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Phospholipase A2 (PLA2) activity was measured and partially characterized in prostasomes isolated from human seminal plasma. The results show high PLA2 activity in seminal plasma with a threefold enrichment in the isolated prostasomes. Highest activity was found at pH 8.0 and 10.5, and there was an absolute requirement for calcium with almost total inhibition of PLA2 activity in the presence of 1 mM EDTA. Analysis with two-dimensional gel electrophoresis of prostasome material revealed a complex protein pattern with most of the proteins in the molecular weight interval of 10,000–90,000 daltons. The possible role of PLA2 in prostasomes is discussed.

The electrophoretic seminal plasma protein pattern changes obviously within a few hours if the plasma is not frozen after the ejaculation. Mainly cathodal proteins disappear. Addition of serine protease inhibitors has no apparent affect, but o-phenanthroline markedly retards the conversions seen on electrophoresis. The subcellular organelles in seminal plasma are shown to contain a zinc ion dependent peptidase with optimal activity at pH 7.8. Estimations of enzyme activity may be performed using the elastase substrates n-tert butyloxycarbonyl-L-alanine-paranitrophenylester (Boc(Ala)) and succinyl(alanine)3-paranitroanilide (Suc(Ala)3pNA). The enzyme is inactivated by addition of o-phenanthroline and is completely reactivated by the addition of zinc ions.

Liquefaction of coagulated human semen was inhibited by o-phenanthroline; subsequent addition of Zn2+ reversed this inhibition, but not if the coagulum was repeatedly washed before Zn2+ was added. No liquefaction of the coagulum occurred when Fe2+ was added (in a 1:3 molar ratio to o-phenanthroline), and the gel repeatedly washed. This o-phenanthroline-depleted coagulum was liquefied by resuspended pellet from ultracentrifuged pooled seminal plasma. In denatured and reduced semen coagulate, we identified the 52 kDa, 71 kDa, and 76 kDa protein bands of the predominant seminal vesicle protein. The protein was not present in the supernatant after centrifugation of coagulated semen, and it was degraded to several minor basic proteins when semen liquefied. These findings imply that the predominant seminal vesicle protein functions as the structural protein of coagulated semen. In much the same way as in ejaculated semen, the 52 kDa, 71 kDa, and 76 kDa protein bands in seminal vesicle secretion collected postmortem were digested to minor basic proteins after incubating the secretion with resuspended pellet from ultracentrifuged seminal plasma. This pellet contained the membrane-bound succinyl(alanine)3-p-nitroanilide hydrolysing peptidase of prostatic origin which, like the liquefaction process, was active in the presence of EGTA, inhibited by non-chelated Zn2+, and inactivated by o-phenanthroline--an inactivation that was reversed by Zn2+.

The Mg2+- and Ca2+-dependent ATPase system in human seminal fluid is linked to membranes encasing small organelles denoted 'prostasomes'. This activity was completely pelleted after ultracentrifugation at 105,000 X g, provided the seminal plasma was diluted 1:10. On the contrary, prostatic acid phosphatase activity remained in the supernatant and was inhibited to about 75% by tartrate (0.5 mmol l-1) contrasting with the ATPase system that was not inhibited. Calmodulin, in the concentration interval 0.30-0.90 mumol l-1, did not further activate the ATPase system. Calmidazolium, which is a highly lipophilic substance, was a competitive inhibitor of the Mg2+- and Ca2+-dependent ATPase and half maximal inhibition was attained at 2-3 mumol l-1 calmidazolium. This inhibition was antagonized to a moderate degree (25%, P less than 0.02) by calmodulin, 0.60 mumol l-1. Quercetin, at concentrations of 30-60 mumol l-1, did not influence the prostasome membrane ATPase in any direction. Oleate was an inhibitor of the ATPase system at any concentration up to 50 mumol l-1. This inhibition was not counteracted to an appreciable extent by calmodulin. These results displayed a pattern giving support to the view that the Mg2+- and Ca2+-dependent ATPase of the prostasome membrane may be the molecular basis for vectorial transport of Ca2+ into prostasomes.

Semen was collected under standardized conditions from men who were divided into three groups: a control group from normal spermiograms, men with abnormal spermiograms, and azoospermic men following vasectomy. Ultracentrifuged seminal plasma was analyzed for uridine, xanthine, urate, and the pelleted material for Mg2+- and Ca2+-dependent ATPase and protein. No significant intergroup differences were noted except the significant elevation of xanthine of vasectomized men. Uridine that occurs in high concentration in seminal plasma displayed a positive correlation to percentage motile sperms from the 26 men with normal and abnormal spermiograms. It was concluded that an optimal secretory function of uridine may parallel the increasing percentage of sperm with a better quality of motility. A linear relationship between ATPase activity and sperm penetration ability existed only when taken into consideration those 13 specimens with lowest enzyme activity.

Seminal plasma from 22 men attending an infertility clinic was subjected to preparative ultracentrifugation for 2 h at 105,000 g. The pelleted material as well as the supernatant thus obtained were investigated with regard to prostasome membrane-linked enzyme activities in relation to other semen parameters. The mean activity of Zn2+-dependent adenosine triphosphatase in the sedimented prostasome fraction was 1.45 +/- 1.02 mumol (range 0.29-4.79) orthophosphate released per milligram protein and 20 min, while the corresponding figures for the supernatant were 0.56 +/- 0.30 (range 0.12-1.29). Hence, 72% of the specific activity was sedimented, and 28% remained in the supernatant. The same pattern was recognized with regard to the other two enzymes investigated, although they displayed individual characteristics with regard to distribution after ultracentrifugation. The pelleted prostasome-linked mean aminopeptidase activity was 0.39 U/mg protein (81.9%), with only 0.087 U (18.1%) remaining in the supernatant. The corresponding figures for gamma-glutamyltransferase were 7.89 (60.4%) and 5.17 (39.6%) mu kat/g protein, respectively. The different enzyme activities in the prostasome fraction and supernatant, respectively, were interrelated to each other and correlated significantly with r values between 0.73 and 0.93 (p less than 0.001). It was concluded that a minor fraction of prostasomes remained in the supernatant after ultracentrifugation. A relationship existed between prostasomes and semen volume revealing a rather consistent pattern in that small volumes favoured the presence of comparatively more prostasomes in the supernatant and less prostasomes in the pelleted fraction than large volumes. In addition, the sperm concentration seemed to be another determinant of the distribution of prostasomes in seminal plasma on subsequent ultracentrifugation.

A method for the determination of selenium in human spermatozoa and prostasomes is described. The samples were digested with 25% (w/v) tetramethylammonium hydroxide (TMAH) in methanol and analyzed by atomic absorption spectrometry with electrothermal atomization and Zeeman background correction (ET-AAS). Nickel was used as a matrix modifier. Calibration was performed using the matrix-based calibration curve. The TMAH-digestion method agreed well with a conventional digestion procedure using concentrated nitric acid. The TMAH-digestion does not require heating or strong acids and it was suitable for small biological samples. The average recovery of added selenium in spermatozoan digests was 95.1 +/- 5.2% (n = 5). The coefficient of variation was 9.1% (n = 21). The accuracy of the method tested with the NBS standard 1577 (bovine liver, certified at 1.1 +/- 0.1 micrograms Se/g) resulted in a value of 0.98 +/- 0.10 micrograms Se/g (n = 16). The method was further tested in an interlaboratory comparison study.

Seminal plasma constituents were separated on Sephadex G200 gel columns. The column eluate was analysed with regard to protein content, ATPase activity and promotive activity on sperm progressive motility. Two different chromatographic fractions were also subjected to electron microscopy after sedimentation by preparative ultracentrifugation. A maximum promotive value on sperm progressive motility coincided with a maximum ATPase activity value in a single peak from seminal plasma eluted first on the column and containing less protein than the other peaks appearing later in the chromatogram. This first peak was the only one containing ATPase activity and membrane-surrounded organelles named prostasomes. Other peaks, rich in protein but lacking ATPase and prostasomes, displayed a moderate and rather irregular pattern in reference to promotive activity on sperm progressive motility. Evidence is given that the positive effect by prostasomes is specific on sperm progressive motility. Hence, procedures aiming at a change of membrane integrity of the prostasomes resulted in diminished effects on sperm progressive motility. This could be explained by a probable dissipation of the electrochemical gradient of calcium ions.

Lipid analysis and ESR studies were carried out on prostasomes isolated from human semen. Cholesterol plus phospholipids amounted to approximately 0.80 mumol per mg protein with a striking quantitative domination of cholesterol over the phospholipids, the molar ratios of cholesterol/sphingomyelin/glycerophospholipids being 4:1:1. Saturated and monounsaturated fatty acids were dominating both in the glycerophospholipids and in sphingomyelin. The order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes order parameters, S, deduced from ESR spectra of spin-labelled fatty acids incorporated into prostasome membranes were very high, viz. 0.75 for 5-doxylstearic acid and 0.30 for 16-doxylstearic acid at 25 degrees C. Slightly lower values were obtained for the spin-labelled fatty acids when they were incorporated into dispersions of extracted prostasome lipids or into synthetic lipid mixtures of similar composition. The highly ordered lipids in the prostasome membrane thus seemed to be minimally perturbed by proteins in the membrane and ESR spectra showed no signs of immobilized lipids.

A stimulation by zinc ions of the hydrolysis of ATP by prostasomes prepared from human semen has been observed. Stimulation was maximal at a Zn2+/ATP stoichiometry of 0.5/l, and increasing this ratio resulted in a gradual decrease in ATPase activity. The pH optimum was 6.0. The apparent Km for Zn2+-dependent ATPase was 0.43 mmol/l and apparent Vmax 5.60 mumol/mg protein/20 min. Other divalent cations could replace Zn2+ as cofactor more or less effectively in the order Mn2+ greater than Cd2+ greater than Ba2+ greater than Sr2+. Potassium ions produced a further activation of the Zn2+-dependent ATPase system by about 10%. Such a stimulation was also attained to some extent by other monovalent cations as Rb+, NH4+, Li+ and to a lesser extent by Cs+. Orthovanadate in the concentration interval 5-1,000 mumol/l was inhibitory of the Zn2+-dependent ATPase system in a dose-dependent fashion. An aminopeptidase activity was also linked to the prostasomes. This enzyme activity was dramatically inhibited by 2 mmol/l orthophenantroline. A reactivation of the orthophenantroline-inhibited aminopeptidase activity was possible by adding Zn2+ to the reaction mixture. Hence, prostasomes contained ATPase as well as aminopeptidase activities both of which being dependent upon Zn2+. These two activities did not seem to be expressions of an ATP-dependent protease activity associated with prostasomes.

An intact organelle, the prostasome, is secreted by the acinar epithelial cell of the human prostate gland. The ultrastructural location of the prostasome is within membrane-bound storage vesicles in the epithelial cells. Prostasomes are delivered into the glandular lumen by an exocytotic event, which is preceded by fusion of adjacent membranes belonging to the storage vesicle and the epithelial cell. Alternatively, the storage vesicle can be translocated in toto from the cell interior into the acinar lumen through the plasma membrane. This latter event has been designated diacytosis. Both phenomena seem to occur with approximately equal frequency in the human prostate gland. An ATPase system that is Mg2+ and Ca2+-dependent is firmly linked to the membranes encasing the prostasomes. The ATPase system may be the molecular basis for vectorial transport of calcium into these organelles. Also a protein kinase activity is located in the membranes. An increase in membrane thickness was observed on phosphorylation. The physiologic function of the prostasomes is not known. They may be important for promoting forward motility of spermatozoa.

Secretory granules and vesicles were demonstrated within human prostatic cells and in the acinar lumen. In size and ultrastructure the granules and vesicles were the same as those previously isolated from human prostatic fluid and seminal plasma. "Prostasomes" is suggested as the designation for these secretory granules and vesicles. As a rule they were found in storage vesicles within the secretory cells. Two mechanisms for translocation of the prostasomes from the cell interior to the acinar lumen are described. One mechanism involves exocytosis with binding of storage vesicles to, and fusion with the plasma membrane, resulting in release of prostasomes directly into the acinar lumen. The other mechanism implies displacement of storage vesicles in toto from the cell interior to the acinar lumen. This process differs from exocytosis and is here designated "diacytosis". Both phenomena appear to be of roughly equal frequency.

Two cases of a previously unreported sperm defect appearing in boar studs in Finland are presented. Spermatozoa showed small particles scattered on their surface with a prevalence decreasing with boar age. Semen samples, either stained with eosin-nigrosin or examined with phase contrast optics on formaldehyde-fixed spermatozoa, revealed the presence of multiple particles attached to the surface of spermatozoa counted as dead cells at fixation. Transmission electron microscopy revealed these were multivesicular and multilamellar vesicles, built up by phospholipid membranes. The case is classified as a post-epididymal multivesicular sperm defect with a favorable prognosis.

Glycolytic enzymes are among the most frequently identified proteins in proteomics of exosomes/extracellular vesicles. This review brings up the possibility that exosomes/extracellular vesicles during their life-time in bodily fluids power important energy-consuming functions by glycolytic conversion of glucose or fructose into ATP. It was seen that prostasomes (exosomes of the prostate) could produce ATP by glycolysis and that the produced ATP quickly was consumed by adjacent prostasomal ATPases. The glycolytic ATP production appeared to be coupled to self-sustaining energy requirements. It will also be discussed how a failure in this machinery (lowered activity of ATPases) with a resultant polluting leakage of extracellular ATP could affect cancer development.

Prostasomes, which are prostate-derived organelles, were purified from human seminal plasma for inclusion in Earle's balanced salt solution (EBSS) medium with or without human serum albumin. These media were used for swim-up experiments and the subsequent analyses of sperm motility parameters at different incubation times. The yield of motile spermatozoa after swim-up in EBSS medium enriched with boiled prostasomes was increased by 32% compared with EBSS containing albumin. Native prostasomes were less active. Combinations of albumin and either prostasomes or boiled prostasomes significantly increased the recovery of motile spermatozoa and also increased the percentage of spermatozoa displaying progressive motility after 1 h of incubation. Media lacking albumin showed lower values regarding progressive motility after 22 h of incubation. A beneficial effect of prostasomes was noted on lateral head displacement and percentage of hyperactive spermatozoa during the first 6 h of incubation. These results suggest that inclusion of prostasomes, especially boiled prostasomes, in swim-up media may improve the recovery of hyperactive motile spermatozoa for up to 6 h in cases of established male factor infertility, and consequently increase the opportunities for fertilization.

Extracellular vesicles (EVs) are membrane-coated objects such as exosomes and microvesicles, released by many cell-types. Their presence in body fluids and the variable surface composition and content render them attractive potential biomarkers. The ability to determine their cellular origin could greatly move the field forward. We used multiplex proximity extension assays (PEA) to identify with high specificity and sensitivity the protein profiles of exosomes of different origins, including seven cell lines and two different body fluids. By comparing cells and exosomes, we successfully identified the cells originating the exosomes. Furthermore, by principal component analysis of protein patterns human milk EVs and prostasomes released from prostate acinar cells clustered with cell lines from breast and prostate tissues, respectively. Milk exosomes uniquely expressed CXCL5, MIA, and KLK6, whereas prostasomes carried NKX31, GSTP1, and SRC, highlighting that EVs originating from different origins express distinct proteins. In conclusion, PEA provides a powerful protein screening tool in exosome research, for purposes of identifying the cell source of exosomes, or new biomarkers in diseases such as cancer and inflammation.

Extracellular Vesicles (EVs) are membrane-limited particles containing proteins, lipids, metabolites and nucleic acids that are secreted by healthy and cancerous cells. These vesicles are very heterogeneous in size and content and mediate a variety of biological functions. Three subtypes of EV have been described in the male genital tract: microvesicles, myelinosomes and exosomes. Each type of EVs depends on the location of secretion such as the testis, prostate or epididymis. It has been shown that EVs can fuse together and deliver information to recipient cells, for example spermatozoa in the male genital tract. Cryo-electron microscopy remains the reference technique for determining EV morphology, but quantifying the absolute concentration of these EVs in biological fluids remains a challenge from a clinical point of view. The field of bio detection has considerably increased with the introduction of nanomaterials in biosensors and will provide a better understanding of the impact of these EVs. However, functional modifications of male gametes result from interactions with the components of the intraluminal fluid all along the genital tract and depend on the secretion and absorption of proteins and lipids from the local microenvironment. We cannot therefore exclude the possibility of epigenetic modulation of the information that will be transmitted to the embryo and therefore to the next generation via EVs.

Background One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype.