Preliminary Studies On The Proteomics Of Buffalo Oocytes And Follicular Fluid

In this study, two-dimensional electrophoresis, liquid chromatography, and mass spectrometry were used to analysis the different protein expression in buffalo oocytes during in vitro maturation and during development. The thesis consists two sections:the first is a literature review, and the second describes the research in three parts.Part I:The conditions for two-dimensional protein expression of buffalo oocytes were established and optimized, and the differences during in vitro maturation were compared. The results indicated that lysis buffer A (7M urea,2M thiourea,4%CHAPS and0.5%DTT) was suitable for protein extraction. A clear2DE map was obtained in13cm pH4-7dry strips, and about300spots were detected using Image Master2D platinum software. It was found that there were27different proteins in GV and MⅡstage of buffalo oocytes, and8of them were successfully identified by mass spectrometry, including the HSP60, HSC71, MVP, GEMIN8, and NMP2proteins, which may be used as oocyte maturation markers.Part Ⅱ:In this part, the conditions for the two-dimensional analyse of buffalo oocytes were established, and some related parameters such as different methods of protein extraction, IPG strips, loading volume were optimized. The results indicated that the TCA-acetone precipitation method produced more spots and higher resolution than that of acetone and2-D clean-up kit in2-DE maps, which reflects a more abundant proteome. When150μg proteins were used in the24cm nonlinear (NL) IPG strip (pH3～10), about300spots were detected using the Image Master2D platinum software.Part III:The objective of this study was to detect the protein and peptide expression profiles in follicles from small follicles (?<4mm), medium-sized follicles (4mm<?<8mm) and big follicles (?>8mm). First, matrix-assisted laser desorption ionization mass spectrometry was used to establish a model of the peptide expression profile and to compare peptide expression changes. The results showed that there were12significantly different peptides in the three forms of follicle. The expression of6peptides increased and2peptides decreased in mature follicles; a1746Da peptide was expressed specifically in big follicles, and may be used as a biomarker of follicle maturation. Second, the different proteins from differential diameter follicular fluid were compared using liquid chromatography and mass spectrometry (LC-MS). The differential fraction was collected and subjected to further treatments, including reduction, alkylation and enzyme digestion; the peptides were identified after second time liquid chromatography separation. The results indicated that there was a remarkable protein peak from transferrin (TRF) in the dominant follicular fluid, and this could be a marker of potential ovulation. At the same time, the LC-MS technique provides a basis for buffalo follicular fluid separation and proteome profile construction.In summary, the two-dimensional map of buffalo oocytes before and after in vitro maturation, and different diameters follicular fluid proteins conditions was established, and the important regulatory proteins were screened. Some known and unknown functional proteins that may be involved in follicular growth and cocyte development were selected by analysis the diffirence, and a theoretical foundation laid down for the production of a comprehensive developmental mechanism for germ cells and for the improvement of buffalo reproductive efficiency.