孤雌激活猪囊胚玻璃化冷冻后氧化应激水平研究

Effect of Vitrification on the Level of Oxidative Stress in Parthenogenetic Porcine Blastocysts

本实验以孤雌激活猪扩张囊胚为对象,旨在研究胚胎玻璃化冷冻后的氧化应激水平。选择体外培养至第5天的扩张囊胚采用Cryotop法玻璃化冷冻保存,解冻后恢复培养48 h,分析囊胚扩张率、囊胚细胞数、胞内活性氧(ROS)、超氧化物阴离子(O^(2-))和谷胱甘肽(GSH)的水平及抗氧化酶相关基因(SOD1、SOD2、CAT、GPX4)的mRNA表达量。结果表明:相比于新鲜囊胚,冷冻囊胚在24 h和48 h的囊胚扩张率明显下降(P<0.05),但囊胚细胞数无显著性变化(P>0.05);冷冻囊胚内ROS和O^(2-)水平均显著高于新鲜囊胚(P<0.05),GSH含量显著低于新鲜囊胚(P<0.05);玻璃化冷冻导致囊胚内SOD2 mRNA表达水平升高、CAT和GPX4 mRNA表达水平降低,但不影响SOD1的mRNA表达水平。综上可见,玻璃化冷冻加重孤雌激活猪囊胚的氧化应激水平,表现为ROS和O^(2-)水平升高,GSH含量下降,部分抗氧化酶相关基因mRNA的表达水平异常。

This study was conducted to investigate the effect of vitrification on the level of oxidative stress in parthenogenetic porcine blastocysts.On day 5 blastocysts were vitrified by Cryotop method and recovered for 48 h after warming.The vitrified blastocysts were evaluated by their expansion rate,cell number,levels of reactive oxygen species(ROS),superoxide anion(O2-)and glutathione(GSH),and mRNA expression of antioxidant enzyme related genes(SOD1,SOD2,CAT and GPX4).The results showed that the percentages of expanded blastocysts at 24 and 48 h after warming were significantly lower in the vitrified group than in the fresh groups.However,total cell numbers in blastocysts were not significantly different between groups.The levels of ROS and O2-in vitrified blastocysts were significantly higher than those in fresh blastocysts.GSH content in vitrified blastocysts was significantly lower than that in fresh blastocysts.Moreover,Vitrification significantly increased SOD2 mRNA level,decreased mRNA levels of CAT and GPX4,and did not affect SOD1 expression level.In conclusion,our data demonstrated that vitrification increase the oxidative stress of parthenogenetic porcine blastocysts according to higher levels of ROS and O2-,lower content of GSH and abnormal expression levels of SOD2,CAT and GPX4.