超低温冷冻对牛卵母细胞组蛋白乙酰化和膜蛋白CD9表达的影响

The effect of bovine oocyte cryopreservation on the histone acetylation and CD9 expression

本试验旨在探讨玻璃化冷冻对牛体外成熟卵母细胞(MⅡ期)组蛋白乙酰化和膜蛋白CD9表达的影响。牛MⅡ期卵母细胞采用OPS法冷冻,即卵母细胞于10%EG+10%DMSO溶液中预处理30s,然后再移入玻璃化溶液EDFSF30中处理25s,以OPS为承载器投入液氮中。毒性组卵母细胞未投入液氮,其他过程与冷冻组相同,新鲜牛MⅡ期卵母细胞为对照组。卵母细胞解冻后,利用免疫荧光染色法检测存活细胞DNA组蛋白乙酰化;qRT-PCR和Western blot检测CD9mRNA蛋白的表达。结果表明:冷冻组卵母细胞形态正常率(93.8%)和存活率(92.7%)显著低于毒性组(100.0%,97.2%)和对照组(100.0%,98.5%)(P<0.05),而毒性组与对照组之间无显著差异。超低温冷冻后,卵母细胞DNA组蛋白乙酰化水平显著上升(P<0.05),CD9mRNA与蛋白表达显著降低(P<0.05)。以上显示,玻璃化冷冻不但降低了牛体外成熟卵母细胞的存活率,而且改变了DNA组蛋白乙酰化和膜蛋白CD9表达水平。

This study was conducted to investigate effect of cryopreservaion on histone acetylation and CD9 expression in bovine metaphaseⅡ(MⅡ)oocytes.The oocytes were randomly allocated into three groups:untreated(control),exposed to vitrification solution without being plunged into liquid nitrogen(toxicity),or vitrified by open-pulled straw method(vitrification).After vitrification and warming,the oocytes were morphologically evaluated and their viability were assessed using fluorescein diacetate(FDA)stainning.Histone acetylation were examined by fluorescence microscopy,CD9expression in oocytes was assessed by qRT-PCR and Western blot.The results showed that after oocytes warming,the rates of morphologically normal and surviving oocytes(93.8% and 92.7%,respectively)in the vitrification group were lower(P<0.05)than those in both the Toxicity(100.0% and 97.2%,respectively)and control(100.0% and 98.5%,respectively)groups.There were no significant difference(P>0.05)in either rate between the toxicity and control groups.The relative histone acetylation was significantly increased in oocytes after vitrification.The vitrification/warming procedures significantly decreased CD9 expression at leveles of mRNA and protein.In conclusion,vitrification of bovine oocytes caused a decrease in their survival,and changed the expression of histone acetylation and CD9as well.