Polymorphism of rat seminal vesicle secretory proteins: Characterization of svp-1 and svp-2 and their identification with the major secretory proteins IV and V

The proteins secreted by the rat seminal vesicle can be separated into five major fractions (namely, RSV-I through V) by gel electrophoresis in denaturing conditions. Two polymorphic proteins, svp-1 and svp-2, also present in the mouse, are produced by the seminal vesicle as well, but the procedure used for their identification makes it impossible to ascertain whether they correspond to any of the major fractions mentioned above. We show here that, on the basis of molecular weight measurements and of amino acid composition determinations, svp-1 and RSV-V are indeed the same protein. We also show that svp-2 is strictly related to another major secretory protein, RSV-IV, whose amino acid composition is almost identical, but for a few amino acid residues, to that of svp-2. We thus conclude that the latter protein is a variant of RSV-IV that can be expressed only in rats homozygous for a given allele at the svp-2 locus. This paper thus brings together published information on the genetics of the loci coding for svp-1 and for svp-2 and on the molecular biology of RSV-IV and RSV-V and of their corresponding gene.This work was supported by a CNR grant from Progetto Finalizzato Ingegneria Genetica e Basi Molecolari delle Malattie genetiche.     

A protein family immunorelated to a sperm-binding protein and its regulation in human semen

In human seminal plasma a family of proteins that is immunologically related to the RSV-IV protein secreted under androgen control from the epithelium of the rat seminal vesicles was detected by a radioimmunoassay. Evidence for the origin of these antigens from human seminal vesicle is presented. Quantitative measurements of this family of proteins were performed in men with low levels of serum testosterone (idiopathic hypogonadotropic hypogonadism) and in individuals having serum testosterone in the normal range of values but carrying sex chromosome aberrations (Klinefelter's syndrome). In the first case we have found a marked decrease in the total amount of the RSV-IV-related proteins. An increase of about 40% in the total amount of these antigens was obtained in these subjects by gonadotropin treatment. A decreased amount of these proteins was also detected in the subjects affected by Klinefelter's syndrome. The possibility that some factor(s) under genetic control is involved, in addition to testosterone, in the regulation of this family of proteins is discussed.     

The 11S rat seminal vesicle mRNA directs the in vitro synthesis of two precursors of the major secretory protein IV

The 11s mRNA extracted from the rat seminal vesicles directs the synthesis of two different precursors of the major secretory protein RSV-IV. These two precursors are not interconvertible and seemingly originate from different translational events. Sucrose gradients, polyacrylamide gel electrophoresis and positive hybridization translation experiments do not allow the separation of the two putatively different mRNAs. It is concluded that the two RSV-IV precursors either derive from two extremely similar, but physically not separable mRNA species, or from two different modes of translation of the same mRNA molecule.     

Synthesis of a testosterone-dependent secretory protein by rat seminal vesicle-derived cell lines.

Primary cell cultures were established from explants of rat seminal vesicle. The establishment of primary cell cultures required, among other factors, the presence of testosterone. Two cell populations were detected in such primary cultures: fibroblast-like cells and epithelial-like cells; the latter encompassed a subtype of small cells and a subtype of large squamous cells (most likely the result of a degenerative process acting upon the former). Histochemical, as well as electron-microscopical observations, indicated the presence of a persistent secretory activity in the small epithelial cells; fibroblast and large squamous epithelial cells were inactive in this respect. Staining of the cells with a peroxidase-conjugated antibody and analysis of the proteins produced in the presence of labelled methionine, showed that one of the major rat seminal vesicle secretory proteins, namely RSV-IV, was also produced. Conditions which favoured the growth of epithelial cells, rather than of fibroblasts, were determined. The use of nearly homogeneous cell populations and the use of collagen-coated Petri dishes, allowed the cloning of two independently obtained permanent cell lines, namely SVC-1 and SVC-2. The in vitro growth rate of both cell lines was modulated by the amount of testosterone in the medium. Both cell lines were able to synthesize a significant amount of RSV-IV protein under testosterone control.     

Immunologically reactive albumin-like protein in human testis and seminal plasma

An immunologically reactive albumin-like protein (albumin) was localized, by an immunostaining technique, in the testis of infertile men (normal spermatogenesis, obstructive azoospermia) at the level of the Sertoli cells and in some cells of the germinal epithelium (secondary spermatocytes and early spermatids). No positive reaction was detectable in prepubertal testis. In vasectomized men, mean seminal albumin values were drastically reduced (by about 80%) in comparison to fertile controls, indicating a probable testicular origin. Mean seminal albumin values were also decreased in patients affected by azoospermia due to a seminiferous tubular lesion (about 40%) and in oligozoospermic patients (about 30%). In the same seminal samples transferrin, an index of Sertoli cell function, was also measured. Albumin and transferrin results were well correlated in the seminal plasma of each group (with the exception of vasectomized subjects), including a group of men with abnormally high concentrations of seminal transferrin. A weak correlation was found between seminal albumin and sperm count. We suggest that the presence of albumin in the human adult testis and in seminal plasma could be related to its ability to transport androgens.     

Seminal prostaglandins in men with subnormal sperm motility and therapeutic treatment

The contents of prostaglandins in seminal plasma from a total of 73 men were evaluated. The subjects were grouped as follows: normospermic men, patients with impaired motility, patients with small untreated varicocele and patients with impaired motility and Kallikrein therapy. Sperm density, morphology and motility were examined. High performance reversed phase liquid chromatography (HPLC) in combination with specific radioimmunoassays were used for the determination of PGE2, PGI2 and PGF2 alpha. There was a significant difference (p less than 0.025; F-test) between the PGI2 concentrations in patients with impaired motility (5.6 +/- 1.4 pg/mg protein) and normal men (8.8 +/- 3.7 pg/mg protein). PGE2 and PGF2 alpha were significantly different in patients with varicocele (p less than 0.025, F-test). Wide ranges of prostaglandins occurred in the Kallikrein-group with no significant differences. We conclude that: a) PGI2 is an additional prostaglandin compound in seminal plasma, b) its measurement may not be useful as diagnostic parameter in subfertile men and c) Kallikrein has no influence on the prostaglandin content in seminal plasma and other seminal parameters as motility, motility index and sperm counts.     

Seminal prostaglandins in men with subnormal sperm motility and therapeutic treatment

The contents of prostaglandins in seminal plasma from a total of 73 men were evaluated. The subjects were grouped as follows: normospermic men, patients with impaired motility, patients with small untreated varicocelle and patients with impaired motility and Kallikrein therapy. Sperm density, morphology and motility were examined. High performance reversed phase liquid chromatography (HPLC) in combination with specific radioimmunoassays were used for the determination of PGE₂, PGI₂ and PGF_2α. There was a significant difference (p < 0, 025; F-test) between the PGI₂ concentrations in patients with impaired motility (5,6 ± 1,4 pg/mg protein) and normal men (8,8 ± 3,7 pg/mg protein). PGE₂ and PGF_2α were significantly different in patients with varicocele (p < 0,025, F-test). Wide ranges of prostaglandins occured in the Kallikrein-group with no significant differences. We conclude that: a) PGI₁ is an additional prostaglandin compound in seminal plasma. b)its measurement may not be useful as diagnostic parameter in subfertile men and c) Kallikrein has no influence on the prostaglandin content in seminal plasma and other seminal parameters as motility, motility index and sperm counts.     

Leukocyte migration inhibitory factor (LIF) to sperm from autoimmune men in infertile couples

Leukocyte migration inhibitory factor (LIF) is produced by lymphocytes with receptors specific to sensitizing antigens. This principle was used to detect possible antigenic differences between sperm of autoimmune and nonautoimmune men. Sixteen fertile and 91 infertile couples were screened for cytotoxic and hemagglutinating antibodies to sperm from their husbands and controls. Their lymphocytes were tested for the production of LIF to sperm extracts and seminal plasma from the husbands and controls by a direct leukocyte migration inhibition assay. Twenty-nine of 35 men producing LIF to sperm and/or seminal plasma were positive for sperm antibodies (p = 0.0004, vs sperm antibody-negative controls). Twenty-three of 29 wives with LIF production had sperm-autoimmune husbands (p = 0.04). Leukocyte migration was significantly inhibited in sperm-autoimmune men by autologous sperm extracts and seminal plasma in contrast to control sperm extracts and seminal plasma (p = 0.0006 and 0.001, respectively). The wives of autoimmune men had significantly higher LIF responses to their husbands' sperm extracts than to other antigens (p = 0.02). Men with cytotoxic antibodies in their seminal plasma produced LIF to autologous sperm (p = 0.001). It is suggested that certain sperm and seminal plasma antigens of autoimmune men may lead to specific humoral and cell-mediated immune responses in both partners.     

Pattern of neutral and phospholipids in the semen of normospermic, oligospermic and azoospermic men

Total lipid concentration was elevated in the seminal plasma of oligo- and azoospermic men. The total cholesterol content was comparatively more in the seminal plasma of azoospermic men than in that of normo- and oligospermic men. In general, infertility was associated with increased seminal concentrations for most of the neutral lipid classes. However, total phospholipids and most of the phospholipid classes were diminished in the seminal plasma of oligo- and azoospermic men and in the spermatozoa of oligospermic men. We suggest that there is a positive correlation between seminal phospholipids and fertility and a negative correlation between seminal neutral lipids and fertility.     

Membrane-associated protein kinase activities in seminal plasma from vasectomized men

Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2+; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these membrane subfractions. Electron microscopic examination showed that there are several kinds of membrane-limited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.     

The major protein of bull seminal plasma is a secretory product of seminal vesicle

We isolated the major protein with apparent molecular weight, Mr, 15,000-16,000 from seminal plasma as well as from seminal vesicle secretion of bull and proved by amino acid analysis and tryptic peptide mapping that the two proteins were identical. An antiserum against this major protein was employed to quantitate and identify the major protein in seminal plasma as well as in seminal vesicle secretion. The antiserum did not cross-react with proteins from bovine or human plasma or follicular fluid, respectively. Cell-free translation of poly(A+)RNA isolated from seminal vesicle tissue resulted in formation of one major species with apparent Mr 18,000. Using the anti-major protein antiserum, this major species was specifically immuno absorbed. We thus provided evidence that the major protein component of bull seminal plasma is a secretory protein of seminal vesicles. Furthermore, it appeared that the isolated major protein may be closely related to the protein PDC109, purified from bull seminal plasma and sequenced by Esch et al. (Biochem. Biophys. Res. Commun. 113, 861-867 (1983).     

Characterization of seminal plasma proteins stabilizing the sperm viability in rainbow trout (Oncorhynchus mykiss)

In seminal plasma of the rainbow trout 12 proteins were detected by SDS-PAGE, ranging in their molecular weight from 135 to 16 kDa. Only those proteins with a molecular weight of 65, 54, 47 and 16 kDa occurred in all investigated seminal plasma samples. The 65 and the 54 kDa protein were found in highest quantities (34-45% of the total quantified protein content) followed by the 47 and the 16 kDa protein (6-7% of the total quantified protein content). The 65 and the 48 kDa protein were glycoproteins as they stained positively with Periodic-Acid-Schiff reagent (PAS) specific for carbohydrates as well as with Coomassie Blue. The 90 and 19 kDa protein were found in 82-91% of the investigated samples, all other proteins in lower frequencies of 36-73%. Seminal plasma contained no lipoprotein as staining with Sudan black B was negative. To find out which proteins positively affected the sperm viability (defined as sperm motility which could be activated) spermatozoa were incubated in sperm motility inhibiting saline solution containing different seminal plasma protein fractions. Sperm motility which could be activated after an incubation period of 48 h was highest in those fractions which shared the 54, 47, and the 16 kDa protein. When spermatozoa were incubated in untreated seminal plasma sperm viability was still higher than in the seminal plasma protein fractions indicating that other components of the seminal plasma positively affected sperm viability, too. The possible influence of seminal plasma proteins on sperm physiology is discussed.     

The major basic proteins of bull seminal vesicle secretion

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.     

Gelatinolytic proteinase activities in human seminal plasma

Proteinase activities in human seminal plasma were detected using gelatin-containing sodium dodecyl sulphate-polyacrylamide gel electrophoresis zymography. Three prominent bands of activity of Mr 60,000, 66,000 and 90,000 were observed as well as 9 other bands of less intensity (34,000-158,000). These proteinases were dependent upon calcium for optimal activity, did not hydrolyse casein, and were predominantly in the soluble portion of seminal plasma. Examination of seminal plasma of men with different sperm concentrations, split ejaculates, and prostatic secretions indicated that the prostate gland was a source of most of these activities. Proteinase activities of Mr 34,000, 37,000, 82,000 and 120,000 were expressed more frequently in seminal plasma from normozoospermic men than from seminal plasma of oligo- or azoospermic men, indicating that they may also arise from spermatozoa in the semen sample. The proteinases of Mr 60,000 and 66,000 were found in all seminal plasmas whereas there was variation in the expression of the other molecular forms of enzyme, even in the normozoospermic samples. There are multiple forms of gelatinolytic proteinase activities in human seminal plasma which appear to arise from multiple sources in the reproductive tract including the Cowper's/urethral glands, the prostate gland, seminal vesicle and/or spermatozoa. Their function(s) in semen remains to be established.     

Impact of seminal and serum zinc on semen quality and hormonal status: A population-based cohort study of Russian young men

BackgroundTrace elements are important factors in human reproductive health. Among them, special attention is paid to zinc, which is an essential trace element and is necessary for the normal functioning of the male reproductive system and the process of spermatogenesis. The aim of the study was to investigate the association between seminal and serum zinc concentrations and semen quality and reproductive hormone levels in population of Russian young men.MethodsThe study population consisted of 626 young Russian men (median age 22.5 years), recruited from the general population, regardless of their fertility status. Each participant provided semen and blood sample, information about his lifestyle and ethnicity. Semen quality (sperm concentration, motility and morphology), reproductive hormone levels (testosterone, estradiol, LH, FSH and inhibin B), and serum and seminal zinc concentrations were evaluated. The semen samples were analyzed according to the WHO laboratory manual (WHO, 2010). Serum hormones were measured by enzyme immunoassay, zinc concentrations were determined using spectrophotometry and direct colorimetry without deproteinization.ResultsZinc was present in the seminal plasma in a significantly higher concentration than in the blood serum (median serum Zn concentration was 23.6 μmol/L vs seminal Zn concentration 1571.8 μmol/L). The seminal zinc concentration was positively related to the total sperm count, sperm concentration, progressive motility and normal morphology (Spearman’s test: 0.221; 0.286; 0.269; 0.183, respectively, p < 0.001), while the serum Zn concentration was negatively related to serum testosterone and estradiol levels (r = −0.249 and r = −0.096, respectively, p < 0.001−0.05). It was found that the seminal Zn content in men with normal semen quality was higher compared to men with lowered semen quality (means: 6.37 and 5.03 μmol/ejaculate, respectively, p < 0.001). Similarly, the semen volume, total sperm count, sperm concentration, progressive motility, normal morphology and the serum testosterone level in men with the seminal Zn deficiency were lower than in men with the normal seminal Zn content.ConclusionBased on the results of our population-based study, seminal Zn levels were closely associated with semen parameters in young men, so Zn deficiency may be an important risk factor for lowered semen quality. Seminal Zn determinations should be considered as a useful tool in addition to other parameters in assessing male fertility.     

Selenium,Copper and Zinc in Seminal Plasma of Men with Varicocele,Relationship with Seminal Parameters

Varicocele has been associated with decrease in seminal parameters. Selenium (Se), copper (Cu), and zinc (Zn) are trace elements essential for normal spermatogenesis of mammals and play a critical role as antioxidant defense system enzymes. Se, Cu, and Zn are associated with sperm quality in fertile and infertile men. However, there is little information about Se, Cu, and Zn concentrations in semen in patients with varicocele and its association with seminal parameters. The purpose of this study was to determine the concentrations of Se, Cu, and Zn in semen of patients with varicocele and the relationship with seminal parameters. Total Reflection X-Ray Fluorescence was used for the fist time in the seminal fluid analysis. The concentration of selenium in men with varicocele was smaller than the normozoospermic group, while no differences were observed for both concentrations of zinc and copper. A significant positive correlation between zinc and selenium concentration was observed. Selenium in seminal plasma correlates with a good spermatozoa concentrations, motility, and morphology. Additionally, a significant positive correlation was observed between zinc levels and sperm count. In conclusion, a decrease in selenium concentration was associated with detriment of seminal parameters. A study should be conducted to evaluate the benefits of both zinc and selenium supplementation to improve seminal parameters in patients with varicocele.     

Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans

In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 ± 13 nm vs. 105 ± 25 nm) but similar equilibrium buoyant density (～1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostate-specific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals.     

Adsorption of seminal plasma proteins by boar spermatozoa

Seminal plasma protein adsorption by boar spermatozoa was examined using ejaculated sperm from vesiculectomized boars and seminal plasma from vasectomized boars. Sperm adsorbed 14 pg protein/sperm in 10 min. When seminal plasma proteins were radiolabeled, most of the adsorbed radiolabel was present in low M(r) proteins, particularly a 12700 M(r) protein. A 349300 M(r) seminal plasma protein was also readily adsorbed. Three radiolabeled seminal plasma proteins (307600, 165400 and 7400 M(r)) were not detected on the sperm; either they are not adsorbed by the sperm or the sperm were previously exposed to these proteins in other accessory sex gland fluids and had already adsorbed them. A 29100 M(r) sperm protein was also radiolabeled (4.9% of the adsorbed radiolabel), although there was no corresponding seminal plasma protein. Large quantities of seminal plasma protein (particularly low M(r) proteins) are adsorbed by sperm not previously exposed to seminal vesicle secretion. The functions of these proteins are yet to be determined.     

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.     

Exploration du plasma seminal humain: Nouvelle approche par la resonance magnetique nucleaire du proton (RMN-1H)

Human seminal plasma glycerylphosphorylcholine (GPC), glycerylphosphorylethanolamine (GPE), citric acid (Cit) and lactic acid (Lac) were analzed by measuring the peak area of proton nuclear magnetic resonance (NMR) spectra in four groups of patients: 21 men with secretory azoospermia; 14 men with excretory azoospermia after vasectomy; seven patients presenting with very severe oligoasthenozoospermia; and 18 normozoospermic subjects. When comparing secretory and excretory azoospermia cases with normozoospermic men, seminal plasma GPC, Cit and Lac peak areas were lower in azoospermic cases compared to normozoospermic men (16.79, 8.18 and 2.28 versus 23.38, 10.58 and 4.3. arbitrary units respectively, P <0.01). The peak area ratios Cit/Lac and GPC/lac were significantly different between normozoospermic men and either secretory or excretory azoospermic groups (P<0.01). A significant difference was also found in the GPE/GPC peak intensity ratio between secretory and excretory azoospermia cases (P<0.001). These results demonstrate that quantitative markers such as GPC, GPE, Cit and Lac may be useful in the examination of human seminal plasma by proton NMR in infertility investigations.