes.After 12 hours culture in vitro,the PBE rate in the inhibitor group was similar to that in the control group,while the PBE rate in the agonist group was significantly lower than that in the control group(P<0.05).With the increase of XMU-MP-1 concentration,the PBE rate decreased gradually.Immunofluorescence assay showed that the oocytes in the agonist group had abnormal chromosome arrangement at the first meiotic metaphase,the spindle was shortened and deformed,and the normal actin caps structure were not be formed on the oocyte membrane.Conclusions:Enhanced activ

关 键 词：卵母细胞 减数分裂 Hippo信号通路 YAP蛋白 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]