Interaction between prostasomes and spermatozoa from human semen

Prostasomes are prostate-derived organelles that occur freely in human seminal plasma. They promote forward motility of spermatozoa probably by closely interacting with them in an unknown manner. We have studied the interaction between human prostasomes and spermatozoa by applying them as two separate samples in free-zone electrophoresis. During the run these samples approached each other and finally fused into one single peak that was not further dissociated. Both the spermatozoa and prostasomes displayed a net-negative surface charge, the latter being less negative. This discrepancy in charge was even more pronounced by pretreatment of prostasomes with neuraminidase, which, however, did not affect the interaction. This implies a strong interaction of a probable hydrophobic character between cells and organelles. The presence of prostasomes and spermatozoa in the fused, single peak was confirmed by transmission electron microscopy. Evidence for interaction was apparent in transmission electron microscopy after embedding in a hydrophilic, but not in a hydrophobic, resin. This observation supports the view that the bonds between prostasomes and spermatozoa are of hydrophobic character. This type of interaction enables the prostasomes to act in close vicinity to spermatozoa and may create the prerequisites for a proper microenvironment of the spermatozoa favoring their forward motility.     

Paracrine stimulation of vascular smooth muscle proliferation by diadenosine polyphosphates released from proximal tubule epithelial cells

The purinergic receptor system plays an important role in the regulation of both vascular and tubular functions within the kidney; however, the release of purinergic agonists other than ATP by renal tissue is not known. In this investigation, we determine if kidney tissue is a source of diadenosine polyphosphates, which have high affinity for the P(2X) and P(2Y) receptors. Both diadenosine pentaphosphate and hexaphosphate were identified by matrix-assisted laser desorption ionization-mass spectrometry in extracts purified from both whole porcine kidney and from cloned cells of the LLC-PK1 cell line. Both polyphosphates in nanomolar concentrations were found to significantly stimulate the proliferation of vascular smooth muscle cells derived from rat thoracic aortas. The purinergic-receptor antagonist, suramin, did not significantly affect the growth-stimulatory properties of the polyphosphates. The growth stimulation of vascular smooth muscle cells by platelet-derived growth factor was potentiated by both diadenosine polyphosphates. We conclude that diadenosine polyphosphates are endogenous purinergic agonists of the kidney that have physiologic and pathophysiologic relevance. These epithelial cell metabolic products have vasoregulatory properties while linking the energy supply and tubular function.     

Diadenosine tetraphosphate (Ap4A) and triphosphate (Ap3A) signaling of human sperm motility

The ubiquitous dinucleotide polyphosphate, diadenosine tetraphosphate (Ap4A), has been shown to be a signal molecule for DNA replication in mammalian cells. In this study, Ap4A and a related compound, diadenosine triphosphate (Ap3A), were tested for possible signaling functions in human spermatozoa. A computerized automated semen analyzer was used to detect changes in spermatozoa motility parameters. Cryopreserved-thawed donor spermatozoa were washed and incubated in 0.1 mM Ap4A, 0.1 mM Ap3A, or control medium. The data indicated that both Ap4A and Ap3A decreased the percentage of motile spermatozoa after 4 or more hours of incubation in vitro. The two dinucleotide polyphosphates caused an increase in the amplitude of lateral spermatozoa head displacement parameter only at the start of incubation. The other spermatozoa kinematic parameters were unaffected. No opposing ying-yang dual actions of Ap4A to Ap3A were seen. From the results, Ap4A and Ap3A were observed to be potential inhibitory signals of spermatozoa motility after prolonged exposure.     

A minor fraction of a high-affinity folate binding protein from the epididymis is associated with membranous vesicles and spermatozoa in human semen

A glycolipid linked high-affinity folate binding protein (FBP) is present in human semen at a concentration of 1-2 nmol/L. The association between FBP and seminal components as well as the cellular source of FBP was analysed. Immunoblotting of human seminal plasma, with and without prostasomes, prostasomal fractions and spermatozoa with antibodies against human FBP revealed a single distinct band similar to that observed with purified human FBP. Flow cytometry identified FBP on the surface of ejaculated spermatozoa. Immunohistochemistry showed positive immunostaining of epididymal epithelium, vas deferens and ejaculated spermatozoa, whereas the prostate gland, seminal vesicles, testicular spermatozoa and seminiferous tubules of testis stained negatively. Electron microscopy immunocytochemistry with antibodies against rat FBP showed labelling located to luminal microvilli and intracellular vesicles of rat epididymal epithelial cells and the surface of spermatozoa in the epididymal duct. High-affinity binding of picomolar amounts of tritiated folate to fractions of human prostasomes or prostasome-like vesicles was completely depressed by excess amounts of unlabelled folate. The study indicates that FBP is secreted from the epithelia of epididymis and vas deferens, and that a small fraction of FBP is associated with prostasome-like vesicles which adhere to spermatozoa in the epididymal duct. FBP could have a bacteriostatic function by depriving folate-requiring bacteria of folate and/or ascertain a normal DNA replication subsequent to fertilization by vectorial transfer of folate to the inner compartment of the spermatozoa.     

Expression profile and distribution of Annexin A1, A2 and A5 in human semen

Annexins (ANXAs) have been identified in different seminal components mainly by proteomic methods. The presence and distribution of Annexin A1, A2 and A5 (ANXA1, ANXA2, ANXA5) in human semen was analysed and the corresponding mRNAs studied in spermatozoa. All three ANXAs were present in prostasomes and spermatozoa, but only ANXA1 in prostasomes‐free seminal plasma. Immunofluorescence showed ANXA1 and ANXA5 in the sperm head, mid‐piece and flagellum. The amount of mRNAs corresponding to ANXA1 and A2 decreased with increasing levels of the corresponding proteins indicating a probable regulation of their expression at the translational level during spermatogenesis. Additionally, DNA fragmentation was assessed by the sperm chromatin dispersion test. Lower amounts of ANXA1 and A2 with higher levels of the corresponding mRNAs were noted in poor quality semen samples. ANXA5 was detected in spermatozoa from all semen samples, but no particular trend was noted. The corresponding mRNA were detected both in excellent and poor quality semen samples. Results showed that ANXA1 and A2 expressions appear to be related with DNA fragmentation suggesting their possible use as new biomarkers for sperm DNA quality. ANXA5’s natural presence in spermatozoa suggest that revision of high‐quality sperm selection by binding to this protein is needed.     

PC3 prostasomes对精细胞上游筛选后活力的影响

目的 探讨从PC3前列腺癌细胞株中分防出的prostasome样颗粒（PC3prostasome）对上游筛选后精细胞活力的。方法 将新鲜或冻融精细胞置入含PC3prostasome(0.10mg/ml或0.25mg/ml）及人血白蛋白（HSA，10mg/ml）的EBSS液中上游筛选后，测试比较活力的差别。结果 新鲜标本不同孵育时间和孵育1h不同参数间比较，PC3prostasome0.10mg/ml组促精细胞活力人中于HSA组（P＜0.01），且此作用可持续6h。冻融复温标本PC3prostasome组恢复活力的细胞比HSA组增加63％（P＜0.001）。结论 PC3prostasome具有与精浆prostasome相同的刺激精细胞活力作用，且此作用强于HSA。     

The Janus-faced nature of prostasomes: their pluripotency favours the normal reproductive process and malignant prostate growth

Prostasomes are submicron secretory granules synthesized, stored and secreted by the epithelial cells of the human prostate gland. They are membrane-surrounded also in their extracellular appearance and the membrane architecture is composite. They are believed to be life-giving and act as protectors of the spermatozoa in the lower and upper female genital tract on their way to the ovum. Hence, the prostasomes are immunosuppressive and inhibitory of complement activation. Further, they promote sperm's forward motility and have antioxidant and antibacterial capacities. The prostasomes with their many composite abilities seem to turn against the host cell after the age of 50 y being conducive to the transition of the normal prostate epithelial cell into a neoplastic cell and therewith lay the foundations of the very high prevalence of prostate cancer of men of more than 50 y of age.     

Prothrombotic effect of prostasomes of metastatic cell and seminal origin

BACKGROUND: Prostasomes are secretory granules produced by the glandular epithelial cells of the prostate. Seminal prostasomes contain high amounts of Tissue Factor (TF) but no studies of TF on malignant cell prostasomes have been made. Here we compare the expression, phosphorylation, and function of TF on prostasomes of different origin. METHODS: TF was detected on prostasomes isolated from seminal fluid and human prostate cancer cell lines (PC-3, DU145, and LNCaP) using FACS and enzyme immunoassay (EIA). Incubation of prostasomes with radioactive ATP under conditions favoring protein kinase A activity led to phosphorylation of TF as detected by immunoprecipitation and SDS-PAGE. The prothrombotic effect of prostasomes was investigated in whole blood and recalcified plasma. Blocking experiments were performed using anti-TF antibodies and corn trypsin inhibitor. RESULTS: TF was expressed on all tested prostasome preparations with lowest values found for seminal ones. Prostasomal TF was the main endogenous substrate for prostasomal protein kinase A. All tested prostasome preparations greatly enhanced the rate of clot formation in a dose-dependent fashion, that is, the clotting capability of prostasomes seemed to be related to the extent of their expression of TF. In addition, the density of the clot varied between different prostasome preparations. When incubated in whole blood, prostasomes were found to associate to WBC thereby inducing them to express and release TF. CONCLUSIONS: These data show that TF is overexpressed and also subjected to phosphorylation by malignant cell prostasomes. This suggests major roles for prostasomes in thrombotic events that occur in some advanced cases of prostate cancer.     

Zinc ion stimulation of ATP cleavage by prostasomes from human seminal plasma

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.     

Prostasomes: current concepts

BACKGROUND: Prostasomes are membranous vesicles secreted by prostate gland, and they contain large amounts of cholesterol, sphingomyelin, calcium, and several enzymes. Prostasomes are involved in a number of biological functions. At ejaculation, these prostasomes are expelled with prostate secretions and are to be found in the seminal plasma as seminal prostasomes, which facilitate sperm function in various ways. METHODS: In this review, we discuss the structural and functional role of prostasomes, the various enzyme systems associated with these vesicles, and the biological role prostasomes play in male reproduction. RESULTS AND CONCLUSIONS Prostasomes are pluripotent and well-organized organelles secreted by the prostate gland. Prostasomes are ascribed to have many physiologiocal functions, the primary function being enhancement of sperm capacity. The several enzyme systems, small signaling molecules, and neuroendocrine markers associated with prostasomes reveal the complex nature of these vesicles in regulating sperm viability and vitality. The functional significance of these molecules that regulate complex pathways in these small vesicles is still a matter of dogma. Critical evaluation of the biological processes associated with prostasomes might be helpful in modeling new contraceptive agents, improving the techniques of in vitro fertilization, and in furthering our understanding and treatment of male factor infertility.     

Nucleic acid association to human prostasomes.

Human prostasomes isolated from seminal plasma were subjected to phenol extraction and then to absorbance (A) measurements at 260 nm (A260) and 280 nm (A280). The A260/A280 ratio was about 2 for prostasome extract and lower for seminal plasma extract, indicative of the presence of nucleic acid. The ratio of nucleic acid to protein in prostasomes was about 1:100, and the ratio in seminal plasma was 1:1,000. Hence nucleic acid is enriched in prostasomes (compared to seminal plasma of 10). Treatment of prostasome samples with 2% sodium dodecyl sulfate resulted in an efficient dissociation of nucleic acid from prostasomes as demonstrated by electrophoresis. The association of nucleic acids of various sizes (range; 200 to 20,000 base pairs) to prostasome membranes was most probably genuine and not the result of contamination from spermatozoa, erythrocytes, leukocytes, or bacteria. The results of experiments employing nucleic acid-degrading enzymes favored the concept that double-stranded DNA but not RNA is present at the prostasome membrane surface.     

Overexpression of ecto-protein kinases in prostasomes of metastatic cell origin

BACKGROUND: Prostasomes are secretory granules produced, stored, and released by the glandular epithelial cells of the prostate. They express numerous enzymes whose physiological roles have so far not been fully evaluated. In this study, we investigated the expression and function of prostasomal protein kinases and ATPase. METHODS: The protein kinase activities of prostasomes isolated from seminal fluid and malignant prostate cell lines (PC-3, DU145, and LNCaP) were investigated using the model phosphorylation substrates histone and casein, as well as the plasma proteins C3 and fibrinogen, in combination with specific protein kinase inhibitors. The prostasomal ATPase activity was also evaluated. The expression of protein kinases and ATPase on prostasomes was verified by flow cytometry. RESULTS: Prostasomes (intact or solubilized with octylglucoside or saponin) from prostate cancer cells had higher expression of protein kinases A, C, and casein kinase II compared to prostasomes isolated from seminal plasma, resulting in higher phosphorylation of both exogenous and endogenous substrates. Using intact prostasomes, it was found that prostasomes of metastatic origin had lower ATPase activity, resulting in higher residual ATP available for the phosphorylation reaction. Finally, complement component C3 and fibrinogen (two proteins whose activities are modulated by phosphorylation) were identified as physiologically relevant phosphorylation substrates. CONCLUSIONS: These results indicate that prostasomes are capable of modifying proteins possibly involved in the innate response by extracellular phosphorylation mediated by ecto-kinases. This is a novel mechanism by which prostatic malignant cells may interact with their environment.     

Localization of met-enkephalin on human spermatozoa and evidence for its physiological role

Pro-opio-melano-cortino-derived peptides have been identified in rat testicular extracts and in human seminal plasma, but their physiological role is still unknown. We report that met-enkephalin is localized on human spermatozoa, by means of an indirect immunofluorescent technique. Furthermore, we demonstrate that a met-enkephalin analog (D-Ala2-Mephe4-Met-(o)-ol-enkephalin, FK 33-824, Sandoz, Basel, Switzerland: DAMME) inhibits in a dose-dependent manner the acrosome reaction induction. The hypothesis of a physiological role of seminal met-enkephalin on human spermatozoa fertilizing ability is briefly discussed.     

The Effect of Zinc, Arginine, Fructose and Seminal Supernatant of Normal Semen on the Triple Adenosine Triphosphatase Activities of the Spermatozoa from Males with Oligoasthenozoospermia

When the spermatozoa of oligoasthenozoospermic patients were suspended with the supernatant of normal semen, an increase in triple ATPase enzyme activities besides enhancement of spermatozoa motilities were observed. This suggests that factor or factors present in the supernatant of normal semen that effects spermatozoa motility also have a positive effect on triple ATPase enzyme activities. In an attempt to produce such an effect, zinc, arginine and fructose were added to the incubation media where the spermatozoal ATPase enzyme activities were determined. Zinc increased Ca2+-Mg2+ ATPase enzyme activity without affecting Na+/K+-Mg2+ and Mg2+ ATPase activities. Triple ATPase enzyme activities remained unchanged after arginine and fructose additions. As a result zinc is thought to be one of the factors that affect spermatozoa motility in seminal plasma.     

Transfer of functional prostasomal CD59 of metastatic prostatic cancer cell origin protects cells against complement attack

BACKGROUND: Prostasomes are secretory granules produced, stored, and released, by the glandular epithelial cells of the prostate. They express the glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein CD59, which has been shown to be transferred to spermatozoa and erythrocytes. METHODS: The CD59 content of prostasomes isolated from seminal fluid and malignant prostate cells (PC-3, DU145, and LNCaP) and the transfer of prostasomal CD59 to rabbit erythrocytes (RE) and to PIPLC-treated and unmanipulated cancer cells were investigated using FACS. All prostasomes were also incubated with RE and tested in a hemolytic assay. RESULTS: Prostasomes from cancer cells had higher expression of CD59 than those of normal cells. Prostasomal CD59 of different origin could be transferred to RE, malignant cell lines stripped of CD59 by PIPLC, or unmanipulated LNCaP cells. Malignant cell prostasomes had an increased ability to inhibit complement-mediated lysis compared to those from non-malignant cells. CONCLUSIONS: These results point to a novel mechanism by which prostasomes can protect prostatic malignant cells from complement attack.     

Prostasomes as zinc ligands in human seminal plasma

Prostasomes are small vesicles, containing zinc, secreted by prostate in human seminal plasma and showing a physiological role on sperm properties. In this study, the possible correspondence between prostasomes and a prostatic high molecular weight protein complex, recently indicated as zinc ligand, has been investigated. Isolated prostasomes, examined by scanning electron microscopy, were dialysed to evaluate their zinc binding capacity. Furthermore, seminal plasma Sephadex G-75 elution was carried out before and after prostasome removal. Prostasome preparations, containing typical vesicles of 50-500 nm, showed a positive correlation between their zinc and protein levels. They were able to take up zinc against gradient. Furthermore, the seminal zinc amount, bound to the high molecular weight proteins, was strongly reduced in the free-prostasome sample with respect to the total seminal plasma. This study suggested the correspondence between the prostasomes and a high-sized zinc ligand complex of prostatic origin. Therefore, it demonstrated, for the first time, the zinc binding capacity of prostasomes, a new property which could be related to their biological functions.     

Acrosome reaction in the head-attached human sperm

Summary: The physiological acrosome reaction of the sperm occurs on the surface of the oocyte. Although its occurrence on the zona pellucida is considered to be a specific response to zona compounds, whether the simple attachment itself could influence the reaction of motile spermatozoa to a fixed surface and promote onset of the reaction is questioned. A motile human sperm head fixation method was developed recently, through which a group of head-attached sperm can be established easily. These head-attached sperm were investigated for their acrosome reactions. After staining by fluorescein iso-thiocyanate conjugated peanut lectin (FITC-PNA), many head-attached spermatozoa (75.7%) were found to have undergone an acrosome reaction.     

In vitro inhibition of angiogenesis by prostasomes

Prostasomes are biologically active organelles that are secreted by human prostate epithelial cells, and it is believed that they have a role in prostatic disease. We studied the effect of prostasomes on the human umbilical vein endothelial cell (HUVEC)/Matrigel model of angiogenesis, and the association of labelled prostasomes with HUVECs. The growth inhibitory effect of prostasomes on HUVECs was assayed by spectrophotometric measurement of residual biomass. Preparations of HUVECs on a Matrigel base were exposed to prostasomes, and the development of capillary-like networks was quantified. Prostasomes were labelled with PKH-26, and cultured with HUVECs. Prostasomes were not shown to have a significant effect on HUVEC survival. Angiogenesis assays showed inhibition. The PKH-26-labelled particles were shown to have adhered to the HUVECs. This study adds the inhibition of an in vitro correlate of angiogenesis to the known actions of prostasomes.     

Affinity sites for β-glucuronidase on the surface of human spermatozoa

Summary. Glycosidases secreted by the epididymis become bound to the surface of spermatozoa during their transit through the epididymal duct. They are believed to play a role in mammalian fertilization. In the present report, we demonstrate that β-glucuronidase binds to the surface of ejaculated human spermatozoa with high affinity and in a saturable manner. The binding is Ca²⁺-independent, inhibited by either mannose-6-phosphate, phosphomannan fragments from the yeast Hansenula holstii and α-mannosidase from the Dictyostelium discoideum , suggesting that phospho-mannosyl receptors are involved in the recognition of the enzyme. The catalytic site of the enzyme is not involved in the binding. The localization of the β-glucuronidase binding-sites is restricted to the surface of the sperm head. These results suggest that the spermatozoa could be the target for glycosidases present in the seminal plasma.     

Growth-inhibitory effect of prostasomes on prostatic cancer cell lines in culture

OBJECTIVES: Prostasomes, small corpuscular organelles derived from the prostate gland, were isolated from human seminal plasma for incubation with three human prostatic cancer cell lines: DU145, PC3, and LNCaP. The aim of this study was to establish any possible growth-inhibitory effect of prostasomes on prostatic cancer cell lines. METHODS: Prostasomes were isolated from human seminal plasma. We determined their growth-inhibitory effect on the prostatic cancer cell lines by a fluorimetric cytotoxic assay technique. The results obtained were presented as survival index, defined as a percentage of control cultures, after compensation for blank values. RESULTS: The prostasomes exhibited a dose-dependent growth-inhibitory effect on the cells, cell line DU145 being the most sensitive one displaying a survival index (mean +/- SD) of 46+/-10% at 200 microg prostasomes (protein)/ml while the other two cell lines displayed survival indices of 93+/-6 and 93+/-9%. Heat treatment of the prostasomes abolished their growth-inhibitory effect on the cell lines, whereas 24-hour dialysis of the prostasomes did not affect their activity in this regard. Seminal plasma devoid of prostasomes exhibited less effect on the growth of these cancer cell lines, and heat treatment of the plasma did not influence the result. However, dialysis of the seminal plasma for 24 h abrogated its growth-inhibitory effect. CONCLUSION: The prostasomal growth-inhibitory effect on the cancer cell lines was different from that of the prostasome-free seminal plasma. The former effect was most probably of a proteinaceous nature while the latter effect was most likely due to the divalent cation composition of seminal plasma. In this regard, the zinc ion content seemed to be decisive.