玻璃化冷冻对小鼠卵巢组织DNA甲基化的影响

Effect of vitrification on DNA methylation in mouse ovarian tissue

目的:探讨小鼠卵巢组织玻璃化冷冻对DNA甲基化水平的影响。方法:选择C57BL/6J雌性小鼠为研究对象,以卵巢组织玻璃化冷冻后复苏为冷冻组,新鲜的卵巢组织为对照组,观察卵巢组织的光镜结构和电镜超微结构,实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测DNA甲基转移酶(DNMT1、DNMT3a、DNMT3b)和印记基因H19的mRNA表达。结果:冷冻组卵巢组织及卵泡的结构与对照组相似,细胞器完整;冷冻组DNA甲基转移酶DNMT1、DNMT3a、DNMT3b的表达较对照组增高,H19的表达较对照组稍有下降,但差异均无统计学意义(均P>0.05)。结论:玻璃化冷冻可较好地保存卵巢组织且对卵巢的超微结构及DNA甲基化状态无明显影响。

Objective:To explore the effects of vitrification of ovarian tissue on DNA methylation level.Methods:Female mice of C57BL/6J were used as the research object.The frozen group was vitrified frozen ovarian tissue,and the control group was fresh ovarian tissue.The mRNA expression of methyltransferase(DNMT1,DNMT3a,DNMT3b)and imprinted gene H19 were detected by quantitative real-time polymerase chain reaction(qRT-PCR).We observed ovarian tissue by optical microscope and ultrastructure of ovarian tissue by electron microscope.Results:The structure of ovarian tissue and follicles in the frozen group was similar to that in the control group.The organelles were intact.The expression of DNA methyltransferases DNMT1,DNMT3a and DNMT3b were higher in the frozen group than in the control group,and the expression of H19 in the frozen group was slightly lower than that in the control group,but all differences were not statistically significant(all P>0.05).Conclusion:Vitrified cryopreservation can preserve ovarian tissue and has no significant effect on the ultrastructure and DNA methylation status of ovary.