title: 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.
abstract: 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.
  
  
  METHODS
  Experimental animal study.
  
  
  METHODS
  Academic institution.
  
  
  METHODS
  Abattoir-derived bovine ovaries.
  
  
  METHODS
  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.
  
  
  METHODS
  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).
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Xue-Ming
authors.initials: XM
authors.lastname: Zhao
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Ren
authors.forename: Wei-Hua
authors.initials: WH
authors.lastname: Du
authors.forename: Hai-Sheng
authors.initials: HS
authors.lastname: Hao
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Tong
authors.initials: T
authors.lastname: Qin
authors.forename: Hua-Bin
authors.initials: HB
authors.lastname: Zhu
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002417:Cattle / Q000196:embryology; D002478:Cells, Cultured; D002970:Cleavage Stage, Ovum / Q000187:drug effects / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D004353:Drug Evaluation, Preclinical; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000187:drug effects* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D018392:Genomic Imprinting / Q000187:drug effects; D019901:Locus Control Region / Q000187:drug effects / Q000235:genetics; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D058989:Vitrification
chemical_list: D004791:Enzyme Inhibitors; C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D000077209:Decitabine; D001374:Azacitidine