添加Ge-132对牛体外胚胎发育潜能的影响

Effect of Adding Ge-132 on Bovine Embryo Development Potential in vitro

试验旨在研究添加羧乙基锗倍半氧化物(carboxyethylgermanium sesquioxide,Ge-132)对牛孤雌激活后胚胎发育率、胚胎细胞数、早期胚胎内活性氧(reactive oxygen species,ROS)水平及胚胎内相关凋亡基因的影响。在牛早期胚胎体外培养基中添加不同浓度的Ge-132(0、10、100和200μg/mL),观察其对牛体外孤雌激活胚胎发育的影响;应用Hoechst对孤雌激活后第8天胚胎进行染色后制作装片,在显微镜下对细胞进行计数;用DCFH-DA染色检测早期胚胎内ROS水平,并用Image J测量荧光强度后对数据进行统计分析,用RT-PCR对胚胎内细胞凋亡相关基因(Caspase-3、Bax、Bcl-xl和Survivin)进行分析。结果显示,10μg/mL Ge-132处理组胚胎率与对照组间无显著差异(P>0.05),但可降低1细胞期的ROS水平;10μg/mL Ge-132处理组较对照组早期胚胎细胞数显著增加(P<0.05);通过检测细胞凋亡相关基因mRNA转录水平发现,与对照组相比,10μg/mL Ge-132处理组胚胎促凋亡基因Caspase-3表达水平显著降低(P<0.05),抑制细胞凋亡基因Survivin表达水平显著升高(P<0.05)。结果表明,在早期胚胎培养基中添加10μg/mL Ge-132可降低细胞内ROS水平,减少胚胎中氧化应激诱导的细胞凋亡,从而提高孤雌激活后牛胚胎的发育潜能。

This study was aimed to investigate the effect of carboxyethylgermanium sesquioxide(Ge-132)on the development rate of embryos,the number of embryonic cells,the early embryonic reactive oxygen species(ROS)level and the related apoptotic genes in the embryo after activation of bovine parthenogenesis.The effects of different concentrations of Ge-132(0,10,100 and 200μg/mL)on the embryo development of parthenogenetically activated bovine embryos were studied,Hoechst staining was performed on the 8th day embryos,the number of cells was counted under the microscope.The ROS level was detected in the early embryos by DCFH-DA staining,and the data were analyzed statistically after the measurement of fluorescence intensity by Image J.In addition,the apoptosis related genes(Caspase-3,Bax,Bcl-xl and Survivin)in the embryo were analyzed by RT-PCR method.The results showed that there was no significant difference in the embryo rate between 10μg/mL Ge-132 treatment and control groups(P>0.05),but 10μg/mL Ge-132 could reduce the ROS level in the one cell stage.The number of total cells increased significantly in 10μg/mL Ge-132 treatment group than that of control group(P<0.05).Furthermore,by detecting the mRNA transcriptional level of apoptosis related genes,it was found that compared with control group,the expression level of the apoptotic gene Caspase-3 was significantly decreased in 10μg/mL Ge-132 treatment group(P<0.05),whereas the expression level of anti-apoptotic gene Survivin was significantly increased in 10μg/mL Ge-132 treatment group(P<0.05).In conclusion,the supplementation of 10μg/mL Ge-132 to the early embryo culture medium could reduce the intracellular ROS level and the apoptosis induced by oxidative stress in the embryo,thereby improving the developmental potential of the bovine embryos after parthenogenetic activation.