在玻璃化牛双细胞胚胎体外发育过程中5-aza-2′-脱氧胞苷对推定的H19印记控制区甲基化的影响

Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan Western Road, Haidian District, Beijing 100193, China

Objective: To determine the effect of vitrification and 5-aza-2′-deoxycytidine (5-aza-dC) on the methylation levels of the putative imprinted control region (ICR) of H19 and H19 expression in bovine two-cell embryos and their derived blastocysts. Design: Experimental animal study. Setting: Academic institution. Animal(s): Abattoir-derived bovine ovaries. Intervention(s): Vitrified two-cell embryos were cultured in vitro to blastocysts with 0.01 μM 5-aza-dC (5-aza-dC group) or without 5-aza-dC (vitrification group). Fresh embryos and their derived blastocysts were used as control. Main Outcome Measure(s): Putative ICR methylation of H19 was measured by bisulfate mutagenesis and sequencing, blastocyst development rate; total cell number were determined; and H19 expression was measured by real-time reverse transcriptase-polymerase chain reaction (PCR). Result(s): Vitrification significantly increased putative ICR methylation of H19 in two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 in vitrified two-cell embryos and their derived blastocysts. The H19 expression level was significantly higher in blastocysts from the 5-aza-dC group than the vitrification group. The blastocyst development rate and total cell number in the 5-aza-dC and vitrification groups were similar. Conclusion(s): Putative ICR methylation levels of H19 significantly increased in vitrified two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced putative ICR methylation of H19 and increased H19 expression in blastocysts derived from vitrified two-cell embryos. © 2012 American Society for Reproductive Medicine, Published by Elsevier Inc.

Zhu, H.-B.; Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuan Western Road, Haidian District, Beijing 100193, China; email: huabinzhu@yahoo.com.cn