Until recently, oocyte development was mostly followed by visual inspection of the different stages of the oocytes, either under a microscope (Selman et al., 1993; Maack and Segner, 2003), or by following the activity of enzymes involving with the maturation process with their corresponding maturation stages. Oocyte growth and development are accompanied by changes in the opacity of the oocyte, as well as changes in color, size, and density. These changes are mostly attributed to the accumulation of Vtg products, to their aggregation into protein aggregates, to their processing into small peptides, and finally into free amino acids. Molecular staging of the oocytes can be performed using transcriptomics and proteomics. It is based on establishing genes and proteins expression patterns as representing specific stages of oocyte development. The gene expression patterns discussed in Chapter 3 in this book (Knoll-Gellida et al., 2006) are example of the transcriptomic-based approach and its comparison with proteomic profiling (recent review of molecular staging in farm animals (Sirard et al., 2003). Molecular staging based on proteomics approach was demonstrated in pigs by Ellederova et al. (2004). Proteomics-based molecular staging is able to include in the analysis those proteins produced outside the oocytes (such as Vtg products, which are synthesized in the liver).
Ellederova et al. (2004) used proteomics tools to analyze pig oocyte proteins during in vitro maturation (IVM) using 2D-PAGE for separation, and visualization of oocyte proteins and MS for the identifications. Comparative analysis was used to identify unique protein patterns of different staged in vitro mature oocytes. They noticed that the expression of antiquitin (D7A1) increased during first meiosis and second meiosis compared to germinal vesicle in vitro maturated oocytes. Such differentially expressed proteins may be useful as bio-markers of oocyte IVM and quality.
We have also followed the changes in the forms of Vtg-derived proteins in sea bream and in zebrafish during oocyte development and identified by MS/MS many of the Vtg-derived protein spots on a 2D-PAGE. We therefore propose 2D-PAGE as a useful approach for establishing comprehensive maps of Vtg-derived proteins in oocytes during their development (Gattegno et al., in preparation).
The exquisite sensitivity of modern genomics and proteomics technologies enables the analyses of the transcriptomes and the proteomes of single oocytes. The significance of this ability relates to the possibility of studying the variability among oocytes of the same apparent stage and correlating the visible changes taking place during maturation with molecular level changes in protein repertoires. The oocytes are visualized and photographed under the microscope, followed by extraction and analysis of their proteins. Each of the different maturation stage proteins are resolved by 2D-PAGE, with each gel image representing the protein repertoire of a single oocyte. Each gel reveals the differences among individual oocytes and correlates protein patterns with morphology. We have analyzed single oocytes of zebrafish and of sea bream that were morphologically defined at developmental stage III. They differed significantly in their protein repertoires, most notably, in their processing of the Vtg-derived proteins. Earlier stage oocytes are more difficult to analyze due to their small size and limited amount of protein content. Oocytes that were morphologically defined at stage IV displayed a very similar protein repertoire, since Vtg is very abundant at this stage, and as previously mentioned, it masks many of the other proteins in the sample (Gattegno et al., in preparation).
The use of proteomics for cataloguing of oocyte proteins, following the changes in the proteins repertoire during development and determining the variability between individual oocytes is just beginning. It is likely to enhance our understanding of oocyte biology in the near future and to facilitate medical and biotechnology advances in reproductive biology and animal farming as the technology will be implemented into routine use.
Funding for the research in A.A. lab was from EC contract # Q5RS-2002-00784 - CRYOCYTE.
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