Effect of 5-aza-2′-deoxycytidine on methylation of the putative imprinted control region of H19 during the in vitro development of vitrified bovine two-cell embryos

Capsule:
H19 imprinted control region methylation levels significantly increased in vitrified two-cell embryos and their derived blastocysts; 5-aza-dC significantly reduced H19 imprinted control region methylation and increased H19 expression in blastocysts derived from vitrified embryos.

Authors:
Xue-Ming Zhao, Ph.D., Jing-Jing Ren, M.Sc., Wei-Hua Du, Ph.D., Hai-Sheng Hao, Ph.D., Dong Wang, Ph.D., Yan Liu, Ph.D., Tong Qin, Ph.D., Hua-Bin Zhu, Ph.D.
Volume 98, Issue 1 , Pages 222-227, July 2012

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.

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