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

相当一部分由核移植（NT）衍生和体外生产（IVP）胚胎出生的后代受到多种异常的影响，其中高出生体重和延长妊娠期是主要特征；这种现象被称为“大后代综合症”。其潜在的机制目前尚不清楚，但主要由甲基化改变引起的胚胎和胎儿基因表达模式的表观遗传修饰被认为与该综合征有关。关于胚胎基因表达模式，与体内基因相比，在IVP和nt衍生的胚胎中发现了许多畸变，从表达抑制到从头开始过表达，或者更频繁地到特定基因的显著上调或下调。本研究的目标将是开发一个模型系统，其中甲基化可以通过实验进行操作。这为研究IVP和nt衍生的牛胚胎中基因表达普遍失调的潜在机制提供了基础。我们将通过分析单系胚胎中的基因表达来鉴定牛的印迹基因。这些基因表达模式将与体外、体内（“对照组”）和NT后产生的胚胎的基因表达模式进行比较。为了获得表观遗传调节的信息，这些印迹基因的甲基化状态将通过亚硫酸盐测序来确定。此外，基因产物的定位将通过原位杂交进行研究。此外，我们打算研究明显不受印迹影响的发育重要基因的表达模式，以及它们的修饰是否由于甲基化。在未来的实验中，将在牛胎儿和新生儿中分析表观遗传修饰基因的表达。本研究结果将证明，牛胚胎着床前是研究哺乳动物异常发育的合适模型，因此也是评估辅助生殖技术后人类发育的一个有价值的系统。

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.