Strukturelle, molekulare und funkfionale Analyse der frühen Embryogenese beim Rind: Parameter für das Potenfial von Oozyten nach in vitro vs. in vivo Reifung

• Applicants: Dr. Georg Josef Arnold; Dr. Felix Andreas Habermann; Professor Dr. Eckhard Wolf
• Subject Area: Veterinary Medical Science ; Gynaecology and Obstetrics
• Term: from 2008 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 58733678

• Final Report Year: 2017

Final Report Abstract

This project aimed at structural, molecular and functional investigations of early mammalian development using bovine oocyte maturation and embryogenesis as a model system. Based on a large number of in vitro matured and fertilized oocytes, the time course of oocyte maturation, fertilization and of the first cleavage divisions as well as their relation to developmental potential of the embryo were established. In addition, a comprehensive catalogue of developmental abnormalities in these individual steps was compiled. For a detailed analysis of molecular correlates associated with oocyte maturation and embryo development, a holistic transcriptome study of oocytes in the germinal vesicle (GV) and metaphase II (MII) stages and of 4-cell, 8-cell, 16-cell embryos and blastocysts was performed. Using three different strategies, i) detection of de novo transcripts that are not present in oocytes; ii) detection of transcripts from the paternal allele; and iii) detection of incompletely spliced primary transcripts, we were for the first time able to map the onset of embryonic transcription for more than 7,400 genes. These studies were complemented by holistic proteome studies using an iTRAQ-nano-LC-MS/MS approach. A number of proteins with significant changes in their abundance during early development were discovered, demonstrating the dynamism of the proteome during early embryogenesis. A set of highly specific and sensitive assays based on selected reaction monitoring (SRM) were established for nine selected proteins, and their absolute concentrations in the embryo were determined in nine different stages. A Principal Component Analysis (PCA) based on these measurements revealed a characteristic and stage discriminating panel of protein concentrations for all stages analysed. In summary, this established new structural and molecular readouts for normal and disturbed oocyte maturation and early development. These will be important for future attempts to evaluate and improve assisted reproductive techniques (ART) and to provide new insight into fertility problems cause by genetic, epigenetic an environmental factors.

Publications

• (2010) Creating new knowledge for ruminant reproduction from rapidly expanding and evolving scientific databases. Soc Reprod Fertil Suppl 67:29-40
  Bauersachs S, Blum H, Krebs S, Fröhlich T, Arnold GJ, Wolf E
  
• (2011) Cell arrest and cell death in mammalian preimplantation development: lessons from the bovine model. PLoS One 6:e22121
  Leidenfrost S, Boelhauve M, Reichenbach M, Güngör T, Reichenbach HD, Sinowatz F, Wolf E, Habermann FA
  
• (2011) Galectins as tools for glycan mapping in histology: comparison of their binding profiles to the bovine zona pellucida by confocal laser scanning microscopy. Histochem Cell Biol 135:539-52
  Habermann FA, André S, Kaltner H, Kübler D, Sinowatz F, Gabius HJ
  
• (2014) Fine mapping of genome activation in bovine embryos by RNA sequencing. Proc Natl Acad Sci USA 111:4139-44
  Graf A, Krebs S, Zakhartchenko V, Schwalb B, Blum H, Wolf E
  
• (2014) Genome activation in bovine embryos: review of the literature and new insights from RNA sequencing experiments. Anim Reprod Sci 149:46-58
  Graf A, Krebs S, Heininen-Brown M, Zakhartchenko V, Blum H, Wolf E
  
• (2014) Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos. Nucleus 5:542-54
  Popken J, Koehler D, Brero A, Wuensch A, Guengoer T, Thormeyer T, Wolf E, Cremer T, Zakhartchenko V
  
• (2014) Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos. Nucleus 5:555-89
  Popken J, Brero A, Koehler D, Schmid VJ, Strauss A, Wuensch A, Guengoer T, Graf A, Krebs S, Blum H, Zakhartchenko V, Wolf E, Cremer T
  
• (2014) Stage-specific proteome signatures in early bovine embryo development. J Proteome Res 13:4363-76
  Deutsch DR, Fröhlich T, Otte KA, Beck A, Habermann FA, Wolf E, Arnold GJ
  
• (2015) Proteome analysis of early lineage specification in bovine embryos. Proteomics 15:688-701
  Demant M, Deutsch DR, Fröhlich T, Wolf E, Arnold GJ
  
• (2015) Remodeling of the nuclear envelope and lamina during bovine preimplantation development and its functional implications. PLoS One 1;10:e0124619
  Popken J, Graf A, Krebs S, Blum H, Schmid VJ, Strauss A, Guengoer T, Zakhartchenko V, Wolf E, Cremer T