三种抗氧化剂对斑马鱼卵母细胞冷冻保存效果比较

Comparison of the effects of three antioxidants on the cryopreservation of zebrafish oocytes

为研究抗氧化剂对斑马鱼卵母细胞冷冻保存效果的影响,在基础冷冻保护液配方中分别添加褪黑素、谷胱甘肽和甜菜碱等3种抗氧化剂进行了冷冻保存试验,比较冷冻后卵母细胞的活性氧(ROS)含量、总抗氧化能力(T-AOC)、腺嘌呤核苷三磷酸(ATP)含量、琥珀酸脱氢酶(SDH)活性、凋亡基因表达水平和存活率。试验结果显示:经过筛选,3种抗氧化剂的最优添加浓度分别为褪黑素1×10^(-3) mol/L、谷胱甘肽5×10^(-4) mol/L、甜菜碱1×10^(-3) mol/L。与对照组相比,添加最优浓度抗氧化剂的3个试验组(褪黑素组、谷胱甘肽组和甜菜碱组)的ROS含量均显著降低,ATP含量和SDH活性均显著提高,褪黑素组和甜菜碱组的T-AOC水平显著提高(P<0.05);褪黑素组卵母细胞的caspase3、caspase9和bax基因表达量显著降低,bcl-2基因表达量则显著升高(P<0.05);谷胱甘肽组卵母细胞的caspase3基因表达量显著下降,bcl-2基因表达量显著上升(P<0.05);甜菜碱组卵母细胞的caspase9和bax基因表达量显著降低(P<0.05)。复苏后的卵母细胞在体外培育120 min,褪黑素组、谷胱甘肽组、甜菜碱组卵母细胞的存活率分别比对照组提高10.13%、14.20%和15.99%;冷冻保存1、7、30、60、90 d,3个试验组卵母细胞的存活率均显著高于对照组(P<0.05);冷冻保存90 d,褪黑素组、谷胱甘肽组、甜菜碱组的存活率分别提高了9.05%、7.34%、6.68%。试验结果说明,在斑马鱼卵母细胞冷冻保存过程中添加1×10^(-3) mol/L褪黑素或5×10^(-4) mol/L谷胱甘肽或1×10^(-3) mol/L甜菜碱,均能够增强冷冻保存后卵母细胞的线粒体功能,降低细胞氧化应激水平,减少细胞凋亡,从而提高斑马鱼卵母细胞冷冻效率。

To investigate the effects of antioxidants on the cryopreservation of zebrafish oocytes,melatonin,glutathione and betaine were added to the basic cryoprotective solution in this study.The antioxidant capacity,mitochondrial function,expression of apoptosis-related genes and survival rate were tested.The results showed that the optimal concentration of the three antioxidants were melatonin 1×10^(-3) mol/L,glutathione 5×10^(-4) mol/L,and betaine 1×10^(-3) mol/L.ATP content and SDH activity were significantly increased in the three experimental groups(melatonin group,glutathione group and betaine group)supplemented with the optimal concentration of antioxidants,while ROS content was significantly decreased(P<0.05).The level of T-AOC in betaine and melatonin groups significantly increased(P<0.05).The expression of caspase3,caspase9 and bax genes in melatonin group significantly decreased,while the expression of bcl-2 significantly increased(P<0.05).The expression of caspase3 in glutathione group was significantly decreased,while the expression of bcl-2 was significantly increased.The expression of caspase9 and bax in betaine group was significantly decreased.The survival rate of oocytes in melatonin,glutathione and betaine groups increased by 10.13%,14.20%and 15.99%,respectively after 120 min vitro incubation.After 1,7,30,60 and 90 days of cryopreservation,the survival rate of oocytes in three experimental groups were significantly higher than that of the control group.After 90 days of cryopreservation,the survival rate was increased by 9.05%,7.34%and 6.68%,respectively.The results indicated that the addition of antioxidants melatonin,glutathione and betaine during zebrafish oocyte cryopreservation could protect mitochondrial function,reduce oxidative damage and cell apoptosis,and improve the quality of zebrafish oocytes.