Stage specific proteome signatures in bovine oocyte maturation and early embryogenesis

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.