The correlation between human seminal plasma sialoproteins and ejaculate parameters.

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.

Contrasting effects of molecular crowding on the membrane-perturbing and chaperone-like activities of major bovine seminal plasma protein, PDC-109.

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.

Semenogelin, a coagulum macromolecule monitoring factor involved in the first step of fertilization: A prospective review.

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.

Blocking serine protease activity prevents semenogelin degradation leading to hyperviscous semen in humans.

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.

Promotion of osteogenesis by Sweroside via BMP2-involved signaling in postmenopausal osteoporosis.

Phlomis umbrosa has been traditionally used for bone diseases in traditional Korean Medicine. Sweroside (SOS), marker compounds of P. umbrosa, has been known to promote osteoblast differentiation. In this study, ameliorative effects of SOS on osteoporosis and potential target pathway were investigated. Ovariectomized mice were administered three doses of SOS three times a week for 4 weeks after inducing osteoporosis. Bone mineral content (BMC) and bone mineral density (BMD) were analyzed by dual energy X-ray absorptiometry. A human osteosarcoma cell line (SaOS-2) was differentiated to clarify the promoting effects of SOS on osteoblast differentiation and bone formation. Osteoblastic bone-forming markers were evaluated in lumbar vertebrae (LV) and mineralized SaOS-2 cells. SOS markedly elevated BMC and BMD levels and attenuated the bone marrow adipocytes in the femoral shaft. SOS increased the formation of bone matrix in SaOS-2 cells. Bone morphogenetic protein-2 (BMP2) and runt-related transcription factor 2 (CBFA1) in LV and SaOS-2 cells were up-regulated by SOS. SOS increased alkaline phosphatase (ALPL), osteopontin (SPP1), and bone sialoprotein-1 (BSPH1). In conclusion, SOS induced the formation of mineralized bone matrix by regulating BMP2/CBFA1-mediated molecules. Therefore, SOS could be a therapeutic compound of treatment for osteoporosis by producing the new bone matrix.

Identification of an antibacterial polypeptide in mouse seminal vesicle secretions.

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.

Evaluation of ubiquitination and sumoylation of acrosin inhibitor during in vitro capacitation of porcine sperm.

The main objective of this study was to investigate the expression of acrosin inhibitor (AI), ubiquitin (Ub), and small ubiquitin-related modifier 1 (SUMO1) proteins during in vitro capacitation of pig sperm. Duroc pig sperm was divided into fresh sperm and capacitation treatment groups. Protein expression was evaluated using computer-assisted sperm analysis (CASA) systems, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and immunofluorescence. The results showed that the expression of AI (30 kDa) incapacitated sperm was significantly lower than that in fresh sperm (P < 0.05), and that the levels of ubiquitinated and SUMO1-ylated proteins in capacitated sperm were significantly higher than those in fresh sperm (P < 0.05). Immunofluorescence results showed that AI, Ub, and SUMO1 were located in the acrosome region of the fresh and capacitated sperm heads. After capacitation, the fluorescence intensity of AI and SUMO1 decreased, while that of Ub increased. The protein band at 30 kDa represented the AI-Ub-SUMO1 complex, indicating that this complex was involved in sperm capacitation. Furthermore, SUMO1 increased the stability of AI at 30 kDa, preventing its complete decomposition, while at 46 kDa, in the absence of SUMO1, AI is bound to ubiquitin, and was completely degraded.

Identification of coding region SNPs from specific and sensitive mRNA biomarkers for the deconvolution of the semen donor in a body fluid mixture.

mRNA markers provide a very promising method for the identification of human body fluids or tissues in the context of forensic investigations. Previous studies have shown that different body fluids can be distinguished from each other according to their specific mRNA biomarkers. In this study, we evaluated eight semen-specific mRNA markers (KLK3, NKX3-1, CKB, KLK2, PRAC1, SEMG1, TGM4, and SORD) that encompass 12 coding single nucleotide polymorphisms (cSNPs) to identify the semen contributor in a mixed stain. Five highly specific and sensitive mRNA markers for blood, menstrual blood, saliva, vaginal secretions, and skin were also incorporated into the PCR system as body fluid-positive controls. Reverse transcription polymerase chain reaction (RT-PCR), multiplex PCR and SNaPshot mini-sequencing assays were established for the identification of semen-specific mRNA. The amplicon size ranged from 133 to 337 bp. The semen-specific system was examined against blood, menstrual blood, saliva, vaginal secretions, and skin swabs. The eight mRNA biomarkers were semen-specific and could be successfully typed in laboratory-generated mixtures composed of different body fluids supplemented with 1 ng of semen cDNA. This system possessed a high sensitivity that ranged from 1:10-1:100 for detecting trace amounts of semen in semen-containing body fluid mixtures. Additionally, our results demonstrated that the cSNPs polymorphisms included in the mRNA markers were concordant with genomic DNA (gDNA). Despite the presence of other body fluids, the system exhibited high sensitivity and specificity to the semen in the mixture. In future studies, we will add other cSNPs from the semen-specific genes using massively parallel sequencing to further improve our system.

Direct seminal fluid identification by protease-free high-resolution mass spectrometry.

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.

Effect of chronic stress on expression and secretion of seminal vesicle proteins in adult rats.

Chronic stress (CS) is known to affect men's health especially fertility by reducing semen quality. Although the effects of CS on testicular function and sperm parameters are documented, changes of substances and secreting proteins in the seminal vesicle (SV) have never been reported. This study aimed to demonstrate the alterations of contents and expressions of proteins in seminal vesicle fluid (SVF) under CS. Fourteen adult rats were divided into control and CS groups (n = 7/each). Control rats were not exposed to stressor, while the CS animals were immobilised by restraint cage (4 hr/day) and followed by forced swimming (15 min/day) for consecutive 60 days. Biochemical substances and malondialdehyde (MDA) level in SVF were examined. Expressions of heat-shock protein 70 (Hsp70), caspases (Casp) 3 and 9, and tyrosine-phosphorylated (TyrPho) proteins were investigated in seminal vesicle tissue (SVT) and SVF. It was found that CS caused reductions of seminal epithelial height and secreted substance levels. Significantly, MDA levels in SVF and expressions of Hsp70, Casp and TyrPho proteins were increased in of CS animals. It was concluded that CS affected seminal secretion. Low quality of CS seminal plasma may associate with increase of MDA and expressions of secreted proteins.

SEMG1/2 augment energy metabolism of tumor cells.

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.

[Effects of silencing the SEMG1 protein on the cycle and apoptosis of GC-1 spg cells].

OBJECTIVE: To investigate the effects of silencing the semenogelin 1 (SEMG1) protein on the cycle and apoptosis of the spermatogonia germ cell line (GC-1 spg). METHODS: SEMG1-specific siRNA was transfected into GC-1 spg cells by lipofectamine 2000 (the siRNA-SEMG1 group), the relative expression levels of the SEMG1 protein in the GC-1 spg cells of the siRNA-SEMG1, blank control and negative control groups were detected by Western blot, and the apoptosis and cycle of the cells in different groups were determined by flow cytometry. RESULTS: The expression of the SEMG1 protein in the GC-1 spg cells was dramatically decreased in the siRNA-SEMG1 group compared with those in the blank and negative control groups (1.80±0.05 vs 2.51±0.13 and 2.50±0.12， P < 0.01), but the apoptosis rate was remarkably higher in the former than in the latter two groups (ï¼»6.77 ± 0.15ï¼½% vs ï¼»0.70 ± 0.06ï¼½% and ï¼»0.8 ± 0.06ï¼½%, P < 0.01). No statistically significant difference was observed in the cell cycles among the three groups (P > 0.05). In addition, Western blot showed that the expression of the caspase-3 protein was significantly higher and that of the BCL2 protein markedly lower in the siRNA-SEMG1 than in the blank and negative control groups (P < 0.05). CONCLUSIONS: SEMG1-specific siRNA can effectively silence the expression of the SEMG1 protein in GC-1 spg cells and promote their apoptosis.

Identification of the major rabbit and guinea pig semen coagulum proteins and description of the diversity of the REST gene locus in the mammalian clade Glires.

The seminal vesicle secretions of guinea pig and rabbit were analyzed for semen coagulum proteins. Using SDS-PAGE we discovered a previously not fully recognized semen coagulum protein, Svp5, in the guinea pig and a single predominant component, SVP200, in the rabbit. Potential genes of these proteins were identified in genome databases by their homology with human and murine genes. The structure of their fullength transcripts was determined using seminal vesicle cDNA and sequencing primers based on genomic sequences. Homology searching indicated that both Svp5 and SVP200 were synthesized from composite genes that were the result of merger between two genes showing homology with human SEMG2 and PI3. For a deeper understanding of the evolution of the genes, we retrieved and analyzed genome sequences from the REST gene loci, encompassing genes of semen coagulum proteins and related rapidly evolving seminal vesicle-transcribed genes, of 14 rodents and 2 lagomorphs. The analysis showed that rodents of the suborders myomorpha, hystricomorpha, and castorimorpha had unique sets of REST genes, whereas sciuromorpha seemed to be lacking such genes. It also indicated a closer relationship between myomorpha and castorimorpha than to rodents of the two other analyzed suborders. In the lagomorph species, the pika appeared to be devoid of REST genes, whereas the rabbit had a single expressed REST gene, SVP200, and two pseudogenes. The structural similarity of semen coagulum proteins in rabbit and hystricomph species suggests that they are closely related. This was also supported by other similarities at their REST gene loci, e.g. the finding of a PI3-like gene in the rabbit that also had features in common with caltrin2 of hystricomorph rodents. The homologies indicate that hystricomorpha may have separated from myomorpha and castorimorpha before the separation of hystricomorpha from lagomorpha.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

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.

Epididymal proteins Binder of SPerm Homologs 1 and 2 (BSPH1/2) are dispensable for male fertility and sperm motility in mice.

The binder of sperm family of proteins has been reported to be indispensable for sperm maturation and capacitation. However, their physiological functions in fertility have only been studied in vitro. CRISPR/Cas9 genome editing was utilized to generate double knockout (DKO) mice by simultaneously targeting the two murine binder of sperm genes, Bsph1 and Bsph2. To confirm that the homologous genes and proteins were completely eliminated in the DKO mice, different methods such as reverse transcription polymerase chain reaction, digital droplet-polymerase chain reaction and liquid chromatography tandem mass spectrometry were applied. Bsph1/2 DKO male mice were bred by intercrossing. Compared to wild type counterparts, male Bsph1/2 null mice, lacking BSPH1/2 proteins, were fertile with no differences in sperm motility and sperm count. However, the weights of male pups were significantly increased in Bsph1/2 double knockout mice in a time dependent manner spanning days 6 and 21, as well as 6 weeks of age. No change was detected in the weights of female pups during the same period. Taken together, these data indicate that BSPH1/2 proteins are dispensable for male fertility in mice but may influence growth.

Human Semenogelin 1 Promotes Sperm Survival in the Mouse Female Reproductive Tract.

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.

Role of Binder of SPerm homolog 1 (BSPH1) protein in mouse sperm-egg interaction and fertilization.

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.

Identification of Urinary Biomarkers for Exercise-Induced Immunosuppression by iTRAQ Proteomics.

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.

Epididymal protease inhibitor (EPPIN) is a protein hub for seminal vesicle-secreted protein SVS2 binding in mouse spermatozoa.

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.

Conserved core tryptophans of FnII domains are crucial for the membranolytic and chaperone-like activities of bovine seminal plasma protein PDC-109.

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.