Search

PDC-109, one of the most abundant proteins in bovine seminal plasma, has detrimental effect on spermatozoa in a time- and concentration-dependent manner. Therefore, we hypothesized that sequestration of detrimental protein from ejaculates would be beneficial following cryopreservation of sperm cells. To this aim, we evaluated the effect of sequestration of PDC-109 either by anti-PDC-109 antibodies (Ab) or egg yolk (EY) alone or by the synergistic action of EY + Ab in minimizing cryoinjury to bull spermatozoa. PDC-109 protein was purified by applying two-step chromatography procedures. The purified protein was injected in rabbits to raise antibodies which were isolated using ion-exchange chromatography. After checking the Ab cross-reactivity, they were quantitated and added to ejaculates, either alone or in addition to EY in Tris-glycerol (TG) extender. Thus, ejaculates were processed in extender containing EY + TG (group I), Ab + TG (group II) or EY + Ab + TG (group III). Semen quality parameters (SQPs) viz. viability and acrosome integrity (FITC-PSA), cryoinjury to spermatozoa (chlortetracycline, CTC assay) and in vitro fertility of protein-sequestered-semen (zona-penetration assay) were evaluated. A significant (p < 0.05) improvement in post-thaw SQPs as well as in non-capacitated spermatozoa observed at pre-freeze and post-thaw stages of cryopreservation in group III compared with other groups indicated reduction in protein-mediated cryoinjury. From this study, it can be concluded that sequestration of PDC-109 by synergistic action of EY+Ab as compared to either of them alone significantly improve sperm quality and minimize cryoinjury to bull spermatozoa upon storage at ultra-low temperatures., (© 2013 Blackwell Verlag GmbH.)

Objectives: To investigate the presence of manserin in human prostate cancers and to correlate manserin expression with pathologic outcomes and progression-free survival., Methods: Eighty-seven patients with recent prostate cancer were classified into 4 groups based on Gleason score, and manserin immunohistochemistry was correlated with Gleason sum grade. To investigate the validity of manserin as a prognostic factor, the Cox proportional hazards regression model was performed on 48 patients in our cohort with T3 or T4 prostate cancer who were initially treated with androgen deprivation therapy., Results: The manserin-positive rates of patients with Gleason sums of 6, 7, 8, and ≥9 were 0%, 20.0%, 35.0%, and 48.1%, respectively. Manserin-positive rates were positively correlated with Gleason sums (P = 0.0001). Median times to cancer progression in groups with (n = 8) and without (n = 40) manserin expression were 8 months and 28 months, respectively (P = 0.01). Univariate Cox analysis revealed that manserin expression, clinical stage T4, and high Gleason sum were significantly associated with progression. Multivariate analysis revealed that only 2 factors, manserin expression (hazard ratio (HR) 4.99, P = 0.01) and clinical stage T4 (HR 4.77, P = 0.03), were independent risk factors for progression., Conclusions: This is the first report of manserin expression in human prostate cancers. Manserin may serve as a marker of prostate cancer progression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10-15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To compare the sperm protein profile between men with and without varicocele., Methods: The present study was designed as a case-control study. The research patients were recruited from the Infertility Unit of the Royan Institute in 2009. We included 20 sperm samples from normozoospermic men without varicocele (control group) and 20 sperm samples from oligozoospermic patients with varicocele, grade 3 (varicocele group) in the present study. The sperm protein profile in the 2 groups was characterized using 2-dimensional gel electrophoresis. Differences in protein expression were established using gel analysis software, and protein identification was performed using mass spectroscopy analysis., Results: In the varicocele group, we noted 15 consistent differences in protein expression (1, spots missing; 12, less abundant; and 2, more abundant) compared with the control group (P < .01). The findings revealed that heat shock proteins, mitochondrial proteins, and cytoskeleton proteins are the proteins mainly affected by varicocele disease., Conclusion: To our knowledge, the present study is a novel study, with few studies describing the correlation between sperm protein in men with and without varicocele obtained using a 2-dimensional proteomic approach. It could be an important prerequisite to the development of diagnostic tests to predict varicocelectomy outcomes in patients with varicocele and abnormal findings on a spermogram in the clinical environment., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Isothermal titration calorimetry is a highly sensitive technique for the study of molecular interactions. This method has been applied quite extensively to investigate the interaction of proteins with small ligands, other proteins, and nucleic acids as well as with drugs and metal ions. In this chapter, we describe the application of ITC for the investigation of thermodynamics of protein-lipid interaction. A number of parameters such as enthalpy of binding (ΔH), entropy of binding (ΔS), association constant (K (a)), binding stoichiometry (n), and free energy of binding (ΔG) can be obtained from a single calorimetric titration, providing a complete thermodynamic characterization of the interaction. The method is described in detail taking the major protein of the bovine seminal plasma, PDC-109, which exhibits a high preference for interaction with choline-containing lipids, as an example. The method can be applied to investigate the thermodynamics of the interaction of other soluble proteins with lipid membranes.

Identification of protein-protein interactions is vital for complete understanding of a biological process and for functional characterization of a protein in related biochemical pathways. In this study, we performed analysis of prolactin inducible protein (PIP) interactions in human seminal plasma. PIP and its interacting partners were co-immunoprecipitated, analyzed by SDS-PAGE and identified by MALDI-TOF mass spectrometry. Three major interacting partners were identified, viz. human serum albumin, zinc-α-2 glycoprotein and semenogelin I fragments. This is the first report of interaction between PIP and semenogelin I fragments in human seminal plasma or elsewhere with a suggestive role in reproductive physiology which might be helpful for spermatozoa to acquire their motility., (Copyright © 2012 Elsevier B.V. All rights reserved.)

EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine-rich protein containing both Kunitz and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down-regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen-dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility., (Copyright © 2012 Wiley Periodicals, Inc.)

Semenogelins and eppin are seminal plasma proteins that form a complex and inhibit sperm motility. However, the role of these proteins in prostate cancer is poorly understood. We immunohistochemically stained for semenogelins I and II and eppin in 291 radical prostatectomy specimens. We then evaluated the association between their expressions in nuclei, cytoplasms, or intraluminal secretions of benign/high-grade prostatic intraepithelial neoplasia/carcinoma cells and clinicopathologic profile available for our patient cohort. Stains were positive in 32%/77%/84% (nuclear semenogelin I), 87%/94%/84% (nuclear semenogelin II), 56%/64%/37% (nuclear eppin), 7%/15%/11% (cytoplasmic semenogelin I), 6%/11%/9% (cytoplasmic semenogelin II), 68%/74%/95% (cytoplasmic eppin), 97%/98%/13% (secreted semenogelin I), 98%/97%/11% (secreted semenogelin II), and 97%/98%/48% (secreted eppin) of benign/prostatic intraepithelial neoplasia/carcinoma, respectively. The levels of nuclear semenogelin I/cytoplasmic eppin were significantly higher in carcinoma than in benign (P<.001/P<.001) or prostatic intraepithelial neoplasia (P<.001/P<.001) and in prostatic intraepithelial neoplasia than in benign (P<.001/P=.006). Significantly higher nuclear semenogelin II expression was found in prostatic intraepithelial neoplasia than in benign (P<.001) or carcinoma (P<.001). Significantly lower nuclear eppin expression was seen in carcinoma than in benign (P<.001) or prostatic intraepithelial neoplasia (P<.001). Secreted semenogelin I, secreted semenogelin II, and secreted eppin were all significantly lower in carcinoma than in benign (P<.001) or prostatic intraepithelial neoplasia (P<.001). There were no statistically significant correlations between each stain and clinicopathologic features except significantly lower nuclear eppin expression in Gleason score 8 or higher tumors. Kaplan-Meier and log-rank tests further revealed that patients with nuclear semenogelin I-positive tumor had a significantly higher risk for biochemical recurrence (P=.046). Multivariate Cox model showed a trend toward significance (P=.093) in nuclear semenogelin I positivity as an independent predictor for recurrence. These results suggest that nuclear semenogelin I expression could be a reliable prognosticator in men who undergo radical prostatectomy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dose-dependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.

Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.

Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10 000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation.

Background: Studies have shown that perinatal exposure to the synthetic estrogen diethylstilbestrol (DES) leads to feminization of the seminal vesicle (SV) in male mice, as illustrated by tissue hyperplasia, ectopic expression of the major estrogen-inducible uterine secretory protein lactoferrin (LF), and reduced expression of SV secretory protein IV (SVS IV)., Objectives: The present study was designed to evaluate the role of the estrogen receptor (ER) in this action by using ER-knockout (ERKO) mice., Methods: Wild-type (WT), ERα-null (αERKO), and ERβ-null (βERKO) male mice were treated with either vehicle or DES (2 μg/day) on neonatal days 1-5. These mice were divided into two groups: In the first group, intact mice were sacrificed at 10 weeks of age; in the second group, mice were castrated at 10 weeks of age, allowed to recover for 10 days, treated with dihydrotestosterone (DHT) or placebo, and sacrificed 2 weeks later. Body weights and SV weights were recorded, and mRNA expression levels of Ltf (lactoferrin), Svs4, and androgen receptor (Ar) were assessed., Results: In DES-treated intact mice, SV weights were reduced in WT and βERKO mice but not in αERKO mice. DES-treated WT and βERKO males, but not αERKO males, exhibited ectopic expression of LF in the SV. DES treatment resulted in decreased SVS IV protein and mRNA expression in WT males, but no effect was seen in αERKO mice. In addition, DES-treated βERKO mice exhibited reduced Svs4 mRNA expression but maintained control levels of SVS IV protein. In DES-treated castrated mice, DHT implants restored SV weights to normal levels in αERKO mice but not in WT mice, suggesting full androgen responsiveness in αERKO mice., Conclusions: These data suggest that DES-induced SV toxicity and feminization are primarily mediated by ERα; however, some aspects of androgen response may require the action of ERβ.

Background: Cell-free seminal mRNA (cfs-mRNA) exists in human ejaculate at high concentrations and with high stability, and contains many tissue-specific transcripts secreted from the male reproductive system. Owing to the sensitivity of RNA technology, cfs-mRNAs are ideal candidates for non-invasive biomarkers of physiopathological conditions. This study applied cfs-mRNA in identifying the presence of either germ cells or complete obstruction in men with azoospermia., Methods: RT-PCR was performed to amplify the germ cell-specific (DDX4), seminal vesicle-specific (SEMG1) and prostate-specific (TGM4) mRNAs from cfs-mRNAs, which were isolated from the seminal plasma of men with non-obstructive azoospermia (NOA) or obstructive azoospermia (OA). The 39 patients with NOA, diagnosed by testicular biopsy, included 8 men with maturation arrest (MA), 3 men with incomplete sertoli cell only (iSCO) syndrome and 28 men with complete SCO (cSCO). The 29 patients with OA, confirmed by the presence of sperm in the testis or epididymis, included 8 men with congenital bilateral absence of the vas deferens (CBAVD) and 21 men with non-CBAVD. Healthy individuals and vasectomized men were enrolled as controls., Results: TGM4 was detected in all participants. Consistent with their diagnosis, DDX4 was detected in all patients with MA or iSCO but was absent in most cases of cSCO (n = 21, 75.0%) or non-CBAVD (n = 18, 85.7%), and in all men with vasectomy or CBAVD. The presence of DDX4 in the other seven men with cSCO and three patients with non-CBAVD suggests the presence of germ cells in the testis, and incomplete obstruction, respectively. SEMG1 was undetectable in three patients with CBAVD with bilateral absence of the seminal vesicles, and in two non-CBAVD cases with low ejaculate volume., Conclusions: These results suggest that, with high sensitivity and representativity, cfs-mRNA could be non-invasive biomarkers for identifying the presence of germ cells or complete obstruction in azoospermia.

A study was carried out to assess the effect of sequestration of PDC-109 protein, a majority constituent of heparin binding proteins (HBP) of seminal plasma, on freezability and in vitro fertilizing ability of crossbred bull spermatozoa after cryopreservation. The study consisted of isolation and characterization of PDC-109 protein to raise anti-sera against it in rabbits. Following which, raised antibodies against PDC-109 protein was quantitated and coated in tubes used for collection of ejaculates. Semen ejaculates thus collected were cryopreserved using EYTG extender. Physico-morphological characteristics, viz. motility, viability, acrosomal integrity and HOS response as an indicator of freezability of cryopreserved spermatozoa were determined at pre freeze as well as post thaw stage. At pre freeze stage, a significant (p<0.05) improvement in viability (83.83 ± 2.18 vs 75.17 ± 2.42) and acrosome integrity (81.33 ± 2.38 vs 72.83 ± 2.39) in antibodies treated group than control was observed. Similarly, increase in HOS responsive spermatozoa was highly significant (p<0.01) than control (78.83 ± 1.69 vs 67.5 ± 1.75). At post thaw stage, significant (p<0.05) improvement in viability (69.50 ± 2.16 vs 60.33 ± 2.19) and HOS responsive spermatozoa (68.67 ± 1.62 vs 58.50 ± 1.32) and highly significant (p<0.01) increase in individual motility (56.17 ± 1.83 vs 47.00 ± 1.86) and acrosome integrity (75.17 ± 2.38 vs 61.83 ± 2.1) was observed in antibodies treated group when compared to control was observed. The results from the study revealed that sequestration of PDC-109 protein from semen samples leads to significant improvement in pre-freeze and post-thaw values of above parameters in cryopreserved spermatozoa. It is thus concluded that sequestration of PDC-109 protein from ejaculates improves freezability of crossbred bull spermatozoa., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The development of a new male contraceptive requires a transition from animal model to human and an understanding of the mechanisms involved in the target's inhibition of human spermatozoan fertility. We now report that semenogelin (SEMG1) and anti-EPPIN antibodies to a defined target site of 21 amino acids on the C terminal of EPPIN cause the loss of intracellular calcium, as measured by Fluo-4. The loss of intracellular calcium explains our previous observations of an initial loss of progressive motility and eventually the complete loss of motility when spermatozoa are treated with SEMG1 or anti-EPPIN antibodies. Thimerosal can rescue the effects of SEMG1 on motility, implying that internal stores of calcium are not depleted. Additionally, SEMG1 treatment of spermatozoa decreases the intracellular pH, and motility can be rescued by ammonium chloride. The results of this study demonstrate that EPPIN controls sperm motility in the ejaculate by binding SEMG1, resulting in the loss of calcium, most likely through a disturbance of internal pH and an inhibition of uptake mechanisms. However, the exact steps through which the EPPIN-SEMG1 complex exerts its effect on internal calcium levels are unknown. Anti-EPPIN antibodies can substitute for SEMG1, and, therefore, small-molecular weight compounds that mimic anti-EPPIN binding should be able to substitute for SEMG1, providing the basis for a nonantibody, nonhormonal male contraceptive.

Semen serves as a vehicle for HIV and promotes sexual transmission of the virus, which accounts for the majority of new HIV cases. The major component of semen is the coagulum, a viscous structure composed predominantly of spermatozoa and semenogelin proteins. Due to the activity of the semen protease PSA, the coagulum is liquefied and semenogelins are cleaved into smaller fragments. Here, we report that a subset of these semenogelin fragments form amyloid fibrils that greatly enhance HIV infection. Like SEVI, another amyloid fibril previously identified in semen, the semenogelin fibrils exhibit a cationic surface and enhance HIV virion attachment and entry. Whereas semen samples from healthy individuals greatly enhance HIV infection, semenogelin-deficient semen samples from patients with ejaculatory duct obstruction are completely deficient in enhancing activity. Semen thus harbors distinct amyloidogenic peptides derived from different precursor proteins that commonly enhance HIV infection and likely contribute to HIV transmission., (Copyright © 2011 Elsevier Inc. All rights reserved.)

PDC-109, the major bovine seminal plasma protein, binds to sperm plasma membrane and modulates capacitation in the presence of heparin. In view of this, the PDC-109/heparin interaction has been investigated employing various biophysical approaches. Isothermal titration calorimetric studies yielded the association constant and changes in enthalpy and entropy for the interaction at 25 °C (pH 7.4) as 1.92 (±0.2) × 10(5) M(-1), 18.6 (±1.6) kcal M(-1), and 86.5 (±5.1) cal M(-1) K(-1), respectively, whereas differential scanning calorimetric studies indicated that heparin binding results in a significant increase in the thermal stability of PDC-109. The affinity decreases with increase in pH and ionic strength, consistent with the involvement of electrostatic forces in this interaction. Circular dichroism spectroscopic studies indicated that PDC-109 retains its conformational features even up to 70-75 °C in the presence of heparin, whereas the native protein unfolds at about 55 °C. Atomic force microscopic studies demonstrated that large oligomeric structures are formed upon binding of PDC-109 to heparin, indicating an increase in the local density of the protein, which may be relevant to the ability of heparin to potentiate PDC-109 induced sperm capacitation.

The Laboratories for Reproductive Biology at the University of North Carolina at Chapel Hill began collaboration with Human Genome Sciences (Rockville, Maryland) to sequence a human epididymal library and identify epididymal-specific genes. Among the first clones obtained from Human Genome Sciences was a clone for EPPIN (official symbol, SPINLW1). Our laboratory has described EPPIN (epididymal protease inhibitor) as a novel gene on human chromosome 20q12-13.2 that encodes a cysteine-rich protein containing both Kunitz-type and WAP-type 4-disulfide core consensus sequences that characterize it as a protease inhibitor. EPPIN expresses 3 mRNA splice variants that encode 2 protein isoforms found in the testis and epididymis. Of the 2 isoforms, 1 is secreted and 1 lacks a secretory signal piece. EPPIN is predominantly a dimer, although multiples often exist, and in its native form, EPPIN is found on the sperm surface complexed with lactotransferrin and clusterin. During ejaculation, semenogelin from the seminal vesicles is bound to the EPPIN protein complex, initiating a series of events that define EPPIN's function: modulating prostate-specific antigen (PSA) activity, providing antimicrobial protection, and binding semenogelin, thereby inhibiting sperm motility. As PSA hydrolyzes semenogelin in the ejaculate coagulum, spermatozoa gain progressive motility. Using immunization as a tool to study antigen function, we demonstrated that EPPIN is essential for fertility because immunization of male monkeys with recombinant EPPIN results in complete, but reversible, contraception. To exploit our understanding of EPPIN's function, we have developed a high-throughput screen to look for compounds that inhibit EPPIN-semenogelin interaction and mimic anti-EPPIN, inhibiting sperm motility. These compounds are now being developed into a nonhormonal male contraceptive.

Our laboratory has characterized EPPIN [epididymal protease inhibitor; SPINLW1] as a novel gene on human chromosome 20q12-13.2, which encodes a cysteine-rich protein of 133 amino acids with a calculated molecular mass of 15.283 kDa, containing both Kunitz-type and WAP (whey acidic protein)-type four-disulfide core consensus sequences. Eppin is secreted by Sertoli cells in the testis and epididymal epithelial cells; it is predominantly a dimer, although multimers often exist, and in its native form eppin is found on the human sperm surface complexed with LTF (lactotransferrin) and clusterin. During ejaculation SEMG (semenogelin) from the seminal vesicles binds to the eppin protein complex, initiating a series of events that define eppin's function. Eppin's functions include (i) modulating PSA (prostate-specific antigen) enzyme activity, (ii) providing antimicrobial protection and (iii) binding SEMG thereby inhibiting sperm motility. As PSA hydrolyses SEMG in the ejaculate coagulum, spermatozoa gain progressive motility. We have demonstrated that eppin is essential for fertility because immunization of male monkeys with recombinant eppin results in complete, but reversible, contraception. To exploit our understanding of eppin's function, we are developing compounds that inhibit eppin-SEMG interaction and mimic anti-eppin, inhibiting sperm motility. These compounds should have potential as a male contraceptive.

Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.

Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress.

Background: Little is known about the role of semenogelins, seminal plasma proteins that play critical roles in semen clotting and subsequent liquefaction in the presence of zinc and prostate-specific antigen, in human malignancies., Methods: We investigated the expression of semenogelins in four human prostate cancer lines by RT-PCR and Western blotting as well as in 70 radical prostatectomy specimens by immunohistochemistry. Effects of semenogelin overexpression on prostate cancer cell proliferation were also assessed., Results: mRNA/protein signals for semenogelins I (SgI) and II (SgII) were detected only in androgen-sensitive LNCaP cells cultured with zinc. Transfection of SgI/SgII increased/decreased cell growth of androgen receptor (AR)-positive/semenogelin-negative CWR22Rv1 in the presence of zinc, whereas it showed marginal effects in AR-negative/semenogelin-negative PC-3 and DU145. Immunohistochemical studies showed that SgI and SgII stain positively in 55 (79%) and 31 (44%) cancer tissues, respectively, which was significantly higher than in corresponding benign tissues [SgI-positive in 13 (19%) cases (P < 0.0001) and SgII-positive in 15 (21%) cases (P = 0.0066)]. Among the histopathological parameters available for our patient cohort, there was an inverse association only between Gleason score (GS) and SgII expression (GS ≤ 7 vs. GS ≥ 8: P = 0.0150; GS7 vs. GS ≥ 8: P = 0.0111). Kaplan-Meier and log-rank tests further revealed that patients with SgI-positive/SgII-negative tumor have the highest risk for biochemical recurrence (P = 0.0242)., Conclusions: These results suggest the involvement of semenogelins in prostate cancer and their prognostic values in predicting cancer progression after radical prostatectomy. Additional functional analyses of semenogelins are necessary to determine their biological significance in prostate cancer., (Copyright © 2010 Wiley-Liss, Inc.)

The major protein of bovine seminal plasma, PDC-109 binds to choline phospholipids present on the sperm plasma membrane upon ejaculation and plays a crucial role in the subsequent events leading to fertilization. PDC-109 also shares significant similarities with small heat shock proteins and exhibits chaperone-like activity (CLA). Although the polydisperse nature of this protein has been shown to be important for its CLA, knowledge of other factors responsible for such an activity is scarce. Since surface exposure of hydrophobic residues is known to be an important factor which modulates the CLA of chaperone proteins, in the present study we have probed the surface hydrophobicity of PDC-109 using bisANS and ANS. Further, effect of phospholipids on the structure and chaperone-like activity of PDC-109 was studied. Presence of DMPC was found to increase the CLA of PDC-109 significantly, which could be due to the considerable exposure of hydrophobic regions on the lipid-protein recombinants, which can interact productively with the nonnative structures of target proteins, resulting in their protection. However, inclusion of DMPG instead of DMPC did not significantly alter the CLA of PDC-109, which could be due to the lower specificity of PDC-109 for DMPG as compared to DMPC. Cholesterol incorporation into DMPC membranes led to a decrease in the CLA of PDC-109-lipid recombinants, which could be attributed to reduced accessibility of hydrophobic surfaces to the substrate protein(s). These results underscore the relevance of phospholipid binding and hydrophobicity to the chaperone-like activity of PDC-109.

The objective of the present study was to investigate the effect of Single-Layer Centrifugation (SLC) on boar spermatozoa, namely the effect of removal of seminal plasma proteins and cholesterol from the surface of spermatozoa. The presence of porcine seminal plasma proteins I and II (PSP-I/PSP-II) before and after SLC was studied using immunofluorescence, whereas the removal of cholesterol was shown qualitatively by thin-layer chromatography (TLC). Finally, the integrity of the sperm plasma membrane was observed by electron microscopy. It was shown that the seminal plasma proteins PSP-I and -II were removed from spermatozoa during SLC but could be restored by adding seminal plasma to the SLC-selected sperm samples. Some cholesterol was also lost from the spermatozoa during SLC but the plasma membrane itself appeared to be morphologically intact. Further studies are underway to examine the relevance of these findings to boar sperm cryopreservation and sperm fertility.

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Approximately 1 in 10 couples is infertile. No definite cause can be found in about 25% of those cases. Studies have indicated that seminal vesicle secretion functions as an optimizer of fertilization. The Zn(2+) binding protein semenogelin I (SgI) represents a major fraction of the proteins present in seminal vesicle fluid, and it serves as a structural component of the coagulum that is formed after ejaculation. Cleavage of SgI by prostate-specific antigen results in liquefaction of the coagulum. Fragmented SgI has antibacterial effects and inhibits spermatozoa mobility. SgI has also been found complexed to eppin on spermatozoa, and this complex has been suggested to be of importance for fertility. Here, we used flow cytometry and surface plasmon resonance to study SgI regarding its association with spermatozoa and the interaction dependency on Zn(2+). The concentration of Zn(2+) in seminal plasma is approximately 100 times higher than in blood plasma, and the metal ion is known to change the structure of SgI. We found that SgI binds to spermatozoa in a concentration-dependent and saturable manner. In solution, SgI bound to spermatozoa in a non-Zn(2+)-dependent way, whereas immobilized SgI interacts with spermatozoa only in the presence of Zn(2+). It indicates that SgI must exhibit a specific structure or free flexibility to be able to interact with that ligand. Our results indicate that the association of SgI to spermatozoa is conformation dependent and specific. These findings could constitute a basis for the development of a male contraceptive.

Seminal plasma proteins of the fibronectin type II (Fn2) family modulate mammalian spermatogenesis by triggering the release of the lipids phosphatidylcholine and cholesterol from sperm cells. Whereas the specific interaction of these proteins with phosphatidylcholine is well-understood, their selectivity for cholesterol is unknown. To characterize the interaction between the bovine Fn2 protein PDC-109 and cholesterol, we have investigated the effect of PDC-109 on the dynamics of fluorescent cholesterol analogues in lipid vesicles by time-resolved fluorescence anisotropy. The data show that PDC-109 decreases the rotational mobility of cholesterol within the membrane and that the extent of this impact depends on the cholesterol structure, indicating a specific influence of PDC-109 on cholesterol. We propose that the cholesterol recognition/interaction amino acid consensus (CRAC) regions of PDC-109 are involved in the interaction with cholesterol.

Background: Semenogelin (Sg), the main protein of human semen coagulum, prevents sperm capacitation. The objective of this study was to examine the role of Sg and its mechanism of action., Methods and Results: Sg blocked sperm capacitation triggered by various stimuli, via inhibition of superoxide anion (O(2)*-; luminescence assay) and nitric oxide (NO*; tested using diaminofluorescein) generation. Triton-soluble and -insoluble sperm fractions contained Sg and Sg peptides (immunoblotting), the level of which decreased with initiation of capacitation. This drop was prevented by superoxide dismutase and NO* synthase inhibitor and was reproduced by addition of O(2)*- and NO*. Zinc (Zn(2+)) blocked and a zinc chelator (TPEN) promoted the decline in Sg levels. There was a decreased labelling of Sg on the head in capacitating spermatozoa with the two fixation techniques tested (immunocytochemistry). Reactive oxygen species (ROS) (O(2)*- and NO*) caused, these changes, and zinc prevented them. Spermatozoa quickly internalized Sg upon incubation and Sg was then rapidly degraded in a zinc-inhibitable manner., Conclusions: Sg blocked capacitation mainly via inhibition of ROS generation. Spermatozoa appeared permeable to Sg and processed Sg in a zinc-inhibitable fashion. ROS themselves could promote sperm disposal of Sg which maybe one of the mechanisms that allows initiation of capacitation.

The major protein of bovine seminal plasma, PDC-109, binds to choline phospholipids on the sperm plasma membrane and induces the efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. The high abundance, polydisperse nature and reversibility of thermal unfolding of PDC-109 suggest significant similarities to chaperone-like proteins such as spectrin, alpha-crystallin, and alpha-synuclein. In the present study, biochemical and biophysical approaches were employed to investigate the chaperone-like activity of PDC-109. The effect of various stress factors such as high temperature, chemical denaturant (urea), and acidic pH on target proteins such as lactate dehydrogenase, alcohol dehydrogenase, and insulin were studied in both the presence and absence of PDC-109. The results obtained indicate that PDC-109 exhibits chaperone-like activity, as evidenced by its ability to suppress the nonspecific aggregation of target proteins and direct them into productive folding. Atomic force microscopic studies demonstrate that PDC-109 effectively prevents the fibrillation of insulin, which is of considerable significance since amyloidogenesis has been reported to be a serious problem during sperm maturation in certain species. Binding of phosphorylcholine or high ionic strength in the medium inhibited the chaperone-like activity of PDC-109, suggesting that most likely the aggregation state of the protein is important for the chaperone function. These observations show that PDC-109 functions as a molecular chaperone in vitro, suggesting that it may assist the proper folding of proteins involved in the bovine sperm capacitation pathway. To the best of our knowledge, this is the first study reporting chaperone-like activity of a seminal plasma protein.

Bovine BSP5 belongs to the Binder of SPerm (BSP) family. BSP5 plays a role in the bovine sperm capacitation by promoting cholesterol and phospholipid efflux. The variable N-terminal part in the BSP proteins is the uncharacterized region with no known function. Full-length, N-terminal part, and individual fibronectin type II domains of bovine BSP5 were cloned, expressed and purified from Escherichia coli. His-S tagged N-terminal part showed large variation in migration on SDS-PAGE in comparison to other constructs. Using mass spectrometry it was demonstrated that the His-S-N-terminal part has the expected molecular mass (13 kDa). The recombinant N-terminal part was sensitive to E. coli endogenous proteases during purification. Denaturing purification involving boiling lysis of cells was carried out, as the protein was thermostable. The His-S-N-terminal part lacked structure as determined by CD analysis. Bioinformatics analyses confirmed that the N-terminal part of bovine BSP5 is intrinsically disordered. In addition, bioinformatics analysis indicated that rabbit BSP and multiple forms of BSP proteins of bovine and equine species possess partially or completely disordered N-terminus. The conservation of disorder at the N-terminus in BSP members belonging to different species suggests a role in biological process such as sperm capacitation and/or sperm-egg interactions., (2010 Elsevier Inc. All rights reserved.)

PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2) repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs), a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD) simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M(-1), estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.

Seminal plasma (SP) is a complex fluid which exerts biological actions in the female reproductive tract. In pigs, SP elicits endometrial inflammation and consequent immune changes after mating. This study tested whether heparin-binding spermadhesins (HBPs) and the heterodimer of porcine sperm adhesions I and II (PSP-I/PSP-II) in SP recruit different lymphocyte subsets (CD2(+), CD4(+) and CD8(+) T cells) or polymorphonuclear leukocytes (PMNs) to the superficial endometrium or luminal epithelium and lumen, respectively, of oestrous sows. In Experiment 1, endometrial biopsies were taken between 2 and 120 min after infusion of uterine horns with HBPs, PSP-I/PSP-II or saline and evaluated by immunohistochemistry or histology. In Experiment 2, the uterus of oestrous sows was infused with PSP-I/PSP-II or saline to assess PMN numbers in the uterine lumen 3h later. PSP-I/PSP-II elicited CD2+ T cell recruitment from 10 min, and CD8(+) T cells from 60 min after infusion, while HBPs increased CD4(+) T cell recruitment by 120 min. PSP-I/PSP-II but not HBPs induced PMN migration to the surface epithelium by 10 min. PMN numbers were elevated 5-fold by 30 min and 7-fold from 60 min, with PMNs detectable in the lumen from 30 min after infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls at 3h after infusion. These data show that PSP-I/PSP-II heterodimer in seminal plasma has a predominant role in triggering the recruitment of uterine PMNs and T cells after mating, initiating a cascade of transient and long-lasting immunological events., (2009 Elsevier Ireland Ltd. All rights reserved.)

Seminal plasma motility inhibitors (SPMIs) are proteinase-resistant fragments of semenogelin I and II (Sgs), which are the major proteins of semen coagulum. SPMIs inhibit the motility of spermatozoa, and Sgs are thought to be natural regulators of human sperm function. The mechanism underlying sperm motility regulation and its association with defective motility in infertile men remain unclear. The purpose of this study was to investigate the association between SPMIs and spermatozoa in infertile men with asthenozoospermia. Fifty-four semen samples from 37 asthenozoospermic patients and 17 samples from 9 normal healthy subjects were analyzed. Spermatozoa, washed by Percoll density gradients, were immunostained with anti-SPMI antibody and subjected to flow cytometric analysis. The proportion of spermatozoa labeled with the antibody and the average intensity of fluorescence labeling per spermatozoa were analyzed in relation to the parameters used for semen analysis. A significant negative correlation was found between sperm motility and the proportion (R = -0.68) and intensity (R = -0.38) of labeling. These results suggest that SPMIs remain on the sperm surface after liquefaction. This might account for some disorders of sperm motility observed in infertile men with asthenozoospermia., (Copyright © 2010 S. Karger AG, Basel.)

The microenvironment and accessibility of the tryptophan residues in domain B of PDC-109 (PDC-109/B) in the native state and upon ligand binding have been investigated by fluorescence quenching, time-resolved fluorescence and red-edge excitation shift (REES) studies. The increase in the intrinsic fluorescence emission intensity of PDC-109/B upon binding to lysophosphatidylcholine (Lyso-PC) micelles and dimyristoylphosphatidylcholine (DMPC) membranes was considerably less as compared to that observed with the whole PDC-109 protein. The degree of quenching achieved by different quenchers with PDC-109/B bound to Lyso-PC and DMPC membranes was significantly higher as compared to the full PDC-109 protein, indicating that membrane binding afforded considerably lesser protection to the tryptophan residues of domain B as compared to those in the full PDC-109 protein. Finally, changes in red-edge excitation shift (REES) seen with PDC-109/B upon binding to DMPC membranes and Lyso-PC micelles were smaller that the corresponding changes in the REES values observed for the full PDC-109. These results, taken together suggest that intact PDC-109 penetrates deeper into the hydrophobic parts of the membrane as compared to domain B alone, which could be the reason for the inability of PDC-109/B to induce cholesterol efflux, despite its ability to recognize choline phospholipids at the membrane surface.

Semenogelin I and II (Sgs) are the major component of human semen coagulum. The protein is rapidly cleaved after ejaculation by a prostate-specific antigen, resulting in liquefaction of the semen coagulum and the progressive release of motile spermatozoa. Sgs inhibit human sperm motility; however, there is currently no information on its effect on the sperm membrane. This study investigated the role of Sgs on human sperm motility through regulation of membrane potential and membrane permeability. Fresh semen samples were obtained from normozoospermic volunteers, and studies were conducted using motile cells selected using the swim-up method. Sgs changed the characteristics of sperm motion from circular to straightforward as evaluated by a computer-assisted motility analyzer, and all parameters were decreased more than 2.5 mg/mL. The results demonstrate that Sgs treatment immediately hyperpolarized the membrane potential of swim-up-selected sperm, changed the membrane structure, and time-dependently increased membrane permeability, as determined through flow cytometric analysis. The biphasic effects of Sgs were time- and dose-dependent and partially reversible. In addition, a monoclonal antibody against Sgs showed positive binding to cell membrane proteins in fixed cells, observed with confocal fluorescence microscopy. These results demonstrate that Sgs modifies the membrane structure, indirectly inhibiting motility, and provides suggestions for a therapy for male infertility through selection of a functional sperm population using Sgs.

Epididymal protease inhibitor (eppin [official symbol, SPINLW1]) is of interest as a male contraceptive target because of its specificity and location on the human sperm surface. We have examined the effect of anti-eppin antibodies from infertile male monkeys and the effect of recombinant human semenogelin on human sperm motility. Anti-eppin antibodies significantly decreased the progressive motility of human spermatozoa as measured by decreased total distance traveled, decreased straight-line distance, and decreased velocity. Anti-eppin treatment of spermatozoa significantly increased the amount of cAMP present in nonprogressive spermatozoa; however, approximately 25% of antibody-treated spermatozoa could be rescued by the addition of cAMP-acetoxymethyl ester, indicating that anti-eppin-treated spermatozoa have a compromised ability to utilize cAMP. Addition of recombinant human semenogelin has a concentration-dependent inhibitory effect on progressive motility (increased tortuosity and decreased velocity). We tested the hypothesis that anti-eppin antibodies bound to eppin would subsequently block semenogelin binding to eppin. Anti-eppin antibodies from infertile monkeys inhibited eppin from binding to semenogelin. Addition of affinity-purified antibodies made to the dominant C-terminal epitope of eppin had an inhibitory effect on progressive motility (increased tortuosity, decreased velocity, and straight distance). Our results suggest that the eppin-semenogelin binding site is critical for the removal of semenogelin in vivo during semen liquefaction and for the initiation of progressive motility. We conclude that the eppin-semenogelin binding site on the surface of human spermatozoa is an ideal target for a nonsteroidal male contraceptive.

Mammalian sperm undergo a series of maturation steps before acquiring fertilization competence. Our previous work demonstrated the importance of binder of sperm (BSP) proteins in bovine sperm capacitation. Recent studies identified a BSP-homologous DNA sequence in the human genome (BSPH1) and mRNA expression in the epididymis. The aim of this study was to develop an efficient method to express and purify recombinant human BSPH1. BSPH1 accumulates in inclusion bodies when expressed with an N-terminal hexahistidine tag in BL21 (DE3) Escherichia coli cells. Similar to other BSP proteins, BSPH1 contains two fibronectin type-II (Fn2) domains, each consisting of two disulfide bonds. Therefore, when expressed in Origami B (DE3)pLysS cells, a strain favouring disulfide bond formation, an improvement in soluble protein yield was observed. However, protein was aggregated, which complicated subsequent purification steps. Expression of glutathione-S-transferase-tagged BSPH1 in both cell types also led to accumulation in inclusion bodies. Finally, successful production of soluble and active protein was achieved when BSPH1 was expressed as a His(6)-thioredoxin-tagged protein. Recombinant protein bound phosphatidylcholine liposomes, low-density lipoproteins and human sperm, therefore displayed binding activities common to all BSP-family proteins, which may indicate similar biological function(s). This approach was also successful in producing the murine orthologue of BSPH1 in the soluble and active form. Thus, fusion to thioredoxin and expression in Origami B (DE3)pLysS cells may constitute a strategy applicable to all BSP-family proteins, and possibly to other proteins containing Fn2 domains. This work is important to elucidate the role of BSPH1 in human sperm functions and fertility.

The male reproductive tract of ungulates contains two protein families bearing tandemly arranged fibronectin II (Fn2) modules; one (small Fn2 proteins) bears two modules (e.g. BSP-A1/2), the other (long Fn2 proteins) bears four (e.g. epididymal sperm-binding protein 1 (ELSPBP1)). While it is well known that small Fn2 proteins are present in bull semen, nothing is known about long Fn2 proteins. In the present study, the presence of ELSPBP1 proteins in the bull epididymis and their association with maturing spermatozoa were investigated using a specific antibody against canine ELSPBP1. Analysis of western blots showed ELSPBP1 to be present in the caput, corpus and cauda regions of the epididymis. The protein, which bound phosphorylcholine (PC) strongly, appeared to associate with the spermatozoa during maturation because it was absent from caput spermatozoa but present on cauda spermatozoa. Immunocytochemistry of cauda spermatozoa showed the protein to be bound to the post-acrosomal and midpiece regions. ELSPBP1 could not be detected on freshly ejaculated spermatozoa but was revealed after a capacitating treatment. Our previous studies have shown differences between bovine caput and cauda spermatozoa in terms of their ability to control cell volume. Because of the close homology of BSP-A1/2 PC binding regions with Fn2 regions in ELSPBP1, BSP-A1/2 was used as a model to investigate the effect of a PC-binding Fn2 protein on cell volume control. While the protein had no effect on cauda spermatozoa, it caused caput spermatozoa to swell more in response to hypotonic stress, similarly to untreated cauda spermatozoa.

Background Aims: Chronic lymphocytic leukemia (CLL) is an indolent disease. It is currently recommended that patients with CLL stages 0 and I follow a watchful waiting strategy. These patients are, therefore, a suitable group for testing immunotherapeutic approaches to avoid problems of immunosuppression as a result of disease progression and chemotherapy. In this study, we investigated the expression of SEMG-1 in early CLL to determine the suitability of SEMG-1 as a target for further development of tumor vaccines for early CLL., Methods: A combination of reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunocytochemistry was used to evaluate the expression of SEMG-1 in early CLL. The results were correlated with Zap 70 expression. Recombinant SEMG-1 protein was used in an enzyme-linked immunosorbent assay (ELISA) to determine the presence of SEMG-1 antibodies (Ab) in serum from these patients., Results: The SEMG-1 gene was expressed in 19/41 (46%) patients with early CLL. Gene expression was associated with protein synthesis in CLL cells. Protein expression, however, was heterogeneous within individual patients. Only transcripts encoding the SEMG-1(50) variant and not SEMG-1(43) were detected. SEMG-1(50) was expressed irrespective of the Zap 70 status. High-titer SEMG-1 IgG but not IgM Ab were detected in some of these patients, suggesting that SEMG-1-reactive immune responses are intact within the immune repertoire of early CLL patients., Conclusions: SEMG-1 is expressed in nearly half of patients with early CLL and may be a target for further investigations into its use for immunotherapy of early CLL.

A 75-kDa protein secreted from mouse coagulating gland was purified to homogeneity by a series of isolation steps including ion exchange chromatography on a DEAE-Sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. It was identified to be Type IV transglutaminase (TG(4)), based on the establishment of N-terminal sequences by automated Edman degradation together with partial sequences by MS analysis. Its cross-linking activity was tested on the reduced sample of mouse seminal secretion which contained seven major monomer proteins tentatively designated as SVS I-VII. The enzyme was able to cross-link any of SVS I-III but failed to cross-link the other SVS proteins with a M(r) value less than 14 kDa. SVS I and SVS III showed comparable substrate activity, but were much weaker than SVS II during the TG(4) catalysis.

Proteins involved in reproductive fitness have evolved unusually rapidly across diverse groups of organisms. These reproductive proteins show unusually high rates of amino acid substitutions, suggesting that the proteins have been subject to positive selection. We sought to identify seminal fluid proteins experiencing adaptive evolution because such proteins are often involved in sperm competition, host immunity to pathogens, and manipulation of female reproductive physiology and behavior. We performed an evolutionary screen of the mouse prostate transcriptome for genes with elevated evolutionary rates between mouse and rat. We observed that secreted rodent prostate proteins evolve approximately twice as fast as nonsecreted proteins, remarkably similar to findings in the primate prostate and in the Drosophila male accessory gland. Our screen led us to identify and characterize a group of seminal vesicle secretion (Svs) proteins and to show that the gene Svs7 is evolving very rapidly, with many amino acid sites under positive selection. Another gene in this group, Svs5, showed evidence of branch-specific selection in the rat. We also found that Svs7 is under selection in primates and, by using three-dimensional models, demonstrated that the same regions have been under selection in both groups. Svs7 has been identified as mouse caltrin, a protein involved in sperm capacitation, the process responsible for the timing of changes in sperm activity and behavior, following ejaculation. We propose that the most likely explanation of the adaptive evolution of Svs7 that we have observed in rodents and primates stems from an important function in sperm competition.

We have previously shown that seminal vesicle protein IV (SV-IV) and its 1-70 N-terminal fragment have anti-inflammatory activity and modulate anti-thrombin III (AT) activity. Moreover, mass spectrometry analysis of purified SV-IV has shown that the protein was found to be highly heterogeneous and 14% of the total SV-IV molecules are truncated forms, of particular interest the 1-16, 1-17, and 1-18 peptides. In this work we report experimental data which demonstrate that the 1-16 peptide (P1-16) possesses a marked effect on the AT activity by preventing the formation of the thrombin-AT complex. We found that the formation of thrombin-AT complex is markedly decreased in the presence of P1-16 used at equimolar concentration with thrombin as evaluated with SDS-PAGE. We also monitored the conformational changes of thrombin in the presence of different P1-16 concentrations, and calculated the K(d) of thrombin/P1-16 system by circular dichroism technique. The probable interaction sites of P1-16 with thrombin have been also evaluated by molecular graphics and computational analyses. These results have potential implications in the treatment of sterility and thrombotic diseases.

Aim: To study the molecular mechanism of epididymal protease inhibitor (Eppin) modulating the process of prostate specific antigen (PSA) digesting semenogelin (Sg)., Methods: Human Sg cDNA (nucleotides 82-849) and Eppin cDNA (nucleotides 70-723) were generated by polymerase chain reaction (PCR) and cloned into pET-100D/TOPO. Recombinant Eppin and Sg (rEppin and rSg) were produced by BL21 (DE3). The association of Eppin with Sg was studied by far-western immunoblot and radioautography. In vitro the digestion of rSg by PSA in the presence or absence of rEppin was studied. The effect of anti-Q20E (N-terminal) and C-terminal of Eppin on Eppin-Sg binding was monitored., Results: Eppin binds Sg on the surface of human spermatozoa with the C-terminal of Eppin (amino acids 75-133). rSg was digested with PSA and many low molecular weight fragments were produced. When rEppin is bound to rSg, then digested by PSA, incomplete digestion and a 15-kDa fragment results. Antibody binding to the N-terminal of rEppin did not affect rSg digestion. Addition of antibodies to the C-terminal of rEppin inhibited the modulating effect of rEppin., Conclusion: Eppin protects a 15-kDa fragment of rSg from hydrolysis by PSA., ((c) 2008, Asian Journal of Andrology, SIMM and SJTU. All rights reserved.)

The epithelial localization and expression of the spermadhesin PSP-I and PSP-II subunits were determined in the testis, ductus epididymes (caput, corpus and cauda), seminal vesicles and bulbourethral glands of mature boars, using immunohistochemical, western blotting and RT-PCR methods. Immunohistochemistry showed positive labelling for PSP-I and PSP-II antibodies in the epithelium of seminal vesicles in all males tested. Positive immunolabelling, but with variable intensity, was also present in the epididymal epithelium (caput, corpus and cauda), although varying largely among segments and boars. Immunoreactivity was nearly or completely absent in the seminiferous epithelium and the bulbourethral gland, although SDS-PAGE and western blotting revealed the presence of PSP-I and PSP-II immunoreactive bands in all the tissue extracts, including the testis and the bulbourethral gland. mRNA amplification by RT-PCR using primers specific for PSP-I and PSP-II showed a trend similar to that observed for western blotting, i.e. intensity variation between tissues (even between segments of the same epididymis) and among boars. Our results indicate that the seminal vesicles are the main source of PSP-I and PSP-II spermadhesins, although epididymal segments, testis and the bulbourethral gland also participate in the expression of both proteins.

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.

Liquefaction of human semen involves proteolytic degradation of the seminal coagulum and release of motile spermatozoa. Several members of human kallikrein-related peptidases (KLKs) have been implicated in semen liquefaction, functioning through highly regulated proteolytic cascades. Among these, KLK3 (also known as prostate-specific antigen) is the main executor enzyme responsible for processing of the primary components of semen coagulum, semenogelins I and II. We have recently identified KLK14 as a potential activator of KLK3 and other KLKs. This study aims to elucidate the cascade-mediated role of KLK14 ex vivo. KLK14 expression was significantly lower (p = 0.0252) in individuals with clinically delayed liquefaction. Concordantly, KLK14 expression was significantly (p = 0.0478) lower in asthenospermic cases. Specific inhibition of KLK14 activity by the synthetic inhibitor ACT(G9) resulted in a significant delay in semen liquefaction, a drop in the "early" (30 min postejaculation) "chymotrypsin-like" and KLK1 activity, and an increase in the "late" (90 min postejaculation) chymotrypsin-like activity. Conversely, the addition of recombinant active KLK14 facilitated the liquefaction process, augmented the early chymotrypsin-like activity, and lowered late chymotrypsin-like activity. Given that the observed chymotrypsin-like activity was almost completely attributed to KLK3 activity, KLK3 seems to be regulated bidirectionally. Accordingly, a higher level of KLK3 fragmentation was observed in KLK14-induced coagula, suggesting an inactivation mechanism via internal cleavage. Finally, semenogelins I and II were directly cleaved by KLK14. Semenogelins were also able to reverse KLK14 inhibition by Zn2+, providing a novel regulatory mechanism for KLK14 activity. Our results show that KLK14 exerts a significant and dose-dependent effect in the process of semen liquefaction.

The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.