June-Sub Lee

Bisphenol-A Affects Male Fertility via Fertility-related Proteins in Spermatozoa

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.

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa

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.

Sperm Proteomics: Road to Male Fertility and Contraception

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.

Discovery of Predictive Biomarkers for Litter Size in Boar Spermatozoa

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.

Effect of sodium fluoride on male mouse fertility

Sodium fluoride (NaF), an environmental pollutant, has been tested for its impact on fertility in several species of laboratory animals. A literature demonstrated that NaF adversely affects sperm motility, morphology, capacitation, and the acrosome reaction. However, the molecular mechanisms underlying these alterations have not yet been elucidated. Therefore, present study was designed to evaluate the regulatory pathways involved in the effect of NaF on sperm function and fertilization. In this in vitro study, mouse spermatozoa were incubated with a range of concentrations (2.5, 5, and 10 mm) of NaF for 90 min in media that support in vitro fertilization. Our results showed that NaF was associated with reduced intracellular ATP generation, motility, and motion kinematics. Likewise, short‐term exposure of spermatozoa to NaF significantly reduced the intracellular calcium concentration, protein kinase‐A activity, and tyrosine phosphorylation of sperm proteins, which were associated with a significant decrease in the rate of capacitation and the acrosome reaction. Finally, NaF significantly reduced the fertilization and blastocyst formation during early embryonic development. On the basis of these results, we propose that NaF reduces sperm motility, capacitation, and the acrosome reaction leading to poor fertilization and suppressed embryonic development.

Sodium nitroprusside suppresses male fertility in vitro

Sodium nitroprusside is a nitric oxide donor involved in the regulation of the motility, hyperactivation, capacitation, and acrosome reaction (AR) of spermatozoa. However, the molecular mechanism underlying this regulation has not yet been elucidated. Therefore, this study was designed to evaluate the molecular basis for the effects of sodium nitroprusside on different processes in spermatozoa and its consequences on subsequent oocyte fertilization and embryo development. In this in vitro study, mouse spermatozoa were incubated with various concentrations of sodium nitroprusside (1, 10, and 100 μm) for 90 min. Our results showed that sodium nitroprusside inhibited sperm motility and motion kinematics in a dose‐dependent manner by significantly enhancing intracellular iron and reactive oxygen species (ROS), and decreasing Ca2+, and adenosine triphosphate levels in spermatozoa. Moreover, short‐term exposure of spermatozoa to sodium nitroprusside increased the tyrosine phosphorylation of sperm proteins involved in PKA‐dependent regulation of intracellular calcium levels, which induced a robust AR. Finally, sodium nitroprusside significantly decreased the rates of fertilization and blastocyst formation during embryo development. Based on these results, we propose that sodium nitroprusside increases ROS production and precocious AR may alter overall sperm physiology, leading to poor fertilization and compromised embryonic development.

A novel approach to identifying physical markers of cryo-damage in bull spermatozoa.

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.

Actin-related protein 2/3 complex-based actin polymerization is critical for male fertility

The actin‐related protein 2/3 (Arp2/3) complex is critical for regulation of actin polymerization, which is associated with sperm motility and capacitation status. However, the function of the Arp2/3 complex in male fertility has not yet been fully elucidated. Therefore, this study was designed to investigate the role of the Arp2/3 complex in different processes in spermatozoa and its consequences on fertilization and early embryonic development. In this in vitro study, mouse spermatozoa were incubated with different concentrations (10, 100, and 500 μm) of CK‐636, an Arp2/3 complex antagonist. Our results demonstrated that inhibition of the Arp2/3 complex by high concentrations (100 and 500 μm) of CK‐636 induced hyper‐activated motility and acrosomal reaction, whereas intracellular calcium and tyrosine phosphorylation levels in spermatozoa were inhibited. Moreover, exposure of spermatozoa to the highest concentration of CK‐636 reduced fertilization and embryo development. Interestingly, fertilization was significantly increased after treatment with 100 μm CK‐636, whereas embryonic development was significantly decreased. Therefore, we conclude that the Arp2/3 complex plays a decisive role in regulation of sperm function and male fertility via actin polymerization. We anticipate that the Arp2/3 complex may have clinical application as marker for male fertility and male contraceptive targeting.

Improving litter size by boar spermatozoa: application of combined H33258/CTC staining in field trial with artificial insemination

Conventional semen analysis offers basic information on infertility; however, its clinical value in predicting fertility status is unclear. To establish an accurate diagnosis of male fertility, semen analysis under capacitation condition is necessary because only capacitated spermatozoa are capable of fertilizing oocytes. The objective of this study was to verify male fertility based on conventional semen analysis before and after capacitation, including the assessment of motility (%), motion kinematics, and capacitation status of spermatozoa. A computer‐assisted sperm analysis system and chlortetracycline staining were applied to evaluate the motility parameters and capacitation status, respectively. To enable efficacy of the two methods for predicting fertility, correlation analysis was performed with the historic litter size. Our results showed that sperm motility (%), motion kinematics, and their variations before and after capacitation represented a statistical non‐significant correlation with litter size. Litter size showed significant correlation with acrosome reaction (AR) after capacitation (r = 0.375), as well as differences (Δ) in AR (r = 0.333) and capacitated (B) pattern (r = −0.447) before and after capacitation. The overall accuracy of the assay for predicting litter sizes using the AR and differences (Δ) in the AR and B pattern was 70%. On the basis of these results, we propose that capacitation status of spermatozoa is a more reliable indicator for evaluating male fertility status compared to motility parameters. Therefore, we suggest that analysis of capacitation status in company with conventional semen analysis may accept to evaluate more accurate diagnosis or prognosis of male fertility.

Effect of Arp2/3 Complex on Sperm Motility and Membrane Structure in Bovine

Sperm capacitation refers to polymerization of filamentous (F)-actin from globular (G)-actin. While the role of ac- tin-related protein 2/3 (Arp2/3) complex in actin polymerization is well appreciated, the underlying mechanism(s) and its relationship with capacitation are poorly understood. Therefore, to evaluate the potential role of Arp2/3 complex on capacitation, bovine spermatozoa were incubated with multiple doses (1, 10 and 100 μM) of CK-636, an inhibitor of Arp2/3 complex with heparin. The cellular localization of the Arp2/3 complex in spermatozoa was identified by immunohistochemistry, whereas western blot was also applied to detect the protein tyrosine phosphorylation of sperm proteins. Additionally, sperm motility and kinematic parameters were evaluated using a computer-assisted sperm analysis system. CK-636 resulted in significant changes in the ratio of Arp2/3 complex localization between acrosome and equatorial region of the spermatozoa. Short-term exposure of spermatozoa to 100 μM of CK-636 significantly decreased sperm motility, however a non-detectable effect on protein tyrosine phosphorylation was observed during capacitation. On the basis of these results, we propose that Arp2/3 complex is associated with morphological changes during capacitation and compromised sperm motility.