Investigations on epigenetic modulation during pre-implantation embryonic gene expression and its significance for the incidence of the large offspring syndrome

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A considerable proportion of the offspring born from nuclear transfer (NT)-derived and in vitro produced (IVP) embryos are affected by multiple abnormalities of which a high birth weight and an extended gestation length are the predominant features; a phenomenon that has been called "Large Offspring Syndrome (LOS)". The underlying mechanisms are widely unknown at present, but epigenetic modifications of embryonic and fetal gene expression patterns primarily caused by methylation changes are thought to be involved in this syndrome. With regard to embryonic gene expression patterns, numerous aberrations have been found in IVP and NT-derived embryos compared to their in vivo counterparts, ranging from suppression of expression to de novo overexpression or more frequently to a significant up- or downregulation of a specific gene. The objectives of the present study will be the development of a model system in which methylation can be manipulated experimentally. This gives a basis for studying the underlying mechanisms of the widespread dysregulation of gene expression in IVP and NT-derived bovine embryos. We will identify imprinted bovine genes by analysing gene expression in uniparental embryos. These gene expression patterns will be compared with those from embryos generated in vitro, in vivo ("controls") and after NT. To obtain information on epigenetic modulations, the methylation status of these imprinted genes will be determined by bisulfite sequencing. Furthermore, localization of gene products will be investigated by in situ hybridisation. In addition, we intend to study expression patterns of developmentally important genes that are obviously not subject to imprinting and whether their modification is due to methylation. In future experiments, expression of genes identified to be subject to epigenetic modifications will be analysed in bovine fetuses and neonates. Results of this study will demonstrate that the preimplantation bovine embryo is a suitable model for studying abnormal mammalian development and therefore is also a valuable system for assessing human development following assisted reproductive technologies.