白蛋白浓度对开放式细管玻璃化冷冻猪卵母细胞线粒体基因和凋亡基因表达水平的影响

Effect of albumin concentration on mitochondrial genes and apoptosis gene expression in porcine oocytes during open pulled straw vitrification cryopreservation

为了探讨不同浓度白蛋白在猪卵母细胞开放式细管(OPS)玻璃化冷冻保存中对线粒体基因(SOD1、Mfn2)和凋亡基因(BAX)表达和甲基化的影响,试验采集新鲜的健康长白猪卵巢中的猪卵丘-卵母细胞复合体(COCs),将其分为空白对照组和试验1,2,3组,分别用含0、1.5%、2.0%、2.5%白蛋白的冷冻保护液(TCM-199+10%胎牛血清+5%乙二醇)进行OPS玻璃化冷冻、解冻,通过RT-qPCR、激光共聚焦方法分析SOD1、Mfn2和BAX基因的表达水平及甲基化的情况。结果表明:与空白对照组比,试验1,2组SOD1基因表达水平上调,其中试验2组差异显著(P<0.05),试验1组差异不显著(P>0.05);试验3组SOD1基因表达水平下调,但与空白对照组差异不显著(P>0.05)。试验1,2,3组Mfn2基因表达水平均下调,其中试验1,2组与空白对照组均差异显著(P<0.05),试验3组与空白对照组差异不显著(P>0.05)。试验1,2组BAX基因表达水平下调,其中试验2组与空白对照组差异显著(P<0.05),试验1组与空白对照组差异不显著(P>0.05);试验3组BAX基因表达水平上调,但与空白对照组差异不显著(P>0.05)。试验1,2,3组5-mC、5-hmC的AO值均降低,其中试验1组5-mC的AO值与空白对照组差异显著(P<0.05),试验2,3组5-mC的AO值与空白对照组均差异不显著(P>0.05);试验1,2,3组5-hmC的AO值均显著减低(P<0.05)。说明白蛋白在猪卵母细胞OPS玻璃化冷冻中可减少超低温对细胞造成的损伤,对细胞有保护作用,其中添加2.0%白蛋白对细胞的保护效果最显著。

In order to investigate the effects of different concentrations of albumin on the expression and methylation of mitochondrial genes(SOD1,Mfn2)and apoptosis gene(BAX)during open pulled straw(OPS)vitrification cryopreservation of porcine oocytes,porcine cumulus oocyte complexes(COCs)from fresh healthy Landrace ovaries were collected and divided into blank control group and test 1,2 and 3 groups.OPS vitrification freezing and thawing procedures were carried out with cryoprotectant(TCM-199+10%fetal bovine serum+5%ethylene glycol)containing 0,1.5%,2.0%and 2.5%albumin respectively.The expression levels and methylation of SOD1,Mfn2 and BAX genes were analyzed by RT-qPCR and laser confocal methods.The results showed that,compared with the blank control group,the level of SOD1 gene expression in test 1 and 2 groups was up regulated,and there was significant difference between test 2 group and blank control group(P<0.05),but there was no significant difference between test 1 group and blank control group(P>0.05);the level of SOD1 gene expression in test 3 group was down regulated,but there was no significant difference between test 3 group and blank control group(P>0.05);the expression level of Mfn2 gene in test 1,2 and 3 groups were down regulated,and there were significant differences between test 1 and 2 groups and the blank control group(P<0.05),but there was no significant difference between test 3 group and the blank control group(P>0.05).The BAX gene expression level of test 1 and 2 groups was down regulated,and there was significant difference between test 2 group and blank control group(P<0.05),but there was no significant difference between test 1 group and blank control group(P>0.05).The expression of BAX gene in the test 3 group was up regulated,but there was no significant difference compared with the blank control group(P>0.05).The AO values of 5-mc and 5-hmc in test 1,2 and 3 groups were decreased,and the AO value of 5-mc in test 1 group was significantly different from that in the blank control group(P<0.05),and there was no significant difference in AO value of 5-mc between test 2 and 3 groups and blank control group(P>0.05);the AO value of 5-hmc in the experimental test 1,2 and 3 groups was significantly reduced(P<0.05).The results indicated that albumin could reduce the damage caused by ultra-low temperature in porcine oocytes during OPS vitrification cryopreservation and has a protective effect on the cells.Among them,the protective effect of 2.0%albumin on cells was the most significant.