Retrotransposon Activity and DNA Methylation Control in Bovine Preimplantation Embryos Retrotransposon Activiteit en DNA-methylatie Controle in Bovine Preimplantatie Embryo's Wenwen Li Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Veterinary Medicine, Faculty of Veterinary Medicine Ghent University 2016 Promoter Co-promoter Prof. Dr. Luc Peelman Prof. Dr. Ann van Soom Department of Nutrition, Genetics and Ethology Faculty of Veterinary Medicine Ghent University Members of the Jury Prof. Dr. Geert Opsomer (Chairman) Faculty of Veterinary Medicine Ghent University Prof. Dr. Peter Bols Department of Veterinary Science University of Antwerp Prof. Dr. Filip Van Nieuwerburgh Faculty of Pharmaceutical Sciences Ghent University Prof. Dr. Ward De Spiegelaere Faculty of Veterinary Medicine Ghent University Dr. Karen Goossens Institute for Agricultural and Fisheries Research (ILVO) Dr. Bart Leemans (Secretary) Faculty of Veterinary Medicine Ghent University iii iv Members of the Jury .................................................................................................................. iii List of figures ............................................................................................................................ ix List of abbreviations .................................................................................................................. xi Chapter 1 General introduction .................................................................................................. 1 1.1 Introduction of retrotransposons ............................................................................................... 3 1.1.1 Classification of retrotransposons .......................................................................................... 3 1.1.2 Retrotransposons in the bovine genome ................................................................................. 9 1.1.3 Mechanisms of retrotransposition ........................................................................................ 11 1.1.4 Expression and mobilization of retrotranposons in mammalian genomes ........................... 15 1.1.5 Impact of retrotransposon activity on the mammalian genome ........................................... 19 1.1.6 Regulation of retrotransposition ........................................................................................... 26 1.1.7 DNA methylation of retrotransposons as marker for global cell methylation status ........... 34 1.2 Introduction of early embryo development ............................................................................. 37 1.2.1 In vivo bovine preimplantation embryo development.......................................................... 37 1.2.2 Transcriptional dynamics in preimplantation embryo .......................................................... 39 1.2.3 Epigenetic dynamics in preimplantation embryo ................................................................. 40 1.2.4 Activation of retrotransposons in early development ........................................................... 42 1.2.5 ARTs used in bovine embryo production ............................................................................. 46 1.2.6 Influence of IVC on embryo development ........................................................................... 47 1.2.7 Oxidative stress in early embryo development .................................................................... 49 1.3 References ............................................................................................................................... 52 Chapter 2 Aims of the study ..................................................................................................... 83 Chapter 3 Retrotransposon Expression Profiling and Reference Gene Selection for qPCR Analysis in Bovine Preimplantation Embryos ......................................................................... 87 Chapter 3.1 Retrotransposon Expression Profiling in Bovine Preimplantation Embryos ............. 89 v 3.1.1 Abstract ......................................................................................................................... 90 3.1.2 Introduction .................................................................................................................. 90 3.1.3 Material and methods ................................................................................................... 91 3.1.4 Results and discussion .................................................................................................. 98 3.1.5 Conclusions ................................................................................................................ 100 3.1.6 Acknowledgements .................................................................................................... 100 Chapter 3.2 Reference Gene Selection for Normalization of RT-qPCR Data in Sexed Bovine Preimplantation Embryo under Low and High Oxygen Tension ................................................ 101 3.2.1 Abstract ....................................................................................................................... 102 3.2.2 Introduction ................................................................................................................ 102 3.2.3 Material and methods ................................................................................................. 103 3.2.4 Results and discussion ................................................................................................ 108 3.2.5 Conclusions ................................................................................................................ 114 3.2.6 Acknowledgements .................................................................................................... 115 3.3 References ............................................................................................................................. 115 Chapter 4 High Oxygen Tension Increases Global Methylation in Bovine 4-cell Embryos and Blastocysts but does not Affect General Retrotransposon Expression .................................. 119 4.1 Abstract .................................................................................................................................. 121 4.2 Introduction ........................................................................................................................... 122 4.3 Materials and methods ........................................................................................................... 124 4.4 Results ................................................................................................................................... 129 4.4 Discussion .............................................................................................................................. 135 4.5 Acknowledgements ............................................................................................................... 140 4.6 References ............................................................................................................................. 141 Chapter 5 Repeats as Global DNA Methylation Marker in Bovine Preimplantation Embryos .......... 149 5.1 Abstract .................................................................................................................................. 151 Preimplantation embryo; DNA methylation; retrotransposon; bisulfite sequencing ................... 151 vi 5.2 Introduction ........................................................................................................................... 152 5.3 Materials and Methods .......................................................................................................... 154 5.4 Results ................................................................................................................................... 159 5.5 Discussion ............................................................................................................................. 165 5.6 Conclusion ............................................................................................................................. 167 5.7 Acknowledgements ............................................................................................................... 168 5.8 References ............................................................................................................................. 168 Chapter 6 General discussion and conclusions....................................................................... 173 6.1 Correlation of DNA methylation and expression of retrotransposons during preimplantation development ................................................................................................................................ 175 6.2 Oxidative stress influence on methylation and retrotransposon expression .......................... 178 6.3 Difficulties encountered in the immunofluorescence staining .............................................. 180 6.4 Comparison of DNA methylation experiments ..................................................................... 183 6.5 DNA methylation reprogramming during preimplantation embryo development ................ 186 6.6 Retrotransposons as DNA methylation marker ..................................................................... 188 6.7 General conclusions .............................................................................................................