Establishment Of Proteomics Methods And Initial Analysis Of The Differential Proteins Between Dairy Cow X And Y Spermatozoa

In the last few years, the completion of genome project has provided a broader platform for life science. However, biodiversity and complexity suggested that the information from genome was still not enough to explain the mechanism of life. So proteomics was more and more useful to understanding of the mechanism of life.In order to establish the proteomic research platform of bovine sperm, the optimizing methods including preparation of sperm protein, 2-DE protocols and in-gel digestion were explored. With the help of proteomic platform, the differentially expressed proteins of X and Y bovine spermatozoa were isolated and identified. It provided scientific evidence for the differential protein between X and Y bovine spermatozoa. Some immunological methods would be used to separate X and Y spermatozoa. Also it provides a new method to study the mechanisms of sex determination.The sperm protein was prepared by the method of heat TRIzol. Two-dimensional gel electrophoresis(2-DE) was used to separate the total proteins of bovine X and Y spermatozoa. ImageMaster 6.0 software was applied to analyze 2-DE images. The differential proteins between X and Y spermatozoa were identified by both MALDI-TOF -MS and bioinformatics.Good 2-DE pattern was obtained by optimizing the isoelectric focusing protocols and staining methods. The in-gel digestion protocols which could digest the silver-staing spots for mass spectrum was established through optimize condition. Finally, 16 spots from the sample of frozedn-thawed bovine spermatozoa were identified. Twelve differential proteins in the X and Y spermatozoa were identified, six of which only expressed or up-regulated in X spermatozoa and the others only expressed or up-regulated in Y spermatozoa. Differentially expressed proteins identified in this study may be associated with spermatozoa production and metabolism.