PLCγ1在小鼠卵母细胞减数分裂恢复中的作用

The role of PLCγ1 in mouse oocyte meiotic resumption

目的探究磷脂酶(PLC)Cγ1在卵母细胞减数分裂恢复中的作用及机制,进一步阐明卵母细胞成熟的分子机理,为与相关的人体临床应用提供理论基础。方法利用Real-time PCR检测PLC不同亚型在颗粒细胞与卵丘细胞中的表达情况,构建体外卵泡培养模型及COC培养模型研究PLCγ1特异抑制剂U73122对LH及EGF诱导的减数分裂恢复的影响,利用钙离子荧光探针Fluo 3-AM检测胞内钙离子水平变化,酶联免疫法检测胞内cGMP水平变化,利用Real-time PCR、Western blot以及免疫组化检测不同亚型1,4,5-三磷酸肌醇受体(IP 3R)的表达变化情况。结果研究表明,颗粒细胞与卵丘细胞中Plcγ1 mRNA的表达均显著高于其它亚型(P<0.05),U73122能够有效抑制LH和EGF诱导的卵母细胞减数分裂恢复(P<0.05)以及EGF诱导的卵丘细胞内钙离子水平升高(P<0.05),U73122还显著逆转了EGF介导的胞内cGMP水平下降(P<0.05),此外,研究还发现在颗粒细胞,卵丘细胞与卵母细胞中Itpr1 mRNA的表达量显著高于其它亚型(P<0.05),Western blot与免疫组化的结果也表明IP 3R1在三种细胞中均有表达。结论LH-EGFR信号通过PLCγ1-IP 3R1升高胞内钙离子水平诱导卵母细胞减数分裂恢复。

Objective To explore the effect and mechanism of PLCγ1 in mouse oocyte meiotic resumption and to provide a reference for its study and clinical application in humans.Methods Real-time PCR was used to detect the mRNA expression levels of PLC isoforms in granulosa cells and cumulus cells.Models of follicle culture and cumulus-oocyte complexes(COCs)culture in vitro were generated to study the effect of U73122(PLCγ1 specific inhibitor)on LH-and EGF-induced oocyte meiotic resumption.Changes in intracellular calcium levels were monitored using Fluo 3-AM,and cGMP levels were measured by ELISA.The expressions of IP 3R isoforms(mRNA and protein levels)were detected by Real-time PCR,Western blot,and immunohistochemistry.Results The expression of Plcγ1 mRNA levels in granulosa cells and cumulus cells was significantly higher than that of other isoforms(P<0.05).Furthermore,U73122 inhibited LH/EGF-induced oocyte meiotic resumption(P<0.05)and decreased EGF-induced calcium levels in cumulus cells(P<0.05).In addition,U73122 significantly reversed the decline of cGMP levels mediated by EGF(P<0.05).The expression of Itpr1 mRNA levels was significantly higher than other isoforms in granulosa cells,cumulus cells,and oocytes(P<0.05).Western blot and immunohistochemistry showed that IP 3R1 protein was expressed in all the three cell types.Conclusions LH-dependent EGF receptor signaling increases calcium levels in cumulus cells through PLCγ1-IP 3R1,which ultimately induces meiotic resumption.