Abstract
BackgroundMammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0.ResultWe identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction.ConclusionThe results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.
Highlights
Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization
The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality
The acrosome reacted (AR) and capacitated (B) patterns were significantly increased in after-capacitation (P < 0.05), while the non-capacitated (F) pattern was significantly decreased in after-capacitation spermatozoa (P < 0.05)
Summary
Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. In this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. Ejaculated spermatozoa undergo marked structural and biochemical changes within the female reproductive tract before fertilization. It has been demonstrated that freezethawing of human spermatozoa results in differential expression of twenty-seven proteins compare to their fresh ejaculate [20]. This study suggested that cryopreservation may be induced spermatozoa dysfunction due to protein degradation and protein phosphorylation [20,21]. It is a matter of paramount importance to detect a set of differentially expressed proteins associated with capacitation as well as other functional state of spermatozoa
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