短链脂肪酸对体外氧化应激诱导的卵母细胞成熟障碍的保护作用

Protective effect of short-chain fatty acids on oocyte maturation disorder induced by oxidative stress in vitro

目的短链脂肪酸(SCFAs)对卵母细胞体外成熟的作用及其可能的机制尚不明确。文中探讨短链脂肪酸对小鼠卵母细胞体外诱导氧化应激后发育成熟的影响及其作用机制。方法收集6~8周龄ICR雌鼠生发泡(GV)期的卵母细胞,分为对照组(不干预)、H_(2)O_(2)处理组(50μmol/L H_(2)O_(2)处理1.5 h)、H_(2)O_(2)+乙酸组(50μmol/L H_(2)O_(2)处理1.5 h后,1 mmol/L乙酸干预)、H_(2)O_(2)+丙酸组(50μmol/L H_(2)O_(2)处理1.5 h后,10μmol/L丙酸干预)和H_(2)O_(2)+丁酸组(50μmol/L H_(2)O_(2)处理1.5 h后,2μmol/L丁酸干预)。并于培养3 h后观察生发泡破裂(GVBD)率,于培养16 h后观察第一极体排出(PBE)率;免疫荧光化学染色法检测体外培养9 h进入第一次减数分裂中期(MI期)和16 h排出第一极体进入第二次减数分裂中期(MII期)的卵母细胞内染色体的排列和纺锤体的形态;Western blot检测不同处理组Pi3k/Akt通路相关蛋白表达量。结果成熟率结果显示,与对照组GVBD率[(86.87±1.76)%]和PBE率[(85.37±1.98)%]相比,H_(2)O_(2)处理组[(66.73±1.25)%、(50.57±4.22)%]明显下降(P<0.05),而与H_(2)O_(2)处理组相比,H_(2)O_(2)+乙酸组[(80.67±2.62)%、(78.07±1.78)%],H_(2)O_(2)+丙酸组[(74.50±2.91)%、68.03±1.84)%]和H_(2)O_(2)+丁酸组[(75.93±2.12)%、(66.40±3.46)%]卵母细胞的成熟率显著改善(P<0.01)。免疫荧光结果显示,在添加了SCFAs的M2培养液中成熟的卵母细胞减数分裂的超微结构较H_(2)O_(2)处理组明显改善,主要体现在染色体排列和纺锤体形态的改变。Western blot结果显示,H_(2)O_(2)处理组卵母细胞中的p-Akt和Akt蛋白表达量的比值较对照组明显增多(P<0.01),而H_(2)O_(2)+乙酸组和H_(2)O_(2)+丙酸组较H_(2)O_(2)处理组明显降低(P<0.01)。结论在体外培养液中添加短链脂肪酸可以改善氧化应激后小鼠卵母细胞的发育潜能,为改善体外氧化应激诱导的卵母细胞成熟障碍提供新思路。

Objective The role of short-chain fatty acids(SCFAs)on oocyte maturation in vitro and their possible mechanisms are not clear.To investigate the effect and underlying mechanism of SCFAs on compromised maturation of mouse oocytes induced by in vitro oxidative stress(OS).Methods 6-8-week-old female ICR mice were used to collect oocytes.Oocytes at germinal vesicle(GV)stage were isolated and released into M2 medium.They were divided into control group(no intervention),H_(2)O_(2)treatment group(50μmol/L H_(2)O_(2)treatment for 1.5 h),H_(2)O_(2)+acetic acid group(50μmol/L H_(2)O_(2)treatment for 1.5 h,1 mmol/L acetic acid intervention),H_(2)O_(2)+propionic acid group(50μmol/L H_(2)O_(2)treatment for 1.5 h,10μmol/L propionic acid intervention)and H_(2)O_(2)+butyric acid group(50μmol/L H_(2)O_(2)treatment for 1.5 h,2μmol/L butyric acid intervention).Oocytes were cultured for 3 h to evaluate the ratio of germinal vesicle breakdown(GVBD)and 16 h to evaluate the ratio of polybody exclusion(PBE).For immunofluorescence analysis,GV oocytes were cultured for 9 h to metaphase I(MI)and 16 h to metaphase II(MII)in vitro,and then the chromatin and spindle structures were analyzed.For Pi3K/Akt pathway analysis,the expression of relative proteins were detected by Western blot.Results Compared with the control group,the GVBD rate(66.73±1.25)and PBE rate(50.57±4.22)of oocytes in the H_(2)O_(2)-treated group were significantly decreased,while the maturation rate of oocytes in the H_(2)O_(2)+acetic acid group(80.67±2.62,78.07±1.78),H_(2)O_(2)+propionic acid group(74.50±2.91,68.03±1.84)and H_(2)O_(2)+butyric acid group(75.93±2.12,66.40±3.46)were significantly improved compared with the H_(2)O_(2)-treated group(P<0.01).Immunofluorescence results showed that SCFAs significantly rescued impaired meiotic structures of oocytes in H_(2)O_(2)-treated group.The protein level of p-Akt was significantly increased in H_(2)O_(2)-treated group,which was reversed after further treatment of acetic acid and propionic acid(P<0.01).Conclusion SCFAs improve the compromised maturation of mouse oocytes induced by in vitro OS,which provide new solutions for improving OS-related impairment in oocyte maturation.