| The testicular seminiferous tubules are the important site of spermatogenesis containing somatic Sertoli cells and different types of germ cells. The Sertoli cells provide nutrients for germ cells and play a vital role in formation of mature sperm developed from spermatocyte. Spermatogenesis is a lengthy, remarkably sophisticated process involved in the transmission of genetic heritage. Mammalian spermatogenesis includes three major divisions:spermatocytogenesis, meiosis, and spermiogenesis, the process controlled by a variety of excreted factors and conditioned by the successive activation and/or repression of thousands of genes and proteins between Sertoli cells and germ cells. Thus the detailed molecular mechanisms of spermatogenesis are not yet fully understood.Water buffalo (Bubalus bubalis) is an economically important livestock species in Guangxi with the characteristics of roughage-resistance, high feed conversion rate, and high dairy nutrition value, but its reproductive efficiency is low compared to other domesticated ruminants. Therefore an in-depth basic research in this ruminant species is highly desirable to achieve greater economic value. Here, we will use for the first time comparative proteomics methods to explore the protein expression from buffalo testicular seminiferous tubules at three different developmental stages (prepuberty, puberty and postpuberty), and then identify spermatogenesis-associated and male reproductive physiology related proteins. The thesis were mainly divided into the following contents according to the two different types of protein separation methods:two-dimensional electrophoresis (2-DE) and liquid chromatography (LC).Chapter one describes the comparative proteomic analysis of three different developmental stages (prepuberty, puberty and postpuberty) of buffalo testicular seminiferous tubules using 2-DE method and includes two parts. Firstly, a suitable 2-DE system for separating proteins of buffalo testicular seminiferous tubules was established. This involved comparing and optimizing various parameters, such as IPG strips and protein loading volume. The results indicated that a higher resolution and a better 2-DE map with about 486 protein spots could be obtained in a loading volume of 350 μg in 24 cm IPG strips (pH 4-7). Secondly, the proteins from three different developmental stages (prepuberty, puberty and postpuberty) of buffalo testicular seminiferous tubules were separated using the established and optimized 2-DE system. ImageMaster 2.0 software was used to compare and analyze 2-DE maps, and MALDI-TOF-MS and bioinformatics were applied to identify the different proteins. The results showed that 150 different protein spots were obtained between prepubertal and pubertal buffalo testicular seminiferous tubules, and 92 different protein spots were obtained between pubertal and postpubertal buffalo testicular seminiferous tubules. Finally,4 different protein spots between prepubertal and pubertal buffalo testicular seminiferous tubules were successfully identified by MS as belonging 4 proteins, which were all down-regulated in the prepubertal buffalo testicular seminiferous tubules; and 15 different protein spots between pubertal and postpubertal buffalo testicular seminiferous tubules were successfully identified by MS as belonging 13 unique proteins,8 of which were up-regulated in the postpubertal buffalo testicular seminiferous tubules,4 proteins were down-regulated, and one protein was only expressed in the postpubertal buffalo testicular seminiferous tubules. Taken together, the results establish an appropriate 2-DE system for separating buffalo testicular seminiferous tubules proteins and identify a group of different proteins may be related to buffalo spermatogenesis, such as Superoxide dismutase, Heat shock-related 70 KDa protein 2, Vimentin, Prohibitin, KRT8 protein, and Galectin-1, and so on.Chapter two describes the comparative proteomic analysis of three different developmental stages (prepuberty, puberty and postpuberty) of buffalo testicular seminiferous tubules using tandem mass tag (TMT) coupled to LC-MS/MS method and includes three parts. Firstly, the peptides from three different developmental stages (prepuberty, puberty and postpuberty) of buffalo testicular seminiferous tubules proteins digested with trypsin were labeled with TMT reagents, separated by two-dimensional liquid chromatography and identified using LTQ-Orbitrap Elite MS. In total,1286 proteins were successfully identified and quantified by TMT reporter ions in three biological and four technological replicates. Of these proteins,304 showed significant differential expression (p<0.05, ratio≥2.0 fold) among the three developmental stages. Compared with the pubertal buffalo testicular seminiferous tubules proteins,252 proteins were up-regulated in prepubertal buffalo testicular seminiferous tubules,16 proteins were down-regulated; one protein was up-regulated in postpubertal buffalo testicular seminiferous tubules,26 proteins were down-regulated; and 9 proteins were down-regulated both in prepubertal and postpubertal buffalo testicular seminiferous tubules. Secondly, bioinformatics analysis including GO analyses and pathway analyses of the identified differential expression proteins were carried out using jointly UniProt and the SpermatogenesisOnline 1.0 database. The results showed that 27 proteins were theoretically found to be involved in spermatogenesis, and 26 proteins may be involved in cell aging, proliferation, and developmental processes. Finally, seven selected proteins (SPERT, EEF1G, ROPN1, AKAP3, HSPA2, ODF2, and CABS1) were validated by Western blot, quantitative RT-PCR, and further subcellular localization analysis. The results of WB analysis of seven proteins were consistent with those of TMT quantification and the expression patterns of four genes(SPERT, EEF1G, AKAP3, and HSPA2) correspond to those of the protein levels. The immunohistochemical analysis showed that EEF1G was mainly expressed in the nucleus of round spermatids, as well as moderately expressed in the nucleus of Sertoli cells and Leydig cells, HSPA2 was localized in both the cytoplasm and nucleus of spermatogenic cells and at the basement membrane of seminiferous tubules, and CABS1 was expressed in the cytoplasm of the late spermatids in buffalo seminiferous tubules. In addition, immunofluorescence analyses of four sperm-located proteins showed that SPERT was located in the region of the acrosome, neck, and the beginning of the midpiece, AKAP3 and ODF2 were mainly located in the region of acrosome, whilst ROPN1 was located in the principal piece. Taken together, the results isolated and identified different proteins on a large scale of buffalo testicular seminiferous tubules at different developmental stages by TMT-coupled 2D LC-MS/MS method, and comprehensively displayed the protein expression and change regularity.In conclusion, by performing comparative proteomic analyses of buffalo testicular seminiferous tubules at three different developmental stages for the first time by 2-DE/MS and LC/MS methods, the results help to develop an understanding of the molecular mechanisms involved in spermatogenesis and provide clues for finding molecular markers associated with buffalo spermatogenesis, whilst improving our understanding of the mechanisms of male reproduction regulation. |
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