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

从核移植（NT）衍生的和体外产生的（IVP）胚胎出生的相当大比例的后代受到多种异常的影响，其中高出生体重和延长的妊娠期长度是主要特征;这种现象被称为“大后代综合征（LOS）"。其潜在机制目前还不清楚，但认为主要由甲基化变化引起的胚胎和胎儿基因表达模式的表观遗传修饰与该综合征有关。关于胚胎基因表达模式，与其体内对应物相比，在IVP和NT衍生胚胎中发现了许多畸变，范围从表达抑制到从头过表达或更频繁地到特定基因的显著上调或下调。本研究的目的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衍生的牛胚胎中广泛的基因表达失调的潜在机制提供了基础。我们将通过分析单亲胚胎中的基因表达来鉴定印记牛基因。将这些基因表达模式与来自体外、体内（“对照”）和NT后产生的胚胎的基因表达模式进行比较。为了获得关于表观遗传调节的信息，将通过亚硫酸氢盐测序来确定这些印迹基因的甲基化状态。此外，将通过原位杂交研究基因产物的定位。此外，我们打算研究发育重要基因的表达模式，这些基因显然不受印记的影响，以及它们的修饰是否是由于甲基化。在未来的实验中，将在牛胎儿和新生儿中分析鉴定为表观遗传修饰的基因的表达。本研究的结果将证明，植入前牛胚胎是一个合适的模型，用于研究异常哺乳动物的发展，因此也是一个有价值的系统，用于评估人类的发展后，辅助生殖技术。

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.