Expression, Localization Of SUMO-1 And Preliminary Identification Of SUMO-1 Target Proteins In Bovine Spermatozoa

Reproductive ability of Bovine is critical for its production and reproduction. Therefore, the study of spermatogenesis, spenn maturation and semen quality control could provide important basis for improving the reproductive ability of bovine. Mature spermatozoa have highly condensed DNA that is virtually silent both transcriptionally and translationally. Therefore, post-translational modifications and their ability to regulate important sperm functions, including capacitation and the acrosome reaction, which are both, essential for oocyte fertilization, play a major role in male fertility. SUMOylation is one of the important post-translational protein modifications involved in the regulation of essential cell functions. In order to preliminary evaluate the potential roles of SUMOylation during sperm maturation, capacitation and acrosome reaction in water buffalo and bull, this project was designed to detect the cellular localization and protein content of small ubiquitin-related modifier-1 (SUMO-1) in sperm by immunofluorescence staining and western blot analysis, respectively; preliminarily identified the potential SUMO-1 target proteins by Co-immunoprecipitation and Mass Spectrometry analysis,analyzed the potential functions and pathways involved of these proteins by bioinformatics analysis, predicted the potential SUMO-1 targets' sites in those proteins. The results are as follows.(1) We examined the expression and localization of SUMO-1 in sperm of water buffalo and bull using immunofluorescence staining analysis. We confirmed the expression of SUMO-1 in the acrosome. In addition, Western blot analysis revealed changes of SUMO-1 conjugates after capacitation and when the acrosome reaction was induced by calcium ionophore A23187 in the buffalo sperm. After capacitation, protein bands intensity (SUMO-1 conjugates) at approximately 130, 37 and 27 kDa showed a lower intensity, a band at 30 kDa appears and bands at 85 and 50 kDa are enhanced. Furthermore, after capacitation, the spermatozoa were induced to undergo the acrosome reaction, and a band at 37 kDa decreased and a smear of bands below 25 kDa disappeared. This result implied that SUMO-1 mediated SUMOylation modification changes during sperm capacitation and acrosome reaction, which could be involved the regulation and function of these processes.(2) The potential proteins modified or conjugated by SUMO-1 in water buffalo sperm were pulled down and analyzed by mass spectrometry. Sixty proteins were preliminarily identified,including proteins important for sperm morphology and motility, such as relaxin receptors and cytoskeletal proteins, including tubulin chains, actins, and dyneins. Forty-six proteins were predicted as potential sumoylation targets.In conclusion, the presence of SUMO-1 in buffalo and bull sperms and changes to SUMO-1 expression during capacitation and the acrosome reaction opens up a new avenue of research to characterize the physiological roles of SUMO-1 and evaluate its contribution to fertility in bovines. The identification of SUMO-1 potential targets could provide important basis to further understand the regulation of SUMOylation modification during sperm maturation, capacitation and acrosome reaction, however, further experiments are still need to confirm the SUMO-1 mediated modification of these potential target proteins, and related function analysis.