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.

Semen searching when sperm is absent.

Sexual assault cases have varying factors that may mask semen findings when analysing evidence at the forensic laboratory. Semenogelin (Sg) is a potential marker for the identification of semen even at azoospermy or when few sperm cells are found. The current study examined Sg in normospermic and azoospermic donors as an internal evaluation of sensitivity, specificity and interference. The impact of a historical review of 53 judicial sexual assault cases over a five-year period was also analysed. The use of varying tests was of importance to prioritize certain samples within cases. Semen findings by Sg were then compared to prostate-specific antigen (PSA), phosphatase enzyme (AP) and Y-chromosome presence, the latter being used in an attempt to link semen fluid identification with obtaining a male DNA profile. Test findings were the highest ever registered for Sg (1:400,000), PSA (1:800,000), AP (1:25,000) and sperm cytology (SC) (1:50,000). Our results demonstrated the usefulness of using the Sg marker to avoid a false semen-negative result (6% cases), particularly in cases where sperm was absent or scarce (11% spermatozoa positive cases). Results were expressed in categories according to the set: Sg-PSA-AP. Thus, categories I (full positive, 46%), VI (full negative, 27%) and III (Sg/PSA positive; 11%) were the most frequent and Y-chromosome was obtained in 59%, 12% and 12% ratios, respectively. In conclusion, Sg was recommended for the workflow procedure of semen investigation when sperm absence is expected either from azoospermic/oligospermic or normospermic semen, especially before/after ejaculation.

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.

Mass spectrometry-based proteomics as a tool to identify biological matrices in forensic science.

In forensic casework analysis, identification of the biological matrix and the species of a forensic trace, preferably without loss of DNA, is of major importance. The biological matrices that can be encountered in a forensic context are blood (human or non-human), saliva, semen, vaginal fluid, and to a lesser extent nasal secretions, feces, and urine. All these matrices were applied on swabs and digested with trypsin in order to obtain peptides. These peptides were injected on a mass spectrometer (ESI Q-TOF) resulting in the detection of several biomarkers that were used to build a decision tree for matrix identification. Saliva and blood were characterized by the presence of alpha-amylase 1 and hemoglobin, respectively. In vaginal fluid, cornulin, cornifin, and/or involucrin were found as biomarkers while semenogelin, prostate-specific antigen, and/or acid phosphatase were characteristic proteins for semen. Uromodulin or AMBP protein imply the presence of urine, while plunc protein is present in nasal secretions. Feces could be determined by the presence of immunoglobulins without hemoglobin. The biomarkers for the most frequently encountered biological matrices (saliva, blood, vaginal fluid, and semen) were validated in blind experiments and on real forensic samples. Additionally, by means of this proteomic approach, species identification was possible. This approach has the advantage that the analysis is performed on the first "washing" step of the chelex DNA extraction, a solution which is normally discarded, and that one single test is sufficient to determine the identity and the species of the biological matrix, while the conventional methods require cascade testing. This technique can be considered as a useful additional tool for biological matrix identification in forensic science and holds the promise of further automation.

Levels of semenogelin in human spermatozoa decrease during capacitation: involvement of reactive oxygen species and zinc.

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.

Identification of protamine 1 as a novel cancer-testis antigen in early chronic lymphocytic leukaemia.

Early chronic lymphocytic leukaemia (CLL) is an ideal disease for immunotherapy. We previously showed that SEMG 1 is a cancer-testis (CT) antigen in CLL. In this study, SEMG 1 was applied as the bait in a yeast two-hybrid system of a testicular cDNA library. Seven clones were isolated and Protamine (Prm) 1 was identified as a novel CT antigen in early CLL. PRM1 transcripts were detected in 11/41 (26.8%) patients. Prm 1 protein was also expressed but heterogeneously within individual patients. Of the 11 patients expressing Prm 1, four expressed Zap 70 protein and seven did not. These results, therefore, indicate that Prm 1 could potentially be a suitable target for the design of tumour vaccine for patients with early CLL, including for those with poor risk CLL. High titres of Prm 1 IgG antibodies could be detected in 20 of these 41 CLL patients but not in any of the 20 healthy donors (P = 0.0001), suggesting the presence of Prm 1-reactive immune responses within the immune repertoire of patients with early CLL. Further work is warranted, especially in approaches to upregulate Prm 1 expression, and to determine the role of Prm 1 as an immunotherapeutic target for early CLL.

Proximity Ligation Real-Time PCR: A protein-based confirmatory method for the identification of semen and sperm cells from sexual assault evidence.

The positive identification of seminal fluids in sexual assault crimes is considered crucial evidence to determine whether a sexual act occurred or not. However, current presumptive methods lack specificity and sensitivity. Confirmation of semen by microscopic examination of spermatozoa is laborious, time consuming, and can sometimes lead to negative or inconclusive results. Here we report the use of the Proximity Ligation Real-Time PCR (PLiRT-PCR) assay as an attractive and promising confirmatory method for the identification of semen and sperm proteins using two polyclonal antibodies, Prostate Specific Antigen (PSA) and Sperm-Specific Protein (SP10), respectively. PLiRT-PCR, relies on protein recognition by pairs of proximity probes (antibody-DNA conjugates) that give rise to a ligated DNA strand. The ligated DNA strand is then amplified and detected by qPCR.

Identification and detection of protein markers to differentiate between forensically relevant body fluids.

The identification of body fluids at a crime scene is an important aspect of forensic casework analysis, being a source for investigative leads and contributing to case evidence. Yet, current methods for the forensic identification of body fluids suffer from several limitations, ranging from poor sensitivity and specificity, to sample destruction and interference with subsequent DNA analysis. Moreover, current identification assays target only one body fluid at the time. Besides being inefficient in terms of time, money and sample consumption, poor identification methods can also negatively influence the outcome of a (court) case. In this study, eleven potential protein biomarkers and antibodies were selected and assessed on their suitability for serving as identification markers, as a first step towards the development of a new multiplex protein-based body fluid identification assay relying on antigen-antibody interactions. Performing antibody-based dot blot assays, the specificity of the biomarkers for their target body fluids was evaluated, and biomarker detection was studied in diluted, mixed, aged and simulated casework samples. Hereby, nine out of eleven markers were identified as promising biomarkers to identify blood, semen, saliva, urine and sweat. With the identification of these targets and detection antibodies, a major step forward has been taken towards the development of a highly sensitive and specific, fast and non-labour-intensive protein-based body fluid identification assay, suitable for on-site analysis and able to test for multiple body fluids in a single reaction.

Abnormal Expression of Sg I is Closely Related to Seminal Vesiculitis.

OBJECTIVE: To investigate firstly the relationship between semenogelin I (Sg I) expression and seminal vesiculitis. Seminal vesiculitis is one of the most common diseases in male urogenital system. However, the cause and the pathogenesis of seminal vesiculitis remain unknown. Sg I, mainly synthesized and secreted by seminal vesicle, is abundant in human seminal plasma and has antibacterial activity. MATERIALS AND METHODS: Tissue samples were collected from 15 normal cases and 28 patients with seminal vesiculitis. Reverse transcription-polymerase chain reaction was performed to detect the expression difference of Sg I messenger ribonucleic acid (RNA) between normal seminal vesicle tissues and seminal vesiculitis tissues. Immunohistochemistry was applied to detect the expression difference of Sg I protein between the 2 groups. RESULTS: Reverse transcription-polymerase chain reaction showed the expression of Sg I messenger RNA in seminal vesiculitis tissues to be significantly lower than in normal seminal vesicle tissues. In most cases with seminal vesiculitis (78.6%), the same result was observed upon immunohistochemical analysis at the protein level. CONCLUSION: Abnormal expression of Sg I is closely related to seminal vesiculitis. Low expression of Sg I may play an important role in the occurrence and the development of seminal vesiculitis through weakening the antibacterial activity of seminal vesicle.

Differential expression of bone matrix regulatory proteins in human atherosclerotic plaques.

In the present study, we examined the expression of regulators of bone formation and osteoclastogenesis in human atherosclerosis because accumulating evidence suggests that atherosclerotic calcification shares features with bone calcification. The most striking finding of this study was the constitutive immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein in nondiseased aortas and the absence of bone morphogenetic protein (BMP)-2, BMP-4, osteopontin, and osteonectin in nondiseased aortas and early atherosclerotic lesions. When atherosclerotic plaques demonstrated calcification or bone formation, BMP-2, BMP-4, osteopontin, and osteonectin were upregulated. Interestingly, this upregulation was associated with a sustained immunoreactivity of matrix Gla protein, osteocalcin, and bone sialoprotein. The 2 modulators of osteoclastogenesis (osteoprotegerin [OPG] and its ligand, OPGL) were present in the nondiseased vessel wall and in early atherosclerotic lesions. In advanced calcified lesions, OPG was present in bone structures, whereas OPGL was only present in the extracellular matrix surrounding calcium deposits. The observed expression patterns suggest a tight regulation of the expression of bone matrix regulatory proteins during human atherogenesis. The expression pattern of both OPG and OPGL during atherogenesis might suggest a regulatory role of these proteins not only in osteoclastogenesis but also in atherosclerotic calcification.

Rapid detection of semenogelin by one-step immunochromatographic assay for semen identification.

To identify semen in forensic samples, we developed an analytical system for one-step immunoassay that has been constructed using the concept of immunochromatography and can identify semenogelin (Sg), which originates in the seminal vesicles. The system employed monoclonal antibody (mAb) and polyclonal antibody (pAb) against recombinant Sg-II (63 kDa), which has been synthesized in insect cells using baculovirus. The two antibodies bound with the seminal plasma motility inhibitor (SPMI; 14 kDa) as a final fragment peptide of Sg. The test stick is based on the sandwich technique using the above antibodies. When serial dilutions of seminal plasma were analyzed using this test stick, the intensity of a clear immunoreactive signal peaked at 2000-fold dilution. Thereafter, the signals decreased slowly but still persisted up to 400,000-fold dilution. The Sg antigen was undetectable in saliva, urine, breast milk, serum or vaginal secretions. Also, the test stick shown did not react with animal semen samples, such as those from horses, dogs, swine and bulls. When semen samples, diluted 100,000-fold from 100 men were tested, the Sg antigenic activity was detectable in all samples. In addition, the specificity and sensitivity of the test stick for identification of semen were demonstrated by comparative forensic studies. We conclude that this immunoassay method is a useful confirmatory test for the identification of semen. The immunochromatographic system for forensic testing or research use will become available commercially soon.

Quantification of seminal plasma motility inhibitor/semenogelin in human seminal plasma.

Semenogelin I and II (Sg I and II) are the major components of human semen coagulum. The protein is rapidly cleaved after ejaculation by the chymotrypsin-like protease prostate-specific antigen (PSA), which results in the liquefaction of the semen coagulum and the progressive release of motile spermatozoa. One of the cleavage products of the protein, a 14-kDa protein, is a sperm motility inhibitor (seminal plasma motility inhibitor [SPMI]). We developed a monoclonal antibody (mAb) that is specific to the fragment of Sgs, SPMI, and a sandwich enzyme-linked immunosorbent assay (ELISA) system for the quantification of Sgs using this mAb. Then, we measured SPMI/Sg levels in human seminal plasma from healthy male volunteers (n = 100, aged 18-24 years). The mean level of SPMI/Sg in seminal plasma was 19 +/- 13 mg/mL (range, 4-68 mg/mL). Log-transformed SPMI/Sg levels were negatively correlated with the sperm motility (r = -0.229, P =.0220) and positively correlated with the total protein concentration (r = 0.793, P <.0001). This result supports that SPMI, one of the fragments of Sg, has its inhibitory effect on ejaculated spermatozoa in liquefied semen under physiological conditions.

Mouse seminal vesicle secretory protein of 99 amino acids (MSVSP99): characterization and hormonal and developmental regulation.

Polyclonal antibodies have been generated to investigate the localization, tissue and species distribution, androgen regulation, and ontogeny of a protein secreted by mouse seminal vesicle, designated as MSVSP99 (ie, mouse seminal vesicle secretory protein of 99 amino acids). MSVSP99 is a polymorphic compound with a molecular weight of around 14 kilodaltons and a positive immunoreactivity range of 5.23 to 5.70. Positive immunoreactivity was restricted to the epithelial cells of the seminal vesicle. Western blot analysis showed organ specificity for MSVSP99, which could not be detected in several organs in the mouse. Time course decrease of MSVSP99 after castration closely followed that of its mRNA. In contrast, the length of time required to restore control levels after testosterone treatment was higher for the protein than it was for its mRNA. Whereas the MSVSP99 gene is already active in 10-day-old males, MSVSP99 is first detected at 27 days. Then, we conclude that factors other than the accumulation of the mRNA regulate MSVSP99 expression.

Screening biological stains with qPCR versus lateral flow immunochromatographic test strips: a quantitative comparison using analytical figures of merit.

Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit-including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)-were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 µL of saliva for the RSID™ Saliva cards, 0.03 µL of saliva for Quantifiler(®) Duo, and 0.001 µL of semen for Quantifiler(®) Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection.

Seminal hyperviscosity is not associated with semenogelin degradation or sperm deoxyribonucleic acid damage: a prospective study of infertile couples.

OBJECTIVE: To investigate the association between seminal hyperviscosity, the extent of semenogelin degradation, and sperm DNA integrity (DNA fragmentation index [DFI] and high DNA stainability [HDS]) in semen from infertile couples. DESIGN: Prospective study. SETTING: University-affiliated fertility center. PATIENT(S): Twenty-four consecutive infertile couples with moderate or high seminal viscosity (hyperviscosity group) and 25 consecutive infertile couples with normal semen viscosity (control group) undergoing standard IVF. INTERVENTION(S): Semen volume and seminal hyperviscosity, sperm concentration, motility, and morphology, level of semenogelin degradation (by immunoblotting), and sperm chromatin damage (by sperm chromatin structure assay and expressed as %DFI and %HDS) were evaluated. MAIN OUTCOME MEASURES(S): Sperm %DFI and %HDS in the hyperviscosity group and the control group and the relationship between the extent of semenogelin degradation and seminal viscosity. RESULT(S): Semen volume in couples with moderate and high seminal viscosity was significantly lower as compared with the control group. In addition, total motility and normal morphology were significantly lower in the couples with high seminal viscosity as compared with the control group; however, there were no significant differences in sperm %DFI and %HDS between the hyperviscosity group and the control group. In addition, there was no relationship between the extent of semenogelin degradation and seminal viscosity. CONCLUSION(S): Our data suggest that seminal hyperviscosity (a posttesticular factor) is not an important cause of sperm DNA damage. Moreover, seminal hyperviscosity is not related to the degree of semenogelin degradation.

Top-down mass spectrometry reveals new sequence variants of the major bovine seminal plasma protein PDC-109.

The major protein of bovine seminal plasma, PDC-109, is a 109-residue polypeptide that exists as a polydisperse aggregate under native conditions. The oligomeric state of this aggregate varies with ionic strength and the presence of lipids. Binding of PDC-109 to choline phospholipids on the sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. In this study, Fourier transform ion cyclotron resonance mass spectrometry was used to analyze PDC-109 purified from bovine seminal plasma. In addition to the previously known PDC-109 variants, four new sequence variants were identified by top-down mass spectrometry. For example, a protein variant containing point mutations P10L and G14R was identified along with another form having a 14-residue truncation in the N-terminal region. Two other minor variants could also be identified from the affinity-purified PDC-109. These results demonstrate that PDC-109 is naturally produced as a mixture of several protein forms, most of which have not been detected in previous studies. Native mass spectrometry revealed that PDC-109 is exclusively monomeric at low protein concentrations, suggesting that the protein oligomers are weakly bound and can easily be disrupted. Ligand binding to PDC-109 was also investigated, and it was observed that two molecules of O-phosphorylcholine bind to each PDC-109 monomer, consistent with previous reports.

Evaluation of sperm proteins in infertile men: a proteomic approach.

In this study, the sperm protein profile was compared between fertile and infertile men using 2-dimensional gel electrophoresis, liquid chromatography mass spectrometer analysis, and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry. Four unique proteins, semenogelin II precursor, prolactin-induced protein, clusterin isoform 1, and prostate-specific antigen isoform 1 preproprotein, were predominantly present in the semen of healthy men; however, semenogelin II precursor and clusterin isoform 1 were not seen in the semen of infertile men, suggesting unique differences in the spermatozoa protein profiles of fertile and infertile men.

Vaginal swab specimen processing methods influence performance of rapid semen detection tests: a cautionary tale.

BACKGROUND: Detection of semen biomarkers in vaginal fluid can be used to assess women's recent exposure to semen. Quantitative tests for detection of prostate-specific antigen (PSA) perform well, but are expensive and require specialized equipment. We assessed two rapid immunochromatographic strip tests for identification of semen in vaginal swabs. STUDY DESIGN: We tested 581 vaginal swabs collected from 492 women. Vaginal secretions were eluted into saline, and PSA was measured using the quantitative IMx (Abbott Laboratories, Abbott Park, IL, USA) assay. Specimens were also tested using the ABAcard p30 test (Abacus Diagnostics, West Hills, CA, USA) for detection of PSA and RSID-Semen test (Independent Forensics, Hillside, IL, USA) for detection of semenogelin (Sg). RESULTS: Vaginal swab extraction using saline was compatible with direct assessment of vaginal swab eluates using ABAcard for PSA detection, but not for Sg detection using RSID. The rapid PSA test detected 91% of specimens containing semen compared to 74% by the rapid Sg test. CONCLUSION: Investigators are urged to optimize vaginal swab specimen preparation methods for performance of RSID or other tests to detect semen components other than PSA. Previously described methods for PSA testing are not uniformly applicable to other tests.

Identification of PDC-109-like protein(s) in buffalo seminal plasma.

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

Semenogelins in the human retina: Differences in distribution and content between AMD and normal donor tissues.

The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retinas from AMD donor eyes. Semenogelins I (SgI) and II (SgII) are the major structural protein components of semen coagulum, but have been recently found in non-genital tissues as well. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with a specific antibody. The presence of SgI was analyzed in retina and RPE total lysates and SgI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. SgI is expressed in the normal human retina and in the retina of AMD donor eyes, where localization was detected in the photoreceptors and in a few ganglion cells. We find the distribution of SgI in the AMD retinas substantially lower than observed in normal retina. SgI localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc.