Sung-Jae Yoon

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.

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.

Gestational Exposure to Bisphenol-A Affects the Function and Proteome Profile of F1 Spermatozoa in Adult Mice.

Background: Maternal exposure to the endocrine disruptor bisphenol A (BPA) has been linked to offspring reproductive abnormalities. However, exactly how BPA affects offspring fertility remains poorly understood. Objectives: The aim of the present study was to evaluate the effects of gestational BPA exposure on sperm function, fertility, and proteome profile of F1 spermatozoa in adult mice. Methods: Pregnant CD-1 mice (F0) were gavaged with BPA at three different doses (50 μg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on embryonic days 7 to 14. We investigated the function, fertility, and related processes of F1 spermatozoa at postnatal day 120. We also evaluated protein profiles of F1 spermatozoa to monitor their functional affiliation to disease. Results: BPA inhibited sperm count, motility parameters, and intracellular ATP levels in a dose-dependent manner. These effects appeared to be caused by reduced numbers of stage VIII seminiferous epithelial cells in testis and decreased protein kinase A (PKA) activity and tyrosine phosphorylation in spermatozoa. We also found that BPA compromised average litter size. Proteins differentially expressed in spermatozoa from BPA treatment groups are known to play a critical role in ATP generation, oxidative stress response, fertility, and in the pathogenesis of several diseases. Conclusions: Our study provides mechanistic support for the hypothesis that gestational exposure to BPA alters sperm function and fertility via down-regulation of tyrosine phosphorylation through a PKA-dependent mechanism. In addition, we anticipate that the BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa. Citation: Rahman MS, Kwon WS, Karmakar PC, Yoon SJ, Ryu BY, Pang MG. 2017. Gestational exposure to bisphenol-A affects the function and proteome profile of F1 spermatozoa in adult mice. Environ Health Perspect 125:238–245; http://dx.doi.org/10.1289/EHP378

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.

Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome.

Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.

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.

Increased frequency of aneuploidy in long-lived spermatozoa.

Aneuploidy commonly causes spontaneous abortions, stillbirths, and aneuploid births in humans. Notably, the majority of sex chromosome aneuploidies in live births have a paternal origin. An increased frequency of aneuploidy is also associated with male infertility. However, the dynamics and behavior of aneuploid spermatozoa during fertilization in humans have not been studied in detail. Therefore, we compared the frequency of aneuploidy and euploidy in live spermatozoa from normozoospermic men over a 3-day period. To assess the dynamics and behavior of aneuploid spermatozoa, we simultaneously evaluated sperm viability using the hypo-osmotic swelling test and sperm aneuploidy using fluorescence in situ hybridization. Whereas the frequency of viable euploid spermatozoa significantly decreased over 3 days, the frequency of viable spermatozoa with aneuploidy interestingly showed a time-dependent increase. In addition, spermatozoa with abnormal sex chromosomes survived longer. To compared with spermatozoa with other swelling patterns, those with tail-tip swelling patterns had a lower frequency of aneuploidy at all time points. This study revealed the novel finding that the frequency of aneuploid spermatozoa with fertilization capability significantly increased compared to that of euploid spermatozoa over 3 days, suggesting that aneuploid spermatozoa can survive longer than euploid spermatozoa and have a greater chance of fertilizing oocytes.