胚胎玻璃化冷冻解冻技术对子代胚胎和胎盘中印记基因H19表达及甲基化水平的影响

Expression and methylation level of imprinted gene H19in fetuses and placentas derived from vitrified embryos

目的建立玻璃化冷冻解冻移植小鼠模型,探究玻璃化冷冻解冻对子代胚胎及胎盘组织中印记基因H19表达及甲基化水平的影响。方法实验动物选择ICR清洁级小鼠,根据处理不同分为3组,自然妊娠(NC)组,胚胎体外培养(IVC)组和胚胎玻璃化冷冻解冻(VET)组,每组收集7只孕鼠。观测3组孕鼠孕9.5d的胎鼠存活率。取3组孕鼠孕9.5d的胚胎及胎盘标本,采用实时定量PCR技术分析H19基因在胎鼠及胎盘中的表达情况,采用焦磷酸测序技术检测H19基因在胎鼠和胎盘中的甲基化水平。结果NC组的平均窝仔数(10.43±2.07)显著高于IVC组的(6.43±1.51)和VET组的(6.00±1.16)(P<0.05)。IVC组和VET组的存活率分别为40.2%和37.5%,与NC组(100.0%)相比,具有显著性差异(P均<0.05)。在胚胎组织中,与NC组相比,IVC组和VET组的H19表达水平显著增加(P<0.05);在胎盘组织中,3组间H19基因的表达水平比较无显著性差异(P>0.05);在胚胎和胎盘组织中,3组间H19基因甲基化水平比较均无显著性差异(P>0.05)。结论胚胎的玻璃化冷冻解冻和胚胎体外培养均可导致小鼠存活率下降,胚胎中H19基因表达水平明显增高。但并未发现胎盘组织中H19基因水平的变化,且无论在胚胎或胎盘组织中,H19基因甲基化水平均未发现变化,后期需要更多的研究加以探索。

Objective:To investigate the effect of vitrification on expression and methylation level of imprinted gene H19 in fetuses and placentas derived from vitrified embryos in mouse.Methods:The ICR clean grade mice were selected and divided into three groups according to different treatments:NC group(natural mating),IVC(in vitro culture)group and VET(vitrified embryo transfer)group,7 mice for each group.The fetal survival rate at 9.5 days gestation was observed in three groups.Embryos and placenta specimens of pregnant mice of 3 groups were collected at 9.5 days of gestation.Expression and methylation level of imprinted gene H19 in fetuses and placentas were investigated by reverse transcription-quantitative polymerase chain reaction(qRT-PCR)and pyrosequencing.Results:The average litter size in IVC group(6.43±1.51)and VET group(6.00±1.16)was significantly decreased compared to NC group(10.43±2.07).The fetal survival rate at 9.5 days gestation was significantly decreased in IVC(40.2%)and VETS(37.5%)groups compared to NC group(100%).In fetuses tissues,the expression level of H19 was significantly increased both in IVC group(2.34±0.41)and VET group(2.17±0.35)compared to NC group(1.40±0.46).There was no significantly difference of H19 expression in placentas in three groups(P>0.05).Furthermore,there was no significant difference in the methylation level of H19 both in fetuses and placentas among the three groups(P>0.05).Conclusions:Both vitrification&thawing and culture in vitro for embryos may lead to decrease the survival rate of fetuses.The expression levels of H19 gene in embryos are increased significantly,but no significant change in H19 gene level was found in placenta.Moreover,the methylation levels of H19 are not significantly changed both in fetuses and placentas in the three groups.More researches are needed in the future.