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Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Katerina Komrskova reports financial support was provided by Ministry of Health of the Czech Republic. Veronika Palenikova reports financial support was provided by Grant Agency of the Charles University. Pavla Postlerova reports financial support was provided by Grant Agency of the Czech Republic. Veronika Palenikova, Hana Pavlova, Daniel Kraus, Katerina Komrskova, Pavla Postlerova reports financial support was provided by Institute of Biotechnology of the Czech Academy of Sciences. Veronika Palenikova, Hana Pavlova, Daniel Kraus, Katerina Komrskova, Pavla Postlerova reports financial support was provided by ERDF of BIOCEV project. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Crowded environments inside cells and biological fluids greatly affect protein stability and activity. PDC-109, a polydisperse oligomeric protein of the bovine seminal plasma selectively binds choline phospholipids on the sperm cell surface and causes membrane destabilization and lipid efflux, leading to acrosome reaction. PDC-109 also exhibits chaperone-like activity (CLA) and protects client proteins against various kinds of stress, such as high temperature and low pH. In the present work, we have investigated the effect of molecular crowding on these two different activities of PDC-109 employing Dextran 70 (D70) - a widely used polymeric dextran - as the crowding agent. The results obtained show that presence of D70 markedly increases membrane destabilization by PDC-109. Isothermal titration calorimetric studies revealed that under crowded condition the binding affinity of PDC-109 for choline phospholipids increases approximately 3-fold, which could in turn facilitate membrane destabilization. In contrast, under identical conditions, its CLA was reduced significantly. The decreased CLA could be correlated to reduced surface hydrophobicity, which was due to stabilization of the protein oligomers. These results establish that molecular crowding exhibits contrasting effects on two different functional activities of PDC-109 and highlight the importance of microenvironment of proteins in modulating their functional activities., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Musti J. Swamy reports financial support was provided by University Grants Commission (India). Musti J. Swamy reports financial support was provided by University of Hyderabad., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Human male infertility affects approximately 1/10 couples worldwide, and its prevalence is found more in developed countries. Along with sperm cells, the secretions of the prostate, seminal vesicle and epididymis plays a major role in proper fertilization. Many studies have proven the functions of seminal vesicle secretions, especially semenogelin protein, as an optimiser for fertilization. Semenogelin provides the structural components for coagulum formation after ejaculation. It binds with eppin and is found to have major functions like motility of sperm, transporting the sperm safely in the immune rich female reproductive tract until the sperm cells reach the egg intact. The capacitation process is essential for proper fertilization and semenogelin involved in mediating capacitation in time. Also, it has control of events towards the first step in the fertilization process. It is a Zn ions binding protein, and Zn ions act as a cofactor that helps in the proper motility of sperm cells. Therefore, any imbalance in protein that automatically affect sperm physiology and fertility status. This review sheds a comprehensive and critical view on the significant functions of semenogelin in fertilization. This review can open up advanced proteomics research on semenogelin towards unravelling molecular mechanisms in fertilization., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Prostate-specific antigen (PSA) is a prostate-specific serine protease enzyme that hydrolyzes gel-forming proteins (semenogelins) and changes the semen from gel-like to watery viscosity, a process called semen liquefaction. Highly viscous semen and abnormal liquefaction reduce sperm motility and contribute to infertility. Previously, we showed that nonspecific serine protease inhibitor (AEBSF) prevented proteolytic degradation of semenogelin in mice. However, it is unclear whether similar effect could be recapitulated in fresh human ejaculates. Therefore, in this study we evaluated the effect of AEBSF on the degradation of semenogelin (SEMG1) and its subsequent impact on semen liquefaction and sperm motility in fresh semen ejaculates collected from healthy men. We found that AEBSF showed a dual contraceptive action where it effectively 1) prevented degradation of SEMG1 resulting in viscous semen and 2) decreased sperm motility in human semen samples. However, the impact of AEBSF on sperm motility and viability could be due to its inhibitory activity toward other serine proteases or simply due to its toxicity. Therefore, to determine whether inhibition of PSA activity alone could disrupt SEMG1 degradation and contribute to hyperviscous semen, a neutralizing PSA antibody was used. We found that PSA antibody effectively prevented SEMG1 degradation with a subtle impact on sperm motility. These findings suggest that the target inhibition of PSA activity can prevent proteolytic degradation of SEMG1 and block liquefaction process, resulting in hyperviscous semen. As it is currently unknown if blocking semen liquefaction alone could prevent pregnancy, it needs further extensive studies before drawing any translational conclusions., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In both men and women, pathogenic bacteria enter the reproductive tract and cause harmful symptoms. Intrauterine and oviductal inflammation after copulation may have severe effects, such as infertility, implantation failure, oviduct obstruction, and robust life-threatening bacterial infection. Human seminal plasma is considered to be protective against bacterial infection. Among its components, Semenogelin-I/-II proteins are digested to function as bactericidal factors; however, their sequences are not conserved in mammals. Therefore, alternative antibacterial (bactericidal and/or bacteriostatic) systems may exist across mammals. In this study, we examined the antibacterial activity in the seminal plasma of mice lacking a gene cluster encoding Semenogelin-I/-II counterparts. Even in the absence of the majority of seminal proteins, antibacterial activity remained in the seminal plasma. Moreover, a combination of gel chromatography and liquid chromatography coupled with tandem mass spectrometry revealed that the prostate and testis expressed 4 protein as a novel antibacterial (specifically, bacteriostatic) protein, the sequence of which is broadly conserved across mammals. Our results provide the first evidence of a bacteriostatic protein that is widely present in the mammalian seminal plasma., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Serological screening of sexual assault evidence has traditionally focused on enzyme activity and immunochromatographic assays that provide only a presumptive indication of seminal fluid and have limited sensitivity relative to DNA testing. Seminal fluid detection based on protein mass spectrometry represents a "Next Gen" serological technology that overcomes the specificity and sensitivity limitations of traditional serological screening but requires time-consuming sample preparation protocols. This paper describes a novel "peptidomics" approach to seminal fluid detection that eliminates the need for lengthy trypsin digestion. This streamlines sample preparation to a one-step process followed by high-resolution mass spectrometry to identify naturally occurring seminal fluid peptides and low-molecular weight proteins. Multiple protein biomarkers of seminal fluid were consistently and confidently identified based on the multiplexed detection of numerous endogenous peptides. These included Semenogelin I and II (90% and 86% sequence coverage, respectively); Prostate Specific Antigen/p30 (29% sequence coverage); and Prostatic Acid Phosphatase (24% sequence coverage). The performance of this streamlined peptidomics approach to seminal fluid identification in a forensic context was also assessed using simulated casework samples of the type typically collected as part of a sexual assault examination (e.g., oral and vaginal swabs stained with semen). The resulting data demonstrate that sub-microliter quantities of seminal fluid on cotton swabs can be recovered and reliably detected. This supports the forensic applicability of a peptidomic assay for seminal fluid identification with same-day sample preparation and analysis. Future development and streamlined multiplex peptidomic assays for additional biological stains can easily be envisaged., (© 2020 American Academy of Forensic Sciences.)

SEMG1 and SEMG2 genes belong to the family of cancer-testis antigens (CTAs), whose expression normally is restricted to male germ cells but is often restored in various malignancies. High levels of SEMG1 and SEMG2 expression are detected in prostate, renal, and lung cancer as well as hemoblastosis. However, the functional importance of both SEMGs proteins in human neoplasms is still largely unknown. In this study, by using a combination of the bioinformatics and various cellular and molecular assays, we have demonstrated that SEMG1 and SEMG2 are frequently expressed in lung cancer clinical samples and cancer cell lines of different origins and are negatively associated with the survival rate of cancer patients. Using the pull-down assay followed by LC-MS/MS mass-spectrometry, we have identified 119 proteins associated with SEMG1 and SEMG2. Among the SEMGs interacting proteins we noticed two critical glycolytic enzymes-pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Importantly, we showed that SEMGs increased the protein level and activity of both PKM2 and LDHA. Further, both SEMGs increased the membrane mitochondrial potential (MMP), glycolysis, respiration, and ROS production in several cancer cell lines. Taken together, these data provide first evidence that SEMGs can up-regulate the energy metabolism of cancer cells, exemplifying their oncogenic features.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

In mice, the Binder of Sperm Homolog 1 protein is exclusively expressed in the epididymis. BSP proteins play a role in the membrane modification events that occur during sperm capacitation. In the current study, we investigated the role of mouse recombinant BSP homolog 1 (rec-BSPH1) in sperm-egg interaction. Mouse oocytes were co-incubated with different concentrations of rec-BSPH1 or control proteins and then inseminated with sperm. To establish whether rec-BSPH1 interfered with in vitro fertilization of mouse oocytes, rec-BSPH1 binding to egg and sperm was first tested using an immunodetection assay. In separate experiments, sperm were immuno-neutralized by anti-rec-BSPH1 antibodies to indirectly verify the implication of BSPH1 in sperm-egg interaction and fertilization. The study revealed a dose-dependent inhibition of fertilization when oocytes were pre-incubated with rec-BSPH1. Moreover, sperm immuno-neutralization with anti-rec-BSPH1 antibodies led to dramatic motility changes, followed by compromised fertilization. In view of these results, we conclude that BSPH1 could be a marker of sperm fertility and thus an eventual target for male contraceptive development., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Semenogelin 1 (SEMG1), a main component of human seminal plasma, is a multi-functional protein involved in the regulation of sperm motility and fertility. SEMG1 is orthologous to mouse seminal vesicle secretion 2 (SVS2), required for sperm survival in the female reproductive tract after copulation; however, its in vivo function remains unclear. In this study, we addressed this issue by examining the effect of recombinant SEMG1 on intrauterine mouse sperm survival. SEMG1 caused a dose-dependent decrease in mouse sperm motility, similar to its effect on human sperm, but SVS2 had no effect on mouse sperm motility. Mouse epididymal sperm in the presence of 100 µM SEMG1, a concentration that does not affect mouse sperm motility, were injected into the mouse uterus (intrauterine insemination, IUI). IUI combined with SEMG1 significantly increased the survival rate of intrauterine mouse sperm. The effect of SEMG1 on intrauterine sperm survival was comparable with that of SVS2. For clinical applications, three potentially sperm-protecting polypeptides that are easy to handle were designed from SEMG1, but their individual use was unable to mimic the ability of SEMG1. Our results indicate that SEMG1 has potential clinical applications for effective IUI and thereby for safe, simple, and effective internal fertilization., Competing Interests: The authors declare no conflicts of interest.

EPPIN is a sperm-surface drug target for male contraception. Here we investigated EPPIN-interacting proteins in mouse spermatozoa. We showed that EPPIN is an androgen-dependent gene, expressed in the testis and epididymis, but also present in the vas deferens, seminal vesicle and adrenal gland. Mature spermatozoa presented EPPIN staining on the head and flagellum. Immunoprecipitation of EPPIN from spermatozoa pre-incubated with seminal vesicle fluid (SVF) followed by LC-MS/MS or Western blot revealed the co-immunoprecipitation of SVS2, SVS3A, SVS5 and SVS6. In silico and Far-Western blot approaches demonstrated that EPPIN binds SVS2 in a protein network with other SVS proteins. Immunofluorescence using spermatozoa pre-incubated with SVF or recombinant SVS2 demonstrated the co-localization of EPPIN and SVS2 both on sperm head and flagellum. Our data show that EPPIN's roles in sperm function are conserved between mouse and human, demonstrating that the mouse is a suitable experimental model for translational studies on EPPIN., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Messenger RNA profiling for body fluid identification (bfID) is a useful approach to collect contextual information associated with a crime. Current methods require costly fluorescent probes, lengthy amplification protocols and/or time-consuming sample preparation. To simplify this process, we developed a bfID method that has the potential to be rapid in analysis time, inexpensive and fluorescence-free, combining a universal operating procedure with a high-throughout (microwell plate) platform for simultaneous detection of mRNA markers from whole blood, semen, saliva, and vaginal fluid. Full bfID sample preparation and analysis of 23 samples was completed in under 3 h using smart phone optical detection and analysis and show efficacy of the method in a validated blind study. The results provide an efficient, sensitive and specific approach to supplement the current biochemical tests in a forensic laboratory., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The fibronectin type II (FnII) domain, present in diverse vertebrate proteins, plays crucial roles in several fundamental biological processes. PDC-109, the major bovine seminal plasma protein, contains two FnII domains that bind to choline phospholipids on sperm plasma membrane and induce lipid efflux crucial for successful fertilization. PDC-109 also exhibits chaperone-like activity and protects other proteins against various types of stress. Here, we show that a core tryptophan residue is highly conserved across species in the FnII domains. Mutation of conserved tryptophan residues W47, W93, and W106 in the FnII domains of PDC-109 to alanine leads to drastic decrease or complete abolition of membrane-binding and chaperone-like activities. These observations suggest that conserved tryptophans are important for the function of FnII proteins., (© 2019 Federation of European Biochemical Societies.)

Purpose: To identify noninvasive immune biomarkers of exercise-induced immunosuppression using the iTRAQ proteomics technique., Methods: Fifteen healthy males were recruited and subjected to a four-week incremental treadmill running training program. After each week of training, WBC counts and CD4 + and CD8 + lymphocytes were measured to monitor the immune function status. iTRAQ proteomics technology was used to identify differential proteins and their characteristics in urine., Results: Our data showed that the WBC counts, CD4 + lymphocytes, and CD4 + /CD8 + ratio decreased by more than 10% after four weeks of training, suggesting exercise-induced immunosuppression. A total of 1854 proteins were identified in urine during the incremental running using the iTRAQ technology. Compared with the urine before training, there were 89, 52, 77, and 148 proteins significantly upregulated and 66, 27, 68, and 114 proteins significantly downregulated after each week, respectively. Among them, four upregulated proteins, SEMG-1, PIP, PDGFRL, and NDPK, increased their abundance with the increased exercise intensity. Bioinformatics analysis indicates that these proteins are involved in stress response and immune function., Conclusion: Four weeks of incremental treadmill running induced immunosuppression in healthy males. By using iTRAQ proteomics, four proteins in the urine, SEMG-1, PIP, PDGFRL, and NDPK, were found to increase incrementally with the increased exercise intensity, which have the potential to be used as noninvasive immune biomarkers of exercise-induced immunosuppression., Competing Interests: The authors have declared no conflicts of interest., (Copyright © 2020 Guoqin Xu et al.)

In recent years, genomic and proteomic biomarkers have been identified for the diagnosis of male fertility to overcome the limitations of conventional semen analysis. Owing to the limited genes available so far, the single gene approach is commonly adopted for analyzing the phenotype of interest. However, the single-gene approach is less effective than multiple-gene strategies for diagnosing a desirable phenotype. Herein, we investigate the ability of two fertility-related genomic markers (porcine seminal protein (PSP)-I and PSP-II) in spermatozoa to predict boar litter size in addition to conventional semen parameters. First, we examined different semen parameters (motility, motion kinematics, and capacitation status) and gene expression in high- and low-litter size boar spermatozoa. Then, we evaluated the correlation of these parameters with the fertility of 21 Yorkshire boars. Finally, we investigated the efficacy of single/combined markers to predict male fertility using a comprehensive statistical model. Our result showed that there were no significant differences in sperm motility, motion kinematics, or capacitation status, however, the mRNA expression of PSP-I and PSP-II in spermatozoa was significantly different in high- and low-litter size boars. In the individual screening test, the expression of both genes was negatively correlated with boar fertility (r = 0-0.578 and -0.456, respectively), whereas only hyperactivation (HYP) showed a positive correlation (r = 0.444) among the tested semen parameters. As single markers, PSP-I and PSP-II have a better diagnostic power to predict boar fertility, regardless of HYP, in quality assessment analyses. In addition, when these markers were combined, the positive predictive value, negative predictive value, and overall test effectiveness for fertility detection were improved. Surprisingly, when PSP-I and PSP-II were considered together, the deviation of the predicted average litter size between high- and low-litter size boars was 1.77. Based on the findings, we suggest that the use of genomic markers in spermatozoa rather than commonly analyzed semen parameters may be more accurate for evaluating male fertility. Moreover, using a combination of markers could increase the overall accuracy of (in)fertility predictions, and thus, could be considered for field application., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2 -deficient ( Svs2 -/- ) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster ( Svs2-6 -/- mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2 -/- male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.

Purpose: Based on prior reports demonstrating that neutral endopeptidase (NEP) inhibitors increase sperm motility, the goal of our studies was to identify endogenous seminal peptides that inhibit NEP and investigate their potential effect on sperm motility., Methods: Peptidomic analysis was performed on human seminal fluid, identifying 22 novel peptides. One peptide, named RSIY-11, derived from semenogelin-1, was predicted through sequence analysis to be a substrate and/or potential inhibitor of NEP. Enzymatic analysis was conducted to determine the inhibitory constant (Ki) of RSIY-11 as an inhibitor of NEP. Total and progressive sperm motility was determined at baseline and 30 and 60 min following addition of RSIY-11 to seminal fluid in 59 patients undergoing an infertility workup at an urban medical center. Additionally, the effects of RSIY-11 on sperm motility were evaluated in 15 of the 59 patients that met criteria for asthenospermia., Results: RSIY-11 was shown to act as a competitive inhibitor of NEP with a Ki of 18.4 ± 1.6 μM. Addition of RSIY-11 at concentrations of 0.75 μM, 7.5 μM, and 75 μM significantly increased sperm motility at all time points investigated, with increases of 6.1%, 6.9%, and 9.2% at 60 min, respectively. Additionally, within the subgroup of patients with asthenospermia, RSIY-11 at concentrations of 0.75 μM, 7.5 μM, and 75 μM significantly increased sperm motility at all time points investigated, with increases of 7.6%, 8.8%, and 10.6% at 60 min, respectively., Conclusions: RSIY-11 is a newly identified semenogelin-1-derived peptide present in seminal fluid. RSIY-11 acts as a potent competitive inhibitor of NEP, which when added to seminal fluid significantly increases sperm motility. RSIY-11 could play a potential role in the treatment for male factor infertility related to asthenospermia and improve intrauterine insemination outcomes.

A number of viruses causing sexually transmissible diseases are transmitted via mammalian seminal plasma. Several components of seminal plasma have been shown to influence those viruses and their physiological impact. To unravel whether components of seminal plasma could affect viruses transmitted via other pathways, it was investigated here whether the bovine seminal plasma protein PDC-109, belonging to the Fn-type 2 protein family, influences the activity of influenza A viruses, used as a model for enveloped viruses. We found that PDC-109 inhibits the fusion of influenza virus with human erythrocyte membranes and leads to a decreased viral infection in MDCK cells. In the presence of the head group of the phospholipid phosphatidylcholine, phosphorylcholine, the inhibitory effect of PDC-109 was attenuated. This indicates that the impact of the protein is mainly caused by its binding to viral and to erythrocyte membranes thereby interfering with virus-cell binding. Our study underlines that Fn-type 2 proteins have to be considered as new antiviral components present in mammalian seminal plasma.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA 352-372 ) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 μM) and PACAP (0.1 μM) decreased intracellular norepinephrine (NE) content and increased 3 H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2 μM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronically increasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

The major bovine seminal plasma protein, PDC-109, is a mixture of glycosylated (BSP-A1) and non-glycosylated (BSP-A2) isoforms of a 109-residue long polypeptide. It binds to spermatozoa by specifically recognizing choline phospholipids on the plasma membrane and destabilizes it by penetrating the hydrophobic interior, resulting in lipid efflux, which is necessary for sperm capacitation and successful fertilization. PDC-109 also acts as a molecular chaperone and protects target proteins from denaturation and aggregation induced by various types of stress. In order to investigate the role of glycosylation in these activities, we have separated BSP-A1 and BSP-A2 from PDC-109, and also cloned and expressed BSP-A2 in E. coli and purified the recombinant BSP-A2 (rBSP-A2) to homogeneity. Employing biophysical and biochemical approaches we have investigated the membrane-perturbing and chaperone-like activities (CLA) of PDC-109, BSP-A1, BSP-A2 and recombinant BSP-A2 (rBSP-A2). The results obtained demonstrate that glycan-lacking wild-type BSP-A2 and rBSP-A2 exhibit higher membrane-perturbing activity but decreased CLA as compared to PDC-109. In contrast, BSP-A1 exhibits significantly higher CLA than PDC-109, but its ability to destabilize membranes is considerably lower. This differential modulation of the membrane-perturbing and chaperone-like activities has been explained on the basis of higher membrane-penetrating ability and lower solubility of glycan-lacking BSP-A2 as compared to the glycosylated BSP-A1., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: To compare the differentially expressed proteins in mice with kidney-yang deficiency and those with kidney-yin deficiency induced by hydrocortisone, and explore the similar and different material bases of male infertility caused by the two types of kidney deficiency., Methods: Thirty Kunming mice were equally randomized into a normal control, a kidney-yang deficiency and a kidney-yin deficiency group. The animals of the normal control group were injected intraperitoneally with normal saline at 0.2 ml qd for 7 days, while those of the latter two groups with hydrocortisone at 25 mg/kg/d for 10 days and 50 mg/kg/d for 7 days, respectively, for establishment of kidney-yang deficiency and kidney-yin deficiency models. Then the pathological changes in the testicular tissue of the mice were observed by HE staining and the differentially expressed proteins were compared among different groups using isobaric tags for relative and absolute quantitation (iTRAQ) and the bioinformatics method., Results: Sod1 was found to be a reproduction-related node protein differentially expressed in the testis tissues of the two types of kidney-deficiency mice, more highly expressed in the kidney-yin than in the kidney-yang deficiency group (P < 0.05). Five reproduction-associated node proteins were co-expressed in the testes of the two groups of kidney-deficiency mice, with significantly up-regulated expression of Rps28 and down-regulated expressions of Rpl11, Rplp2, Svs2 and Svs3a (P < 0.01)., Conclusions: Sod1 may be one of the key material bases for the differentiation of male infertility caused by kidney-yang deficiency from that induced by kidney-yin deficiency, while Rps28, Rpl11, Rplp2, Svs2 and Svs3a may be the common material bases of male infertility caused by the two types of kidney deficiency.

Members of the Binder of SPerm (BSP) superfamily have been identified in both human and mouse epididymis. These proteins are known to bind sperm membrane and promote sperm capacitation. Studies suggest that BSPH2 might play a different role in sperm functions from its counterparts; however, the role of BSPH2 remains mainly unexplored. To investigate whether the absence of one member of the BSP family could affect fertility, mice lacking Bsph2 expression were generated using clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) technology. Knockout (KO) male mice were mated with wild-type (WT) females, and the number and weight of the pups were determined. Sperm motility in WT and KO was assessed using sperm class analyzer (SCA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for protein identification. Fertility analysis of null Bsph2 mice did not reveal any phenotype. No differences were noticed on average litter size or average pup weight. Normal testis weight and morphology were observed in Bsph2 +/- and Bsph2 -/- compared to the WT. Quantitative polymerase chain reaction analyses revealed that Bsph1 messenger RNA expression was increased in mutant mice, whereas LC-MS/MS analysis displayed no increase in protein expression level. Taken together, we show the existence of redundant function for murine BSPH2 and the lack of BSPH2 itself does not lead to sterility., (© 2018 Wiley-Liss, Inc., A Wiley Company.)

Smoking releases cadmium (Cd), the metal toxicant which causes an imbalance in reactive oxygen species level in seminal plasma. This imbalance is envisaged to impair the sperm DNA morphology and thereby result in male infertility. In order to correlate this association, we performed in vitro and in silico studies and evaluated the influence of reactive oxygen species imbalance on sperm morphology impairments due to smoking. The study included 76 infertile smokers, 72 infertile non-smokers, 68 fertile smokers and 74 fertile non-smokers (control). Semen samples were collected at regular intervals from all the subjects. Semen parameters were examined by computer assisted semen analysis, quantification of metal toxicant by atomic absorption spectrophotometer, assessment of antioxidants through enzymatic and non-enzymatic methods, diagnosis of reactive oxygen species by nitro blue tetrazolium method and Cd influence on sperm protein by in vitro and in silico methods. Our analysis revealed that the levels of cigarette toxicants in semen were high, accompanied by low levels of antioxidants in seminal plasma of infertile smoker subjects. In addition the investigation of Cd treated sperm cells through scanning electronic microscope showed the mid piece damage of spermatozoa. The dispersive X-ray analysis to identify the elemental composition further confirmed the presence of Cd. Finally, the in-silico analysis on semenogelin sequences revealed the D-H-D motif which represents a favourable binding site for Cd coordination. Our findings clearly indicated the influence of Cd on reactive oxygen species leading to impaired sperm morphology leading to male infertility., (Copyright © 2018 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Seminal plasma proteins are relevant for sperm functionality and some appear responsible for establishing sperm interactions with the various environments along the female genital tract towards the oocyte. In recent years, research has focused on characterizing the role of these proteins in the context of reproductive biology, fertility diagnostics and treatment of related problems. Herein, we focus on the main protein of bovine seminal plasma, PDC-109 (BSP-A1/-A2), which by virtue of its lectin properties is involved in fertilization. By means of surface plasmon resonance, the interaction of PDC-109 with a panel of the most relevant glycosidic epitopes of mammals has been qualitatively and quantitatively characterized, and a higher affinity for carbohydrates containing fucose has been observed, in line with previous studies. Additionally, using the orthogonal technique of Carbohydrate REcognition Domain EXcision-Mass Spectrometry (CREDEX-MS), the recognition domain of the interaction complexes between PDC-109 and all fucosylated disaccharides [(Fuc-&alpha;1,(3,4,6)-GlcNAc)] has been defined, revealing the specific glycotope and the peptide domain likely to act as the PDC-109 carbohydrate binding site.

Background: We previously demonstrated that a seminal plasma protein, semenogelin I (SgI), functioned as an androgen receptor (AR) coactivator. Meanwhile, several short sequence motifs in AR coregulators, such as LxxLL (L=leucine), have been shown to mediate specific interactions with AR., Methods: We investigated the role of the LxxLL motif within SgI in the interactions with AR and cell growth in prostate cancer lines in vitro., Results: A full-length SgI with mutations in the motif (i.e., LxxAA; A=alanine) failed to significantly increase cell proliferation/migration as well as androgen-mediated AR transcription. Co-immunoprecipitation showed no physical interactions between AR and the mutant SgI. In addition, transfection of an 18-amino acid peptide of SgI containing LxxLL, but not LxxAA, resulted in considerable reduction in cell growth and prostate-specific antigen expression in LNCaP and C4-2 lines., Conclusions: The LxxLL motif of SgI could be a novel therapeutic target for both androgen-sensitive and castration-resistant prostate cancers.

Binder of sperm-5 (BSP-5) is one of the fertility-associated proteins of cattle seminal plasma. Binding of sperm to the oviductal epithelium is mediated by BSP group of proteins. However, it is not clear, whether this protein is also involved in sperm motility. In the present study, attempts were made to characterize BSP-5 protein in both normozoospermic (NS) and asthenozoospermic (AS) Murrah buffalo (n = 18; Bubalus bubalis), Holstein Friesian (n = 8, Bos taurus) and Jersey cattle (n = 8; Bos taurus) bull seminal plasma and also study its expression pattern in these species. 1-D Western blot demonstrated three major BSP-5 immunoreactive protein bands (24.2 kDa, 20.5 kDa, and 12.3 kDa) in buffalo seminal plasma. Of these, the intensities of 24.2 and 20.5 kDa protein bands reduced significantly (P ≤ 0.05) in seminal plasma of AS group compared to that of NS group. On the contrary, the expression of 12.3 kDa protein band did not vary significantly between the groups. In Holstein Friesian seminal plasma, at least six BSP-5 immunoreactive protein bands (25.1, 23.6, 19.5, 13.8, 13.1 and 12.3 kDa) could be detected. Of these, the intensities of 23.6, 13.8/13.1 and 12.3 kDa protein bands decreased (P = 0.058, 0.111, 0.053) in AS group bulls compared to NS bulls. Holstein Friesian bull seminal plasma demonstrated a BSP-5 immunoreactive duplex protein band of 13.8/13.1 kDa, which was not evident in buffalo seminal plasma. In 2-D Western blot, a train of five BSP-5 immunoreactive duplex protein spots (Mr 21.0-27.6 kDa, pI of ∼3.9-5.1) was detected. Mass spectrometry of one of the representative duplex spot confirmed that these were BSP-5 and BSP-3 proteins, respectively. Indirect immunofluorescence studies showed that BSP-5 is primarily localized to the mid-piece/mitochondrial region of buffalo spermatozoa. To conclude, the findings of the present study could establish the significance and association of BSP-5 proteins in sperm motility and how their level differ in semen from two different clinical groups of buffalo bull (NS vs. AS). Further, the study also demonstrated that the expression pattern of BSP-5 and other BSP variants in seminal plasma of bulls is species-specific., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The major bovine seminal plasma protein, PDC-109, binds to choline phospholipids of the sperm plasma membrane and induces an efflux of cholesterol and choline phospholipids (cholesterol efflux), which is crucial for sperm capacitation. PDC-109 also exhibits chaperone-like activity and protects target proteins against various kinds of stress. Here we show that the polyamines spermine and spermidine, present in high concentration in the seminal plasma of various mammals, increase the ability of PDC-109 to perturb membrane structure as well as its chaperone-like activity. Interestingly, spermine/spermidine alone did not perturb membrane structure but exhibited chaperone-like activity by protecting target proteins against thermal and oxidative stress. When spermine/spermidine was used along with PDC-109, the observed chaperone-like activity was considerably higher than that expected for a simple additive effect, suggesting that PDC-109 and the polyamines act in a synergistic fashion. These results indicate that at the high concentrations present in the seminal plasma spermine/spermidine exhibit a positive modulatory effect on the chaperone-like activity of PDC-109 and may also function as chemical chaperones and protect other seminal plasma proteins from various kinds of stress., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Seminal plasma Binder of SPerm (BSP) proteins bind to sperm at ejaculation and promote capacitation. When in excess, however, BSP proteins damage the sperm membrane. It has been suggested that milk components of semen extenders associate with BSP proteins, potentially protecting sperm. Thus, this study was conducted to investigate if milk proteins interact with BSP proteins and reduce BSP binding to goat sperm. Using gel filtration chromatography, milk was incubated with goat seminal plasma proteins and loaded onto columns with and without calcium. Milk was also fractionated into parts containing mostly whey proteins or mostly caseins, incubated with seminal plasma proteins and subjected to gel filtration. Eluted fractions were evaluated by immunoblot using anti-goat BSP antibodies, confirming milk protein-BSP protein interactions. As determined by ELISA, milk proteins coated on polystyrene wells bound to increasing of goat BSP proteins. Far-western dot blots confirmed that BSP proteins bound to caseins and β-lactoglobulin in a concentration-dependent manner. Then, cauda epididymal sperm from five goats was incubated with seminal plasma; seminal plasma followed by milk; and milk followed by seminal plasma. Sperm membrane proteins were extracted and evaluated by immunoblotting. The pattern of BSP binding to sperm membrane proteins was reduced by 59.3 % when epididymal sperm were incubated with seminal plasma and then with skimmed milk (p < 0.05). When epididymal sperm were treated with milk followed by seminal plasma, coating of sperm with BSP proteins was not significantly reduced (57.6 %; p > 0.05). In conclusion, goat BSP proteins have an affinity for caseins and whey proteins. Milk reduces BSP binding to goat sperm, depending whether or not sperm had been previously exposed to seminal plasma. Such events may explain the protective effect of milk during goat sperm preservation.

Mammalian spermatozoa acquire their fertilizing ability in the female reproductive tract (sperm capacitation). On the other hand, seminal vesicle secretion, which is a major component of seminal plasma, inhibits the initiation of sperm capacitation (capacitation inhibition) and reduces the fertility of the capacitated spermatozoa (decapacitation). There are seven major proteins involved in murine seminal vesicle secretion (SVS1-7), and we have previously shown that SVS2 acts as both a capacitation inhibitor and a decapacitation factor, and is indispensable for in vivo fertilization. However, the effects of SVSs other than SVS2 on the sperm have not been elucidated. Since mouse Svs2-Svs6 genes evolved by gene duplication belong to the same gene family, it is possible that SVSs other than SVS2 also have some effects on sperm capacitation. In this study, we examined the effects of SVS3 and SVS4 on sperm capacitation. Our results showed that both SVS3 and SVS4 are able to bind to spermatozoa, but SVS3 alone showed no effects on sperm capacitation. On the other hand, SVS4 acted as a capacitation inhibitor, although it did not show decapacitation abilities. Interestingly, SVS3 showed an affinity for SVS2 and it facilitated the effects of SVS2. Interaction of SVS2 and spermatozoa is mediated by the ganglioside GM1 in the sperm membrane; however, both SVS3 and SVS4 had weaker affinities for GM1 than SVS2. Therefore, we suggest that separate processes may cause capacitation inhibition and decapacitation, and SVS3 and SVS4 act on sperm capacitation cooperatively with SVS2., (© 2016 Society for Reproduction and Fertility.)

The Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P < 0.05). The protein spot weighing 15.52 ± 0.53 kDa with a pI value of 5.78 ± 0.12 is highlighted by its high abundance in bulls with low semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The present study evaluated functional aspects of binder of sperm 1 (BSP1) in the bovine species. In a first experiment, cumulus-oocyte complexes (n = 1274) were incubated with frozen-thawed ejaculated sperm (18 hours) in Fert-TALP medium containing: heparin, 10, 20, or 40 μg/mL BSP1. Heparin followed by gelatin affinity chromatography was used for purification of BSP1 from bovine seminal vesicle fluid. With ejaculated sperm, cleavage rates were similar when Fert-TALP medium was incubated with heparin (74.1 ± 2.7%), 10 μg/mL BSP1 (77.8 ± 3.1%), or 20 μg/mL BSP1 (74 ± 2.0%). Day-7 blastocyst rates were equivalent after incubations with heparin (40.8 ± 5.0%) and 10 μg/mL BSP1 (34.1 ± 4.4%), but reduced after 20 μg/mL BSP1 (22.4 ± 2.9%) and 40 μg/mL BSP1 (19.3 ± 4.1%; P < 0.05). In the second experiment, cumulus-oocyte complexes (n = 1213) were incubated with frozen-thawed cauda epididymal sperm (18 hours) in Fert-TALP medium containing: no heparin, heparin, 10, 20, or 40 μg/mL. Cleavage and blastocyst rates were similar after treatments with heparin (68.5 ± 1.3% and 24.7 ± 3.2%, respectively) or without heparin (65.5 ± 1.8% and 27.3 ± 1.6%, respectively). Cleavage was higher after treatment with any BSP1 concentrations (74.2 ± 2.7%-79.0 ± 1.1%) than without heparin (P < 0.05). Also, cleavage was better after Fert-TALP medium incubation with 40 μg/mL BSP1 (79.0 ± 1.1%) than with heparin (68.5 ± 1.3%; P < 0.05). Embryo development was higher (P < 0.05) after treatment with 20 μg/mL BSP1 (35.6 ± 2.5%) and 40 μg/mL (41.1 ± 2%) than after incubations with heparin (24.7 ± 3.2%) or without heparin (27.3 ± 1.6%). Interestingly, BSP1 did not cause reductions in blastocyst rates after fertilization with epididymal sperm, as observed with ejaculated sperm. On the basis of immunocytochemistry, there was BSP1 binding to frozen-thawed ejaculated but not to epididymal sperm. Also, anti-BSP1 reaction remained on ejaculated sperm (as expected) and appeared on epididymal sperm after incubation with purified BSP1. Acrosome reaction of ejaculated and epididymal sperm was induced after incubation with purified BSP1 as well, indicating an effect of BSP1 on capacitation. In conclusion, purified BSP1 from bull seminal vesicles was able to bind to and induce capacitation of ejaculated and epididymal sperm. Also, BSP1 added to fertilization media and allowed proper cleavage and embryo development, with the effects being modulated by previous exposure or not of spermatozoa to seminal plasma., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Binder of sperm (BSP) proteins are ubiquitous among mammals and have been extensively investigated over the last three decades. They were first characterized in bull seminal plasma and have now been identified in more than 15 different mammalian species where they represent a superfamily. In addition to sharing a common structure, BSP proteins share many characteristics. They are expressed by seminal vesicles and epididymides, interact with similar ligands and bind to the outer leaflet of sperm membranes via an interaction with choline phospholipids. In addition to playing a major role in sperm capacitation, they are implicated as molecular chaperones in sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume and possibly in the interaction between sperm and oocytes, making them crucial multifunctional proteins. Furthermore, BSP proteins can bind to egg yolk low-density lipoproteins and milk components, an interaction important for the protection of sperm during semen preservation in liquid or frozen state. Our current knowledge of BSP proteins strongly emphasizes their fundamental importance in male fertility and in the optimization of semen preservation techniques. Much work is still ahead in order to fully understand all the mysteries of BSP proteins.

Binder of SPerm (BSP) proteins from ungulates (more specifically bull, bison, buffalo, stallion, boar, goat, and ram) have been extensively studied in the past 30 years. These proteins secreted by seminal vesicles constitute between 1 and 60% of total seminal plasma proteins depending on the species. In addition to sharing many biochemical characteristics such as the ability to bind to gelatin, glycosaminoglycans, choline phospholipids, and lipoproteins, they also share a main function: promoting sperm capacitation. Over the last 10 years, new members of the BSP superfamily have been discovered. These proteins found in bulls, humans, mice, and rabbits are expressed in the epididymides rather than in seminal vesicles and constitute only a minute percentage of the seminal plasma proteins. However, they share many characteristics with BSPs expressed by accessory glands including their structure, their ability to bind to the aforementioned BSP ligands, as well as their ability to promote sperm capacitation. More investigations need to be done on epididymal BSP proteins, but studies described in this review constitute a solid foundation toward deciphering the significance of epididymal BSP proteins in male fertility., (© 2015 American Society of Andrology and European Academy of Andrology.)

Background: Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general., Methods: Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting., Results: Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, β-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1)., Conclusions: These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

Human herpesviruses cause different infectious diseases, resulting in world-wide health problems. Sexual transmission is a major route for the spread of both herpes simplex virus-1 (HSV-1) and -2. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa and, thereby, initiates viral replication. Previously, we demonstrated that the amyloid fibrils semenogelin (SEM) and semen-derived enhancer of viral infection (SEVI), and seminal plasma (SP) augment cytomegalovirus infection (Tang et al., J. Virol 2013). Whether SEM or SEVI amyloids or SP could also enhance other herpesvirus infections has not been examined. In this study, we found that the two amyloids as well as SP strongly enhance both HSV-1 and -2 infections in cell culture. Along with SP, SEM and SEVI amyloids enhanced viral entry and increased infection rates by more than 10-fold, as assessed by flow cytometry assay and fluorescence microscopy. Viral replication was increased by about 50- to 100-fold. Moreover, viral growth curve assays showed that SEM and SEVI amyloids, as well as SP, sped up the kinetics of HSV replication such that the virus reached its replicative peak more quickly. The interactions of SEM, SEVI, and SP with HSVs are direct. Furthermore, we discovered that the enhancing effects of SP, SEM, and SEVI can be significantly reduced by heparin, a sulfated polysaccharide with an anionic charge. It is probable that heparin abrogates said enhancing effects by interfering with the interaction of the viral particle and the amyloids, which interaction results in the binding of the viral particles and both SEM and SEVI.

Binder of sperm (BSP) proteins are ubiquitous among mammals and are exclusively expressed in male genital tract. The main function associated with BSP proteins is their ability to promote sperm capacitation. In mice, two proteins (BSP protein homolog 1 (BSPH1) and BSPH2) have been studied. Using recombinant strategies, BSPH1 was found to bind to epididymal sperm membranes and promote sperm capacitation in vitro. The goal of this study was to evaluate the role of native murine BSPH1 protein in sperm capacitation induced by BSA and HDLs. The effect of antibodies, antigen-binding fragments (Fabs), and F(ab')2 specific for murine BSPH1 on BSA- and HDL-induced capacitation was tested. Results indicate that BSPH1 has no direct role in BSA-induced capacitation. However, antibodies, Fabs, and F(ab')(2) could block capacitation induced by HDLs and could inhibit the HDL-induced increase in tyrosine phosphorylation, suggesting a specific interaction between HDLs and BSPH1. Results indicate that murine BSPH1 proteins in mice could be a new important piece of the puzzle in sperm capacitation induced by HDLs. As murine BSPH1 is orthologous to human BSPH1, this study could also lead to new insights into the functions and the importance of the human protein in male fertility., (© 2015 Society for Reproduction and Fertility.)

The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n = 12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n = 3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1-5.6, 37%) and basic (pI; 7.9-10.0, 27%) proteins and their pattern of expression varied significantly (p < 0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4 kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p < 0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.

Male factors account for 40% of infertility cases. The identification of differentially expressed proteins on spermatozoa from fertile and infertile men can help in the elucidation of the molecular basis of male infertility. The aim of this study was to compare sperm proteomes from 3 different groups: fertile men, normozoospermic men consulting for infertility, and normozoospermic men with an impaired capacity for fertilization (IVF-failure). We used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and LC-MS analysis to identify proteins that are differentially expressed. A total of 348 unique proteins were identified and quantified. The analysis identified 33 proteins that were differentially expressed in the IVF-failure group vs the fertile group. Comparison of the infertile and fertile groups revealed that 18 proteins appeared to be differentially expressed. Four proteins were similarly altered in the IVF-failure and infertile groups: semenogelin 1 (SEMG1), prolactin-induced protein (PIP), glyceraldehyde-3-phosphate dehydrogenase (GAPDHS), and phosphoglycerate kinase 2 (PGK2). These protein markers were selected for validation using multiple reactions monitoring mass spectrometry (MRM-MS) and further confirmed by Western blot analysis. Overall, these results suggest that a panel of proteins may be used as biomarkers for future studies of infertility.

The incidence of renal cell carcinoma (RCC) has been steadily rising each year. There are currently few recognized biomarkers for the diagnosis and prognosis of RCC. We investigated semenogelin I (Sg I) expression and its clinical significance in patients with RCC. The expression levels of Sg I and its protein were measured by qPCR and Western blotting, respectively. Immunohistochemistry was used to investigate the protein expression of Sg I in RCC and normal renal tissue from 53 patients. The Kaplan-Meier method and log-rank test were used to evaluate the data. By qRCR (p < 0.01) and Western blot, the level of Sg I expression in benign tissues was higher than that in RCC tissues. Expression of Sg I was observed in 30 (57 %) RCC cases, which was significantly lower than that observed in benign renal tissues from the same patients [Sg I positive in 53 (100 %) cases (p < 0.0001)] by immunohistochemistry. There was an inverse relation between Sg I expression and clinical stage (pT1-2 vs pT3-4, p < 0.0001). Patients with Sg I-negative tumor had a significantly higher risk of recurrence (Kaplan-Meier and log-rank tests, p < 0.0001). There was low Sg I expression in RCC. Sg I expression has potential value in predicting cancer progression and prognosis. These findings support the use of Sg I as a novel biomarker for RCC.

Background: Hyperresponsiveness to inhaled non-infectious microbial particles (NIMPs) has been associated with illnesses in the airways. Hypersensitivity pneumonitis (HP) is considered to be the prototype for these NIMPs-related diseases; however, there is no consensus on the definitions or diagnostic criteria for HP and the spectrum of related illnesses., Methods and Findings: In order to identify the possible diagnostic markers for illnesses associated with NIMPs in alveolar lining fluid, we performed a proteomic analysis using a two-dimensional difference gel electrophoresis on bronchoalveolar lavage (BAL) fluid from patients with exposure to NIMPs in the context of damp building-related illness (DBRI) or conditions on the borderline to acute HP, designated here as agricultural type of microbial exposure (AME). Samples from patients with HP and sarcoidosis (SARC) were included for reference. Results were compared to results of healthy subjects (CTR). Western blot was used for validation of potential marker proteins from BAL fluid and plasma. Protein expression patterns suggest a close similarity between AME and HP, while DBRI was similar to CTR. However, in DBRI the levels of the inflammation associated molecules galectin-3 and alpha-1-antitrypsin were increased. A novel finding emerging from this study was the increases of semenogelin levels in BAL fluid from patients with AME, HP and SARC. Histone 4 levels were increased in AME, HP and SARC. Elevated plasma levels of histone 2B were detected in HP and SARC, suggesting it to be a potential blood indicator for inflammatory diseases of the lungs., Conclusions: In this study, the proteomic changes in bronchoalveolar lavage of DBRI patients were distinct from other NIMP exposure associated lung diseases, while changes in AME overlapped those observed for HP patient samples. Some of the proteins identified in this study, semenogelin and histone 4, could function as diagnostic markers for differential diagnosis between DBRI and HP-like conditions.

Unlabelled: Semen enhances HIV infection in vitro, but how long it retains this activity has not been carefully examined. Immediately postejaculation, semen exists as a semisolid coagulum, which then converts to a more liquid form in a process termed liquefaction. We demonstrate that early during liquefaction, semen exhibits maximal HIV-enhancing activity that gradually declines upon further incubation. The decline in HIV-enhancing activity parallels the degradation of peptide fragments derived from the semenogelins (SEMs), the major components of the coagulum that are cleaved in a site-specific and progressive manner upon initiation of liquefaction. Because amyloid fibrils generated from SEM fragments were recently demonstrated to enhance HIV infection, we set out to determine whether any of the liquefaction-generated SEM fragments associate with the presence of HIV-enhancing activity. We identify SEM1 from amino acids 86 to 107 [SEM1(86-107)] to be a short, cationic, amyloidogenic SEM peptide that is generated early in the process of liquefaction but that, conversely, is lost during prolonged liquefaction due to the activity of serine proteases. Synthetic SEM1(86-107) amyloids directly bind HIV-1 virions and are sufficient to enhance HIV infection of permissive cells. Furthermore, endogenous seminal levels of SEM1(86-107) correlate with donor-dependent variations in viral enhancement activity, and antibodies generated against SEM1(86-107) recognize endogenous amyloids in human semen. The amyloidogenic potential of SEM1(86-107) and its virus-enhancing properties are conserved among great apes, suggesting an evolutionarily conserved function. These studies identify SEM1(86-107) to be a key, HIV-enhancing amyloid species in human semen and underscore the dynamic nature of semen's HIV-enhancing activity., Importance: Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro, but how long it retains this activity has not been investigated. Semen naturally undergoes physiological changes over time, whereby it converts from a gel-like consistency to a more liquid form. This process, termed liquefaction, is characterized at the molecular level by site-specific and progressive cleavage of SEMs, the major components of the coagulum, by seminal proteases. We demonstrate that the HIV-enhancing activity of semen gradually decreases over the course of extended liquefaction and identify a naturally occurring semenogelin-derived fragment, SEM1(86-107), whose levels correlate with virus-enhancing activity over the course of liquefaction. SEM1(86-107) amyloids are naturally present in semen, and synthetic SEM1(86-107) fibrils bind virions and are sufficient to enhance HIV infection. Therefore, by characterizing dynamic changes in the HIV-enhancing activity of semen during extended liquefaction, we identified SEM1(86-107) to be a key virus-enhancing component of human semen., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Binder of SPerm (BSP) proteins are a family of proteins expressed exclusively in the male reproductive tract (seminal vesicles or epididymis) of several mammalian species. They are known to promote capacitation, a sperm maturation step essential for fertilization. Our recent studies have shown that in human, the Binder of SPerm Homolog 1 (BSPH1) is expressed solely in epididymal tissues. The goal of the current study was to characterize BSPH1 and evaluate its effect on different sperm functions. A human recombinant BSPH1 (rec-BSPH1) was produced, purified and refolded. Rec-BSPH1 was found to share many characteristics with other members of the BSP superfamily, as it was able to bind gelatin and heparin as well as capacitate sperm. Rec-BSPH1 had no effect on sperm acrosome reaction or any sperm motility parameters. Native BSPH1 was localized on the equatorial segment, post-acrosomal segment and neck of ejaculated human sperm. Rec-BSPH1, following incubation with washed ejaculated human sperm, exhibited binding patterns similar to the native protein. These results show that the human epididymal BSPH1 shares many biochemical and functional characteristics with BSP proteins secreted by seminal vesicles of ungulates, and behaves similarly to its murine epididymal orthologue BSPH1. This study of human BSPH1 brings us one step closer to understanding the importance of this protein in male fertility.

Naturally occurring fragments of the abundant semen proteins prostatic acid phosphatase (PAP) and semenogelins form amyloid fibrils in vitro. These fibrils boost HIV infection and may play a key role in the spread of the AIDS pandemic. However, the presence of amyloid fibrils in semen remained to be demonstrated. Here, we use state of the art confocal and electron microscopy techniques for direct imaging of amyloid fibrils in human ejaculates. We detect amyloid aggregates in all semen samples and find that they partially consist of PAP fragments, interact with HIV particles and increase viral infectivity. Our results establish semen as a body fluid that naturally contains amyloid fibrils that are exploited by HIV to promote its sexual transmission.

In mammals, sperm migrate through the female reproductive tract to reach the egg; however, our understanding of this journey is highly limited. To shed light on this process, we focused on defining the functions of seminal vesicle secretion 2 (SVS2). SVS2(-/-) male mice produced sperm but were severely subfertile, and formation of a copulatory plug to cover the female genital opening did not occur. Surprisingly, even when artificial insemination was performed with silicon as a substitute for the plug, sperm fertility in the absence of SVS2 remained severely reduced because the sperm were already dead in the uterus. Thus, our results provide evidence that the uterus induces sperm cell death and that SVS2 protects sperm from uterine attack.

Seminal vesiculitis is the common disease of male urogenital system. However, the pathogenesis of seminal vesiculitis remains unclear. Semenogelin I (Sg I) is mainly synthesized and secreted by seminal vesicle and has antibacterial activity. We thus postulate that Sg I plays an important role during the occurrence and development of seminal vesiculitis. In the present study, we analyzed the expression of Sg I in normal seminal vesicle tissues and seminal vesiculitis tissues through immunohistochemistry. The results showed down-regulated expression of clusterin at protein level in seminal vesiculitis tissues compared with normal seminal vesicle tissues. Our preliminary data suggest that the abnormal expression of clusterin is closely related to seminal vesiculitis. Downregulation of Sg I expression may weaken the antibacterial activity of the seminal vesicle and then induce the occurrence of disease. This is the first study to focus on the relationship between Sg I and human seminal vesiculitis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes., Objectives: We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression., Methods: We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice., Results: The DNA methylation status at four specific CpGs (-160, -237, -306, and -367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (-449 and -459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers-DNMT3A, MBD2, and HDAC2-increased in the SV of DES-exposed WT mice., Conclusion: DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV., Citation: Li Y, Hamilton KJ, Lai AY, Burns KA, Li L, Wade PA, Korach KS. 2014. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle. Environ Health Perspect 122:262-268; http://dx.doi.org/10.1289/ehp.1307351.

Objective: Epididymal protease inhibitor (Eppin) was located on the surface of spermatozoa and modulates the liquefaction of human semen. Here, we identify the correlative protein partner of Eppin to explore the molecular mechanism of liquefaction of human semen., Methods: (1) Human seminal vesicle proteins were transferred on the membrane by Western blotting and separated by 2-D electrophoresis and incubated in recombinant Eppin. The correlative protein was identified by Mass Spectrometry (MS) (2). Western blotting was used to determine the relation of rEppin and rFibronectin(Fn); (3) Co-localization in spermatozoa were detected using immunofluorescence; (4) Correalation of Eppin and Fn was proved by co-immunoprecipitation., Results: Fn was identified as the binding partner of recombinant Eppin by MS. Recombinant of Eppin was made and demonstrated that the Eppin fragment binds the fn 607-1265 fragment. The Eppin-Fn complex presents on the sperm tail and particularly in the midpiece region of human ejaculated spermatozoa. Immunoprecipitation indicated that Eppin in the spermatozoa lysates was complexed with Fn., Conclusions: Our study demonstrates that Eppin and Fn bind to each other in human semen and on human ejaculated spermatozoa. Eppin-Fn complex may involve in semen coagulation, liquefaction and the survival and preparation of spermatozoa for fertility in the female reproductive tract.

Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins.

Among the modes of transmission available to the cytomegalovirus (CMV) is sexual transmission, primarily via semen. Both male-to-female (M-F) and male-to-male (M-M) sexual transmission significantly contribute toward the spread of CMV infections in the global population. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa, thereby initiating viral replication. Both semen and seminal plasma (SP) can enhance HIV-1 infection in cell culture, and two amyloid fibrils, semen-derived enhancer of viral infection (SEVI) and amyloids derived from the semenogelins (SEM amyloids), have been identified as seminal factors sufficient to enhance HIV-1 infection (J. Munch et al., Cell 131:1059-1071, 2007; N. R. Roan et al., Cell Host Microbe 10:541-550, 2011; F. Arnold et al., J. Virol. 86:1244-1249, 2012). Whether SP, SEVI, or SEM amyloids can enhance other viral infections has not been extensively examined. In this study, we found that SP, SEVI, and SEM amyloids strongly enhance both human CMV (HCMV) and murine CMV infection in cell culture. SEVI and SEM amyloids increased infection rates by >10-fold, as determined by both flow cytometry and fluorescence microscopy. Viral replication was increased by 50- to 100-fold. Moreover, viral growth curve assays showed that SP, SEVI, and SEM amyloids sped up the kinetics of CMV replication such that the virus reached its replicative peak more quickly. Finally, we discovered that SEM amyloids and SEVI counteracted the effect of anti-gH in protecting against CMV infection. Collectively, the data suggest that semen enhances CMV infection through interactions between semen amyloid fibrils and viral particles, and these interactions may prevent HCMV from being neutralized by anti-gH antibody.