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Deutsch, Daniela Ramona (2015): Stage specific proteome signatures in bovine oocyte maturation and early embryogenesis. Dissertation, LMU München: Faculty of Chemistry and Pharmacy



One of the most critical periods in mammalian development is early embryogenesis. Its timing and morphology have been well studied, but molecular processes are still poorly understood. Several transcriptomic studies have addressed these molecular events. However, development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome and adequate de/activation and relocation of proteins. Therefore, this thesis addresses early mammalian development, i.e., oocyte maturation and the first steps of embryogenesis, by holistic iTRAQ-based discovery approaches and by a targeted approach based on SRM. Numerous proteins important for oocyte maturation and embryogenesis so far not described in the mammalian system were identified and quantified, and protein profiles for key players previously described in the literature were contributed. In oocyte maturation, bioinformatics analysis revealed proteins increasing in abundance involved in rearrangement of the cytoskeleton, protein transport and cell cycle progression. Supplementation of oocyte maturation media with the gonadotropins follicle stimulation hormone and luteinizing hormone resulted in an increase of developmentally important proteins, but did not change developmental rates. Addition of growth hormone during oocyte maturation led to diverse and opposing effects at the level of proteins and also did not influence subsequent embryo development. During early embryogenesis, a considerable fraction of proteins continuously increased in abundance, despite a strongly attenuated rate of translation reported for this period. Bioinformatics analysis revealed particularly interesting proteins involved in the p53 pathway, lipid metabolism and mitosis. Activation of the unfolded protein response is demonstrated, which may be an indicator of the still lower success rates of in vitro versus in vivo embryo production. Relevant differences between transcript and protein abundance levels were detected, e.g., for WEE2, which highlights the importance of innovative proteomic tools and workflows to complement transcriptome data of early embryogenesis. iTRAQ results of early embryogenesis were successfully verified by targeted 5-plex SRM analysis. By principal component analysis, SRM quantifications comprising a panel of only five proteins were shown to discriminate between all four developmental stages analyzed. Using an expanded 27-plex SRM assay, proteins were quantified in nine developmentally interesting stages and absolute protein contents were established for nine proteins. SRM is a highly sensitive tool for detection of disturbances and disorders of embryonic development at the molecular level, thus complementing morphological analyses by high resolution microscopy.