Proteomic Analysis Of Sheep Testis In Different Developmental Stages

Male fertility is of paramount importance for animal reproduction and for breeding industries. As an important male reproductive organ, testis could produce sperms and androgens. To complete this complex process, lots of proteins are needed to express and regulate coordinately. So it is of great importance to do research on the protein compositions of testis by the method of proteomic. This study used sheep testis as the experimental material. Firstly, establish and optimize the two-dimensional electrophoresis(2-DE) system for testis proteins. Then, the difference of proteins expression in different developmental stages of sheep testis is studied by the method of comparative proteomic, mass spectrometry and bioinformatics. Finally, the q RT-PCR, Western blot and immunohistochemistry were used to detect some of the differential expressed proteins in different developmental stages of sheep testis. The results are as follws:1.Establishment and optimization of two- dimensional gel electrophoresis system in testis of sheep17 cm p H 3—10 IPG strips were used to separate sheep testis proteins. And different extracting methods, loading quantities, isoelectric focusing time and separation gel concentrations were compared of their influence on the 2-DE maps. The results indicated that the best 2-DE maps were obtained through preparing proteins samples by the trichloroacetic acid(TCA)/acetone precipitation, loading 750 μg protein samples, isoelectric focusing with 75 000 Vh or more and 12% sodium dodecyl sulfonate(SDS) gel for protein separation. The optimized 2-DE system gives the maximum number of protein spots with clear background.2.Proteomic analysis of sheep testis in different developmental stagesIn this study, 2-DE was applied to identify proteins expressed in five developmental stages of sheep. In total, the detected spot number in five developmental stages were 382, 522, 567, 596 and 671, respectively. Using PDquest 8.0 changes of the proteins from 5 developmental stages of sheep testis were investigated and 45 differentially expressed protein spots were found. 45 spots were identified by MALDI-TOF-TOF mass spectra, and successfully identified 37 proteins. All the identified proteins were annotated in detail based on their biological process, cellular component and molecular function by Blast2 GO. And discover that the identified proteins w ere mainly involved in ―cellular process and single-organism process‖, ―organelle and cell component‖, ―binding and catalytic activity‖.3.Confirmation of differentially expressed proteins of sheep testisThe transcript level of 15 proteins in sheep testis were also detected through q RT-PCR to compare the 5 developmental stages. 6 genes showed same expression tendency compared with protein, and 9 genes showed different expression tendency compared with protein level. Using Western blot and immunohistochemistry, the tendency of the expression of 4 proteins were proved to be identical to the results of 2-DE.