Proteomic Analysis of Effect of Hyperthermia on Spermatogenesis in Adult Male Mice

Abstract

We characterized cellular and molecular mechanisms involved in spermatogenesis following short-term heat exposure of murine testis. For these studies, we utilized a proteomic approach with two-dimensional gel electrophoresis (2DE) analyses and mass spectroscopic identification of proteins with altered expression in mouse testes at different times after heat shock. We established a proteome reference map from 7-wk-old mouse testis linked to a federated proteome database. We used these tools to analyze quantitative variations in the tissue over a time course of 0.5, 2, 6, and 12 h following heat exposure. We separated 108 protein spots expressed differentially between the heat shock tissues and the control mouse testes. Of these spots, we identified 36 by comparing with the control reference map. We then focused on the heterogeneous nuclear ribonucleoproteins (hnRNPs) and the chaperonins containing t-complex polypeptide-1 (CCT). Further analysis in this heat-shocked model suggests numerous potential mechanisms for heat shock-induced spermatogenic disorder.

Proteomics of spermatogenesis: from protein lists to understanding the regulation of male fertility and infertility

“…Second, with western blotting, antibodies specific for PTMs can identify spots in a gel containing proteins with these modifications. 60 In our studies, 21,22,32,37,39,49,57 most of the proteome profiles related to spermatogenesis were established by 2DE-MS, and many proteins were represented by two or more spots in the gel, which indicated PTMs such as phosphorylation.…”

“…To identify the wholeprotein expression profile of the testis, Zhu et al, 21 in our laboratory, established a proteome reference map containing 504 identified proteins from adult mouse testis using two-dimensional electrophoresis (2DE) and mass spectrometry (MS), which was linked to a nationwide proteome database (full data sets are available on http://reprod.njmu.edu.cn/cgi-bin/2d/2d.cgi). The online reference map hyperlinked the identified protein spots to the individual protein entries.…”

“…However, LC-MS cannot match proteins identified by software with their Mr and pI, and another major limitation is that this technique is not suitable for quantitative analysis. 59,65 To overcome the shortcomings of 2DE and LC as individual techniques, this study 21,30,32 combined 2DE and LC to explore protein changes in cells and tissues. In mouse testis, we used 2DE to construct an overall proteome profile and comparative proteome profiles of different stages of spermatogenesis, which revealed that many proteins appeared as two or more spots.…”

Proteomic Analysis of Testis Biopsies in Men Treated with Injectable Testosterone Undecanoate Alone or in Combination with Oral Levonorgestrel as Potential Male Contraceptive

“…The stained gels were scanned, and the ImageMaster 2D Platinum Software (Version 5.0; GE Healthcare, Swiss Institute of Bioinformatics, Geneva, Switzerland) was used for spot detection, quantification, and comparative and statistical analyses, as previously described. 13 In each experiment, protein expression in the testicular biopsy specimens of the subjects treated with TU or TU + LNG was compared with that in the testicular biopsy specimens obtained from subjects at baseline. The values from 4 independent biopsy samples from each group were pooled for the calculation of the means and their respective standard deviations, and independent t-test was performed to determine the significance of the differences between protein expression in the control group (at baseline, before treatment) and that in the group receiving TU or TU + LNG treatment.…”

“…The search conditions used were as described in our previous work. 13 The peptide mass was compared with the theoretical peptide masses of all available proteins from all species. The search conditions used were as follows: 100 ppm for external calibration, one missed cleavage allowed, and modification of cysteines by iodoacetamide, methionine oxidation, and N-terminal pyroglutamylation as variable modifications.…”