Study on oocyte maturation regulation by epidermal growth factor through connexins in mice
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,1 μg/L,10 μg/L and 50 μg/L of EGF medium to observe the maturity rate.Immature COCs were cultured in 10 μg/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 10 μg/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 0 μg/L,0.1 μg/L,1 μg/L and 10 μg/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 10 μg/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 10 μg/L of EGF(P＜0.05).Immature mice COCs were cultured with 10 μg/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 10 μg/L of EGF(P＞0.05).EGFR-specific inhibitor AG can effectively reverse the recovery of oocyte meiosis promoted by EGF in mice,and 1 μg/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 10 μg/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.
国家科技期刊平台
NSTL
万方数据
维普
