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.

Improving body fluid identification in forensic trace evidence-construction of an immunochromatographic test array to rapidly detect up to five body fluids simultaneously.

Body fluid identification is a substantial part of forensic trace analyses. The correct determination of the origin of a biological stain may give valuable information regarding the circumstances of a crime. A simple way to detect a body fluid in a stain is the use of immunochromatographic strip tests. They are easy to use, user-independent, quick, and cheap. Currently, however, it is only possible to analyze one body fluid at a time, requiring the analyst to make previous, possibly subjective, assumptions on the body fluid at hand. Also, identification of mixed body fluids requires the use of several tests, which results in additional sample and time consumption. To combine a simple approach with the possibility to simultaneously detect several body fluids, we constructed a combined immunochromatographic strip test array based on commercially available tests. The array rapidly detects up to five body fluids with a single analysis, and allowing for subsequent DNA extraction from the same material. With this test it was possible to identify the components of a mixture, the test was easily incorporated into standard laboratory work, and its sensitivity and specificity were shown to be comparable to those of conventional strip tests.

Evaluation of Human Semenogelin Membrane Strip Test for Species Cross-reactivity in Dogs.

Semenogelins are proteins originating in the seminal vesicle and are useful markers for the presumptive identification of human semen. Detection of semenogelin can be done with a commercially available membrane test. In this study, a commercially available membrane test for human semenogelin proteins was used to assess for cross-reactivity in dog bodily fluids to allow for the potential utilization for detection of human semen in dog bodily fluids. The authors analyzed canine semen and other bodily fluids, including urine, saliva, vaginal secretions, fecal material, and blood. They also examined the distribution of human semenogelin I transcripts in the canine testis, prostate, and several bodily fluids by reverse transcription polymerase chain reaction. No cross-reactivity was observed in the canine bodily fluids tested except for a single rectal swab, which was negative on a second test. Further testing should be done to validate the use of this kit for screening samples from dogs suspected to have been victims of sexual abuse.

Central T cell tolerance: Identification of tissue-restricted autoantigens in the thymus HLA-DR peptidome.

Promiscuous gene expression (pGE) of tissue-restricted self-antigens (TRA) in medullary thymic epithelial cells (mTECs) is in part driven by the Autoimmune Regulator gene (AIRE) and essential for self-tolerance. The link between AIRE functional mutations and multi-organ autoimmunity in human and mouse supports the role of pGE. Deep sequencing of the transcriptome revealed that mouse mTECs potentially transcribe an unprecedented range of >90% of all genes. Yet, it remains unclear to which extent these low-level transcripts are actually translated into proteins, processed and presented by thymic APCs to induce tolerance. To address this, we analyzed the HLA-DR-associated thymus peptidome. Within a large panel of peptides from abundant proteins, two TRA peptides were identified: prostate-specific semenogelin-1 (an autoantigen in autoimmune chronic prostatitis/chronic pelvic pain syndrome) and central nervous system-specific contactin-2 (an autoantigen in multiple sclerosis). Thymus expression of both genes was restricted to mTECs. SEMG1 expression was confined to mature HLA-DR(hi) mTECs of male and female donors and was AIRE-dependent, whereas CNTN2 was apparently AIRE-independent and was expressed by both populations of mTECs. Our findings establish a link between pGE, MHC-II peptide presentation and autoimmunity for bona fide human TRAs.

Murine binder of sperm protein homolog 1: a new player in HDL-induced capacitation.

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

Developmental validation of RSID™-Semen: a lateral flow immunochromatographic strip test for the forensic detection of human semen.

Tests for the identification of semen commonly involve the microscopic visualization of spermatozoa or assays for the presence of seminal markers such as acid phosphatase (AP) or prostate-specific antigen (PSA). Here, we describe the rapid stain identification kit for the identification of semen (RSID™-Semen), a lateral flow immunochromatographic strip test that uses two antihuman semenogelin monoclonal antibodies to detect the presence of semenogelin. The RSID™-Semen strip is specific for human semen, detecting <2.5 nL of semen, and does not cross-react with other human or nonhuman tissues tested. RSID™-Semen is more sensitive with certain forensic evidence samples containing mixtures of vaginal secretions and semen than either of the commercially available PSA-based forensic semen detection tests or tests that measure AP activity that were tested in parallel. The RSID™-Semen kit also allows sampling a fraction of a questioned stain while retaining the majority of the sample for further processing through short tandem repeat analysis.

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization.

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer's disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.

An aberrant prostate antigen-specific immune response causes prostatitis in mice and is associated with chronic prostatitis in humans.

Chronic prostatitis is a common disease of unclear etiology and has no specific treatment. Mice deficient in the expression of the autoimmune regulator (Aire) gene, which are defective in thymic expression of self antigens and central tolerance, develop spontaneous prostatitis. In this study, we found that Aire-deficient mice developed spontaneous B and T cell immune responses to a prostate autoantigen, seminal vesicle secretory protein 2 (SVS2), which we believe to be novel. We show that thymic expression of this self antigen was Aire dependent. Moreover, prostatitis was induced in WT mice through immunization with SVS2, demonstrating that immunity to SVS2 was sufficient to induce prostatitis. The clinical relevance of this antigen was highlighted by our observation that patients with chronic prostatitis possessed specific autoantibodies against the human SVS2-like seminal vesicle protein semenogelin. These results provide direct evidence that spontaneous chronic prostatitis is an autoimmune disease and is regulated by both central and peripheral tolerance. Moreover, SVS2 and semenogelin are among the relevant autoantigens in mice and humans, respectively.

Cationic polypeptides contribute to the anti-HIV-1 activity of human seminal plasma.

Mucosal surfaces of the reproductive tract as well as their secretions have important roles in preventing sexual transmission of HIV-1. In the current study, the majority of the intrinsic anti-HIV-1 activity of human seminal plasma (SP) was determined to reside in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multistep chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP.

SEMG-1 expression in early stage chronic lymphocytic leukemia.

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.

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.

Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity.

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.

Essential function of transglutaminase and clotting protein in shrimp immunity.

Blood coagulation is a conserved defense mechanism among invertebrates and it has been well studied in horseshoe crab and freshwater crayfish but is ill defined in shrimp. Transglutaminase (TGase) and clotting protein (CP) are molecules involved in the blood clotting system of shrimp. Here, we demonstrate in vivo the functional involvement of TGase and CP in the shrimp blood coagulation system using RNA interference. Expression of TGase mRNA was inhibited in gills, heart, hemocyte, hepatopancreas, intestine and lymphoid organ while the CP gene was suppressed only in gills and heart tissues on day-1 post-injection with 1 microg and 10 microg of TGase- and CP-dsRNA, respectively. However, at day-7 post-injection, systemic gene silencing was observed for both genes and dosages as shown by mRNA expression. We also show the efficiency of dsRNA silencing the protein expression as well as inhibited blood coagulation. Such silencing at the transcription, translation and phenotypic level is the first to be documented in the shrimp system. Challenge test with white spot virus and Vibrio penaecida revealed that TGase and CP are critical molecules for the immune function of shrimp against bacterial and viral infection.

Eppin: a molecular strategy for male contraception.

New male contraceptives, both hormonal and non-hormonal, have many obstacles to overcome before they reach the market as a product. For hormonal contraceptives the long-term efficacy of oligospermia in a large population of unselected men remains to be determined. For nonhormonal contraception target selection remains a primary goal. Immunocontraception, which showed great promise for many years, has recently lost its appeal. Nevertheless, immunocontraception can be utilised as a strategy, particularly in primates, to discern the function of target molecules in the male. As an example, we discuss Eppin, an epididymal protease inhibitor that coats the surface of human spermatozoa. Because Eppin is predicted to be a serine protease inhibitor with chymotrypsin-like specificity and binds semenogelin, the natural substrate of PSA (prostate specific antigen, a serine protease), we investigated whether Eppin would modulate PSA activity and the hydrolysis of semenogelin. Additionally, because antibodies to Eppin provide contraception in male monkeys, we investigated whether antibodies to Eppin would inhibit the PSA hydrolysis of semenogelin. Eppin is a specific inhibitor of PSA activity that requires leucine 87, Eppin's P1 reactive site. Although Eppin modulates the hydrolysis of semenogelin by PSA, antibodies to Eppin do not inhibit PSA activity.

Seminal vesicle protein IV and its derived active peptides: a possible physiological role in seminal clotting.

Seminal vesicle protein 4 (SV-IV) is a highly flexible molecule that in aqueous solution behaves as a concentration-dependent self-associating system in which the degree of association (monomer <--> dimer <--> trimer equilibrium) seems to be related to its biological activities. This review reports the functional role of SV-IV in seminal clotting exerted through the modulation of inflammation, hemostasis, and sperm protection against the damage induced by immunological or reactive oxygen species during the long journey of spermatozoa in the female genital tract.

Applicability of Nanotrap Sg as a semen detection kit before male-specific DNA profiling in sexual assaults.

A commercially available semen detection kit, Nanotrap Sg, which employs a one-step detection test based on immunochromatographic assay for the semenogelin protein, was evaluated for profiling male-specific DNA in sexual assault casework samples. While semen diluted with phosphate-buffered saline held and kept at 4 degrees C for 1 week showed a relatively strong signal intensity with Nanotrap Sg, the signal intensity was decreased by dilution after storage at 4 degrees C or freezing and thawing repeated more than three times. The reproducibility of Nanotrap Sg was tested on a total of 174 sexual assault casework samples from three forensic laboratories using intra- and interassay and no variation was observed in the semenogelin (Sg) signal. The positive signal ratio was 12.6% higher for prostate-specific antigen immunochromatographic membrane tests than Nanotrap Sg. Although spermatozoa were not confirmed in 61 (35%) out of 174 samples, Sg-positive signals could be detected from 41 (67%) of the 61 samples. Female genetic profiles could be observed in 95% of the samples, which tested negative for Sg on the Nanotrap Sg test, but no male genetic profiles could be observed. These results suggest that Nanotrap Sg can positively identify samples containing male DNA even in the absence of detectable intact spermatozoa. Further, Sg-positive signals identified samples for which male-specific DNA profiling could be performed, even if no sperm could be detected from the sample. The potential of Nanotrap Sg for identifying forensic samples with male-specific DNA was clearly demonstrated.

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.

Involvement of semenogelin-derived peptides in the antibacterial activity of human seminal plasma.

Mechanisms for protecting spermatozoa, and the testes that produce them, from infection are essential, given the importance of these cells and organs for the fertility of the individual and perpetuation of the species. This is borne out by the publication of numerous papers on this subject over the last 50 years. We extended our work and that of others on the anti-infectious defense system of the male genital tract, using a new strategy for the direct identification of antibacterial molecules in human seminal plasma. We subjected a liquefied seminal plasma cationic fraction to reversed-phase HPLC, monitored microbicidal activity by gel overlay and radial diffusion assays, and identified the proteins and/or peptides present in each active fraction by mass spectrometry. In addition to proteins with known potent microbicidal activity--phospholipase A2, lactoferrin, and lysozyme--we also found that peptides produced by cleavage of semenogelin I, the predominant human semen coagulum protein, had high levels of antibacterial activity.

Pattern of gene expression and immune responses to Semenogelin 1 in chronic hematologic malignancies.

Normal testicular-specific proteins are frequently aberrantly expressed by tumor cells. Based on this, we have investigated Semenogelin 1, a major protein of human semen coagulum thought to be highly specific to seminal vesicles, in leukemic cells. Using reverse transcription-polymerase chain reaction, Semenogelin 1 gene was frequently expressed in chronic myeloid leukemia (5 of 8, 62.5%) and chronic lymphocytic leukemia (5 of 12, 41.7%) but rarely in multiple myeloma (2 of 30, 6.7%). The gene was not expressed in bone marrow or peripheral blood from healthy donors. Semenogelin 1 expression is normally confined to the testis, suggesting that it is a novel Cancer-Testis (CT) antigen. Translation of the mRNA to Semenogelin 1 protein was confirmed by Western blot analysis of tumor cell lysates and by immunocytochemistry. The recombinant Semenogelin 1 protein was used with a control Escherichia coli-derived recombinant protein in ELISA and Western blot analysis to show that high titer IgG antibodies against Semenogelin 1 were detected in some patients, suggesting the in vivo immunogenicity of the protein. Immune responses predicted gene expression by the leukemia cells. Semenogelin 1 was also frequently coexpressed with other CT antigens, Sperm protein 17 and SPAN-Xb. These results therefore indicate that Semenogelin 1 is a novel CT antigen capable of inducing B-cell responses in vivo in chronic leukemias.

Semenogelin I and II, the predominant human seminal plasma proteins, are also expressed in non-genital tissues.

Semenogelin I (SgI) and semenogelin II (SgII) are the dominating protein components of the coagulum formed by freshly ejaculated human semen. The primary source of these proteins is the seminal vesicles and, apart from a small production of SgII in epididymis, they have not been detected in other tissues. In this report, we have re-examined the distribution of SgI and SgII transcripts and protein by RT-PCR and immunohistochemistry. Both SgI and SgII transcripts were demonstrated in several tissues, with the strongest signals coming from seminal vesicles, vas deferens, prostate, epididymis and trachea. Transcripts in the gastro-intestinal tract and skeletal muscle almost exclusively encoded SgI, whereas in kidney and testis, SgII transcripts were predominant. By immunohistochemistry, the basal cell layer of the secretory epithelium in prostate, trachea and bronchi was stained by antibodies recognizing both SgI and SgII. This is in contrast to the seminal vesicle and vas deferens, where the luminal cells were stained. The staining of skeletal muscle cells and a few scattered cells in the central nervous system suggests that semenogelin expression is not restricted to epithelial cells.