Studies On Buffalo Semen Proteomics

Semen composition is fundamental to male reproductive diseases. In recent years, the development of proteomics has provided a new effective method for semen characterization. So far, proteomics has been used for semen research in human, mouse and other animals to help us understand the molecular mechanisms of spermatogenesis and for treatment of male reproductive diseases. In this study, research on buffalo semen proteomics has been developed on the basis of previous work in our laboratory. The objectives of this study were twofold; firstly to optimize the proteomic technique systems for buffalo sperm and seminal plasma and thereby lay a good technical basis for subsequent differential proteomic research, and secondly to isolate and identify differentially expressed proteins from buffalo sperm of different motility and abnormality to provide a theoretical foundation for understanding the molecular mechanisms associated with sperm having low motility and abnormal morphology.Chapter1describes a comparison of parameters, such as different IPG strips, loading volume, concentreation of SDS-PAGE, and methods for purifing buffalo sperm proteins, in order to optimize the proteomic system for buffalo sperm. The results showed that a higher resolution and a better2-DE map with about500protein spots could be obtained when buffalo sperm proteins were extracted and purified by acetone precipitation;350μg of proteins were loaded onto24cm pH3-10nonlinear IPG strip followed by12.5%SDS-PAGE. Chapter2describes the separation of proteins from buffalo sperm of high and low motility using the optimized2-DE system. ImageMaster2.0software was used to compare and analyze2-DE maps, and MALDI-TOF-MS and bioinformatics were applied to identify the different proteins.2-DE maps were constructed from the differential proteomic study of buffalo sperm samples with high and low motility, and18differentially expressed proteins were found; five of these were down-regulated in the low motility sperm, eight were up-regulated, one was absent and four were only expressed in the low motility buffalo sperm. Finally, four of these proteins were successfully identified by MS as belonging to three unique proteins, which may be related to sperm motility; outer dense fiber of sperm tails protein2, ATP synthase subunit alpha, and succinyl-CoA synthetase subunit alpha. The outer dense fiber of sperm tails protein2and ATP synthase subunit alpha were down-regulated in the sperm with low molity, whereas succinyl-CoA synthetase subunit alpha was up-regulated.In Chapter3, the methodology was the same as in Chapter2.2-DE images were established from a differential proteomic study between higher abnormal rate and normal buffalo sperm;16differentially expressed proteins were found, six of these were down-regulated, five were up-regulated, three were absent and two were only expressed in the higher abnormal rate buffalo sperm. Six of these were successfully identified as belonging to four unique proteins by MS: L-asparaginase; heat shock protein beta-9; galactokinase; tubulin beta-2C chain. L-asparaginase, heat shock protein beta-9and galactokinase were up-regulated in buffalo sperm with the higher abnormal rate, whereas tubulin beta-2C chain was down-regulated. These differental proteins were associated with sperm structure and metabolism, and are possibly related to sperm morphology.In Chapter4, different loading volumes were compared in order to optimize the2-DE system for buffalo seminal plasma proteome, and MS was used to identify some of the proteins. The results showed that the use of300μg of proteins was better than either200μg or400μg; when300μg of proteins were loaded onto24cm pH3-10nonlinear IPG strip followed by12.5%SDS-PAGE, a high-resolution2-DE map with more than500protein spots was obtained.103of these protein spots were identified by MS;27of them were successfully identified for15unique proteins:serum albumin, serotransferrin, T-complex protein1subunit beta, phosphoglycerate kinase, sorbitol dehydrogenase, actin cytoplasmic2, fructose-1,6-bisphosphatase1, zinc-alpha-2-glycoprotein, allergen Bos d2, seminal plasma protein BSP-30kDa; peroxiredoxin-5mitochondrial, epididymal secretory protein E1, lysozyme C-3, ribonuclease RNase A family4, and phosphoglycerate mutase.In summary, the2-DE systems for buffalo sperm and seminal plasma were optimized, and these then laid a good foundation for a semem differential proteomics study. Additionally, it was possible to identify and analyze some of the proteins on the2-DE map that were expressed differently between buffalo sperm with different motility and abnormality. Finally, these results help to develop an understanding of the molecular mechanisms involved in sperm with low motility and abnormal morphology, and provide clues for finding molecular markers associated with sperm motility and morphology.