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Graf, Alexander (2015): Analysis of genome activation in early bovine embryos by bioinformatic evaluation of RNA-Seq data. Dissertation, LMU München: Faculty of Veterinary Medicine



During maternal-to-embryonic transition, control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation. Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts and to date there is no systematic study addressing the activation of specific genes during several stages of early bovine embryo development. Using the bovine model system, comparative analyses of RNA-seq data set were performed. The sequencing libraries had been constructed starting with germinal vesicle (GV) and metaphase II (MII) oocytes and embryos at the four-cell, eight-cell, 16-cell and blastocyst stage. The embryos had been generated in vitro by fertilization of Bos taurus taurus oocytes with sperm of a Bos taurus indicus sire. In total, approximately 13,000 RNA species could be identified in oocytes and each embryonic stages. The number of identified differential abundant transcripts increased in the course of development from roughly 100 to several thousands, with a sharp rise at the eight-cell stage. A bioinformatic approach could be developed to capture maternally delivered and de novo synthesized RNA species separately. It sensitively identified actively transcribed genes despite the fact that comparative analyses failed due to presence of the huge amount of RNA provided by the oocyte. Actively transcribed RNA species could be identified for approximately 8,000 genes, the majority of them at the eight-cell stage. This finding indicated, that the majority of all RNA species provided by oocytes was de novo transcribed during early embryonic development. Furthermore, it could be shown that the de novo transcription of larger genes was initiated later in embryonic development than smaller ones. A procedure was established to identify Bos t. indicus specific SNPs in RNA-Seq datasets which identified more than 60,000 SNPs occurring in 20% of all annotated genes. A major part of these SNPs could be detected at the eight-cell stage. This procedure enables a way to capture and study allele-specific transcription during early embryonic development. The described bioinformatic approaches were used to study major genome activation, an important step in the maternal-to-embryonic transition. More than 4,000 genes were de novo transcribed during major genome activation, which was found to occur at the eight-cell stage. These genes were functionally related to transcription, translation and their regulation. In summary, this thesis created and applied a powerful tool set for bioinformatic dissection of processes occurring during development of early bovine embryos and provided unprecedented insights in major genome activation.