摘要
目的:探究体外成熟(in vitro maturation,IVM)培养液中添加烟酸(niacin,NA)能否改善老龄小鼠卵母细胞质量及其机制。方法:以8周龄C57BL/6J小鼠为年轻对照组,40周龄小鼠为老龄组,取生发泡(germinal vesicle,GV)期卵母细胞,分别添加100、200、300μmol/L NA,观察二细胞和囊胚形成率以确定最适作用浓度。通过第一极体排出(first polar body extrusion,PBE)率、皮质颗粒(cortical granule,CG)分布、纺锤体形态和染色体排列异常比例评估卵母细胞质量。采用免疫荧光分析线粒体膜电位和三磷酸腺苷(adenosine triphosphate,ATP)生成水平来评估线粒体功能;通过检测卵母细胞内和线粒体内活性氧(re⁃active oxygen species,ROS)水平评估氧化应激水平。采用Western blot检测氧化应激相关蛋白:沉默信息调节因子相关酶3(si⁃lent information regulator 3,SIRT3)、超氧化物歧化酶2(superoxide dismutase 2,SOD2)、乙酰化超氧化物歧化酶2(acetylated su⁃peroxide dismutase 2,Ac⁃SOD2)。结果:IVM培养液中NA浓度200μmol/L时囊胚形成率最高(P<0.05),因此选择该浓度为最适作用浓度。NA可改善老龄小鼠卵母细胞PBE率和CG分布,降低异常纺锤体率和染色体排列异常率,提高线粒体膜电位,提高ATP生成水平,降低卵母细胞和线粒体内ROS水平(P均<0.05)。Western blot结果显示添加NA培养后SIRT3和SOD2蛋白表达显著增加(P均<0.05),而Ac⁃SOD2蛋白表达显著下降(P<0.05)。结论:IVM培养液中添加200μmol/L NA可通过改善线粒体氧化应激水平以改善老龄小鼠卵母细胞质量,且可能通过SIRT3⁃SOD2信号通路发挥作用。
Objective:This study aims to investigate whether the addition of niacin(NA)to in vitro maturation(IVM)cultures can improve the quality of oocytes derived from aged mice and its mechanism.Methods:C57BL/6J mice at 8 weeks of age were used as the young control group,and mice at 40 weeks of age were used as the old group.Oocytes at the germinal vesicle(GV)stage were taken and supplemented with 100,200,and 300μmol/L NA,respectively,and the rates of 2-cell and blastocyst formation were observed to determine the optimal concentration.Oocyte quality was assessed by the first polar body extrusion(PBE)rate,cortical granule(CG)distribution,spindle morphology abnormality rate and chromosomal abnormality rate.Mitochondrial function was assessed by mitochondrial membrane potential and adenosine triphosphate(ATP)levels.Oxidative stress levels were assessed by detecting the levels of reactive oxygen species(ROS)in oocytes and mitochondria.The levels of oxidative stress-related proteins:silent information regulator 3(SIRT3),superoxide dismutase(SOD2),and acetylated superoxide dismutase(Ac-SOD2)were detected by Western blot.Results:Under the concentration of 200μmol/L NA culture medium,the blastocyst formation rate of oocytes in the old+NA group reached its highest level(P<0.05),therefore this concentration was selected as the optimal concentration.NA supplementation significantly improved the PBE rate and CG distribution in oocytes,while reducing abnormal spindle rates and chromosomal aberration rates,compared with that of the aged mice.NA supplementation also increased mitochondrial membrane potential,enhanced ATP production,and reduced ROS levels in both oocytes and mitochondria(all P<0.05).Western blot analysis demonstrated that NA supplementation significantly increased SIRT3 and SOD2 protein expression(all P<0.05),while markedly decreasing Ac-SOD2 protein expression(P<0.05).Conclusion:Addition of 200μmol/L NA to IVM culture medium improves oocyte quality in aged mice by ameliorating mitochondrial oxidative stress and may act by affecting the SIRT3-SOD2 signaling pathway.
作者
郭爽
倪曼
程丽
严正杰
高莉
宁松
刘嘉茵
GUO Shuang;NI Man;CHENG Li;YAN Zhengjie;GAO Li;NING Song;LIU Jiayin(Clinical Center of Reproductive Medicine,State Key Laboratory of Reproductive Medicine and Offspring Health,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)
出处
《南京医科大学学报(自然科学版)》
北大核心
2025年第8期1101-1109,1177,共10页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家重点研发项目(2021YFC2700404,2021YFC2700501)。
关键词
烟酸
卵母细胞
体外成熟
老龄
氧化应激
niacin
oocyte
in vitro maturation
aging
oxidative stress
