线粒体靶向抗氧化剂MitoQ对猪孤雌激活囊胚冷冻保存的影响

Application of mitochondria-targeted antioxidant MitoQ in cryopreservation of porcine parthenogenetic blastocysts

在卵母细胞体外成熟、胚胎培养及玻璃化冷冻与解冻过程中全程添加200 nmol∕L MitoQ,观察其对猪孤雌激活囊胚解冻后线粒体膜电位、TUNEL凋亡水平、活性氧(ROS)水平、脂质氧化产物(MDA)水平、总Caspase蛋白表达水平及凋亡氧化相关基因表达水平的影响。结果表明:添加MitoQ冷冻后其囊胚线粒体膜电位(0.83)相比对照组(0.52)显著升高;囊胚凋亡阳性细胞比例(22.36%)相比对照组(36.42%)显著降低;MDA含量(37.64 nmol∕L)与ROS荧光强度(26.32)相比对照组(49.83 nmol∕L与35.70)显著降低;总Caspase活性荧光强度(20.65)相比对照组(42.47)显著降低;相比对照组,Bcl-2、DNM1、CuZnSOD和MFN2基因的表达水平显著提升,Bax和TNF-α基因的相对表达量则显著下降。综上,全程添加MitoQ能够降低猪孤雌激活囊胚的氧化损伤、改善线粒体功能,并通过调节相关基因的表达水平,达到降低其细胞凋亡和提高其抗冻能力的目的。

The aim of this study was to investigate the effect of mitochondria-targeted antioxidant MitoQ on cryopreservation of porcine parthenogenesis blastocysts.MitoQ(200 nmol∕L)was added to culture,vitrification and thawed incubation solutions,and the mitochondrial membrane potential,TUNEL apoptosis rate,reactive oxygen species,lipid oxidation products level,Pan-Caspase protein levels and expression of apoptosis oxidationrelated genes were investigated.The results showed that the mitochondrial membrane potential(0.83)was significantly higher than control group(0.52).TUNEL apoptosis rate(22.36%)was significantly lower than control group(36.42%);MDA content(37.64 nmol∕L)was significantly lower than control group(49.83 nmol∕L);ROS level(26.32)was significantly lower than control group(35.70);the fluorescence intensity of Pan-Caspase(20.65)was significantly lower than control group(42.47);expression levels of DNM1,CuZnSOD and MFN2 were significantly increased,while expression levels of Bax and TNF-αwere significantly decreased.In summary,MitoQ can reduce apoptotic level and enhance anti-freezing ability of porcine parthenogenetic blastocysts by decreasing the oxidative damage,improving mitochondrial function,regulating related gene expression levels.