Retrotransposon activity and DNA methylation control in bovine preimplantation embryos

Retrotransposons, or RNA intermediated transposable elements, have been considered as ‘junk’ or ‘selfish’ DNA and dismissed as uninteresting for a long time. However, they were found active and functional in preimplantation embryos in the last decades. The impact of retrotransposon activity on the genome such as formation of new genetic elements and regulators in the genome is unneglectable. They are also suggested to participate in many activities during early embryo development, like genomic imprinting, X chromosome inactivation, cell proliferation and differentiation. In Chapter 1, the current literature on retrotransposons in mammalian genomes is reviewed. An overview of the classification and composition of retrotransposons in the genome is given, with emphasis of the bovine genome. Furthermore, the mechanisms of retrotransposition are described in detail and the activities of retrotransposons on the level of transcription, translation and mobilization are explained. The impact of retrotransposon activity on the mammalian genome is discussed, illustrating that retrotransposons influence the genome by formatting new genetic elements and working as regulators. The defense mechanisms against retrotransposon to prevent their disturbance of the genomes are also given in Chapter 1. In the latter part of Chapter 1, activation of retrotransposons in early development is discussed. Retrotransposons participate in many embryonic activities, including cell proliferation and differentiation, genomic imprinting, and X-chromosome inactivation. These activities are suggested to be mainly a consequence of DNA methylation loss during epigenetic reprogramming of preimplantation. Therefore, a description of the dynamics of DNA methylation during bovine preimplantation embryo development is included. Those facts reveal the importance of the study of the relation between DNA methylation and retrotransposon expression in embryo development. At last, a short review of using retrotransposons as marker for global DNA methylation status is given. The aims of the study are presented in Chapter 2. The general aim of the thesis was to study retrotransposon expression and their DNA methylation control in bovine preimplantation embryos. The first aim was to find out autonomous retrotransposons with consistent RNA expression, and reliable reference genes for gene expression normalization during bovine preimplantation embryo development. The second aim was to study the relation between DNA methylation and retrotransposon expression under high oxygen. The third aim was to use repetitive sequences as global DNA methylation marker in bovine preimplantation embryos. To achieve these goals, we first profiled autonomous retrotransposon expression in bovine preimplantation embryos. L1_BT, BovB and ERV1-1-I_BT were found to be expressed throughout all stages of preimplantation development (Chapter 3.1). Furthermore, reference genes were evaluated and selected for estimating retrotransposon expression in bovine embryos of different stages and under different conditions (Chapter 3.2). In Chapter 4, the relation between DNA methylation and retrotransposon expression under influence of high oxygen tension as stressor was studied. The global DNA methylation of embryos was estimated by 5-methylcytosine immunofluorescent (5-mC) staining. We found a significant increase in DNA methylation under 20% O2 at the 4-cell stage and in blastocysts. Gene expression of DNA methytransferases (DNMTs) and three retrotransposons (L1_BT, BovB and ERV1-1-I_BT) in 4-cell embryos and blastocysts was analyzed by RT-qPCR. Unexpectedly, the retrotransposon expression was not correlated with the global DNA methylation level estimated by 5-mC staining, but was negatively correlated with the expression of DNMT1. We further studied the DNA methylation level of retrotransposons during embryo development by bisulfite sequencing (Chapter 5). Using this method, a negative correlation between the methylation level (L1_BT BovB > ERV1-1-I_BT) was found. Additionally, we developed a global DNA methylation marker using a combination of repetitive sequences with low mutation rate, taking in to account the proportion of each sequence. The general discussion and the conclusions are presented in Chapter 6. The discussion focuses on the following aspects: 1) correlation of DNA methylation and retrotransposon expression; 2) influence of oxidative stress on methylation and retrotransposon expression; 3) problems in immunofluorescence staining; 4) comparison of the methylation research methods used in the thesis; 5) DNA methylation dynamics during bovine preimplantation embryo development; and 6) retrotransposons used as global DNA methylation marker