Analysis of methylated DNA contents and distribution in bovine placental and foetal tissues after somatic nuclear reprogramming

DNA methylation (methylated (5-mC) and hydroxymethylated (5-hmC) DNA), is highly dynamic during mammalian embryogenesis. After fertilization, a loss of 5-mC of maternal (passive process) and paternal (active process) genomes takes place; 5-hmC accumulates in the paternal pronucleus and decreases progressively with the embryonic cleavage. In blastocyst, the first cell differentiation is associated with higher levels of 5-mC in the inner cell mass compared with the trophectoderm. During foeto-placental development, the patterns of 5-mC and 5-hmC are established according to cell specification and tissue differentiation. In some bovine blastocysts produced by somatic cell nuclear transfer (SCNT), a global DNA hypermethylation has been observed in the trophectoderm. Defaults of placental and foetal development are frequent after SCNT in mammals. Whether these pregnancy pathologies are associated with alterations of global DNA methylation remains to be analysed. In this study, the patterns of DNA methylation were determined in extra-embryonic and foetal tissues during pregnancies obtained by artificial insemination (AI) versus SCNT i) by a global quantification using a LUminometric Methylation Assay and ii) by immunohistochemistry with specific antibodies. Great variations in methylation levels between extra-embryonic and fœtal tissues at D60 of gestation were observed from 30% to 90% (chorionic villi > chorion > liver > heart > brain > amnion > allantois). Chorionic methylation content increased significantly from D18 until term of gestation. Effects of SCNT on trophoblast methylation level in blastocyst were conserved in trophoblast at D18 and D40 and in cotyledons at G60 only. However, by immunocytochemistry, only the mesenchymal part of chorionic villi was stained using 5-mC antibody and an unexpected 5-hmC staining was found in various placental and extra-embryonic cell types, suggesting a role of this epigenetic mark in all lineages in bovine.