Comparative Proteomic Analysis Of Mature And Immature Ovine Oocytes Proteins

Comparative proteomic analysis of mature and immature ovine oocyetes proteins used proteomics method to research the protein composition and difference expression during ovine oocyte maturation from the overall level, which provided a theoretical basis for the molecular mechanism research of ovine oocyte maturation. Thesis contained four parts, there were summary; the construction and preliminary analysis of ovine oocytes proteomic 2-DE map; analysis of the interrelationship of ovine ooeyte nuclear maturation, cumulus expansion and hormone concentrations; comparative proteomic analysis of mature and immature ovine oocytes proteins.GV stage ovine oocytes were solubilized in lysis buffer, and protein concentrations were measured by the method of Bradford and Ramagli, then the 2-DE maps were analyzed through PDQuest8.0. Ovine oocytes were cultured in culture medium consisting hormones to get the mature oocyets, the experiment designed different hormone concentrations to culture the ovine oocytes, and observed the rate of ovine oocyte nuclear maturation and cumulus expansion. After MⅡstage oocytes 2-DE maps were obtained, the differential spots of protein were found and identified by LC-MS/MS.Experiment showed that the protein concentration measured by the Bradford method was higher than Ramagli method; 2-DE map of 180μg protein sample detected by the Ramagli method or 700 oocytes was clearer; E2 could significantly promote the rate of ovine oocyte maturation(P<0.05), but had no effect on the rate of cumulus expansion; and could significantly improve the mature rate of cumulus expanded oocytes(P<0.05), but had no effect on the mature rate of cumulus unexpanded oocytes; Ovine oocyte maturation and cumulus expansion were considered to be no interdependent, but the rate of ovine oocyte maturation and cumulus expansion had a direct ratio relationship at a definitive hormone concentration. Analysis of the gels indicated that 28 protein spots appeared to be differentially expressed between the GV and MII stage, 11 of them appeared to be downregulated and 17 of them appeared to be upregulated. These protein spots were then cored from the 2-DE gels and identified by LC-MS/MS techniques. A total of 4 spots were identified by LC-MS/MS techniques, corresponded to 3 unique proteins, there were truncated VP2, malate dehdrogenase, glutathione transferase M3. Truncated VP2 and malate dehdrogenase were found to be downregulated during oocyte maturation. Glutathione transferase M3 was abundantly expressed in ovine oocytes and appeared to be upregulated.The result suggested that the Ramagli method could reflect concentration of oocyte protein more actually; Each 2-DE map loading 700 oocytes was more reasonable; E2 could stimulated ovine oocytes directly but was not mediated by cumulus cell; Ovine cumulus expansion was possibily involved by the autocrine secretion of cumulus expansion enabling factors by cumulus cell; Cumulus expansion rate could be considered as the standard of the rate of ovine oocyte maturation at a definitive hormone concentration; The synthesis and expression of mRNA and the rate of glucose metabolism were decreased during ovine oocytes maturation, but the levels of antioxidant and detoxication were increased. Experiment provided theoretical basis for the research of molecular mechanism of ovine oocyte maturation.