Establishment Of Two-Dimensional Gel Electrophoresis For Porcine Oocyte

This objective of the study is to establish and optimizetwo-dimensional gel electrophoresis (2-DE) for proteomics of porcineoocyte in vitro maturation (IVM) and analyzed the different proteinexpression during the in vitro maturation. The thesis is arrangedin three chapters: chapter one is a review of literatures; chaptertwo and chapter three are experimental works.The objective in chapter two was to establish 2-DE for proteomicsof pig oocytes and optimize the relating techniques including proteinextraction, sample treatment, sample loading, choice of IPG gels,concentration of SDS gels, preset of electric parameters and protocolfor staining. The results of SDS-PAGE electrophoresis indicated thatthe protein expression pre- and post-IVM was similar.Then one step protocol of 2-DE for protein analysis wasestablished and improved. Porcine oocytes was treatment with 7 M Urea,4%(w/v) CHAP S, 2%(v/v) IPG Buffer (pH 3-10), 40 mM rris, 70 mMdithiothreito (DTT), protease inhibitors, and a trace of bromophenolblue. And then the protein was extracted by ultrasonndandcentrifugation at 4,000G. An Immobiline Dry Strips (IPG) of pH 3-10L, 13 cm was used for the first dimension isoelectric focusing (IEF).initiative to hydrate 100μg sample; the program of IEF with silverstaining was : 30 V, 6 h; 60 V, 6 h; 500 V, 4 h; 1000 V, 1h; 8000V, 7 h. The second dimension was done in 12.5% polyacrylamide gels.Silver staining visualized protein spots. In chapter three, comparative analysis of the oocyte atdifferent stage of maturation by the Imagemaster 2D was carried outto characterize candidate proteins differentially expressed duringin vitro maturation. The results revealed that there are about 800protein spots in each map, 35 spots were difference, thereinto, 22spots were up, 13 spots were fall. The proteins that aredifferentially regulated during IVM may present potential biomarkersof oocyte maturation and quality. It is a useful inventory towarda deeper understanding of the mechanisms underlying reproduction anddevelopment.