Spermatogenesis-associated proteins at different developmental stages of buffalo testicular seminiferous tubules identified by comparative proteomic analysis

Abstract

The testicular seminiferous tubules contain Sertoli cells and different types of spermatogenic cells. They provide the microenvironment for spermatogenesis, but the precise molecular mechanism of spermatogenesis is still not well known. Here, we have employed tandem mass tag coupled to LC-MS/MS with the high-throughput quantitative proteomics technology to explore the protein expression from buffalo testicular seminiferous tubules at three different developmental stages (prepuberty, puberty, and postpuberty). The results show 304 differentially expressed proteins with a ≥2-fold change, and bioinformatics analysis indicates that 27 of these may be associated with spermatogenesis. Expression patterns of seven selected proteins were verified via Western blot and quantitative RT-PCR analysis, and further cellular localizations of these proteins by immunohistochemical or immunofluorescence analysis. Taken together, the results provide potential molecular markers of spermatogenesis and provide a rich resource for further studies on male reproduction regulation.

Comparative proteomic analysis of different developmental stages of swamp buffalo testicular seminiferous tubules

“…We speculate that these proteins may play vital roles in the maturation and cell proliferation of buffalo testicular seminiferous tubules. For example, HSPA2 has been validated to be involved in spermatogenesis and male fertility in previous studies (Govin et al., ; Huang et al., ; Rogon et al., ). The Sertoli cell VIM protein filaments play an important role in the maintenance of spermatogenesis and the vimentin can be reorganized after the germ cells repopulate and spermatogenesis is restored (Kuznetsov, Froberg, Henlon, Reinke, & Fennelly, ).…”

“…Here, we used comparative proteomics to discover the differentially expressed proteins among pre‐pubertal, pubertal and post‐pubertal swamp buffalo testicular seminiferous tubules. Only 25 differentially expressed protein spots were found due to the disadvantage of 2‐DE, and 13 of them were identified by MS showing a very small amount compared to the results of LC/MS (Huang et al., ). Nonetheless, 2‐DE/MS and LC/MS are known to be two complementary strategies and the former is a classical method to display changes in protein spots visually, while the latter can identify total proteins or give a quantitative analysis (Charro et al., ).…”

“…They found 108 differentially expressed proteins in total, and 90 of these proteins were identified successfully by MS and mostly involved in cellular and metabolic processes (Huang et al., ). Recently, comparative proteomic profiles of the whole testis in mouse (Shao et al., ; Zheng et al., ) and cattleyak (Sun et al., ) were constructed using liquid chromatography (LC) technology, and in our group, (Huang et al., ) also employed the same method to generate a comparative proteomic profile of buffalo testicular seminiferous tubules.…”

Novel targets identified by integrated proteomic and phosphoproteomic analysis in spermatogenesis of swamp buffalo (Bubalus bubalis)

“…In this study, 228 DEPs (80 and 148 proteins were up-and down-regulated in prepuberty, respectively) were successfully identified by TMT labeling combined with LC-MS /MS technology, and were then annotated to specific cell processes based on the results of a bioinformatics analysis. In previous studies, the focus has been on the seminiferous tubules of buffalo testes, and not the whole testicle 23,24 . In addition, traditional two-dimensional electrophoresis coupled to tandem mass spectrometry was used to explore the different proteins in developmental stages of swamp buffalo testicular seminiferous tubules, and only 13 differentially expressed proteins were identified 24 .…”

“…Thus, a better understanding of the molecular mechanisms of spermatogenesis in this ruminant species will be valuable. Differential proteomic analysis is a reliable and reproducible high-throughput approach to study the molecular mechanisms of spermatogenesis 18 . Protein phosphorylation plays an important role in cell division, proliferation, differentiation, metabolism, and is closely related to spermatogenesis 15 .…”