表皮生长因子通过间隙连接蛋白调控小鼠卵母细胞成熟的研究

Study on oocyte maturation regulation by epidermal growth factor through connexins in mice

目的探讨表皮生长因子(EGF)促进卵母细胞成熟的作用机制。方法采用逆转录-聚合酶链反应(RT-PCR)技术,检测性未成熟昆明白实验小鼠未成熟卵母细胞复合体(COCs)和成熟COCs中11种间隙连接蛋白(Cx)基因的表达情况;采用细胞免疫荧光实验,确定Cx43在未成熟/成熟COCs及卵母细胞中的表达部位;将未成熟COCs培养于0μg/L、1μg/L、10μg/L、50μg/L EGF培养基中观察其成熟率;将未成熟COCs培养于10μg/L EGF培养基中,观察0 h、4 h、8 h、12 h、16 h、20 h和24 h时间点的成熟率;将未成熟COCs和去颗粒细胞未成熟COCs分别培养于10μg/L EGF培养基中24 h,观察其成熟率;将未成熟COCs培养于10μg/L EGF培养基并分别加入0μg/L、0.1μg/L、1μg/L、10μg/L EGF受体(EGFR)抑制剂(AG),培养22~24 h后观察EGFR对EGF的作用;采用Western blot法检测未成熟COCs培养于10μg/L EGF培养基10 min、30 min、1 h和2 h后Cx43的磷酸化水平。结果RT-PCR结果显示,在小鼠未成熟/成熟COCs中Cx43和Cx45高表达。细胞免疫荧光实验结果显示,Cx43在未成熟COCs中表达于颗粒细胞的细胞膜上,在未成熟卵母细胞的细胞膜和透明带上也有高表达,且呈簇状分布;在成熟的COCs中Cx43在颗粒细胞膜上的表达量降低,在成熟卵母细胞中Cx43的表达量减少,且为均匀分布于细胞膜上,在透明带上不表达。培养于10μg/L EGF中的小鼠未成熟COCs的卵母细胞成熟率最高,达86.9%,显著高于其他不同浓度组(P<0.05)。在10μg/L EGF中培养24 h对小鼠未成熟COCs促进成熟的作用最显著,卵母细胞成熟率达86.2%,显著高于其他不同时长组(P<0.05)。将未成熟COCs去颗粒细胞后,培养于10μg/L EGF培养基中24 h后,未成熟COCs的卵母细胞成熟率没有显著变化(P>0.05)。EGFR特异性抑制剂AG能有效逆转EGF促进小鼠卵母细胞减数分裂恢复的作用,即抑制卵母细胞成熟,且1μg/L AG对EGF作用的逆转效果最显著(P<0.05)。Western blot结果显示,在未成熟COCs中,Cx43以非磷酸化形式为主;添加10μg/L EGF,培养10 min后,Cx43以磷酸化形式为主,持续至2 h时,Cx43又以磷酸化和非磷酸化两种形式存在。结论在小鼠未成熟COCs中,EGF可能通过与颗粒细胞上的EGFR结合,使Cx43快速磷酸化,促进卵母细胞减数分裂恢复和卵母细胞成熟。

Objective:To explore the mechanism of epidermal growth factor(EGF)promoting oocyte maturation.Methods:Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the gene expression of 11 kinds of gap junction proteins(Cx)in immature culumus-oocyte complex(COCs)and mature COCs of Kunming white mice with sexually immature.The expression site of Cx43 in immature/mature COCs and oocytes was determined by cell immunofluorescence experiment.Immature COCs were cultured in 0 g/L,1μg/L,10μg/L and 50μg/L of EGF medium to observe the maturity rate.Immature COCs were cultured in 10μg/L EGF medium,and the maturation rates at 0 h,4 h,8 h,12 h,16 h,20 h and 24 h were observed.Immature COCs and degranulated immature COCs were cultured in 10μg/L of EGF medium for 24 h,respectively,and their maturation rates were evaluated.Immature COCs were cultured in 10 ug/L EGF medium and 0μg/L,0.1μg/L,1μg/L and 10μg/L of EGFR inhibitor(AG)respectively for 22 to 24 h,and the effects of EGF on EGFR were observed.Western blotting was used to detect the phosphorylation level of Cx43 after immature COCs were cultured in 10μg/L of EGF medium for 10 min,30 min,1 h and 2 h.Results:RT-PCR results showed that Cx43 and Cx45 were highly expressed in immature and mature COCs of mice.The results of cell immunofluorescence experiment showed that Cx43 was expressed in granular cell membrane in immature COCs,and also highly expressed in oocyte membrane and zona pellucida and distributed in clusters.The expression of Cx43 in granulosa cells of mature COCs was lower than that in the former.In oocytes,the expression of Cx43 decreased with even distribution on the cell membrane,and was not expressed on the zona pellucida.The highest maturation rate for immature COCs in mice was observed up to 86.9%when cultured in 10μg/L of EGF(P<0.05).Immature mice COCs were cultured with 10μg/L EGF COCs 24 h,and the mature rate reached 86.2%(P<0.05).There was no significant difference in 24 h maturation rate between immature COCs without EGF and immature COCs with degranulation cells added with 10μg/L of EGF(P>0.05).EGFR-specific inhibitor AG can effectively reverse the recovery of oocyte meiosis promoted by EGF in mice,and 1μg/L AG had the best reversal effect(P<0.05).Western blot results showed that Cx43 was mainly in non-phosphorylated form in immature COCs.After 10μg/L of EGF was added,Cx43 was mainly in phosphorylated form after 10 min of culture.After 1 h and 2 h,Cx43 existed in both phosphorylated and non-phosphorylated forms.Conclusions:In immature COCs of mice,EGF combined with EGFR on granulosa cell makes Cx43 phosphorylate rapidly,resulting in meiosis recovery and oocyte maturation.