外源性ROS对绵羊孤雌激活胚胎发育影响的研究

Study on exogenous ROS on the development of sheep parthenogenetic embryos

为了探讨外源性活性氧(ROS)短期孵育绵羊体外成熟后卵丘卵母细胞复合体(in vitro matured cumulus oocyte complexs,IVM-COCs)对孤雌激活(parthenogenetic activation,PA)胚胎发育能力和凋亡的影响,试验以0(对照组),10,100,1000μmol/L的过氧化氢(H2O2)处理液孵育绵羊IVM-COCs 1 h,然后进行孤雌激活并培养,通过荧光探针DCHF-DA测定ROS水平,使用末端脱氧核苷酸转移酶介导的UTP缺口末端标记(TUNEL)法和碘化丙锭(PI)技术评估孤雌激活胚胎囊胚中DNA碎片化程度。结果表明:100μmol/L H2O2处理液孵育IVM-COCs 1 h后孤雌胚胎的囊胚率为(50.50±4.30)%,显著高于对照组及其他浓度处理组(P<0.05);而1000μmol/L H2O2处理液孵育IVM-COCs 1 h后孤雌激活胚胎的卵裂率显著降低且未能发育至囊胚阶段,并诱导永久性的胚胎发育阻滞或死亡。与对照组相比,100μmol/L H2O2处理液孵育IVM-COCs 1 h后ROS水平显著升高(P<0.05),而2-细胞阶段胚胎ROS水平差异不显著(P>0.05);孤雌激活胚胎的卵裂率、囊胚平均细胞数以及囊胚中DNA碎片化比率均差异不显著(P>0.05)。说明用适量的外源性ROS短期孵育绵羊IVM-COCs对孤雌激活胚胎的形成有积极作用。

The objective of this study was to investigate the effect of short-term exogenous reactive oxygen species(ROS)on developmental capacity and apoptosis of parthenogenetic activation(PA)embryos of sheep in vitro matured cumulus oocyte complexes(IVM-COCs).Sheep IVM-COCs were incubated with 0(control group),10,100,and 1000μmol/L H2O2 for 1 h,and subsequently parthenogenetic activation and cultured.The ROS level was determined by fluorescent probe DCHF-DA assay.DNA-fragmented nuclei in blastocysts of PA embryos was assessed using a combination of TUNEL staining and PI nuclear staining.The results showed that the PA blastocyst yield of IVM-COCs incubated with 100μmol/L H2O2 for 1 h was(50.50±4.30)%,which was significantly higher than that of control group and other concentration treatment groups(P<0.05).However,high concentrations(1000μmol/L)H2O2-treated IVM-COCs experienced a greater decrease in cleavage,failed to develop into blastocyst and induced high percentage permanent embryos arrest or demise.Compared with the control group,the ROS level of IVM-COCs incubated with 100μmol/L H2O2 for 1 h was significantly increased(P<0.05),while the ROS level of 2-cell stage embryos was not significantly different(P>0.05).There were no significant differences in the cleavage rate,average cell number and DNA fragmentation ratio in the blastocysts(P>0.05).It indicated that appropriate amount of exogenous ROS short-term incubation IVM-COCs played a positive effect on PA embryo formation.