Forensic Spermatozoa Detection.

Semen is crucial evidence for some sex crimes, with its sole confirmation being sperm detection. The success of sperm detection is dependent on all levels of preanalytic and analytic procedures. Specimen collection must be performed by well-trained and competent forensic physicians as well as forensic nurses, with preservation done properly before laboratory transfer. Laboratory procedures should consider archival sperm identification, by visualization, with adequate amounts separated from other cells to obtain male DNA profiles. Differential extraction is robust and accepted as the forensic standard but is time consuming and may result in male DNA loss. Thus, alternative methods and microdevices have been developed. Challenges in sperm isolation from vaginal or buccal epithelium mixes and discrimination in multiperpetrator cases have been overcome by single-cell profiling; however, problems inherent in identical twin discrimination and azoospermia have yet to be solved. Epigenetics and future molecular biomarkers may hold the key; therefore, all laboratory processes must consider DNA and RNA protection. Long-term specimen preservation should be done when possible in light of future confirmatory tests.

Evidence for mouse sulfhydryl oxidase-assisted cross-linking of major seminal vesicle proteins.

Copulatory plug formation in animals is a general phenomenon by which competition is reduced among rival males. In mouse, the copulatory plug formation results from the coagulation of highly viscous seminal vesicle secretion (SVS) that is rich in proteins, such as dimers of SVS I, SVS I + II + III, and SVS II. These high-molecular-weight complexes (HMWCs) are also reported to be the bulk of proteins in the copulatory plug of the female mouse following copulation. In addition, mouse SVS contributes to the existence of sulfhydryl oxidase (Sox), which mediates the disulfide bond formation between cysteine residues. In this study, flavin adenine dinucleotide (FAD)-dependent Sox was purified from mouse SVS using ion exchange and high-performance liquid chromatography. The purified enzyme was identified to be Sox, based on western blot analysis with Sox antiserum and its capability of oxidizing dithiothreitol as substrate. The pH optima and thermal stability of the enzyme were determined. Among the metal ions tested, zinc showed an inhibitory effect on Sox activity. A prosthetic group of the enzyme was identified as FAD. The Km and Vmax of the enzyme was also determined. In addition to purification and biochemical characterization of seminal vesicle Sox, the major breakthrough of this study was proving its cross-linking activity among SVS I-III monomers to form HMWCs in SVS.

Detection of prostate specific antigen and semenogelin in specimens from female rape victims.

The presence of semen is generally accepted as evidence in sexual assault cases prosecution. Detection of sperm is confirmation of semen; however, sperm cannot always be detected. Prostate specific antigen (PSA) and semenogelin (Sg) are used as semen biomarkers. We compared the detection rate and persistence of sperm, PSA and Sg over a range of time intervals from the time of assault to specimen collection. The results show that sperm had the longest persistence and highest detection rate. The detection rate of the Sg test was significantly better than that of the PSA test overall, whether the sperm test was negative or positive. In conclusion, the detection of sperm should be the first test executed: if sperm is not detected, the Sg test is more suitable than the PSA test and could be used up to 72 h after assault.

Purification of binder of sperm protein 1 (BSP1) and its effects on bovine in vitro embryo development after fertilization with ejaculated and epididymal sperm.

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

Optimization of spermatozoa detection using immunofluorescent staining and laser micro-dissection.

The present study evaluated the use of an immunofluorescence-based assay for the microscopic detection of human spermatozoa, following which the fluorescence-labelled spermatozoa could be excised with a laser micro-dissection system. The Sperm Hy-Liter™ PI kit was able to detect spermatozoa from as little as 20nL of semen. No interference or non-specificity were observed when the kit was used on semen mixed with various body fluids such as blood and urine, as well as when semen was spiked onto different types of fabric. Good results could also be obtained with rectal samples which contain auto-fluorescent fecal materials through the use of dual FITC/PI filters. We also developed a method for concurrent testing of two protein biomarkers of semen (semenogelin and prostate-specific antigen) and detection of spermatozoa. This approach would maximize the evidential value from a single piece of sexual assault exhibit. The results also showed that staining by Sperm Hy-Liter™ PI does not interfere with DNA recovery, facilitating the generation of clear male DNA profiles from dissected spermatozoa, thereby making profile interpretation less complex. In summary, Sperm Hy-Liter™ PI staining was demonstrated to be sensitive, robust and specific.

Murine Binder of SPerm homolog 2 (BSPH2): the black sheep of the BSP superfamily.

Proteins of the Binder of SPerm superfamily are known to bind choline phospholipids on sperm membrane and promote sperm capacitation. The current study focuses on the biochemical and functional characterization of the murine Binder of SPerm homolog 2 (BSPH2). A recombinant protein (rec-BSPH2) was expressed in Escherichia coli Rosetta-gami B (DE3)pLysS cells using pET32a vector. It was purified by immobilized metal ion affinity chromatography and refolded on column using a decreasing urea gradient. Rec-BSPH2 was found to share some binding characteristics with other BSP proteins, such as binding to gelatin, heparin, and epididymal sperm. Rec-BSPH2 as well as murine recombinant BSPH1 were found to have different immunofluorescence patterns when bound to uncapacitated versus capacitated sperm, indicating a rearrangement of these proteins on sperm surface during or following capacitation. Surprisingly, rec-BSPH2 was unable to bind phosphorylcholine liposomes or promote sperm capacitation. It is the first time that such results are reported for proteins of the BSP family. The results indicate that murine BSPH1 and BSPH2 might not have redundant functions, as is the case with bovine BSPs. This study could lead to a better understanding of the role of BSP proteins in sperm functions and the existence of redundant BSP proteins in the reproductive tract.

A comparison of ABAcard(®) p30 and RSID™-Semen test kits for forensic semen identification.

The screening and confirmatory tests available to a forensic laboratory allow evidence to be examined for the presence of bodily fluids. With the majority of evidence being submitted involving sexual assaults, it is important to have confirmatory tests for the identification of semen that are straightforward, quick, and reliable. The purpose of this study was to compare two commonly used semen identification kits utilized by forensic laboratories: ABAcard(®) p30 and Rapid Stain Identification of Human Semen (RSID™-Semen). These kits were assessed with aged semen stains, fresh and frozen post-vasectomy semen, post-coital samples collected on different substrates, post-vasectomy semen mixed with blood, saliva, and urine, a series of swabs collected at increasing time intervals after sexual intercourse, and multiple non-semen samples. The test kits were compared on the basis of sensitivity, specificity, and the cost and time effectiveness of each protocol. Overall, both semen identification tests performed well in the studies. Both kits proved specificity for identifying semen, however the ABAcard(®) p30 test surpassed the RSID™-Semen test in sensitivity, cost per test, and simplified test protocol.

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.

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.

Recombinant expression and affinity purification of a novel epididymal human sperm-binding protein, BSPH1.

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.

[Isolation of low-molecular-mass antibacterial mixtures from human seminal plasma].

OBJECTIVE: To isolate low-molecular-mass antibacterial mixtures from healthy human seminal plasma. METHODS: Semen was obtained by masturbation after at least three days of abstinence from healthy donors. Semen samples were allowed to liquefy at room temperature and then centrifuged at 10,000 r/min for 10 min to separate spermatozoa from seminal plasma. High sensitive antimicrobial activity was measured with radial diffusion assay. Antibacterial activity toward E. coli (ATCC25922) was monitored for each purification steps. The mixture of seminal plasma samples was applied to a SP-Sepharose column. Fractions which showed strong bactericidal activities, were combined and lyophilized. The lyophilized components were dissolved with Milli-Q water and applied to AKTA Superdex 75 column. Peak II of the Superdex 75 column, which showed antibacterial activity and represented the low-molecular-weight cationic fractions of the seminal plasma, was collected and lyophilized. Finally, peak II of the Superdex 75 column was applied to reverse phase HPLC C18 column. Fractions which showed strong antibacterial activity, were lyophilized and store at -20 degrees C. The molecular weight of the low-molecule-mass antibacterial mixtures was determined by Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. RESULTS: The low-molecular-mass mixtures with obviously higher antibacterial activity, which were termed HSLAMs(Human semen low-molecular-mass antibacterial mixtures), were isolated from the healthy human seminal plasma. Based on the mass spectrometry results, some molecules of RP-HPLC peaks were confirmed to be the semenogelin I derived peptides. CONCLUSION: The low-molecule-mass antibacterial mixtures may play an important role in males innate immunity. Semenogelin I derived peptides may be one of the sources of the low-molecule-mass antimicrobial mixtures in human seminal plasma.

Heparin-binding proteins of human seminal plasma: purification and characterization.

Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity-chromatography on Heparin-Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield.

Identification of novel semenogelin I-derived antimicrobial peptide from liquefied human seminal plasma.

Semenogelin I (SgI) is one of the most abundant proteins in human seminal plasma. SgI plays a key role in sperm coagulation and spermatozoon immobilization. In addition, SgI and/or its proteolytic fragments are involved in regulating spermatozoon motility, capacitation and inhibin-like activity. However, little is known about the antibacterial activity of SgI-derived peptides. By a combination of ion-exchange, gel filtration and high-performance liquid chromatography, peptides from liquefied human seminal plasma from 40 healthy donors were isolated and characterized. N-terminal amino-acid sequencing and fast atom bombardment mass spectrometry revealed that four isolated peptides were SgI-derived, namely SgI-29 (85-113), SgI-46 (85-130), SgI-47 (85-131) and SgI-52 (85-136). Interestingly, SgI-29, SgI-46 and SgI-47 are newly identified SgI-derived peptides. Antimicrobial activity assay results indicated that synthesized SgI-29 had strong antibacterial activity toward various bacterial strains. Our results indicate that SgI can be digested into small fragments like newly identified SgI-29, SgI-46 and SgI-47 and may have diversified functions.

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.

Purification, preliminary characterization and immunohistochemical localization of POSVP21 in the sand rat (Psammomys obesus) seminal vesicles.

The sand rat Psammomys obesus is a mammalian species with male seasonal reproduction. Previously Gernigon et al. (1994) [Gernigon T, Berger M, Lecher P. Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a saharian rodent (Psammomys obesus). J Endocrinol 1994;142:37-46.] reported that the seminal vesicles of the adult sand rat contained a major secretory protein band (M.W. 21000) regulated by testosterone. This protein is synthesized in large amounts when the androgen level increases, and accounts for over 22% of soluble proteins from homogenate of seminal vesicles during the breeding season. When analyzed by NepHGE the protein band of 21kDa appeared to be composed of at least 3 visible spots with pHi values varying from 4 to 7. Its partially internal sequence was identified and exhibited five peptides. Polyclonal antibodies against POSVP21 were obtained in rabbits. They were also used to study immunohistochemical antigen localization by the means of an avidin-biotin peroxidase procedure. Observation showed that it is localized in the cytoplasm of epithelial cells and in secretory products in the lumen. The whole RNA of seminal vesicles was translated in a cell-free system derived from rabbit reticulocyte lysate and [35S]-methionine. Two major bands of 14.4 and 21kDa were visualized by means of denaturing gel electrophoresis. SDS-PAGE from medium incubation of seminal vesicle tissue with [35S]-methionine revealed one band with an apparent molecular weight of 21kDa. The results obtained indicate that seminal vesicle epithelium is the site of POSVP21 synthesis and the comparison of the partial amino acid composition of the internal sequence, indicated that POSVP21 constitute a family of most unusual proteins.

Physiological roles of semenogelin I and zinc in sperm motility and semen coagulation on ejaculation in humans.

At ejaculation, human sperm are considered to be mechanically trapped and become immotile in the semen coagulum by binding to semenogelins (Sgs) from the seminal vesicle and zinc ions from the prostate. However, the physiological combined roles of the protein and heavy metal on sperm motility are unknown. Here, we have first demonstrated that Sg I alone, which does not form the semen coagulum without zinc, is an inhibitor of the motility of intact human sperm at physiological concentration. On the other hand, zinc ions alone had no effect on sperm motility, but confer recovery of sperm motility that has been inhibited by Sg I at a concentration equal to or less than 1 mg/ml. These observations suggest that the roles played by Sg I and zinc on sperm motility are not mechanical but physiological. Quartz crystal microbalance analysis suggests that the sperm extract first bind to Sg I and then zinc ions which subsequently increase the protein accumulation, suggesting that Sgs inhibit sperm motility by directly binding to the sperm surface. Further accumulation of Sg I mediated by zinc ions may entrap the quiescent sperm at semen ejaculation.

Localization and expression of spermadhesin PSP-I/PSP-II subunits in the reproductive organs of the boar.

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.

Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir.

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.

Characterization of an eppin protein complex from human semen and spermatozoa.

Eppin (SPINLW1; serine peptidase inhibitor-like with Kunitz and WAP domains 1 (eppin); epididymal protease inhibitor) coats the surface of human ejaculate spermatozoa and originates from Sertoli and epididymal epithelial cells. In this study, we have isolated native eppin from ejaculate supernatants (seminal plasma) and washed ejaculate spermatozoa using column chromatography and two-dimensional SDS-PAGE, and identified by mass spectrometry and Western blots an eppin protein complex (EPC) containing lactotransferrin (LTF; also known as lactoferrin), clusterin (CLU), and semenogelin (SEMG1). To confirm the association of eppin with LTF, CLU, and SEMG1, antibodies to CLU and LTF were used to immunoprecipitate CLU and LTF from human sperm lysates. In both cases identical results were obtained, namely, the immunoprecipitate of the EPC. Additionally, we localized eppin, LTF, and CLU in human Sertoli cells and on human testicular and ejaculate spermatozoa, implying that the EPC is present on spermatozoa from the time they leave the seminiferous tubule. On ejaculate spermatozoa eppin, LTF, and CLU colocalize on the tail. The identification of the EPC components suggests that LTF, CLU, and/or eppin receptors may function as sperm plasma membrane receptors for the EPC, implicating the complex as a central player in a network of protein-protein interactions on the human sperm surface. The EPC may provide a surface network with microbicidal properties that protects spermatozoa as well as regulates the sperm's transition to a motile, capacitated sperm.

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.