title: Improving efficiencies of locus-specific DNA methylation assessment for bovine in vitro produced embryos.
abstract: Characterization of DNA methylation is one assessment of chromatin remodeling in early embryos. Unfortunately, evaluation at specific loci is hindered by their small cell numbers. Our objective was to determine if bisulfite sequencing could be optimized for preimplantation embryos, comparing conversion times, primer design, and DNA amplification methods. Methylation at three loci, SATI, OCT4, and IGF2, was investigated in bovine in vitro produced (IVP) embryos, somatic cells, and no template controls. Bisulfite treatment for 15-16 h gave higher quality DNA than treatment for 18 h. Three step primer design improved bisulfite primer specificity, yielding more PCR product than primers previously reported. Following optimization, methylation data were obtained from as few as 4 cell equivalents. Finally, DNA amplification efficiencies were evaluated using miniprep, TempliPhi, or 96-well glycerol stocks with automated TempliPhi. While TempliPhi was better than standard minipreps, the 96-well format proved most efficient. Preliminary methylation profiles of bovine IVP 2-cell, 8-cell, blastocyst stage embryos and somatic cells were 25, 10, 22, and 74% for SATI and 88, 88, 79, and 88% for OCT4, respectively, suggesting that SATI is demethylated during early embryonic reprogramming, while OCT4 remains hypermethylated. IGF2 methylation was 84, 28, and 84% for bovine IVP 8-cell, blastocyst stage embryos and somatic cells; blastocyst stage embryos exhibited more variability, ranging from 0 to 80%. This new assay will enhance assessment of chromatin remodeling in embryos, and be especially useful for evaluating those produced by assisted reproductive technologies.
authors.affiliation: Department of Animal Sciences, Institute of Food and Agriculture Sciences, University of Florida, Gainesville, FL, USA.
authors.forename: Ewa
authors.initials: E
authors.lastname: Wroclawska
authors.forename: Jason O
authors.initials: JO
authors.lastname: Brant
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Yang
authors.forename: Karen
authors.initials: K
authors.lastname: Moore
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000737:chemistry; D002417:Cattle; D042002:Chromatin Assembly and Disassembly; D003001:Cloning, Molecular; D018899:CpG Islands; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000737:chemistry*; D005260:Female; D005307:Fertilization in Vitro; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry
chemical_list: D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite