title: Effect of vitrification on promoter methylation and the expression of pluripotency and differentiation genes in mouse blastocysts.
abstract: The present study was designed to determine the effects of vitrification on promoter methylation and the expression levels of pluripotency and differentiation genes in mouse blastocysts. Promoter region CpG methylation patterns and the expression levels of octamer-binding transcription factor (Oct4), Nanog homeobox (Nanog), caudal-type homeobox 2 (Cdx2), and heart and neural crest derivatives-expressed transcript 1 (Hand1) were analyzed in fresh and vitrified mouse blastocysts. Methylation was measured by bisulphate mutagenesis and sequencing; gene expression was determined by real-time reverse transcription-PCR. The results showed that vitrification significantly reduced the methylation levels of the Oct4 (85% vs. 62.5%), Nanog (77.5% vs. 55%), and Cdx2 promoters (4.6% vs. 1.4%; P < 0.05) in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog in vitrified blastocysts. Hand1 promoter methylation was not significantly different in the fresh (17.9%) versus vitrification group (21.4%; P > 0.05). The expression levels of Cdx2 and Hand1 were not significantly different in fresh and vitrified blastocysts. In conclusion, vitrification significantly decreased Oct4, Nanog, and Cdx2 promoter methylation in mouse blastocysts, which correlated with increased expression of Oct4 and Nanog.
authors.affiliation: Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Haidian District, Beijing, PR China.
authors.forename: Xue-Ming
authors.initials: XM
authors.lastname: Zhao
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: Tong
authors.initials: T
authors.lastname: Qin
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Hua-Bin
authors.initials: HB
authors.lastname: Zhu
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis / Q000235:genetics; D001755:Blastocyst / Q000502:physiology*; D000071616:CDX2 Transcription Factor; D002454:Cell Differentiation / Q000235:genetics; D015925:Cryopreservation / Q000379:methods; D019175:DNA Methylation; D018507:Gene Expression Regulation, Developmental; D006361:Heating; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D058989:Vitrification
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D000071616:CDX2 Transcription Factor; D018398:Homeodomain Proteins; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D014157:Transcription Factors; C487514:Cdx2 protein, mouse; C095572:Hand1 protein, mouse; C475540:Nanog protein, mouse; C494174:Pou5f1 protein, mouse