牛卵母细胞玻璃化冷冻对基因mRNA表达量的影响

摘要

Objective To evaluate the mRNA expression abundance of genes, which are related with embryonic development and ability to undergo successful fertilization, cleavage and blastocyst development, in the GV stage and in vitro matured ( IVM ) bovine oocytes. Methods The oocytes that vitrified-warmed at GV ( n = 224 ) and IVM ( n = 235 )were in vitro fertilized and cultured up to blastocyst stage under standard conditions. In parallel, we analyzed the relative abundance of Bax, Dnmtl, Hdac1, cyclinb1, Cdc2 and Cu Zn SOD transcripts of the vitrified-warmed oocytes by quantitatire real time-PCR. Result Vitrified oocytes in both GV and IVM stages showed a lower cleavage rate ( 14. 7％ ～ 89.4％,34. 5％ ～ 89.4％, P ＜ 0. 001 ) and development to blastocyst stage (3.0％ ～ 28.4％ , 12. 3％ ～ 28.4％ , P ＜ 0. 001 ) than that in the control group ( n = 238 ). In addition, vitrified oocytes in GV stage showed a lower cleavage rate ( 14. 7％ ～34. 5％ , P ＜0. 001 ) and development to blastocyst stage (3.0％ ～ 12. 3％ , P ＜0. 001 ) than those of vitrified oocytes in IVM period. In the oocytes vitrified in GV period, the Dnmtl and Cu Zn SOD expression abundance was significantly lower than that of controls ( P ＜ 0. 05 ). In the oocytes in M phase, the expression of Bax mRNA was very significantly lower than that in controls ( P ＜ 0. 01 ), and the mRNA expression of cyclinb1, Cdc2 and Cu Zn SOD was significantly lower than that of controls ( P ＜ 0. 05 ). ( P ＜ 0. 05 ). No other significant changes were found in the expression abundance of other genes.Conclusions Following vitrification the bovine oocytes in GV or IVM stage may lead to a lower transcript abundance, that may have negative effects on embryonic development.%目的 将处于生发泡期(GV期)和体外成熟期(IVM)的牛卵母细胞进行玻璃化冷冻.解冻,对其卵裂率和囊胚率以及一些与发育相关基因的mRNA表达量进行评价.方法 玻璃化冷冻GV期(n=224)和IVM期(n=235)牛卵母细胞,解冻后对其进行体外培养并采用quantitative real time-PCR技术对冷冻.解冻后的GV和IVM期卵母细胞中Bax、Dnmtl、Hdacl、Cyclinbl、Cdc2和cu Zn SOD等基因的mRNA表达量进行检测.结果 GV期和IVM期卵母细胞经过冷冻-解冻后,卵裂率(14.7%～89.4%,34.5%～89.4%,P<0.001)及囊胚率(3.0%～28.4%,12.3%～28.4%,P<0.001)均极显著低于对照组(n=238).且冷冻-解冻后的GV期卵母细胞卵裂率(14.7%～34.5%,P<0.001)和囊胚率(3.0%～12.3%,P<0.001)极显著低于冷冻-解冻后的IVM期卵母细胞;GV期卵母细胞经玻璃化冷冻后,Dnmtl和Cu Zn SOD基因的mRNA表达量显著低于对照组(P<0.05),其余基因mRNA表达量变化不显著;对M期卵母细胞而言,Bax基因mRNA表达量极显著低于对照组(P<0.01),Cyclinbl、Cdc2和Cu Zn SOD基因的mRNA表达量显著低于对照组(P<0.05),其余基因表达量没有发生显著变化.结论 对牛GV期或IVM卵母细胞进行玻璃化冷冻可能会导致基因mRNA表达量降低,可能会对胚胎发育产生负面影响.