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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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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.

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.

Over the past 60 years, egg yolk (EY) has been routinely used in both liquid semen extenders and those used to cryopreserve sperm. However, the mechanism by which EY protects sperm during liquid storage or from freezing damage is unknown. Bovine seminal plasma contains a family of proteins designated BSP-A1/-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). These proteins are secretory products of seminal vesicles that are acquired by sperm at ejaculation, modifying the sperm membrane by inducing cholesterol efflux. Because cholesterol efflux is time and concentration dependent, continuous exposure to seminal plasma (SP) that contains BSP proteins may be detrimental to the sperm membrane, which may adversely affect the ability of sperm to be preserved. In this article, we show that the BSP proteins bind to the low-density fraction (LDF), a lipoprotein component of the EY extender. The binding is rapid, specific, saturable, and stable even after freeze-thawing of semen. Furthermore, LDF has a very high capacity for BSP protein binding. The binding of BSP proteins to LDF may prevent their detrimental effect on sperm membrane, and this may be crucial for sperm storage. Thus, we propose that the sequestration of BSP proteins of SP by LDF may represent the major mechanism of sperm protection by EY.

Semenogelin plays an important role in sperm clotting and is degraded into smaller fragments by prostate-specific antigen (PSA) during clot liquefaction. Semenogelin and its fragments inhibit sperm motility in vitro. We studied the expression of semenogelin I mRNA and its localization in various tissues of the male genital tract. We also studied semenogelin concentrations with respect to sperm parameters and the outcome of in vitro fertilization. Semenogelin protein was detected by immunohistochemical staining and semenogelin I mRNA was detected by Northern blot analysis in the seminal vesicles and ampullary part of the vas deferens, whereas specimens from the prostate, epididymis, testis, and the female genital tract were negative. Using monoclonal antibodies against semenogelin, an immunofluorometric assay was developed to measure semenogelin levels in seminal plasma and to evaluate possible correlations with sperm parameters and fertilization in vitro. No correlation was found between the semenogelin concentration and the volume of the ejaculate, sperm concentration, sperm motility, or in vitro fertilization rate. Semenogelin levels were positively correlated with the total protein concentration in seminal plasma, and there was an inverse correlation between the concentration of semenogelin and that of PSA. The levels of semenogelin appear to bear no relationship to the in vitro fertilization capacity of the spermatozoa.

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.

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.