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.

The xenoestrogen bisphenol-A (BPA) is a widespread environmental contaminant that has been studied for its impact on male fertility in several species of animals and humans. Growing evidence suggests that xenoestrogens can bind to receptors on spermatozoa and thus alter sperm function. The objective of the study was to investigate the effects of varying concentrations of BPA (0.0001, 0.01, 1, and 100 μM for 6 h) on sperm function, fertilization, embryonic development, and on selected fertility-related proteins in spermatozoa. Our results showed that high concentrations of BPA inhibited sperm motility and motion kinematics by significantly decreasing ATP levels in spermatozoa. High BPA concentrations also increased the phosphorylation of tyrosine residues on sperm proteins involved in protein kinase A-dependent regulation and induced a precocious acrosome reaction, which resulted in poor fertilization and compromised embryonic development. In addition, BPA induced the down-regulation of β-actin and up-regulated peroxiredoxin-5, glutathione peroxidase 4, glyceraldehyde-3-phosphate dehydrogenase, and succinate dehydrogenase. Our results suggest that high concentrations of BPA alter sperm function, fertilization, and embryonic development via regulation and/or phosphorylation of fertility-related proteins in spermatozoa. We conclude that BPA-induced changes in fertility-related protein levels in spermatozoa may be provided a potential cue of BPA-mediated disease conditions.

Conventional semen analysis has been used for prognosis and diagnosis of male fertility. Although this tool is essential for providing initial quantitative information about semen, it remains a subject of debate. Therefore, development of new methods for the prognosis and diagnosis of male fertility should be seriously considered for animal species of economic importance as well as for humans. In the present study, we applied a comprehensive proteomic approach to identify global protein biomarkers in boar spermatozoa in order to increase the precision of male fertility prognoses and diagnoses. We determined that L-amino acid oxidase, mitochondrial malate dehydrogenase 2, NAD (MDH2), cytosolic 5-nucleotidase 1B, lysozyme-like protein 4, and calmodulin (CALM) were significantly and abundantly expressed in high-litter size spermatozoa. We also found that equatorin, spermadhesin AWN, triosephosphate isomerase (TPI), Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2) were significantly and abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A, TPI, and NDUFS2 were negatively correlated with litter size, whereas CALM and MDH2 were positively correlated. This study provides novel biomarkers for the prediction of male fertility. To the best of our knowledge, this is the first work that shows significantly increased litter size using male fertility biomarkers in a field trial. Moreover, these protein markers may provide new developmental tools for the selection of superior sires as well as for the prognosis and diagnosis of male fertility.

Spermatozoa are highly specialized cells that can be easily obtained and purified. Mature spermatozoa are transcriptionally and translationally inactive and incapable of protein synthesis. In addition, spermatozoa contain relatively higher amounts of membrane proteins compared to other cells; therefore, they are very suitable for proteomic studies. Recently, the application of proteomic approaches such as the two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and differential in-gel electrophoresis has identified several sperm-specific proteins. These findings have provided a further understanding of protein functions involved in different sperm processes as well as of the differentiation of normal state from an abnormal one. In addition, studies on the sperm proteome have demonstrated the importance of spermatozoal posttranslational modifications and their ability to induce physiological changes responsible for fertilization. Large-scale proteomic studies to identify hundreds to thousands of sperm proteins will ultimately result in the development of novel biomarkers that may help to detect fertility, the state of complete contraception, and beyond. Eventually, these protein biomarkers will allow for a better diagnosis of sperm dysfunctions and aid in drug development. This paper reviews the recent scientific publications available from the PubMed database to address sperm proteomics and its potential application to characterize male fertility and contraception.

Cryopreservation is an efficient way to store spermatozoa and plays a critical role in the livestock industry as well as in clinical practice. During cryopreservation, cryo-stress causes substantial damage to spermatozoa. In present study, the effects of cryo-stress at various cryopreservation steps, such as dilution / cooling, adding cryoprtectant, and freezing were studied in spermatozoa collected from 9 individual bull testes. The motility (%), motion kinematics, capacitation status, mitochondrial activity, and viability of bovine spermatozoa at each step of the cryopreservation process were assessed using computer-assisted sperm analysis, Hoechst 33258/chlortetracycline fluorescence, rhodamine 123 staining, and hypo-osmotic swelling test, respectively. The results demonstrate that the cryopreservation steps reduced motility (%), rapid speed (%), and mitochondrial activity, whereas medium/slow speed (%), and the acrosome reaction were increased (P < 0.05). Differences (Δ) of the acrosome reaction were higher in dilution/cooling step (P < 0.05), whereas differences (Δ) of motility, rapid speed, and non-progressive motility were higher in cryoprotectant and freezing as compared to dilution/cooling (P < 0.05). On the other hand, differences (Δ) of mitochondrial activity, viability, and progressive motility were higher in freezing step (P < 0.05) while the difference (Δ) of the acrosome reaction was higher in dilution/cooling (P < 0.05). Based on these results, we propose that freezing / thawing steps are the most critical in cryopreservation and may provide a logical ground of understanding on the cryo-damage. Moreover, these sperm parameters might be used as physical markers of sperm cryo-damage.