Characterization of proteins from boar prostate

PROBLEM: Most components of seminal plasma are secreted by accessory sexual glands: seminal vesicle, prostate gland and bulbourethral gland. The portion of proteins secreted by prostate gland differs in various species. Characterization of boar prostate proteins is the subject of this communication. METHODS OF STUDY: Proteins of boar prostate gland were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H-) and heparin-binding (H+) fractions. The H- and H+ fractions were subjected to reverse phase high performance liquid chromatography (RP HPLC) and their elution profiles were compared with those of the H- and H+ fractions of boar seminal plasma. The isolated proteins were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunodetection, N-terminal amino acid sequencing and mass spectrometry (MALDI). RESULTS: The following proteins of boar prostate secretion were identified: beta-microseminoprotein, serotransferrin, serum albumin, myoglobin and PSP I and PSP II spermadhesins. CONCLUSION: Presented results demonstrate composition of the main proteins of boar prostate secretion. Beta-Microseminoprotein was found to be a major protein of prostate secretion. PSP I and PSP II, major proteins of the H- fraction of boar seminal plasma, were found in boar prostate secretion in lower amounts. The major proteins of the H+ fraction of boar seminal plasma (AQN, AWN) were not detected in prostate secretion.     

Characterization of boar spermadhesins by monoclonal and polyclonal antibodies and their role in binding to oocytes.

PROBLEM: The role of Ala-Trp-Asn (AWN) and Ala-Gln-Asn (AQN) families of spermadhesive sperm proteins in fertilization. METHOD OF STUDY: The preparation and characterization of polyclonal antibodies against AWN and AQN spermadhesins and one monoclonal antibody (MAb), designated Bo.5, against AWN spermadhesin. The use of biochemical and immunocytochemical methods for characterization of spermadhesins on the sperm membrane of boar spermatozoa and in the cryostat sections of boar reproductive organs. RESULTS: Polyclonal anti-AWN and anti-AQN antibodies specifically reacted with AWN and AQN proteins, respectively. MAb Bo.5 detected the 17-, 16-, and 14-kDa protein members of AWN subfamily. The monoclonal, as well as the polyclonal, AWN antibodies remarkably decreased the sperm binding to the egg surface in an in vitro sperm zona pellucida binding assay. CONCLUSIONS: Presented results demonstrate that polyclonal antibodies and MAb Bo.5 against spermadhesins specifically recognize the membrane-associated antigens and inhibit the binding of sperm to oocytes. Reduced binding of sperm to oocytes, due to the antibodies, indicates the role of these spermadhesins in sperm-egg primary binding.     

Inhibition of the antibody responses to rat blood transfusion antigens in mice by boar seminal immunosuppressive fraction

PROBLEM: Immunosuppressive fraction of boar seminal vesicle fluid (ISF) was tested to muffle primary and secondary antibody responses to xenotranfusions. Contemporaneously, heparin non-binding fraction of seminal plasma (H- fraction), presumed to be identical to ISF, was used to support the results. METHOD: To study their similarity, ISF and H- fraction were analyzed by high-performance liquid chromatography and the separated proteins by N-terminal sequencing. In sera of mice treated with ISF or H- fraction, the productions of antibodies against rat erythrocytes and blood serum were evaluated by enzyme-linked immunosorbent assay (ELISA). The productions of IgM, IgA, and IgG subclasses were followed by sandwich ELISA. RESULTS: ISF and H- fraction were proved to be equal complexes of porcine seminal plasma (PSP) proteins PSP I and PSP II. Both inhibited antibody responses to rat erythrocytes and serum and the concentrations of IgM, IgG, IgG1 and IgG2 after the first transfusion with a long-lasting effect. Both suppressed the secondary antibody production if applied before the second transfusion. IgA and IgG3 stayed uninfluenced. ISF and H- fraction had an equal immunosuppressive effect. CONCLUSIONS: ISF was characterized biochemically, found to be identical to H- fraction, and determined to be powerful in overcoming unwanted exaggerated antibody responses to xenotransfusion.     

Semen selenium content and sperm mitochondrial volume in human and some animal species

Selenium (Se) and glutathione peroxidase (GSH-Px) were determinedfrom the seminal plasma samples and spermatozoa of human andfour different animal species. The human sperm Se concentrationwas 1.8±0.8 µg/g dry weight, which was about halfof that in the bull. Abnormal sperm morphology and motilitycorrelated with low sperm Se content. The volume of sperm mitochondrialsheath in human, bull and stallion was measured using transmissionelectron microscopy. In these species the sperm Se content washighly correlated with the volume of mitochondria. Among thefive species studied, the seminal plasma level of Se was lowestin human male and stallion, while the highest levels were encounteredin the bull. No correlation was obtained between human semenquality and seminal plasma Se concentration. The seminal plasmaGSH-Px activity was low in man and ram, absent in boar and stallionbut very high in the bull. The amount of structural sperm Seas well as seminal plasma Se and GSH-Px activity appears tobe highly variable in different species     

Prostatic origin of a zinc binding high molecular weight protein complex in human seminal plasma

The profile of the zinc ligand high molecular weight proteins was investigated in the seminal plasma of 55 normozoospermic subjects by size exclusion high performance liquid chromatography (HPLC). The proteins were recovered from Sephadex G-75 gel filtration of seminal plasma in three zinc-containing fractions which were then submitted to HPLC analysis. The results were, that in all the samples, the protein profiles showed two peaks with apparent molecular weight of approximately 660 and approximately 250 kDa. Dialysis experiments revealed that both approximately 660 and approximately 250 kDa proteins were able to uptake zinc against gradient indicating their zinc binding capacity. The HPLC analysis of the whole seminal plasma evidenced only the approximately 660 kDa protein complex as a single well quantifying peak, furthermore a positive correlation between its peak area and the seminal zinc values (P < 0.001) was observed. This suggested a prostatic origin of the approximately 660 kDa protein complex which was then confirmed by the seminal plasma HPLC analysis of a subject with agenesis of the Wolffian ducts. Finally the study demonstrated the presence of two zinc binding proteins, approximately 660 and approximately 250 kDa respectively, in human seminal plasma and the prostatic origin of the approximately 660 kDa.     

Genomic structure and tissue-specific expression of human and mouse genes encoding homologues of the major bovine seminal plasma proteins

Sperm capacitation is a maturation event that takes place in the female reproductive tract and is essential for fertilization. A family of phospholipid-binding proteins present in bovine seminal plasma (BSP proteins) binds the sperm membrane at ejaculation and promotes bovine sperm capacitation. Homologues of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid, suggesting that BSP proteins and their homologues are conserved among mammals. However, there have been no reports on BSP-homologous proteins in mice and humans to date. A search of the mouse and human genomes, using the nucleic acid sequences of BSP proteins, revealed the presence of three BSP-like sequences in the mouse genome, named mouse BSP Homologue 1 (mBSPH1), mBSPH2 and mBSPH3, and one sequence in the human genome (hBSPH1). Mouse epididymal expressed sequence tags corresponding to partial sequences of mBSPH1 and mBSPH2 were identified. The entire complementary DNA (cDNA) sequences of mBSPH1 and mBSPH2 from mouse epididymis and hBSPH1 from human epididymis were obtained by 5'-/3'-rapid amplification of cDNA ends (RACE) and encode predicted proteins containing two tandemly repeated fibronectin type II domains, which is the signature of the BSP family of proteins. Using RT-PCR, it was revealed that mBSPH1, mBSPH2 and hBSPH1 mRNA are expressed only in the epididymis. Expression of mBSPH3 was not detected in any tissue and probably represents a pseudogene. This work shows, for the first time, that BSP homologues are expressed in mouse and human and may be involved in sperm capacitation in these species.     

Immunofluorescence antigen localization on boar sperm plasma membranes: monoclonal antibodies reveal apparent new domains and apparent redistribution of surface antigens during sperm maturation and at ejaculation

Purified boar sperm plasma membranes (PM) and PM proteins were used as antigens to produce 58 monoclonal antibodies against surface antigens. Fluorescence labelling (biotin-avidin-FITC) was used to determine the distribution of antigens in caput and cauda epididymal and in ejaculated spermatozoa with hybridoma supernatants and/or 1:100 diluted ascites fluid after subcloning. Sixteen areas (subdomains) of apparent restricted antigen mobility were identified and significant differences in the localization of most antigens in caput, cauda, and ejaculated PM were recognized. While localization patterns were highly reproducible with a given protocol for sample preparation and immunolabelling, localization patterns were markedly affected by changes in protocols. Fluorescence patterns were affected by the manner in which sperm were labelled (live sperm or sperm labelled at various steps), by washing, and by temperature or by addition of seminal plasma. These results indicate that the dynamic properties of the sperm PM or the surrounding fluids can easily mask or unmask or reconfigure binding sites for highly site-specific monoclonal antibodies and that antigen distribution is probably under-estimated when these labelling techniques are used. Such changes in the accessibility of antigenic sites to monoclonal antibodies limited determining the extent of distribution of a given antigen on epididymal sperm. However, the reproducibility of patterns when a given protocol is used and the large number of antibodies (39/42) displaying marked differences in localization on caput, cauda, and ejaculated PM suggest that changes in the organization of the PM constituents, whether by addition or subtraction of antigen or through configurational changes in proteins, are a major consequence of sperm maturation in the epididymis.     

Capacitation-dependent concentration of lipid rafts in the apical ridge head area of porcine sperm cells

Lipid architecture of the plasma membrane plays an important role in the capacitation process of the sperm cell. During this process, an increase in membrane fluidity takes place, which coincides with a redistribution of cholesterol to the apical region of the head plasma membrane and subsequently an efflux of cholesterol. Cholesterol is also a major player in the formation of lipid rafts or microdomains in the membrane. Lipid rafts favour specific protein-protein interactions by concentrating certain proteins in these microdomains while excluding others. In this study, we investigated the organization of lipid rafts during in vitro capacitation of boar sperm cells. We report on the presence of the lipid raft-specific proteins caveolin-1 and flotillin-1 in sperm cells. Capacitation induced a change in membrane distribution of these proteins. Lipid analysis on detergent-resistant membranes (DRMs) of sperm cells indicated that capacitation induces a lipid raft concentration rather than a disintegration of lipid rafts, because the total amount of lipid in the DRM fraction remained unaltered. Using a proteomic approach, we identified several major DRM proteins, including proteins involved in capacitation-dependent processes and zona pellucida binding. Our data indicate that sperm raft reorganization may facilitate capacitation-specific signalling events and binding to the zona pellucida.     

Cellular distribution and molecular heterogeneity of MAC393 antigen (clusterin, beta-chain) on the surface membrane of bull spermatozoa

The distribution and size of a surface membrane antigen identified by a monoclonal antibody (MAC9393) have been examined in testicular and epididymal bovine sperm preparations. Western blots indicated a substantial decrease in molecular mass of the antigen during epididymal maturation from approximately 87 kDa in the testis to approximately 35 kDa in the cauda epididymidis. This was accompanied by a change in its cellular localization from the neck and whole head to the acrosomal region. N-terminal microsequencing identified MAC393 antigen as the beta-chain of clusterin. A polyclonal antiserum to the alpha-chain of clusterin recognized both testicular and epididymal forms and revealed that the heterodimer was present on the sperm tail as well as the acrosome. These findings are explained by the co-existence of dimeric and monomeric pools of clusterin on spermatozoa. The polyclonal antiserum recognizes both testicular and epididymal forms of the heterodimer and although the monoclonal antibody binds to the testicular heterodimer, it only recognizes the beta-chain monomer of epididymal clusterin. These findings support previous observations made on human spermatozoa that two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the surface membrane and in seminal plasma.      

Molecular interactions of Leishmania promastigote surface protease with human alpha 2-macroglobulin

The interaction of Leishmania promastigote surface protease (PSP) with the plasmatic protease inhibitor alpha 2-macroglobulin (alpha 2M) was investigated. In plasma, solubilized PSP forms covalent complexes only with alpha 2M, at the exclusion of other protease inhibitors. The formation of complexes is accompanied by the proteolytic cleavage of the alpha 2M subunit and by the transition from the 'slow' to the 'fast' form of alpha 2M. The proteolytic activity of solubilized PSP on azocasein is inhibited by alpha 2M. In contrast, we found no evidence for a specific interaction of alpha 2M with the surface of promastigotes and PSP proteolytic activity on intact cells was not inhibited by alpha 2M.     

Frequent structural chromosome aberrations in immotile human sperm exposed to culture media

BACKGROUND: The influence of culture media or centrifugation on chromosomes of immotile human sperm was examined using ICSI into mouse oocytes. METHODS: In experiment 1, immotile and motile human sperm retrieved directly from ejaculates were injected into mouse oocytes. In experiment 2, immotile human sperm were exposed to seminal plasma or one of four kinds of culture media (HEPES-BWW, modified-BWW, modified-human tubal fluid (HTF) and Dulbecco's phosphate-buffered saline) for 1.5-2.5 h at 18 degrees C in air before microinjection. In experiment 3, immotile human sperm were centrifuged along with HEPES-BWW before microinjection. In experiment 4, frozen-thawed immotile human sperm washed with seminal plasma or HEPES-BWW were injected into mouse oocytes. The hybrid oocytes were prepared for chromosome slides at first cleavage metaphase and were then examined cytogenetically. RESULTS: In experiment 1, there was no significant difference in the incidences of structural chromosome aberrations between motile and immotile sperm (4.3% versus 5.8%). In experiment 2, culture media caused more frequent structural chromosome aberrations (14.3-32.6%) in immotile sperm than did seminal plasma (5.4%). In experiment 3, structural chromosome aberrations were found in 48.1% of the centrifuged immotile sperm, and a live/dead sperm viability test intimated that the aberrant sperm were probably dead. In experiment 4, the incidence of structural chromosome aberrations in frozen-thawed immotile sperm was significantly higher in HEPES-BWW (62.2%) than in seminal plasma (17.2%). CONCLUSIONS: The results indicate that immotile sperm do not have significantly more DNA lesions than motile sperm, although DNA of immotile sperm appears to be vulnerable to damage caused by different culture media.     

ABH Blood Group Antigens in Human Semen

ABSTRACT: Human sperm, erythrocytes, and seminal plasma from the blood and semen of 20 men of known ABH, Lewis, and secretor phenotypes were assayed for ABH blood group antigens. A 1:1 correlation was found between the presence of ABH antigens in seminal plasma and on sperm and if the man had a secretor phenotype. Sperm from nonsecretors or from men of dissimilar ABO blood type could adsorb A antigen when incubated with A antigen-containing seminal plasma. The level of ABH antigens in seminal plasma correlated with the level of ABH antigens on the sperm surface. ABH antigens in semen were present only on a minority of spermatozoa as detected by flow cytometry, and the majority of these sperm were not in the swimup fraction. ABH antigens were not present on sperm within the seminiferous tubules of human testicular material. It was hypothesized that ABH antigens found on human sperm were adsorbed from seminal plasma on a minority of the sperm in an ejaculate.     

Secretory leukocyte protease inhibitor (SLP) concentrations in seminal plasma: SLPI restores sperm motility reduced by elastase

In this study, we quantified secretory leukocyte protease inhibitor (SLPI) and elastase in ejaculates from normal donors and infertile patients with or without leukospermia and investigated the effect of SLPI on sperm motility reduced by elastase. Western blot analysis revealed that SLPI protein was detected in the seminal plasma. The SLPI titre in the seminal plasma with leukospermia was lower than that in the seminal plasma without leukospermia and in the seminal plasma of fertile donors. The elastase concentration in the seminal plasma with leukospermia was significantly higher than that in the seminal plasma without leukospermia. A significant correlation between SLPI and elastase concentrations in the seminal plasma (r = 0.36, P< 0.01) was observed. There was a positive correlation between SLPI titre in the seminal plasma and sperm motility (r = 0.51, P < 0.001). SLPI recovered the sperm motility reduced by elastase in a dose-dependent manner. Our results suggest that SLPI is a potential substance to treat infertile patients with leukospermia.      

Sperm motility inhibitor from human seminal plasma: association with semen coagulum

Human seminal plasma contains a sperm motility inhibitor (SPMI)originating from the seminal vesicles as a 52 kDa precursorform that is rapidly degraded by prostatic proteases after ejaculation.In this study, the distribution of SPMI biological activityand antigens was analysed in chemically induced, as well asnaturally occurring, arrest of semen liquefaction. SPMI activitywas detected exclusively in the coagulated semen fraction at2200 ± 560 IU, whereas total seminal plasma proteinsseparated more evenly between soluble and coagulated components(91 ± 19 and 65 ± 18 mg, respectively). An SPMIantiserum recognized different forms of SPMI precursors at 52,38, 35, 33 and 20 kDa in the coagulum while the soluble proteinfraction contained only one major immunoreactive band at 15kDa. High levels of SPMI activity (1500 ± 180 IU/ml)together with high molecular mass forms of SPMI precursor andlow sperm motility (26%) were detected in semen samples thatfailed to liquefy spontaneously at room temperature. Additionof prostatic secretions to the non-liquefying samples causeda decrease of SPMI activity (330 ± 17 IU/ml) and transformedthe SPMI precursor into low molecular mass forms (14–22kDa) with a concomitant increase in sperm motility to 49%. Theresults suggest that SPMI is highly associated with the seminalcoagulum components as very active forms that may adverselyaffect sperm motilrty when not properly processed after ejaculation. protein precursor/proteolytic processing/prostate/semen/sperm motility     

Sperm motility inhibitor from human seminal plasma: association with semen coagulum

Human seminal plasma contains a sperm motility inhibitor (SPMI)originating from the seminal vesicles as a 52 kDa precursor form that is rapidly degraded by prostatic proteasesafter ejaculation. In this study, the distribution of SPMI biologicalactivity and antigens was analysed in chemically induced, aswell as naturally occurring, arrest of semen liquefaction. SPMIactivity was detected exclusively in the coagulated semen fractionat 2200 ± 560 IU, whereas total seminal plasma proteinsseparated more evenly between soluble and coagulated components(91 ± 19 and 65 ± 18 mg, respectively). An SPMIantiserum recognized different forms of SPMI precursors at 52,38, 35, 33 and 20 kDa in the coagulum while the soluble proteinfraction contained only one major immunoreactive band at 15kDa. High levels of SPMI activity (1500 ± 180 IU/ml)together with high molecular mass forms of SPMI precursor andlow sperm motility (26%) were detected in semen samples thatfailed to liquefy spontaneously at room temperature. Additionof prostatic secretions to the non-liquefying samples causeda decrease of SPMI activity (330 ± 17 IU/ml) and transformedthe SPMI precursor into low molecular mass forms (14–22kDa) with a concomitant increase in sperm motility to 49%. Theresults suggest that SPMI is highly associated with the seminalcoagulum components as very active forms that may adverselyaffect sperm motility when not properly processed after ejaculation.     

Anticomplementary Activity in Human Semen and Its Possible Importance in Reproduction

ABSTRACT: We demonstrated anticomplementary activity on once-washed human sperm, and in normal and vasectomized seminal plasmas. It was demonstrated to be a normal component of human semen. The origin of the activity is proposed to be the seminal plasma with sperm adsorption of activity. The properties of the seminal anticomplementary factor were characterized further, and the molecular size was shown to be less than 3500 daltons. Reduced anticomplementary activity was found to be associated significantly with abnormal semen profiles and infertility in males. The activity in seminal plasma was shown to have no effect on complement-dependent sperm immobilizing antibodies in the serum of an infertile woman, implicating an effect on the post-C₃ components of the complement pathway. The inhibition of complement-dependent haemolysis and the lack of inhibition of complement-dependent sperm immobilization by the anticomplementary factor are considered in the implications of the role of seminal anticomplementary activity in reproduction.     

Purification and characterization of human seminal plasma alpha-L-fucosidase

Human seminal plasma alpha-L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83,000 nmol/min/mg protein) by affinity chromatography on agarose-epsilon-aminocaproyl-fucopyranosylamine. The purified alpha-L-fucosidase appeared to contain a single subunit of 56-57 kDa (as determined by SDS-PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma alpha-L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl-alpha-L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0-4.5) with a second neutral optimum (pH 6.4-7.4) with 60-80% of maximal activity. Apparent K(M) and V(max) values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 micromol/min/mg protein respectively, using Lineweaver-Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma alpha-L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5-7. The acidic isoforms between pI values 5-6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.     

Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis

In men, inhibin B is the circulating isoform involved in the regulation of follicle stimulating hormone (FSH) secretion. Within the testis, inhibin B may have a role in Sertoli and germ cell interactions, thus secretion into seminal plasma may reflect seminiferous tubule function. Using specific immunoassays, inhibin B was present in seminal plasma in fertile men (n = 105) and in unselected men attending an infertility clinic (n = 174) with a wide range in concentration from undetectable (<15 pg/ml) up to 54,100 pg/ml (geometric mean 280 pg/ml). There was a highly significant correlation between seminal plasma inhibin B concentration and sperm concentration (r = 0.46, P < 0.001), but no correlation with percentages of spermatozoa with progressive motility or normal morphology. Inhibin A and isoforms containing pro and alphaC immunoreactivity were not detectable. In post-vasectomy seminal plasma samples (18 of 20) inhibin B was undetectable, indicating that the testis is the predominant source. In unselected men attending an infertility clinic, inhibin B was undetectable in 17% (present in remainder; maximum concentration 26,200 pg/ml; mean 263 pg/ml), with a highly significant correlation between seminal plasma inhibin B and sperm concentration (r = 0.55, P < 0.0001). In men with oligo/ azoospermia (sperm concentration <20 x 10(6)/ml), seminal plasma inhibin B concentrations were lower in those with elevated plasma FSH concentrations (mean values 42 and 205 pg/ml, P < 0.05). Inhibin alpha and betaB subunits were localized predominantly in Sertoli and Leydig cells, using immunohistochemistry. We conclude that inhibin B of testicular origin is present in normal human seminal plasma, but with a very wide range in concentration, and may reflect the functional state of the seminiferous epithelium.      

Mammalian Sperm Fertility Related Proteins

Infertility is an important aspect of human and animal reproduction and still presents with much etiological ambiguity. As fifty percent of infertility is related to the male partner, molecular investigations on sperm and seminal plasma can lead to new knowledge on male infertility. Several comparisons between fertile and infertile human and other species sperm proteome have shown the existence of potential fertility markers. These proteins have been categorized into energy related, structural and other functional proteins which play a major role in sperm motility, capacitation and sperm-oocyte binding. The data from these studies show the impact of sperm proteome studies on identifying different valuable markers for fertility screening. In this article, we review recent development in unraveling sperm fertility related proteins.     

A Human Sperm Coating Antigen Isolated From Sperm Cell Membrane

ABSTRACT: A human sperm cell membrane antigen that is highly specific to sperm and seminal plasma was isolated from plasma membrane fraction of spermatozoa using rabbit antiserum against human seminal plasma. In addition to the high specificity to sperm and seminal plasma, the isolated antigen showed the following characteristics: (1) It is a glycoprotein of approximately 12,000 daltons that has an affinity to lentil lectin; (2) it is distributed in human milk other than in sperm and seminal plasma, but is not found in any other organs and tissues including testis; (3) seminal plasma contains the largest amount of the antigen activity, 60-fold greater than spermatozoa and 900-fold greater than milk, suggesting that this antigen could be a sperm-coating seminal plasma antigen.