title: Limiting dilution bisulfite (pyro)sequencing reveals parent-specific methylation patterns in single early mouse embryos and bovine oocytes.
abstract: To detect rare epigenetic effects associated with assisted reproduction, it is necessary to monitor methylation patterns of developmentally important genes in a few germ cells and individual embryos. Bisulfite treatment degrades DNA and reduces its complexity, rendering methylation analysis from small amounts of DNA extremely challenging. Here we describe a simple approach that allows determining the parent-specific methylation patterns of multiple genes in individual early embryos. Limiting dilution (LD) of bisulfite-treated DNA is combined with independent multiplex PCRs of single DNA target molecules to avoid amplification bias. Using this approach, we compared the methylation status of three imprinted (H19, Snrpn and Igf2r) and one pluripotency-related gene (Oct4) in three different groups of single mouse two-cell embryos. Standard in vitro fertilization of superovulated oocytes and the use of in vitro matured oocytes were not associated with significantly increased rates of stochastic single CpG methylation errors and epimutations (allele methylation errors), when compared with the in vivo produced controls. Similarly, we compared the methylation patterns of two imprinted genes (H19 and Snrpn) in individual mouse 16-cell embryos produced in vivo from superovulated and non-superovulated oocytes and did not observe major between-group differences. Using bovine oocytes and polar bodies as a model, we demonstrate that LD even allows the methylation analysis of multiple genes in single cells.
authors.affiliation: Institute of Human Genetics, Julius Maximilians University, Biozentrum, Am Hubland, Würzburg, Germany.
authors.forename: Nady
authors.initials: N
authors.lastname: El Hajj
authors.forename: Tom
authors.initials: T
authors.lastname: Trapphoff
authors.forename: Matthias
authors.initials: M
authors.lastname: Linke
authors.forename: Andreas
authors.initials: A
authors.lastname: May
authors.forename: Tamara
authors.initials: T
authors.lastname: Hansmann
authors.forename: Juliane
authors.initials: J
authors.lastname: Kuhtz
authors.forename: Kurt
authors.initials: K
authors.lastname: Reifenberg
authors.forename: Julia
authors.initials: J
authors.lastname: Heinzmann
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
authors.forename: Angelika
authors.initials: A
authors.lastname: Daser
authors.forename: Ursula
authors.initials: U
authors.lastname: Eichenlaub-Ritter
authors.forename: Ulrich
authors.initials: U
authors.lastname: Zechner
authors.forename: Thomas
authors.initials: T
authors.lastname: Haaf
mesh_terms: D000818:Animals; D002417:Cattle; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000378:metabolism*; D044127:Epigenesis, Genetic; D018392:Genomic Imprinting; D051379:Mice; D009865:Oocytes; D059705:Polar Bodies; D027724:Reproductive Techniques, Assisted / Q000009:adverse effects; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites
chemical_list: D013447:Sulfites; C042345:hydrogen sulfite