title: Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage.
abstract: The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resulting in loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such as Beckwith-Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamics of establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR was used to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels between biparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprinting of ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse and human, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of the H19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation of these imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage.
authors.affiliation: Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG2 2RD, UK.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Thurston
authors.forename: Jane
authors.initials: J
authors.lastname: Taylor
authors.forename: John
authors.initials: J
authors.lastname: Gardner
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Sinclair
authors.forename: Lorraine E
authors.initials: LE
authors.lastname: Young
mesh_terms: D000481:Allantoin / Q000378:metabolism; D000483:Alleles; D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D002823:Chorion / Q000378:metabolism; D019175:DNA Methylation; D004622:Embryo, Mammalian / Q000502:physiology*; D005260:Female; D005333:Fetus / Q000378:metabolism; D051383:GRB10 Adaptor Protein / Q000235:genetics; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D018392:Genomic Imprinting / Q000235:genetics*; D023421:Models, Animal; D010312:Parthenogenesis / Q000235:genetics; D011247:Pregnancy; D017527:Receptor, IGF Type 2 / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012756:Sheep / Q000196:embryology*
chemical_list: D017527:Receptor, IGF Type 2; D051383:GRB10 Adaptor Protein; D000481:Allantoin