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Prostasomes are small vesicles secreted by the prostate gland to the seminal fluid. In male fertility, the fusion of prostasomes to sperm has been associated with an enhancement of sperm function. Prostasomes also contribute to sperm protection within the female reproductive tract and immunologically relevant proteins have been identified on the prostasomal membrane that may prevent immune-mediated destruction of the sperm.The female genital tract is a potentially hostile environment for spermatozoa and a large number of NK cells have been found in the female reproductive tract. Since it has been suggested that NK cells may represent an important component of innate immunity in the female reproductive tract, we have analyzed the role of prostasomes in the regulation of NK cell activity.Flow cytometric analysis revealed that prostasomes expressed high levels of CD48, the ligand for the activating receptor CD244. The interactions between NK cells and purified prostasomes resulted in a decrease of CD244 expression. Moreover, the decreased NK cell activity observed in NK cells cultured in the presence of prostasomes suggests that prostasomes may immunomodulate the local environment within the female reproductive tract preventing immune-mediated sperm destruction and prolonging their survival rate.

Prostasomes are extracellularly occurring submicron, membrane-surrounded organelles produced by the epithelial cells of the prostate and present in semen after secretion. Even dedifferentiated prostate cancer cells have preserved their ability to produce and export prostasomes to the extracellular space. The precise physiological role of prostasomes is not known, although some of their properties assign them to important physiological and patho-physiological functions that could be exploited in prostate cancer growth and development. In this review, some new properties of seminal and malignant cell line (DU145, PC-3 and LNCaP) prostasomes will be discussed. There are typical differences in the expressions and activities of prostasomal CD59, ATPase, protein kinases and tissue factor (TF) as well as in the transfer of prostasomal CD59 to CD59-deficient erythrocytes (rabbit and human PNH erythrocytes). CD59, protein kinases and TF exhibit characteristic patterns of overexpression by malignant cell prostasomes. A high ATPase activity is recognized on seminal prostasomes with minimal activity on malignant cell prostasomes resulting in more residual ATP available for phosphorylation reactions. Several proteins are phosphorylated by prostasomal protein kinases, namely, complement component C3, fibrinogen, vitronectin and E-cadherin. Furthermore, TF is identified as the main endogenous phosphorylation substrate on prostasomes. In addition, prothrombotic effects of prostasomes are demonstrated. DU145 and PC-3 cell-derived prostasomes exert a higher clotting effect on whole blood and plasma compared to LNCaP cell-derived and seminal prostasomes. In conclusion, malignant cell prostasomes show an increased ability to interact with the biological system in favor of prostate cancer cell promotion and survival. The roles played by prostasomes in this context may improve the understanding of the mechanisms that help the prostate cancer cells to avoid the complement attack (CD59 transfer and phosphorylation and inactivation of C3), to promote angiogenesis (TF) and to metastasize. It may also provide a better understanding of some of the complications usually seen in some terminal prostate cancer patients like thrombotic events and tendency to develop disseminated intravascular coagulation.

PROBLEM: Prostasomes are trilamellar to multilamellar vesicles produced by the acinar cells of the human prostate and are present in appreciable amounts in normal human semen. The aim of this work was to study the effect of prostasomes on human polymorphonuclear cell and monocyte function. METHODS: Functional activity of human neutrophils and monocytes was studied after incubation with prostasomes isolated from normal human seminal plasma. The following functional tests were employed: ability to ingest latex particles and opsonized bacteria (St. aureus) and ability to generate superoxide anion in response to PMA and FMLP. The latter was determined by measuring the super-oxide-dismutase inhibitable reduction of ferricytochrome c to ferrocytochrome c at 550 nm. Expression of cell surface markers and interactions of prostasomes with cells were studied by cytofluorimetry. RESULTS: We show that prostasomes bind rapidly to the leukocyte cell membrane followed by internalization of adsorbed material. Interactions of prostasomes with neutrophils and monocytes inhibits their ability to phagocytose latex particles. The ability to ingest opsonized bacteria is, however, not impaired. Our results also show that incubation of leukocytes with prostasomes effectively inhibits superoxide anion generation in response to activation by PMA and FMLP. CONCLUSIONS: Prostasomes may play a complementary role to other immunosuppressive factors contained in the human semen. They may protect sperm cells from deleterious effects of phagocytosing cells, prolong their life, and consequently enhance the chance of conception. At the same time prostasomes may have a permissive effect on sexually transmitted diseases.

Background: The secretory epithelial cells of the prostate gland use sophisticated vehicles named prostasomes to relay important information to sperm cells in semen. This prostasome-forming and secretory ability of the epithelial cells is also preserved in poorly differentiated prostate cancer cells. Aim: The aim of this thesis was to examine different characteristics of prostasomes, especially those derived from malignant prostate cells, linked to their potential role in diagnosis and prognostication of prostate cancer. Results: Serum samples of prostate cancer patients contained autoantibodies against seminal prostasomes in a higher concentration than did control sera. These autoantibodies were most frequently directed against 25 prostasome-associated proteins, but no one was prostate specific. Clusterin was one of the most frequently occurring prostasomal proteins. Elevated titers were however seen in both patients´ and control sera. Clusterin turned out to be a major antigen of seminal prostasomes. No prostate specific or prostate cancer specific protein was discovered upon proteomic analysis of prostasomes deriving from malignant cells of vertebral metastases of prostate cancer patients. Human chromosomal DNA was identified in both seminal prostasomes and PC-3 cell prostasomes and strong evidence existed that the DNA was localized inside the prostasomes. Four out of 13 DNA clones of seminal prostasomes featured gene sequences (31%). The corresponding figures for PC-3 cell prostasomes were 4 out of 16 clones (25%). Conclusions: Prostasomes are immunogenic and give rise to serum autoantibodies. The most frequently occurring autoantibodies were directed against 25 prostasomal proteins but none of these was exclusively prostate specific. Thirty different proteins were identified in prostate cancer metastasis-derived prostasomes but none of these proteins was prostate cancer specific. Human chromosomal DNA was identified in prostasomes of both normal and malignant cell origin.

Antiprostasome antibodies (APAs) have been identified in serum of patients with prostate cancer and have been proposed as a new marker for metastatic disease. This study reassesses the role of APAs as a prognostic indicator for prostate cancer.Serum samples from healthy controls (n=7) and patients with prostate cancer (n=22) were assayed for APAs using an enzyme-linked immunosorbent assay.APAs in varying amounts were present in healthy individuals as well as in men with prostate cancer. Higher levels were inversely and significantly associated with prostate-specific antigen (PSA). No significant relationships were noted between APA levels and other parameters such as age, time since diagnosis, metastatic status, Gleason histological score and hormonal treatment.The presence of serum APA is unlikely to be a strong prognostic indictor for prostate cancer on an individual basis as false positives will occur. However, such immune reactions which may be associated with PSA in cancer patients are in any case of interest in both the biology of prostate cancer and male fertility. The source of prostasomal antigen may be of critical importance to the outcome of the assay. However, immune reactions to prostasomes may be of considerable interest and warrant continued investigation.

SummaryCancer confers a prothrombotic state and statins are associated with a lowered risk for prostate cancer in vivo by unknown mechanisms. Prostate cancer cells release tissue factor (TF)-bearing, cholesterol-rich prostasomes which are procoagulant in vitro and a possible source for the blood-borne TF found in prostate cancer patients. We investigated the effect of cholesterol depletion on the production of prostasomes and on the TF activity in the conditioned medium of simvastatin-treated PC3 cells. Human PC3 prostate cancer cells were treated with high and low concentrations of simvastatin for different time periods. Caspase-3 was detected with the Array Scan microscope, where-asTF mRNA and protein were analyzed by TaqMan and flow cytometry. TF activity was assessed by measuring the cleavage of a chromogenic thrombin substrate. Prostasomes were isolated by repeated centrifugations and detected and quantified by flow cy tometry. A micromolar dose of simvastatin caused reduction of TF expression and induction of apoptosis in the PC3 cells. The levels of TF on the prostasomes were also decreased but the TF activity in the conditioned medium of the simvastatin-treated PC3 cells was increased due to apoptosis-dependent release of prostasomes. Treatment with a nanomolar dose of simvastatin did not induce apoptosis or alter the expression of TF but instead decreased the production and release of the prostasomes. The TF activity was reduced in parity with the decline in prostasome release. In conclusion, in prostate cancer, a nanomolar dose of simvastatin may have an anti-thrombotic effect due to decreased levels of circulating TF-bearing prostasomes.

BACKGROUND Human seminal fluid contains small exosome-like vesicles called prostasomes. Prostasomes have been reported previously to play an important role in the process of fertilization by boosting survivability and motility of spermatozoa, in addition to modulating acrosomal reactivity. Prostasomes have also been reported to present with sizes varying from 50 to 500 nm and to have multilayered lipid membranes; however, the fine morphology of prostasomes has never been studied in detail. METHODS Sucrose gradient-purified prostasomes were visualized by cryo-electron microscopy (EM). Protein composition was studied by trypsin in-gel digestion and liquid chromatography/mass spectrometry. RESULTS Here we report for the first time the detailed structure of seminal prostasomes by cryo-EM. There are at least three distinct dominant structural types of vesicles present. In parallel with the structural analysis, we have carried out a detailed proteomic analysis of prostasomes, which led to the identification of 440 proteins. This is nearly triple the number of proteins identified to date for these unique particles and a number of the proteins identified previously were cross-validated in our study. CONCLUSION From the data reported herein, we hypothesize that the structural heterogeneity of the exosome-like particles in human semen reflects their functional diversity. Our detailed proteomic analysis provided a list of candidate proteins for future structural and functional studies. Prostate 69: 159–167, 2009. © 2008 Wiley–Liss, Inc.

BACKGROUND: The epididymal epithelium secretes membranous vesicles, called epididymosomes, with which a complex mixture of proteins is associated. These vesicles transfer to spermatozoa selected proteins involved in sperm maturation. Epididymosomes in the human excurrent duct have been described, but their protein composition and possible functions are unknown. METHODS AND RESULTS: Epididymosomes were collected during vasovasostomy procedures, purified and submitted to liquid chromatography with hybrid quadrupole time-of-flight mass spectrometry. From all the mass spectra generated, 1022 peptides allowed the identification of 146 different proteins. Identification of some of these proteins was confirmed by western blots. Furthermore, western blot showed that the protein composition of epididymosomes differed from that characterizing prostasomes; membranous vesicles secreted by the prostate. Organization of the epididymosomes proteome according to common functional features suggests that epididymosomes have multiple functions. In order to understand the origin of epididymosomes collected distally, microarray databases of caput, corpus and cauda epididymidis were analysed to determine where along the excurrent duct the encoded proteins associated to epididymosomes are synthesised. Results suggest that some proteins synthesized in the caput and corpus epididymidis are associated with epididymosomes collected distally. CONCLUSIONS: Epididymosomes thus transit along the excurrent duct, and vesicles collected distally represent a mixed population.

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein known as the main initiator of blood coagulation. TF is over-expressed on many malignant cells and apart from increasing the risk of thrombosis, the presence of TF/FVIIa also promotes the progression of cancer and metastasis by intracellular signaling. TF expressing microparticles (MP) are, moreover, often found in the circulation of cancer patients. Aim: The aim of this thesis was to study different aspects of TF activity, e.g. the importance of procoagulant MP and TF-induced intracellular signaling pathways, with focus on cell migration (chemotaxis) and apoptosis. Results: The TF signaling complexes were shown to prevent apoptosis induced by serum starvation and TRAIL in cancer cells by reduced activation of caspase-8 in a PI3k/AKT-dependent manner. FVIIa also decreased transcription of pro-apoptotic genes in cancer cells treated with TRAIL. Simvastatin triggered apoptosis by transcriptional reduction of BCL-2 due to cytosolic retention of NFκB. Simvastatin also inactivated the PI3k/AKT pathway and reduced the production of the MP-like prostasomes which, respectively, impaired the anti-apoptotic signaling by TF and reduced the procoagulant activity in the vicinity of prostate cancer cells. Intracellular events conducted by the TF/FVIIa complex selectively enhanced PDGF-BB induced chemotaxis which was partly explained by the TF/FVIIa-induced transactivation of the PDGFβ-receptor. This was dependent on Src-family members and engagement of PAR2. Conclusions: The results presented in this thesis extend the current knowledge of TF-mediated signaling. We report the TF complexes to govern the extrinsic pathway of apoptosis, present data on FVIIa-dependent regulation of apoptosis-related genes, and exclude known surface proteins as transmitters of the anti-apoptotic signals. We moreover describe TF/FVIIa to transactivate the PDGFβ-receptor and play a decisive role in the potentiated chemotaxis toward PDGF-BB in a number of cell types. Finally, we explain the mechanism behind simvastatin-induced apoptosis in cancer cells and how statins interfere with TF-dependent signaling and coagulation.

Background The presence of small membranous particles characterizes the male genital fluids of different mammalian species. The influence of semen vesicles, denominated prostasomes, on sperm functional properties has been well documented in humans, but their biological activity is scarcely known in other species. The present work investigated prostasome-like vesicles in pig semen for their ability to interact with spermatozoa and to affect acrosome reaction. Methods Prostasome-like vesicles have been isolated from pig seminal plasma by high-speed centrifugation and Sephadex G-200 gel chromatography. Morphology of purified vesicles has been checked by scanning electron microscopy while their protein pattern has been investigated by SDS-PAGE. Then prostasome- like vesicles have been incubated with pig spermatozoa and their ability to interact with sperm has been tested by the aminopeptidase assay. In addition, the efficiency of vesicles to influence the acrosome reaction has been investigated by assessing the sperm acrosomal status by the PI/FITC-PNA (propidium iodide/fluorescein isothiocyanate-labeled peanut agglutinin) stainings. Results Purified vesicles revealed a complex protein pattern with the occurrence of bands in the high, medium and low molecular weight range. However, the two major bands were observed at ~90 kDa and ~60 kDa. A vesicle-mediated transfer of aminopeptidase to sperm cells has been also detected. Furthermore, a significant increase of acrosome reaction extent has been revealed in spermatozoa incubated with prostasome-like vesicles in comparison to control sperm. Conclusion This is the first report demonstrating that pig prostasome-like vesicles are able, in vitro, to interact with spermatozoa and to stimulate the acrosome reaction. These findings lead to hypothesize a transfer of molecules from vesicles to sperm membrane, thus sensitizing male gametes to undergo the acrosome reaction

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.

Clusterin is a ubiquitous secretory sulphated glycoprotein present in prostasomes. It is an anti-apoptotic mediator in prostate cancer and is among the most frequently occurring prostasomal proteins immunogenic in prostate cancer patients. The aim of the present study was to investigate the occurrence of anti-clusterin antibodies in the serum of patients with prostate cancer and whether there is a relationship between anti-clusterin antibody titres and other clinico-pathological variables.Serum samples were collected from 391 consecutive patients with suspected prostate cancer (150 benign prostate and 241 prostate cancer). The patients' serum samples were used in an ELISA where microtitre wells were coated with purified clusterin from serum of a healthy volunteer. Flow cytometric studies of clusterin and prostasomes were performed.Flow cytometric analyses revealed the presence of clusterin on the surface of seminal prostasomes. Anti-clusterin ELISA titres in sera of patients did not differ significantly from those of a control group. A significant "inverse" correlation existed between anti-clusterin ELISA titres and lymph node metastases (p = 0.047), but only 11 out of 161 patients had metastases. These titres correlated significantly with total prostate (p = 0.021) and transitional zone (p = 0.015) volumes of the patients.The correlation between serum anti-clusterin antibody titres and other clinico-pathological variables was generally weak in prostate cancer patients, although clusterin has been assigned an important role in tumourigenesis and progression of prostate cancer. However, the anti-clusterin antibody titre appeared to be related to prostate volume, correlating to both transitional zone volume and total volume of the prostate.

Thromboembolism is well recognized as a major complication of cancer. Many tumor cells overexpress tissue factor (TF), which activates blood coagulation in cancer patients. Inflammatory cells expressing TF are also contributors to this activation. In prostate cancer, we believe that prostasomes may also be involved in the initiation of blood coagulation. Prostasomes are submicron secretory granules derived from the prostate gland. They are surrounded by membrane and their extracellular appearance and membrane architecture are complex. Seminal prostasomes are believed to be necessary for successful fertilization and act as protectors of the spermatozoa in the lower and upper female genital tract. Cells from prostate cancer and its metastases are able to produce and export prostasomes to the extracellular environment. These prostasomes may differ quantitatively rather than qualitatively from their normal counterparts with regard to protein composition and function. A majority of human prostate cancers have been found to overexpress TF, and we have demonstrated by various methods that prostasomes derived from prostate cancer cells express considerably higher levels of TF compared with prostasomes of nonmalignant cell origin. The mechanism related to thromboembolic disease generated by prostasomes in prostatic cancer patients may be the early release of prostasomes from prostate cancer cells into the blood circulation, where they will evoke their blood-clotting effects.

Prostasomes are membrane-bound secretory vesicles produced by prostatic epithelial cells. They are known to carry many proteins, including tissue factor, and have membranes unusually rich in cholesterol and sphingomyelin. Prostasomes have well-documented effects on fertility, promoting sperm motility, stabilizing the acrosome reaction, and facilitating immunosuppression. This article reviews the evidence of the effects of prostasomes on in vitro angiogenesis assays, and the mechanism by which these effects occur. Seminal prostasomes seem to inhibit angiogenesis, whereas the equivalent particles released by malignant prostate cells promote angiogenesis. In both cases, the effects seem preserved after heat treatment to denature the protein content, suggesting an important role for lipid transfer, in particular, transfer of sphingomyelin.

Understanding the protective mechanisms utilized by metastatic prostate cancer cells in order to avoid attack by complement or other parts of the innate immune system and to affect tumor angiogenesis and metastasis will help us to identify suitable targets for pharmaceutical intervention. We have shown that at least two different complement-attenuating mechanisms are at work in close proximity to prostasomes: transfer of CD59, which inhibits complement at the level of MAC insertion, and phosphorylation of C3 so as to make it resistant to (physiological) activation and thereby regulate complement at the convertase level. In addition, we have shown that both fibrinogen and vitronectin, which play critical roles in cell adhesion, are targets for prostasome-mediated phosphorylation. Given the broad specificity of the various PKs, it is most likely that other relevant substrates such as proteins involved in angiogenesis or different matrix proteins may be found. Finally, we have demonstrated that expression and function of different proteins capable of mediating these effects (CD59 and PKs, particularly PKA) are highly upregulated on prostasomes derived from malignant cell lines as compared to seminal prostasomes, suggesting that the malignant cell-associated prostasomes have a higher potential to interact with neighboring cells.

Prostasomes are submicron-sized, membrane-bound organelles produced by the epithelial cells of the prostate and normally found in the secretion in the gland ducts. Their physiological role is in the promotion of sperm-function in human reproduction. This thesis contains four papers dealing with the production of prostasomes and some possible applications in clinical urology of the prostasome. Paper I and II provided an ultrastructural description of the synthesis, storage and secretion of prostasomes in benign as well as in malignant tissue. Most notable were the extracellular appearances of prostasomes in metastatic lesions whereby the prostasomes become exposed to the immune system of the patient. This supported findings in earlier studies in which patients with advanced prostate cancer had elevated levels of anti-prostasome antibodies. The results of paper III reinforced the view of the prostate-unique origin of the prostasome. In particular, there were no indications in SDS-PAGE patterns or flow-cytometric studies of material from seminal vesicle secretion that it contained components that could be associated with a production of prostasomes. Some possible clinical functions of the prostasomes were investigated in paper IV. Exposure of prostasomes to the immune system through mechanical and thermal trauma to the prostate did not induce an evident formation of anti-prostasome autoantibodies. Furthermore, the serum levels of anti-prostasome antibodies registered by assays with preparations of prostasomes from seminal plasma as antigen did not correlate with existing prostate cancer. Seminal prostasomes seemed not to function as substitute markers for prostate cancer in the test kit used. A possible explanation could be underestimated differences in antigen properties between seminal or prostate gland-derived prostasomes and prostasomes from tumor tissue.

Differently sized microparticles, including exosomes (30–100 nm), prostasomes (50–500 nm), oncosomes (50–500 nm) and other microparticles (100–1000 nm) were found in blood and urine. Exosomes from prostate cancer (PCa) patients can potentially contain cancer-specific nucleic acids, and thus can represent a valuable source of diagnostic material. In this study, we have investigated microvesicles and miRNA from urine of healthy donors and PCa patients. To isolate miRNAs from urine and microparticles, novel methods for miRNA isolation were elaborated (Rus. patent application No 2014137763, priority date 17.09.2014). The study population included 14 patients with PCa (63–82 years, T2-3NxMx1) and control group of 20 healthy volunteers with no previous history of prostate disease (48–73 years). Urine was clarified by two serial centrifugations at 400 g , 20 °C, 20 min and at 17000 g , 20 °C, 20 min. Microparticles were precipitated from the resulting supernatant by high-speed centrifugation at 100000 g , 18 °C, 90 min, the pellet was resuspended and pelleted by centrifugation under the same conditions. To isolate exosomes, total microparticles were filtered through 0.1 μm pore filters and reprecipitated. The resulting pellets were resuspended and exosome samples were investigated by transmission electron microscopy (TEM). MiRNAs were isolated by one-step single-phase protocol and purified using “BioSilica” spin- columns (Zaporozhchenko et al., Anal. Biochem, upcoming, doi: 10.1016/j.ab.2015.03.028) and by recently developed method based on precipitation of excess biopolymers, allowing to isolate miRNAs with better efficiency than commercially available kits. The size and quantity assessment of extracellular RNA were performed using capillary electrophoresis system on Agilent 2100 Bioanalyzer. Concentrations of miRNAs (miR19b, miR25, miR205, miR125b, miR126) were measured by qRT-PCR and normalized to miR-16 using dCq method. TEM demonstrated the presence of 20–300 nm microparticles in urine of healthy donors and PCa patients. Approximately 50–70% of all urine microparticles are represented by 30–100 nm exosomes and residual 30–50% by particles larger than 100 nm. (The pool of urine microvesicles consists of 50–70% exosomes and 30–50% particles larger than 100 nm.). The major part of extracellular RNA found both in exosomes and total microparticles fraction of healthy donors and patients with PCa, is 25–200 nt long and can include tRNA (73–93 n.), 5.8 rRNA (∼150 n.), snoRNK (10–20 n.), snRNA (60–300 n.) piRNAs (29–30 n.), miRNAs (20–25 n.), siRNA (21–25 n.). Concentration of extracellular urine RNA in exosomes and total microparticles of healthy donors and patients with PCa amounts to100 pg/ml of urine on average.Receiver Operating Characteristic (ROC) curve analysis of all miRNAs in samples isolated from total urine did not demonstrate any diagnostic significance. In contrast, in training cohort concentration of miR-19b in exosomes and total microparticles fraction provide 95%/75% and 93%/100% sensitivity and specificity, respectively. Thus, microparticles, isolated from urine of PCa patients represent a valuable source of diagnostically significant miRNAs. To investigate miR-19b, a large testing cohort is required. Search for other potential miRNA-markers by microarray profiling of cell-free miRNA isolated from urine of PCa patients is also required to reveal a sold set of markers for confident PCa diagnostics.

Summary Prostasomes are prostate-derived organelles in seminal plasma exhibiting pluripotent properties to facilitate the fertilization process. Seminal prostasome concentration, size distribution and expression of the prostasomal surface antigens CD10, CD13, CD26 and CD59 were examined by flow cytometry. The study group consisted of 79 men with involuntary infertility. Very strong correlations existed between the prostasome expressions of the different CD markers. Significant correlations between prostasome concentration and CD molecules were weak or lacking. Further, no or weak relationships were observed between the prostasomal CD markers and sperm morphology, seminal fructose, neutral alpha-glucosidase activity, zinc and tumour necrosis factor alpha concentrations. Flow cytometry is a practical way to study prostasomes in seminal fluid without prior separation. This is a new technique for evaluation of the role of prostasomes and their functions in male reproductive physiology.

Lycopene (C(40) H(56)) is a highly lipophilic antioxidant found in human semen in nanomolar concentrations. It has been shown to be one of the most potent carotenoid antioxidant in various human studies. Prostasomes are organelles secreted by glandular prostatic epithelial cells and are known to play an important role in fertility and prostate cancer. They are also known to possess antioxidant activity and aid the functioning of sperm. We studied the ability of these vesicles to adsorb and retain lycopene into their rich lipid environment in vitro. High-performance liquid chromatography analysis confirmed micrograms of lycopene per milligram of prostasomal protein. In view of the prostasomes' lipid-rich nature it is highly likely that these organelles act as delivery vehicles for this highly lipophilic antioxidant substance into human semen.

Prostasomes are membranous vesicles present in ejaculated human semen. They are very rich in cholesterol and can interact with spermatozoa. Their physiological roles are still under study. Prostasomes were mixed with liposomes prepared from various lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), DOTAP/1,2-dipalmytoyl-sn-glycero-3-phosphorylcholine (DPPC, 4:1 molar ratio) and DOTAP/cholesterol (4:1, molar ratio) at different pH values (5-8). The mixing of the lipid phases (fusion) was determined by the relief of octadecyl rhodamine B chloride (R(18)) self-quenching and the radii of the vesicles, by light scattering measurements. The mixing of lipids and the radii of prostasomes were both influenced by the addition of liposome, although in a different manner. The ability of prostasomes (modified by previous treatment with liposomes) to transfer lipid to spermatozoa was also measured. Pretreatment with DOTAP decreased the phenomenon and addition of DPPC abolished it. On the other hand, pretreatment of prostasomes with DOTAP/cholesterol liposomes did not affect the transfer of lipid between prostasome and spermatozoa. Therefore, the ability of vesicles to fuse (or, at least, to exchange the lipid component) was affected by the enrichment in either natural or artificial lipid. This may open new possibilities for the modulation of spermatozoa capacitation and acrosome reaction.

The prostate is a glandular male accessory sex organ vital for normal fertility. It provides the prostatic component of seminal plasma which nourishes and protects sperm following ejaculation. Prostasomes are small (40-500 nm) membrane-bound vesicles produced by epithelial cells lining the prostate acini and are a component of prostatic secretions. Although the existence of these particles has been known for many years, their full function and relevance to reproductive health are largely unknown. Proteomic studies have shown a wide range of proteins (enzymes, structural proteins and novel, unannotated proteins) present in or on the surface of prostasomes providing them with a diverse nature. Interestingly prostasomes are able to fuse with sperm, this event and the associated transfer of proteins lies at the heart of many of their proposed functions. Sperm motility is increased by the presence of prostasomes and their fusion prevents premature acrosome reactions. Prostasomes have been shown to aid protection of sperm within the female reproductive tract because of immunosuppressive, antioxidant and antibacterial properties. Clinically these functions imply a role for prostasomes in male factor infertility. However, the very functions that promote fertility may have negative connotations in later life; recent work has suggested that prostasomes are involved in prostate cancer. Clearly more work is needed to clarify the role of these novel particles and their impact on men's health.

Studies of seminal tissue factor (TF) are few and mostly based on small numbers. Due to the reported lack of factor (F) X in semen, it has been suggested that TF may not have a role in seminal coagulum formation. However, recent identification of a number of haemostatic factors in semen justifies a re-evaluation of its occurrence. Semen specimens were collected from sub-fertile (n = 19), normally fertile (n = 33), semen donors (n = 30) and vasectomized subjects (n = 62), some fractionated into sperm, a prostasome-rich fraction and seminal plasma. Functional and antigenic TF levels were measured and related to conventional fertility parameters. Semen contains high concentration of functional and antigenic TF. Most TF was found in seminal plasma prepared by low-speed centrifugation. When further fractionated by ultracentrifugation much of this may reside in the pellet (prostasomal fraction). It was also detectable on sperm. TF antigen levels were higher in vasectomized subjects than sub-fertile, normally fertile, donor (p = 0.02) and a 'pooled normal semen parameters' (PNSP) stratification (derived from a combination of measurements) (p = 0.06). The sub-fertile group showed a wider variation than normal, donor or the PNSP subjects. Seminal TF antigen levels correlated significantly with sperm agglutination (p = 0.03) and abnormal sperm morphology (p = 0.04). Subjects with anti-sperm antibodies also showed high TF antigen levels. In conclusion, semen contains functional and antigenic TF at high concentrations. A full complement of clotting factors probably exists in semen, so some pro-coagulant role for TF should not be excluded. Decreased seminal TF levels appear to be associated with seminal parameters that are known to favour male fertility.

Two enzymes are involved in the polyol pathway: an aldose reductase that reduces glucose in sorbitol followed by its oxidation in fructose by sorbitol dehydrogenase. It has been previously shown that both enzymes are presented in the bovine epididymis, where they are associated with membranous vesicles called epididymosomes. Based on the distribution of these enzymes, it has been hypothesized that the polyol pathway can modulate sperm motility during the epididymal transit. In the present study, polyol pathway was investigated in semen and along the epididymis in humans in order to determine if sperm maturation can be associated with this sugar pathway. Western blot analysis shows that both aldose reductase and sorbitol dehydrogenase are associated with ejaculated spermatozoa and prostasomes in humans. These enzymes are also associated with epididymosomes collected during surgical vasectomy reversal. Western blot, Northern blot, and reverse transcription-polymerase chain reaction analysis show that aldose reductase and sorbitol dehydrogenase are expressed at the transcriptional and translational levels along the human epididymis. Unlike what occurs in the bovine model, distribution of these enzymes is rather uniform along the human excurrent duct. Immunohistological studies together with Western blot analysis performed on epididymosomes preparations indicate that the polyol pathway enzymes are secreted by the epididymal epithelium. These results indicate that the polyol pathway plays a role in human sperm physiology.

During epididymal transit, mammalian spermatozoa acquire new proteins involved in the acquisition of motility and of male gamete fertilising ability. We have previously shown that membranous vesicles called epididymosomes are involved in the transfer of epididymal-originating proteins to spermatozoa. The cytokine macrophage migration inhibitory factor (MIF) is one of these proteins but the role played by MIF in relation to epididymal sperm maturation still remains unclear. As this protein has already been shown to bear different functions depending on its location, we investigated its distribution along the epididymis and in different compartments of human semen. Northern and Western blot analysis as well as immunohistochemical studies show that MIF is expressed all along the epididymis with a higher level of transcript in the proximal segment. MIF is associated with two types of membranous vesicles, i.e. epididymosomes and prostasomes, the latter being prostate-originating membranous vesicles present in the semen. In semen, MIF is associated with spermatozoa, prostasomes as well as the soluble fraction. The amount of MIF in the seminal fluid varies from one individual to another but does not correlate with the amount of MIF associated with ejaculated spermatozoa. There is a negative correlation between the amount of sperm-associated MIF and the percentage of motility in different semen samples. Sperm separation using discontinuous Percoll gradient centrifugation shows a higher amount of MIF associated with poorly motile spermatozoa compared to highly motile spermatozoa present in the lower Percoll fraction. These results are discussed with regards to the possible involvement of MIF in sperm motility acquisition during the epididymal transit.

SummaryThere is already evidence that a few components of the haemostatic system exist in semen. If these comprise a functional system, they may have a role in seminal clotting and liquefaction processes and ultimately may influence fertility. What might be expected in semen as collected from fertility clinics i.e., after having both coagulated and subsequently liquefied is uncertain. It does however still contain significant amounts of Tissue Factor (TF) although its effect on semen quality remains poorly understood. The present study analyses semen for Tissue Factor Pathway Inhibitor (TFPI). Measurements were made in seminal plasma, swim-up sperm and prostasomes and its relationship with conventional fertility parameters assessed. TFPI antigen levels in seminal plasma were measured in a total of 176 subjects using an Enzyme-linked immunosorbent assay (ELISA). These include sub-fertile (n=37), normally fertile (n=40), fertile sperm donor (n=34), vasectomized subjects (n=65) and in a further group defined by normality in several parameters derived from the World Health Organization (WHO) fertility criteria and termed “pooled normal semen parameters” (PNSP). For characterization studies, both TFPI activity and antigen were measured on whole semen, swim-up sperm and prostasome-rich fraction (n=5). TFPI levels were significantly higher in normal men as compared to sub-fertile (P

Mammalian members of the cysteine-rich secretory protein (CRISP) family are expressed predominantly in the male reproductive tract and are implicated in the process of reproduction from spermiogenesis, posttesticular sperm maturation, and capacitation to oocyte-sperm fusion, and possibly also penetration of the zona pellucida. Rodents express only 2 CRISPs (CRISP-1 and CRISP-2) in their male reproductive system, whereas humans and horses express an additional third member named CRISP-3. We have previously demonstrated that this protein is present in human seminal plasma as well as in other exocrine secretions, in blood plasma, and in neutrophilic granulocytes. To characterize the protein in seminal plasma and localize the production of CRISP-3 in the human male reproductive tract, we performed immunoblotting and enzyme-linked immunosorbent assay measurements of seminal plasma and immunohistochemistry and in situ hybridization of tissue specimens. We were able to show that human CRISP-3 is a quantitatively minor seminal plasma protein not associated with prostasomes. Furthermore, CRISP-3 expression was found in the secretory epithelium throughout the male genital tract, with particularly high expression in the cauda epididymis and ampulla vas deferens. Examination of seminal plasma from vasectomized males indicates that organs downstream of the epididymis are probably the major sources of seminal plasma CRISP-3.

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.

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.

Prostasomes are extracellularly occurring submicron, membrane-surrounded organelles produced by the epithelial cells of the prostate and present in semen. Their precise physiological role is not known, although some of their properties assign them to important physiological and patho-physiological functions. In this thesis, some new properties of seminal and malignant cell line (DU145, PC-3 and LNCaP) prostasomes have been identified. Differences in the expressions and activities of prostasomal CD59, ATPase, protein kinases and tissue factor (TF) have been characterized. The transfer of prostasomal CD59 to CD59-deficient erythrocytes (rabbit and human PNH erythrocytes) has been established. CD59, protein kinases and TF were overexpressed by malignant cell prostasomes. ATPase activity was highest on seminal prostasomes with minimal expression by malignant cell prostasomes resulting in more residual ATP available for phosphorylation reactions. Several proteins were phosphorylated by prostasomal protein kinases, viz. complement component C3, fibrinogen, vitronectin and E-cadherin. Furthermore, TF was identified as the main endogenous phosphorylation substrate on prostasomes. In addition, prothrombotic effects of prostasomes were established. DU145 and PC-3-derived prostasomes exerted a higher clotting effect on whole blood and plasma compared to LNCaP and seminal prostasomes. In conclusion, malignant cell prostasomes showed higher ability to interact with the biological system in favor of prostate cancer cell promotion and survival. The roles played by prostasomes in this context may improve the understanding of the mechanisms that help the prostate cancer cells to avoid the complement attack (CD59 transfer and phosphorylation of C3), to promote angiogenesis (TF) and to metastasize. It may also provide a better understanding of some of the complications usually seen in some terminal prostate cancer patients like thrombotic events and tendency to develop disseminated intravascular coagulation.

Prostasomes are membrane-bound secretory vesicles, in the nanometre diameter range, secreted by the prostatic ductal epithelium [1] into the lumen, where they form part of the ejaculate. Although known to have specific biological properties, their physiological role and overall significance remain far more debatable. There has been comparatively little written about these structures since their detection two decades ago, and this may help explain the relative lack of awareness of prostasomes within the urological community.

MAL, BENE and MAL2 are raft-associated integral membrane proteins of the MAL family of proteins involved in membrane trafficking processes. We show here that the human prostate carcinoma PC-3 cell line expresses the transcripts for the three proteins simultaneously. MAL, BENE and MAL2 co-fractionated with caveolin-1 in the raft fraction of PC-3 cells, and immunofluorescence analysis showed colocalization of these proteins with caveolin-1 in a multivesicular intracellular compartment. Markers of the Golgi apparatus, early and recycling endosomes and lipid droplets were excluded from this compartment. Prostate epithelial cells contain vesicular organelles enriched in raft components named prostasomes that are secreted in the prostate fluid. Interestingly, the prostasome fraction isolated from the culture supernatant of PC-3 cells consisted mainly of 30-130 nm cup-shaped vesicles that were positive for MAL, caveolin-1 and CD59, a glycosylphosphatidylinositol-anchored protein previously found in prostasomes. CD63, an integral membrane protein found in multivesicular bodies/lysosomes and secretory granules was also found in PC-3 cell-derived prostasomes. Prostasome secretion was not inhibited by brefeldin A, a compound that blocks the conventional secretory pathway. However, wortmannin, an inhibitor of phosphatidylinositol-3 kinase, reduced the secretion of prostasomes in PC-3 cells. Our results suggest that MAL family proteins are associated with caveolin-1 in a multivesicular compartment that may be involved in prostasomal secretion in PC-3 cells.

SummarySemen contains enzymes and inhibitors of the haemostatic system as well as the high molecular weight seminal vesicle (HMW-SV) proteins. The former may have roles in seminal clotting and in liquefaction through “fibrinolytic” activity, which may ultimately affect fertility. Although a limited number of studies have addressed the subject, the role of clotting and fibrinolytic factors in semen remains poorly understood. The liquefaction time and the distribution of components vary across split ejaculates. This may have an important bearing on the way clotting/fibrinolytic factors in semen are assessed. Semen contains tissue factor (TF, Thromboplastin, CD142), which originates from the prostate and is associated with prostasomes. The function of TF (and prostasomes) in semen is still a matter for speculation. Recently the presence of minute amounts of factor VII in semen has been demonstrated but its importance is uncertain. Semen also contains a thrombin-like enzyme, prothrombin fragments 1 and 2 (F1+2), D-dimer (DD) and thrombin-antithrombin (TAT) complexes. The presence of several fibrinolytic factors has been demonstrated in semen but few questions about their potential impact on semen quality have been raised. Factors found include tissue plasminogen activator (t-PA), urinary plasminogen activator (u-PA) and plasmin. There are also traces of fibrinogen, plasminogen, plasminogen activator inhibitor-1 (PAI-1), factorVIII coagulant activity (VIII:c) and fibrin monomers. The co-ordinate expression of both TF and PAI-1 by decidual cells of the endometrium is believed to be important in maintaining haemostasis during endovascular trophoblast invasion. Kallikrein-like serine protease inhibitors including prostate specific antigen (PSA) are known to be present in semen at high concentrations. In semen PSA is also found in a complex form with protein C inhibitor (PCI) with mutually inhibitory consequences. A better understanding of the spectrum of coagulating and liquefaction agents in semen to include classical haemostatic processes and the pathogenesis resulting from any imbalances between or within either system may provide the basis for the development of more selective and efficient agents affecting global fertility. Here we review aspects of male reproductive physiology in the light of recent findings concerning conventional clotting/fibrinolytic systems in human semen with a view to stimulating further research.

Prostasomes are small vesicles of prostatic origin contained in human semen. Their composition is peculiar under many aspects. Cholesterol is abundant and many proteins are endowed with enzymatic or other activities. The function of prostasomes has been amply debated and several hypotheses have been put forward. The liquefaction of semen, spermatozoa motility, antibacterial activity and immunological functions have been related to prostasomes. Under certain aspects, prostasomes resemble synaptosomes. The fusion of prostasomes to spermatozoa enriches spermatozoa with cholesterol and causes bursts of cytoplasmic sperm calcium. The interaction of spermatozoa and prostasomes should be limited to vagina since prostasomes are immobile and do not follow spermatozoa in the superior female genital tract. Calcium bursts would increase spermatozoa motility, where cholesterol would decapacitate spermatozoa, so preventing untimely activation. Since spermatozoa receive many different molecules from prostasomes, additional effects are also possible. Prostasomes makes spermatozoa more apt to be activated by progesterone in the proximity of the ovum. Therefore, the fusion between spermatozoa and prostasomes would influence spermatozoa behaviour under many aspects and might be relevant for fecundation. The richness of molecular species in prostasomes is amazing and these small vesicles are expected to lead to many more discoveries in the field of human reproduction.

Cytochrome P450 4F12 (CYP4F12) was originally cloned from human liver and small intestine. CYP4F12 can oxidize arachidonic acid, two stable prostaglandin H2 analogues, and an antihistamine, ebastine, but the tissue distribution and catalytic properties of CYP4F12 have not been fully investigated. An antipeptide polyclonal antibody was raised against the C-terminal of CYP4F12 (PLNVGLQ), evaluated by Western blot analysis and used for immunohistological analysis of 50 human tissues. Western blot analysis of recombinant CYP4F12, expressed in yeast, and microsomal proteins from adult and foetal liver, kidney, placenta at term, seminal vesicles, the prostate gland and purified prostasomes showed that the polyclonal antibody detected a protein of the expected size, approximately 60 kDa. CYP4F12 mRNA could be detected in seminal vesicles and prostate gland by reverse transcription-PCR. Prominent CYP4F12 immunoreactivity occurred, inter alia, in the epithelial cells of the gastrointestinal tract (stomach, small intestine, and colon), collecting tubules, transitional epithelium, ovarian follicles, the endothelium of microvessels of placental villi (first trimester), and epidermis. We screened recombinant CYP4F12 for catalytic activity. Arachidonic acid (20:4n-6) was hydroxylated at C18 and laurate at C11, but significant amounts of metabolites of 18:2n-6, 20:3n-9, 20:5n-3, 22:5n-6, and some prostaglandins could not be detected. We conclude that CYP4F12 is widely distributed in gastrointestinal and urogenital epithelia and exhibits a narrow substrate specificity.

Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using beta-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia.

The lipid composition of germ cell membranes is considerably modified during spermatogenesis, sperm maturation and capacitation. Some of these modifications are caused by exchanges between soluble lipid donors or acceptors and cell membranes. The aim of this study was to assess whether significant lipid transfers between lipoprotein structures are detectable in human seminal plasma. Phospholipid and cholesteryl ester (CE) transfer activities were measured by specific fluorescence and isotopic assays. Seminal plasma samples did not display significant CE transfer. Substantial levels of phospholipid transfer activity were detected in all samples studied, levels were approximately 25% of the phospholipid transfer activity measured in human blood plasma. Concordantly, CE transfer protein was not detected in seminal plasma, while the presence of the phospholipid transfer protein (PLTP) was confirmed by Western blot analysis. Enzyme-linked immunosorbent assay indicated that seminal PLTP concentrations represented 25% of the concentration measured in blood plasma. Blockade of phosphatidylcholine and phosphatidyl-ethanolamine transfer by a 60 min, 56 degrees C heating step or with anti-PLTP antibody revealed that PLTP accounts for almost 80% of the phospholipid transfer activity present in seminal plasma. As shown by gel-permeation chromatography and Western blot analysis, seminal PLTP activity was partially associated with prostasomes. Significantly higher PLTP activity levels were measured in seminal plasma samples with low seminal vesicle secretions. The latter observation may reflect the sustained secretion of active PLTP that is diluted in a variable volume of PLTP-free seminal vesicle secretion. In conclusion, human seminal plasma displays significant phospholipid transfer activity due to the presence of active PLTP.

BACKGROUND Prostasomes are secretory particles in human seminal fluid. Other than a microscopic description of these secretory particles and an incomplete two–dimensional gel electrophoresis (2DE) study, little is known about the composition of proteins in prostasomes. METHODS We employed a direct iterative approach using Gas phase fractionation and microcapillary HPLC-tandem mass spectrometry (μLC-MS/MS) to catalogue the prostasome proteome. RESULTS We identified 139 proteins that can be divided into the following categories: (1) enzymes (33.8% of total), (2) transport/structural (19.4% of total), (3) GTP proteins (14.4% of total), (4) chaperone proteins (5.8% of total), (5) signal transduction proteins (17.3% of total), and (6) unannotated proteins (9.4% of total). A total of 128 of the 139 proteins have not previously been described as prostasomal. CONCLUSIONS The proteins identified can be used as reference dataset in future work comparing prostasome proteins between normal and pathological states such as prostate cancer, benign prostatic hyperplasia, prostatitis, and infertility. Prostate 56: 150–161, 2003. © 2003 Wiley-Liss, Inc.

We used atomic force/lateral force microscopy (AFM/LFM) and the transfer of a lipophilic fluorescent dye (octadecylrhodamine G, R 1 8 ) to study the morphology and fusion of human prostasomes at pH values ranging between 5and 8. The AFM data revealed that the mean diameter of untreated prostasomal vesicles was about 200 nm, but a second prostasome population with a diameter of about 400 nm is also present. This second population increased upon exposing the vesicles to low pH values. The AFM/LFM characterization revealed the smoothness of the prostasome surface and showed that the larger vesicles were fused prostasomes, as also indicated by the relief of R 1 8 self-quenching.

Objective To determine the effect of the fusion of prostasomes to spermatozoa on the acrosome reaction. Design In vitro study of human spermatozoa. Setting Healthy volunteers in an academic research environment. Patient(s) Healthy volunteer men, 25 to 35 years old. Intervention(s) Human semen was fractionated into spermatozoa and prostasomes. Fusion of prostasome to spermatozoa was performed at pH 5.5. Progesterone (1 μM) was added when required. Main outcome measure(s) Evaluation of the acrosome reaction by fluorescence microscopy. Result(s) The percentage of spontaneously acrosome-reacted cells was very low unless the Ca 2+ -ionophore A 23187 was added. The treatment of spermatozoa with 1 μM of progesterone scarcely affected the acrosome reaction; a pretreatment in conditions permitting fusion increased it. The addition of progesterone to prostasome-fused spermatozoa further increased the extent of the acrosome reaction. Conclusion(s) The H + -dependent fusion with prostasomes makes spermatozoa more sensitive to the effect of progesterone on acrosome-reaction induction.