Protiomics Research In The Effect Of Hyperthermia On Spermatogenesis In Adult Men

A great number of efforts have been made at the frontier of male contraceptive research, to find that testicular heat exposure would lead to reversible dyszoospermia and be a prospective strategy.12 Chinese men participated our experiment.The scrota containing the testes were immersed in a water bath at 43°C for 30 minutes once daily for 6 consecutive days. Semen analysis indicated that total sperm counts were suppressed ever since 3 weeks, and reached the lowest level at 6 weeks, while 9 weeks after heat exposure, sperm counts began to recover, and returned to pretreatment levels at 12 weeks. Testicular histologic examinations were performed after 2 weeks, many testicular cells degenerated and detached from the seminiferous epithelium, indicating the abnormal spermatogenesis mode. After 9 weeks, the appearance and structure of seminiferous epithelial almost recovered.The purpose of this study was to apply a proteomic approach with two-dimensional gel electrophoresis (2-DE) to sepertate protein composition of the testis in normal men as well as 2 weeks or 9 weeks after heat shock. After silver staining, we compared and excised the protein spots that altered expression in human testes at different times after heat shock. The MALDI-TOF/TOF mass spectrum was carried out following trypsin digesting. Altogether, we identified 55 spots based on peptide mass fingerprinting corresponding to 50 different proteins undoubtfully by searching the mass spectra against the International Protein Index database expressed differentially between the heat shock testis and the normal ones. All the identified proteins were bioinformatically annotated in detail based on their molecular function, biological process etc.Such analysis gave us an overall impression concerning the molecular mechanism underlying heat shock. Many proteins play roles in bilological regulation and catalytic action. They have important functions, for example, binding protein and nucleotide, having hydrolase and oxireductase activities, binding ion and so on. 8 proteins of them have oxireductase activities, accounting for 16% of annotated ones.By PathwayStudio software analysis, proteins were observed to participate in the categories of cell apoptosis, survival, and proliferation. We then analyze the differential expressed proteins at 2 weeks or 9 weeks after heat shock respectively, from the molecular passway about the abnormal spermatogenesis at 2 weeks and recovered seminiferous epithelial at 9 weeks, we might obtain the key proteins closely related with spermatogenesis. There are 31 proteins changed 2 weeks after heat shock, among which 13 proteins are up-regulated and 19 proteins are down-regulated. PathwayStudio software analysis indicated that such proteins mainly joined in the events of apoptosis, proliferation, and survival. ERP57, PRPF19, STCH, Ppia, Lamin A, Taldo1, hspa4l are the crucial proteins, and participated in most of the cell events of apoptosis, proliferation, and survival. The proteins with anti-apoptotic activities or recovery from injury such as ERP57, PRPF19, STCH, hspa4l were down-regulated, while the Pro-apoptosis or anti-proliferation proteins, take Ppia, Lamin A, Taldo1for example, were up-regulated.9 weeks after heat shock, there were 27 proteins changed their expression level, and among them 20 proteins are up-regulated and 7 proteins are down-regulated. PathwayStudio software analysis indicated that such proteins mainly played roles in apoptosis, proliferation, and survival. TCP1, RBP1, Alad, Vimentin, GAPDH, NPM, Vinculin are the important proteins. TCP1, RBP1, Alad, Vimentin, GAPDH were up-regulated, with anti-apoptotic activities or pro-proliferation while the Pro-apoptosis or anti-proliferation proteins ,take NPM, Vinculin for example, were down-regulated.Western blotting comfermed that the expression tendency of such key proteins was consistant with the 2D results.Following, we reported the expression patterns of these proteins in the human testes. They were expressed in the germ cells and/or the Sertoli, Leydig cells, supplying their possible roles during spermatogenesis by regulating the apoptosis, proliferation of germ cells.All in all, our studies would be helpful to illustrate protein network of abnormal spermatogenesis after testicular heat exposure. Such proteins would be the tagets of heat-induced male germ cell death, and merit clinical diagnosis and therapy of male infertility and provide clues for development of male contraception.