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Objective To review the role of extracellular vesicles (EVs) released from the male reproductive tract and their impact on developing sperm. We discuss how sperm exiting the seminiferous tubules, although developmentally mature, require further modification. Acquisition of various functions including increased motility, transfer of cargoes and ability to undertake the acrosome reaction is mediated through the interaction between sperm and EVs. Methods A review of the literature identified that EVs are released from different portions of the male reproductive tract, notably the epididymis and prostate. These EVs interact with sperm as they pass from the seminiferous tubules to the epididymis and vas deferens prior to ejaculation. Results EVs are small lipid-bound particles carrying bespoke RNA, protein and lipid cargoes. These cargoes are loaded based on the state of the parent cell and are used to communicate with recipient cells. In sperm, these cargoes are essential for post-testicular modification. Conclusions Interactions between developing sperm and EVs are important for the subsequent function of sperm. Prior to ejaculation, these interactions confer important changes for the post-testicular modification and development of sperm. Little is known about the interaction between EVs from the testes and the spermatogonial stem cell niche or developing sperm within the seminiferous tubules. However, the numerous roles of EVs in the post-testicular modification of sperm have led many to suspect that they may also play important roles in developing sperm within the testes. Lay summary Sperm are crucial for successful fertility. In order to do this, they must be able to swim a large distance to meet the egg in the female reproductive tract and fertilise it. Once released from the testes, sperm may appear to be fully developed, but this is not the case. Several important modifications are required in order for them to swim and fertilise an egg. These modifications are carried out by sending sperm small packages from other cells which contain messages and cargo. We discuss the release of these small packages along with different parts of the male reproductive tract and how they change the way sperm behave and function. This article reviews the literature and known functions of these packages called extracellular vesicles, which are released by the male reproductive tract and modify sperm, transforming their function, before they are ejaculated.

Background:Exosomes are extracellular vesicles (EVs) released from cells upon fusion of an intermediate endocytic compartment with the plasma membrane. They refer to the intraluminal vesicles released from the fusion of multivesicular bodies with the plasma membrane. The contents and number of exosomes are related to diseases such as metabolic diseases, cancer and inflammatory diseases. Exosomes have been used in neurological research as a drug delivery tool and also as biomarkers for diseases. Recently, exosomes were observed in the seminal plasma of the one who is asthenozoospermia, which can affect sperm motility and capacitation.Objective:The main objective of this review is to deeply discuss the role of exosomes in spermatozoa after leaving the seminiferous tubule.Methods:We conducted an extensive search of the literature available on relationships between exosomes and exosomes in spermatozoa on the bibliographic database.Conclusion:: This review thoroughly discussed the role that exosomes play in the exchange of spermatozoa after leaving the seminiferous tubule and its potential as a drug delivery tool and biomarkers for diseases as well.

Background: Human seminal prostasomes are intrinsically heterogeneous extracellular vesicles (EVs) whose composition is, additionally, influenced by different physiological conditions. Aiming at the molecular properties of the prostasomal surface exemplified by glycan compositions as a possible distinction factor, we applied lectin-affinity chromatography (LAC) as a new tool for their separation. Since glycans, generally, exhibit various biological activities, introduction of glyco-parameters as reference could upgrade standardization of EVs isolated by different methods and intended for use in biomedicine. Methods: Preparations of seminal prostasomes from normozoospermic (sPro-N) and oligozoospermic (sPro-O) men were subjected to LAC on concanavalin A (Con A) and wheat germ agglutinin (WGA) columns. Prostasomes recovered in LAC-separated fractions were characterized according to the distribution of selected markers: gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), tetraspanin CD63, and total protein/glycoprotein composition. Results: Two CD63-immunoreactive populations exhibiting prostasome signature bands but differing in GGT activity and surface glycans were separated on the WGA column. Additional populations having distinct profiles of total glycoproteins and which can be tracked down by ALP activity were enriched on the Con A column. WGA-separated populations were similar in sPro-N and sPro-O, whereas Con A-separated ones were strikingly different. Conclusions: Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of Con A- and WGA-reactive glycans mark seminal prostasomes populations from normozoospermic and oligozoospermic men.

Background: Extracellular vesicles (EVs), released from the plasma membrane or intracellular compartments, have a specific composition related to their parent cells, but they can, additionally, be modified by the extracellular environment. Although glycans are known to contribute to EV composition and may have biomedical importance as biomarkers and recognition signals, they have not been extensively investigated. In this study, seminal prostasomes, i.e. EVs from seminal plasma (SP) of normo- and oligozoospermic men, were analyzed in order to detect possible changes in their surface glycans under altered physiological conditions. Methods: Prostasomes were isolated from pooled SP by differential centrifugation and gel filtration, followed by glycobiochemical characterization using lectin/immune-transmission microscopy and ion-exchange chromatography. Results: Within the frame of overall similarity in protein composition, surface glycans specifically contributed to the differences between the examined groups of prostasomes in terms of presentation of sialylated and mannosylated moieties. These changes did not affect their anti-oxidative capacity, but implied a possible influence on the accessibility of galectin-3 to its ligands on the prostasomal surface. Conclusions: Subtle differences in the presentation of surface molecules may be helpful for differentiation among vesicles sharing the same physical properties. In addition, this may point to some unexpected regulatory mechanisms of interaction of distinct populations of vesicles with their binding partners.

Background: Prostasomes, extracellular vesicles (EVs) abundantly present in seminal plasma, express distinct tetraspanins (TS) and galectin-3 (gal-3), which are supposed to shape their surface by an assembly of different molecular complexes. In this study, detergent-sensitivity patterns of membrane-associated prostasomal proteins were determined aiming at the solubilization signature as an intrinsic multimolecular marker and a new parameter suitable as a reference for the comparison of EVs populations in health and disease. Methods: Prostasomes were disrupted by Triton X-100 and analyzed by gel filtration under conditions that maintained complete solubilization. Redistribution of TS (CD63, CD9, and CD81), gal-3, gamma-glutamyltransferase (GGT), and distinct N-glycans was monitored using solid-phase lectin-binding assays, transmission electron microscopy, electrophoresis, and lectin blot. Results: Comparative data on prostasomes under normal physiology and conditions of low sperm count revealed similarity regarding the redistribution of distinct N-glycans and GGT, all presumed to be mainly part of the vesicle coat. In contrast to this, a greater difference was found in the redistribution of integral membrane proteins, exemplified by TS and gal-3. Accordingly, they were grouped into two molecular patterns mainly consisting of overlapped CD9/gal-3/wheat germ agglutinin-reactive glycoproteins and CD63/GGT/concanavalin A-reactive glycoproteins. Conclusions: Solubilization signature can be considered as an all-inclusive distinction factor regarding the surface properties of a particular vesicle since it reflects the status of the parent cell and the extracellular environment, both of which contribute to the composition of spatial membrane arrangements.

Prostasomes, a secretory particle from prostate gland in human seminal fluid plays a role in enhancing the fertility and its absence or less presence will lead to male infertility. Very few fertility associated proteins were detected in prostasomes. In order to isolate the prostasomes from ejaculated semen, the characterization was done using biochemical, molecular and

Extracellular vesicles (EVs) are small lipid bound structures released from cells containing bioactive cargoes. Both the type of cargo and amount loaded varies compared to that of the parent cell. The characterisation of EVs in cancers of the male urogenital tract has identified several cargoes with promising diagnostic and disease monitoring potential. EVs released by cancers of the male urogenital tract promote cell-to-cell communication, migration, cancer progression and manipulate the immune system promoting metastasis by evading the immune response.Their use as diagnostic biomarkers represents a new area of screening and disease detection, potentially reducing the need for invasive biopsies. Many validated EV cargoes have been found to have superior sensitivity and specificity than current diagnostic tools currently in use. The use of EVs to improve disease monitoring and develop novel therapeutics will enable clinicians to individualise patient management in the exciting era of personalised medicine.

Zinc (Zn), the second-most necessary trace element, is abundant in the human body. The human body lacks the capacity to store Zn; hence, the dietary intake of Zn is essential for various functions and metabolism. The uptake of Zn during its transport through the body is important for proper development of the three major accessory sex glands: the testis, epididymis, and prostate. It plays key roles in the initial stages of germ cell development and spermatogenesis, sperm cell development and maturation, ejaculation, liquefaction, the binding of spermatozoa and prostasomes, capacitation, and fertilization. The prostate releases more Zn into the seminal plasma during ejaculation, and it plays a significant role in sperm release and motility. During the maternal, labor, perinatal, and neonatal periods, the part of Zn is vital. The average dietary intake of Zn is in the range of 8–12 mg/day in developing countries during the maternal period. Globally, the dietary intake of Zn varies for pregnant and lactating mothers, but the average Zn intake is in the range of 9.6–11.2 mg/day. The absence of Zn and the consequences of this have been discussed using critical evidence. The events and functions of Zn related to successful fertilization have been summarized in detail. Briefly, our current review emphasizes the role of Zn at each stage of human reproduction, from the spermatogenesis process to childbirth. The role of Zn and its supplementation in in vitro fertilization (IVF) opens opportunities for future studies on reproductive biology.

Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein with glutamate carboxypeptidase activity. However, its precise function in the prostate, prostasomes and seminal plasma with regard to male fertility remains unknown. This study was conducted to investigate the seminal plasma PSMA levels in fertile men and patients with oligoasthenoteratozoospermia (OAT) and to analyse its association with sperm parameters. Twenty fertile men and twenty patients admitted at the urology clinic of our institution with the diagnosis of OAT were included in the study. Following semen analysis, seminal plasma was isolated from semen ejaculates. PSMA concentrations in the seminal plasma were determined by ELISA. The correlations between seminal PSMA concentrations and semen parameters were statistically analysed. Seminal plasma PSMA concentration was significantly lower in OAT patients compared to fertile controls (p < .01). In fertile men, PSMA concentration was significantly correlated with the sperm concentration (r = -.481, p < .05), whereas in the patient group no statistically significant correlation was found between the sperm parameters and seminal PSMA level. This is the first study in the literature to investigate PSMA levels in the seminal plasma from infertile men. Decreased levels of seminal plasma PSMA might suggest a role for compromised prostasome function in the pathogenesis of OAT syndrome.

Extracellular vesicles (EVs) are secreted cell-derived membrane structures present in all organisms across animal, bacterial, and plant phyla. These vesicles play important roles in cell-cell communication in many processes integral to health and disease. Recent studies demonstrate that EVs and their cargo have influential and conserved roles in male reproduction. While EVs have been isolated from virtually all specialized tissues comprising the male reproductive tract, they are best characterized in the epididymis (epididymosomes) and seminal fluid (seminal fluid extracellular vesicles or prostasomes). Broadly speaking, EVs promote reproductive success through supporting sperm development and function, as well as influencing the physiology of female reproductive tract cells after mating. In this review, we present current knowledge on the composition and function of male reproductive tract EV populations in both normal physiology and pathology, and argue that their functions identify them as critical regulators of fertility and fecundity.

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction-specific proteins that show demonstrable links to sperm fertility. Many of these membrane-bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.

Prostasomes are exosomes such as extracellular vesicles, produced in the prostatic epithelium and released into the seminal plasma, that play an important role enhancing male fertility. Although some studies have demonstrated that prostasomes have a rich proteomic content, it is still unclear if that proteomic content varies depending on the male fertility status. Prostasomes from 12 normozoospermic and 14 non-normozoospermic seminal samples were isolated by differential ultracentrifugation. Protein content was studied by quantitative mass spectrometry and compared between both cohorts. We identified 1282 proteins with 745 of them (57.8%) being present in all seven prostasome pools. Forty-seven of those commonly present proteins showed differential expression levels in both cohorts. Specifically, prostasomes from non-normozoospermic samples showed a pattern of protein underexpression for a group of proteins including several proteins from the spermatozoa's energy production pathways as well as some proteins directly implicated in sperm activity. Variations in prostasomal protein content levels may have a relevant correlation with male fertility and thus could be of great utility as a biomarker of fertility status.

Infertility has become a global health issue, with approximately 50% of infertility cases generated by disorders in male reproduction. Spermatozoa are conveyed towards female genital tracts in a safe surrounding provided by the seminal plasma. Interestingly, this dynamically changing medium is a rich source of proteins, essential not only for sperm transport, but also for its protection and maturation. Most of the seminal proteins are acquired by spermatozoa in transit through exosomes (epididymosomes and prostasomes). The high number of seminal proteins, the increasing knowledge of their origins and biological functions and their differential expression in the case of azoospermia, asthenozoospermia, oligozoospermia and teratozoospermia or other conditions of male infertility have allowed the identification of a wide variety of biomarker candidates and their involvement in biological pathways, thus to strongly suggest that the proteomic landscape of seminal plasma may be a potential indicator of sperm dysfunction. This review summarizes the current knowledge in seminal plasma proteomics and its potentiality as a diagnostic tool in different degrees of male infertility

Extracellular vesicles (EVs) are gaining attention among the cell biologists and researchers over the last two decades. Prostasomes are considered to be (Evs) secreted by prostate epithelial cells into the semen during emission or ejaculation. Prostasomes contain various proteins required for immune regulation namely, amino and dipeptidyl peptidase; endopeptidase (neutral); decay accelerating factor; angiotensin-converting enzyme. Sperm cells need a few prerequisites in order to fertilize the egg. The role of prostasomes in enhancing the male fertility was reviewed extensively throughout the manuscript. Also, prostasomes have an immunosuppressive, immunomodulatory, antibacterial role in the female reproductive tract, and in some cases they can be used as immunocontraceptive agent to regulate the fertility status. This review will give insights to many active researchers in the field of prostasomal research and male infertility/fertility research. This review will open many unanswered mechanisms of prostasomes with respect to structure-function analysis, fatty acids patterns in diagnosis as well as prognosis of male infertility/fertility. More scientific reports are in need to support the mechanism of prostasomes and its role in immunomodulation. The development of prostasomes as a biomarker for the prostate cancer is still miserable with a lot of controversial results by various researchers.

// Cristiana Pistol Tanase 1,2 , Elena Codrici 1 , Ionela Daniela Popescu 1 , Simona Mihai 1 , Ana-Maria Enciu 1,3 , Laura Georgiana Necula 1,4 , Adrian Preda 5 , Gener Ismail 6,8 and Radu Albulescu 1,7 1 Department of Biochemistry-Proteomics, Victor Babes National Institute of Pathology, Bucharest, Romania 2 Titu Maiorescu University, Faculty of Medicine, Bucharest, Romania 3 Department of Cell Biology and Histology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania 4 Stefan S Nicolau Institute of Virology, Bucharest, Romania 5 Center for Uronephrology and Renal Transplantation, Fundeni Clinical Institute, Bucharest, Romania 6 Center of Internal Medicine-Nephrology, Fundeni Clinical Institute, Bucharest, Romania 7 National Institute for Chemical Pharmaceutical R&D, Bucharest, Romania 8 Carol Davila University of Medicine and Pharmacy, Faculty of Medicine, Bucharest, Romania Correspondence to: Cristiana Tanase, email: // Keywords : prostate cancer, biomarkers, proteomics, mass spectrometry, prostasomes Received : July 29, 2016 Accepted : December 27, 2016 Published : January 04, 2017 Abstract The clinical and fundamental research in prostate cancer - the most common urological cancer in men - is currently entering the proteomic and genomic era. The focus has switched from one single marker (PSA) to panels of biomarkers (including proteins involved in ribosomal function and heat shock proteins). Novel genetic markers (such as Transmembrane protease serine 2 (TMPRSS2)-ERG fusion gene mRNA) or prostate cancer gene 3 (PCA3) had already entered the clinical practice, raising the question whether subsequent protein changes impact the evolution of the disease and the response to treatment. Proteomic technologies such as MALDI-MS, SELDI-MS, i-TRAQ allow a qualitative/quantitative analysis of the proteome variations, in both serum and tumor tissue. A new trend in prostate cancer research is proteomic analysis of prostasomes (prostate-specific exosomes), for the discovery of new biomarkers. This paper provides an update of novel clinical tests used in research and clinical diagnostic, as well as of potential tissue or fluid biomarkers provided by extensive proteomic research data.

The study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI, we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying ∼2,800 individual proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which likely are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure, i.e. diminished abundance of metabolic enzymes related to ATP turnover and those secreted via prostasomes. Further we identify the main outcome related to this fact and that it is intrinsically linked to the low sperm motility in SCI. Together, our data highlights the molecular pathways hindering fertility in SCI and shed new light on other causes of male infertility.

Body fluids contain many populations of extracellular vesicles (EV) that differ in size, cellular origin, molecular composition, and biological activities. EV in seminal plasma are in majority originating from prostate epithelial cells, and hence are also referred to as prostasomes. Nevertheless, EV are also contributed by other accessory sex glands, as well as by the testis and epididymis. In a previous study, we isolated EV from seminal plasma of vasectomized men, thereby excluding contributions from the testis and epididymis, and identified two distinct EV populations with diameters of 50 and 100 nm, respectively. In the current study, we comprehensively analyzed the protein composition of these two EV populations using quantitative Liquid Chromatography-Mass Spectrometry (LC-MS/MS). In total 1558 proteins were identified. Of these, &asymp, 45% was found only in the isolated 100 nm EV, 1% only in the isolated 50 nm EV, and 54% in both 100 nm and 50 nm EV. Gene ontology (GO) enrichment analysis suggest that both originate from the prostate, but with distinct biogenesis pathways. Finally, nine proteins, including KLK3, KLK2, MSMB, NEFH, PSCA, PABPC1, TGM4, ALOX15B, and ANO7, with known prostate specific expression and alternate expression levels in prostate cancer tissue were identified. These data have potential for the discovery of EV associated prostate cancer biomarkers in blood.

The structure and function of normal human prostate is still not fully understood. Herein, we concentrate on the different cell types present in normal prostate, describing some previously unreported types and provide evidence that prostasomes are primarily produced by apocrine secretion. Patients (n = 10) undergoing TURP were prospectively consented based on their having a low risk of harbouring CaP. Scanning electron microscopy and transmission electron microscopy was used to characterise cell types and modes of secretion. Zinc levels were determined using Inductively Coupled Plasma Mass Spectrometry. Although merocrine secretory cells were noted, the majority of secretory cells appear to be apocrine; for the first time, we clearly show high-resolution images of the stages of aposome secretion in human prostate. We also report a previously undescribed type of epithelial cell and the first ultrastructural image of wrapping cells in human prostate stroma. The zinc levels in the tissues examined were uniformly high and X-ray microanalysis detected zinc in merocrine cells but not in prostasomes. We conclude that a significant proportion of prostasomes, possibly the majority, are generated via apocrine secretion. This finding provides an explanation as to why so many large proteins, without a signal peptide sequence, are present in the prostatic fluid.

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. Fil: Munuce, María José. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina Fil: Marini, Patricia Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Teijeiro, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Medicina Reproductiva; Argentina

Seminal plasma (SP) is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles (EVs). The SP interacts with spermatozoa and the inner cell lining of the female genital tract, adsorbing proteins and exosomes that modulate sperm functions and female immune responsiveness. In the present study, boar sperm-free SP was studied using flow cytometry (FC) after membrane tetraspanins (CD9, CD63 and CD81) and membrane receptor CD44 marking of non-enriched (whole SP) or gradient fractions enriched through two-step discontinuous KBr-density-gradient ultracentrifugation, in whole ejaculate or in selected ejaculate fractions. The results, evaluated by transmission electron microscopy, confirmed the presence of exosomes in all fractions of the pig SP. Noteworthy, these pig SP-exosomes were CD44-bearing when analysed by FC, with bands detected by western blotting (WB) at the expected 85 kD size. The two-step discontinuous KBr-density-gradient ultracentrifugation enriched the population of exosomes in two specific gradient fractions, indicating exosomes (either prostasomes or epididymosomes) could be separated from low-density lipoprotein (LDL) but they co-sediment with the high-density lipoprotein (HDL)-bearing fraction. The findings pave for the selective isolation of exosomes in functional studies of their function when interacting with spermatozoa, the oocyte and/or the female genitalia, including hyaluronan-CD44 interplay. (C) 2019 Elsevier Inc. All rights reserved. Funding Agencies|ALF-Research (Region Ostergotland) [LIO-698951]; FORSS (Forskningsradet i Sydostra Sverige) [473121, 745971]; Lions Forskningsfond [DNR LIU-2016-00641]; Swedish Research Council FORMAS, Stockholm, Sweden [2017-00946]

Background The ‘omics’ approach for a noninvasive diagnosis of male reproductive system disorders has gained momentum during the last decade, particularly from a screening and prognosis point of view. Due to the rapid development in assisted reproductive technologies (ART) over the years, the major focus of proteomic studies has been around the ejaculated spermatozoa. Although seminal plasma is not a requirement for ART, the question arose whether the role of seminal plasma is merely to transport spermatozoa. Main body Seminal plasma (SP) contains a large diversity of proteins that are essential not only for sperm transport, but also for sperm protection and maturation. Most of the proteins bind to sperm surface through exosomes (epididymosomes and prostasomes), modulating sperm function, interaction with the female reproductive tract and finally fertilization. This review focuses on the state-of-art discoveries regarding SP proteome and its role in fertilization. Conclusion Tissue-specific proteins in the SP have emerged as fundamental contributors for protein biomarker discovery. This is important for a noninvasive diagnosis of male infertility and development of new therapeutic approaches. Moreover, ART success rates may be improved by taking into account the critical role of seminal proteome in fertilization.

Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13–1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163.

Contents Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow – Zn2 + showed that from 93 to 100% of the prostasome proteins bind zinc ions (P+Zn). SDS-PAGE revealed that canine P+Zn comprised four protein bands, with low molecular weights (10.2–12 kDa). We have also shown a positive effect of prostasomes (p

Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles.We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins.A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles.Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers.

Aminopeptidase N (APN) is a naturally occurring ectopeptidase present in mammalian semen. Previous studies have demonstrated that APN adversely affects male fertility through the alteration of sperm motility. This enzyme constitutes 0.5 to 1% of the seminal plasma proteins, which can be transferred from the prostasomes to sperms by a fusion process. In the present study, we investigated the molecular mechanism of action of APN and its role in regulating sperm functions and male fertility. In this in vitro study, epididymal mouse spermatozoa were incubated in a capacitating media (pH 7) containing 20 ng/mL of recombinant mouse APN for 90 min. Our results demonstrated that the supplementation of recombinant APN in sperm culture medium significantly increased APN activity, and subsequently altered motility, hyperactivated motility, rapid and medium swimming speeds, viability, and the acrosome reaction of mouse spermatozoa. These effects were potentially caused by increased toxicity in the spermatozoa. Further, altered APN activity in sperm culture medium affected early embryonic development. Interestingly, the effect of elevated APN activity in sperm culture medium was independent of protein tyrosine phosphorylation and protein kinase A activity. On the basis of these results, we concluded that APN plays a significant role in the regulation of several sperm functions and early embryonic development. In addition, increased APN activity could potentially lead to several adverse consequences related to male fertility.

Background Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called “storage vesicle” equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization. Methods Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species. Results The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10–150 kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes. Conclusion These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum. General significance This study unravels energy metabolic relationships of prostasomes from four different species.

Summary Galectin-3 is a multifunctional carbohydrate-binding protein that was previously characterized as a proteolytic substrate for prostate-specific antigen (PSA) and was shown to be associated with prostasomes in human semen. Prostasomes are exosome-like vesicles that are secreted by the prostatic epithelium and have multiple proposed functions in normal reproduction and prostate cancer. In the current study, galectin-3 binding ligands in human prostasomes were identified and characterized with the goal to investigate galectin-3 function in prostasomes. Galectin-3 binding proteins were isolated by affinity column chromatography. Candidate ligands identified by MS/MS were PSA, prostatic acid phosphatase (PAP), zinc alpha-2-glycoprotein (ZAG), dipeptidyl peptidase-4 (CD26), aminopeptidase N (CD13), neprilysin, clusterin, antibacterial protein (FALL-39) and alpha-1-acid glycoprotein (ORM1). Biochemical methods were used to characterize the ability of galectin-3 to bind to selected ligands, and galectin-3 cleavage assays were utilized to investigate the protease(s) in prostasomes that cleaves galectin-3. CD26, CD13, PSA, PAP and ZAG immunoreactivity were detected in extracts of purified prostasomes. One-dimensional electroblot analysis of prostasomes demonstrated that CD26, PAP and CD13 immunoreactivity co-migrated with galectin-3-reactive protein bands. PSA and ZAG were found to be associated with the surface of prostasomes. Both intact and cleaved galectin-3 were detected in prostate and prostasome extracts. Cleavage and inhibition assays indicated that PSA in prostasomes proteolytically cleaves galectin-3. The identification of these glycoproteins as galectin-3 ligands lays the groundwork for future studies of galectin-3 and prostasome function in reproduction and prostate cancer.

Recent studies indicate that various types of vesicles, like microparticles (MP) and exosomes, are present in blood, saliva, bone marrow, urine and synovial fluid. These vesicles, which are released upon activation or shear stress, are thought to play a role in coagulation, neovascularisation, inflammation and intercellular signalling. Seminal fluid is a cell-, sperm- and protein-rich suspension. Although seminal fluid is known to contain vesicles like prostasomes, MP and exosomes have never been characterised. Therefore, the aim of our study was to analyse and characterise vesicles in seminal fluid in male partners of patients undergoing controlled ovarian stimulation for IVF/ICSI. MP from seminal fluid of patients during routine IVF/ICSI procedures were detected and analysed with flow cytometry (FACS) and transmission electron microscopy (TEM), using antibodies against tissue factor (TF), CD10, CD13, CD26 and annexin V. The coagulant properties of vesicles were studied using a fibrin generation test. MP were detected in human seminal fluid by both flow cytometry and TEM. Seminal fluid-derived MP expressed CD10, CD13, CD26 and TF, which was highly procoagulant and a powerful trigger of the extrinsic pathway of coagulation. The extent to which the procoagulant activity of MP in seminal fluid contributes to the implantation process itself and therefore affects human reproduction needs to be further elucidated.

Background Angiogenesis is the formation of new blood vessels by capillary sprouting from pre-existing vessels. This process is associated with increased expression of angiogenic factors like vascular endothelial growth factor (VEGF). The VEGF family consists of five members denoted VEGF-A, B, C, D and placenta growth factor (PlGF). Prostasomes are exosome-like extracellular vesicles existing in seminal plasma. The present study aimed at investigating the possible relationship between VEGF-A in seminal fluid and blood plasma and the prostasomal association of VEGF-A. Methods Measurement of VEGF-A concentrations was carried out in seminal plasma from 40 males and in blood plasma from 40 male blood donors utilizing commercial ELISA kits. The prostasomal association of VEGF-A was investigated by flow cytometry. Results We found highly elevated concentrations of VEGF-A in seminal fluid (median value 150000 pg/mL) compared with those of blood plasma. Flow cytometric analysis showed that VEGF-A is bound to the surface of prostasomes. Conclusions Prostasomes and seminal plasma contain the angiogenic factor VEGF-A in high concentrations exceeding that of blood plasma by 1000 times.

New biomarkers are needed to improve the diagnosis of prostate cancer. Similarly to healthy cells, prostate epithelial cancer cells produce extracellular vesicles (prostasomes) that can be isolated from seminal fluid, urine, and blood. Prostasomes contain ubiquitously expressed and prostate-specific membrane and cytosolic proteins, as well as RNA. Both quantitative and qualitative changes in protein, mRNA, long noncoding RNA, and microRNA composition of extracellular vesicles isolated from prostate cancer patients have been reported. In general, however, the identified extracellular vesicle-associated single-marker molecules or combinations of marker molecules require confirmation in large cohorts of patients to validate their specificity and sensitivity as prostate cancer markers. Complications include variable factors such as prostate manipulation and urine flux, as well as masking by ubiquitously expressed free molecules and extracellular vesicles from tissues other than the prostate. Herein, we propose that the most promising methods include comprehensive combinational screening for (mutant) RNA in prostasomes that are immunoisolated with antibodies targeting prostate-specific epitopes.

Semen is a heterogenous and complex fluid with different functions, some of them well known, others still obscure. The aim of this study was to investigate the presence of cathepsins B, S and L in human seminal plasma and their possible associations with other semen variables. Cathepsin B, L and S concentrations were measured in seminal plasma from 99 men utilising commercial ELISA kits. Seminal plasma cathepsin B was approximately 70 times higher, while the cathepsin L values were approximately 500 times higher and the cathepsin S values approximately 40 times higher in seminal plasma than in a group of serum samples. The study shows that seminal plasma contains high levels of cathepsins B, L and S. All three cathepsins were also bound to the surface of prostasomes.

Context: Although progress has been made with regard to types of markers (protein, DNA, RNA, and metabolites) and implementation of improved technologies (mass spectrometry, arrays, and deep sequencing), the discovery of novel biomarkers for prostate cancer (PCa) in complex fluids, such as serum and urine, remains a challenge. Meanwhile, recent studies have reported that many cancer-derived proteins and RNAs are secreted through small vesicles known as exosomes. Objective: This narrative review describes recent progress in exosome research, focusing on the potential role of exosomes as novel biomarkers for PCa. The purpose of this review is to acquaint clinicians and researchers in the field of urology with the potential role of exosomes as biomarker treasure chests and with their clinical value. Evidence acquisition: Medline and Embase entries between 1966 and September 2010 were searched using the keywords exosomes, microvesicles, prostasomes, biomarkers, prostate cancer, and urology. Leading publications and articles constructively contributing to exosome research were selected for this review. Evidence synthesis: Exosomes are small vesicles (50-100 nm) secreted by almost all tissues; they represent their tissue origin. Purification of prostate- and PCa-derived exosomes will allow us to profile exosomes, providing a promising source of protein and RNA biomarkers for PCa. This profiling will contribute to the discovery of novel markers for the early diagnosis and reliable prognosis of PCa. Conclusions: Although the initial results are promising, further investigations are required to assess the clinical value of these exosomes in PCa. (C) 2010 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Prostasomes, vesicles present in human semen, are known to play a role in male fertility. However, the mechanisms involved are still poorly understood. The present study looks at the direct influence of different concentrations of prostasomes on human sperm function in conditions supporting capacitation in vitro. Five million Percoll selected spermatozoa were incubated for 3 h at 37°C under an atmosphere of 5% CO₂ in air, in 100 µl Biggers Whitten Whittingham's medium (BWW) containing polyvinyl alcohol (PVA, 1 mg/ml) and bovine serum albumin (BSA; 3 mg/ml) in the absence or presence of increasing concentrations of prostasomes (expressed in terms of their cholesterol content: 15; 30; 45 nmoles per 100 µL of incubation medium). After in vitro exposure of spermatozoa to prostasomes, our data indicate that i) tyrosine phosphorylation intensity of the 107 KDa protein band was dose dependently lower and ii) the percentage of viable and progressive motile spermatozoa was unchanged and the percentage of non-progressive motility decreased. In addition, the incubation of prostasomes with spermatozoa resulted in an enrichment of their lipid content. Our experiments suggest that adhesion of prostasomes to spermatozoa could be responsible for the decrease in Tyrosine phosphorylation and the alteration of the mean curvilinear velocity (VCL) and the average path velocity (VAP).

Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (≤6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

Background Prostasomes are suggested to be produced in the prostate gland. Although biochemical studies support this, some immunohistochemical findings indicate that also the seminal vesicles could be a source of prostasomes. Therefore, we have compared the secretion of the vesicles with that of the prostate using biochemical and ultrastructural techniques. Methods Ultracentrifuged pellets of substance from seminal vesicle secretions were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and flow cytometry. The secretory cells of the seminal vesicles were examined with transmission electron microscopy. These findings were then compared with published results from similar studies of the prostate secretory cells. Results In SDS-PAGE, the seminal vesicle pellets lacked the three prostasome-characteristic CD-markers, namely CD10, CD13, and CD26, but expressed two proteins of about 55 kDa and 70 kDa, corresponding to clusterin and heat shock protein (HSP70). Flow cytometry showed the presence of secretion particles in the seminal pellet, although of a smaller size than that of the prostasomes. Electron microscopy of the luminal part of the cells in the seminal vesicles demonstrated many secretion granules, each enclosed in a vesicle with a size of about 1 μm. Conclusions Pelleted seminal vesicle secretion is different to prostate secretion in several ways. No prostasome characteristics were detected in the pelleted seminal vesicle secretion.

Galectin-3 is a beta-galactoside-binding protein involved in immunomodulation, cell interactions, cancer progression, and pathogenesis of infectious organisms. We report the identification and characterization of galectin-3 in human semen. In the male reproductive tract, the approximately 30 kDa galectin-3 protein was identified in testis, epididymis, vas deferens, prostate, seminal vesicle, and sperm protein extracts. In seminal plasma, galectin-3 was identified in the soluble fraction and in prostasomes, cholesterol-rich, membranous vesicles that are secreted by the prostate and incorporated into seminal plasma during ejaculation. Two-dimensional immunoblot analysis of purified prostasomes identified five galectin-3 isoelectric variants with a pI range of 7.0 to 9.2. Affinity purification and tandem mass spectrometry of beta-galactoside-binding proteins from prostasomes confirmed the presence of galectin-3 in prostasomes and identified a truncated galectin-3 variant. The intact galectin-3 molecule contains a carbohydrate recognition domain and a non-lectin domain that interacts with protein and lipid moieties. The identification of a monovalent galectin-3 fragment with conserved carbohydrate-binding activity indicates the functional relevance of this truncation and suggests a regulatory mechanism for galectin-3 in prostasomes. Surface biotinylation studies suggested that galectin-3 and the truncated galectin-3 variant are localized to the prostasome surface. Prostasomes are proposed to function in immunosuppression and regulation of sperm function in the female reproductive tract, are implicated in facilitating sexually-transmitted infections, and are indicated in prostate cancer progression. Given the overlap in functional significance, the identification of galectin-3 in prostasomes lays the groundwork for future studies of galectin-3 and prostasomes in reproduction, disease transmission, and cancer progression.

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.

Prostate cancer is the second leading cause of cancer death in men, and early detection is essential to reduce mortality and increase survival. delta-Catenin is a unique beta-catenin superfamily protein primarily expressed in the brain but is upregulated in human prostatic adenocarcinomas. Despite its close correlation with the disease, it is unclear whether delta-catenin presents the potential in prostate cancer screening because it is an intracellular protein. In this study, we investigated the hypothesis of delta-catenin accumulation in the urine of prostate cancer patients and its potential pathways of excretion into extracellular milieu.Prostate cancer cell cultures, human tissue biopsies, and voided urines were characterized to determine extracellular delta-catenin accumulation and co-isolation with exosomes/prostasomes.We identified delta-catenin in culture media and in the stroma of human prostate cancer tissues. In PC-3 cells in culture, delta-catenin was partially co-localized and co-isolated with raft-associated membrane protein caveolin-1 and glycosylphosphatidylinositol-anchored protein CD59, suggesting its potential excretion into extracellular milieu through exosome/prostasome associated pathways. Interference with endocytic pathway using wortmannin did not block prostasome excretion, but delta-catenin overexpression promoted the extracellular accumulation of caveolin-1. delta-Catenin, caveolin-1, and CD59 were all detected in cell-free human voided urine prostasomes. delta-Catenin immunoreactivity was significantly increased in the urine of prostate cancer patients (P0.0005).This study demonstrated, for the first time, the extracellular accumulation of delta-catenin in urine supporting its potential utility for non-invasive prostate cancer detection.

The epididymis is a long, tightly coiled tube within the lumen of which sperm matures. Sperm maturation involves morphological and biochemical changes in the sperm plasma membrane in response to epididymal secretions and their various proteins. Some of these proteins become outer membrane components while others become integral membrane proteins; transfer of some proteins to the sperm plasma membrane may be mediated by epididymosomes. Nevertheless, the molecular pathways by which spermatozoa acquire fertilizing capacity during their transit through the epididymis remain ambiguous. In a recent study of stallion epididymal sperm, we found that sperm harvested from different parts of the epididymis (caput, corpus and cauda) had a varying, but generally poor, ability to undergo the acrosome reaction in vitro. At ejaculation, however, sperm mix with seminal plasma which contains various components, including the small membranous vesicles known as prostasomes, that may enable the sperm to undergo physiological activation. Seminal plasma components may have a 'washing' effect and help to remove 'de-capacitation' factors that coat the sperm during storage in the cauda epididymis; alternatively seminal plasma and prostasomes may contain factors that more directly promote sperm activation. This article reviews current information on the roles of epididymal and accessory gland fluids on the acquisition of fertilizing capacity by stallion sperm.

Objective To investigate the presence of two enkephalin-degrading enzymes (aminopeptidase N [APN] and neutral endopeptidase 24.11 [NEP]) in different fractions of human semen, their distribution in sperm cells, and their effect on sperm motility. Design We performed expression assays for APN and NEP by real-time polymerase chain reaction, Western blot, and immunofluorescence techniques in sperm cells and performed motility analysis after incubation of semen samples with enzyme inhibitors and the opioid receptor antagonist naloxone. Setting Assisted reproduction unit and academic research laboratory. Patient(s) Semen from 50 normozoospermic healthy human donors. Intervention(s) Spermatozoa isolated from semen on a discontinuous Percoll gradient (40%–80%), followed by swim-up, were used for all techniques except for sperm motility analysis, for which fresh semen was used. Main Outcome Measure(s) Immunoblotting blots, indirect immunofluorescence antibody assays, cycle threshold values for real-time polymerase chain reaction, and percentage of motile sperm. Result(s) We found APN in the equatorial segment of the upper post-acrosomal region of the sperm head, in the neck and along the tail of spermatozoa, in prostasomes, and in seminal fluid, whereas NEP was present in a very restricted area of a few sperm cells and in prostasomes. Messenger RNA of both enzymes was detected in spermatozoa. The inhibition of enkephalin-degrading enzymes attenuated the time-dependent decrease of sperm motility; this effect was reversed by naloxone. Conclusion(s) Enkephalin-degrading enzymes are present in human semen and may be involved in the control of sperm motility, mainly by the regulation of endogenous opioid peptides.

Prostasomes are known as extracellular organelles originating from the epithelial cells of the prostate and detected in its secretions where they are mixed with sperm cells after ejaculation. They were first described in men but are also present in the semen of all mammals studied. Since their characterization by Gunnar Ronquist in the late 1970's studies have focused on different aspects which primarily include their molecular composition and structure, and secondly their ability to interact or even fuse with cells, particularly spermatozoa. They have the ability to bring molecules such as lipids or proteins to spermatozoa during their journey to the oocyte, and their role thus seem to be a sort of a "reservoir" that sperm may use depending on the surrounding conditions. Other properties have been suggested but this review will focus on the properties, acquired by sperm cells via prostasomes, that may influence fertility outcome.

Prostasomes are vesicles secreted by epithelial cells of the prostate gland. However, little is known about the mechanism and the regulation of prostasome secretion. Since endocytic organelles may be involved in prostasome release, PC-3-derived prostasomes were investigated by Western blot analysis for the presence of marker proteins normally associated with these organelles. Prostasomes secreted by PC-3 cells contain clathrin, Tsg101, Hrs, Rab11, Rab5, LAMP-1, LAMP-2, LAMP-3/CD63, and annexin II. Moreover, electron microscopy of PC-3 cells revealed the presence of characteristic multivesicular body-like secretory lysosomes containing vesicles with the same size-distribution as released prostasomes. Ultrastructural immunogold labelling showed that LAMP-1, LAMP-2 and LAMP-3/CD63 were associated with these vesicles. In addition, we have investigated whether cholesterol plays a role in prostasome release by the human prostate cancer cell line PC-3. Interestingly, prostasome release was significantly increased when the cholesterol levels of PC-3 cells were reduced by the cholesterol-sequestering agent methyl-beta-cyclodextrin (MBCD), or by treatment with lovastatin and mevalonate. In conclusion, these studies indicate that cholesterol plays an important role in the release of prostasomes by the human prostate cancer PC-3 cells, and suggest that prostasomes may be released after fusion of secretory lysosomes with the plasma membrane.

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.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.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.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.

Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at high [Ca(2+)]c in sperm to down-regulate them, and thus prevent elevated levels of NO, known to induce asthenozoospermia via oxidative stress. Our studies point to the potential underlying cause of infertility in PMCA4's absence, and suggest that inactivating mutations of PMCA4 could lead to asthenozoospermia and human infertility. Screening for these mutations may serve both diagnostic and therapeutic purposes.

The prostate is a principal accessory genital gland that is vital for normal fertility. Epithelial cells lining the prostate acini release in a defined fashion (exocytosis) organellar nanosized structures named prostasomes. They are involved in the protection of sperm cells against immune response in the female reproductive tract by modulating the complement system and by inhibiting monocyte and neutrophil phagocytosis and lymphocyte proliferation. The immunomodulatory function most probably involves small non-coding RNAs present in prostasomes. Prostasomes have also been proposed to regulate the timing of sperm cell capacitation and induction of the acrosome reaction, since they are rich in various transferable bioactive molecules (e.g. receptors and enzymes) that promote the fertilising ability of sperm cells. Antigenicity of sperm cells has been well documented and implicated in involuntary immunological infertility of human couples, and antisperm antibodies (ASA) occur in several body fluids. The propensity of sperm cells to carry attached prostasomes suggests that they are a new category of sperm antigens. Circulating human ASA recognise prostasomes, and among 12 identified prostasomal antigens, prolactin- inducible protein (95 %) and clusterin (85 %) were immunodominant at the expense of the other 10 that were sporadically occurring.

OBJECTIVES To investigate whether cellular exosomes liberated by prostatic cell lines in culture might be acting as the transport vehicles for the dietary antioxidant lycopene, known to be sequestered in the prostate gland and to reduce the risk of developing benign prostatic hyperplasia (BPH) and prostate cancer; its subsequent secretion into seminal plasma also confers protection to spermatozoa against oxidative free-radical damage. MATERIALS AND METHODS Using benign and malignant human prostatic cell culture models, we assessed the role that their exosomes (the putative in vitro analogues of prostasomes) might have in the transport of lycopene. RESULTS Cells exposed to lycopene in vitro accumulated the molecule and secrete lycopene-enriched exosomes. This continued after the lycopene exposure was stopped. Extraction of lycopene from the exosomes, followed by high-performance liquid chromatography, confirmed nanogram quantities of lycopene per milligram of exosomal protein. Packaging into exosomes for export resulted in reduced degradation of this labile antioxidant, and therefore maximized the effectiveness of delivery to the sites of action. CONCLUSION These results support the likelihood that these organelles act as the transport vehicles for this important lipophilic agent known to have a role in the chemoprevention of various urological pathologies such as BPH, prostate cancer and male infertility.

Prostasomes are organelles secreted by prostatic epithelial cells, and are believed to have a role in fertility and prostatic disease. They are known to influence sperm motility and the acrosome reaction, and are thought to have a role in cell transformation, immunosuppression, proliferation, facilitation of local invasion, and angiogenesis. Previously, we have demonstrated the inhibitory effect of prostasomes derived from human semen on angiogenesis using HUVEC cells grown on matrigel. In this study, we use the rat aortic ring assay system, arguably a closer reflection of the in vivo situation. Quantification was by a spectrophotometric method, and underlying mechanisms assessed. Prostasomes demonstrated a clear inhibition of angiogenesis, and this effect persisted after heat treatment of prostasomes to denature protein. This fits with other known effects of prostasomes known to be due to the membrane lipid component, which is unusually high in sphingomyelin and cholesterol.

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 secretory granules synthesized, stored, and secreted by normal and neoplastic human prostate epithelial cells. In prostate cancer, they are anticipated to be released into the blood circulation where they may be immunogenic. The aim of our study was to examine whether prostasome antibody presence in serum bears any prognostic significance for men with prostate cancer. We developed a sensitive and specific immunoassay (enzyme-linked immunosorbent assay) to establish the presence of antiprostasome antibodies in serum. The antiprostasome antibody titer in serum, sampled before any kind of therapy for prostate cancer, was examined together with clinicopathologic variables and outcome over a median follow-up of 350 days in 218 patients with verified prostate cancer. We detected these antibodies in 191 (88%) of these patients. This antibody titer did not correlate to serum values of prostate-specific antigen. Significant, inverse relationships were registered for antiprostasome antibody titer, and metastases to bone and/or lymph nodes (P = 0.035) and pT (P = 0.025). These results indicate that the antiprostasome antibody titer in serum may be a novel marker for prostate cancer liability to metastasize.

Prostate cancer consistently remains a difficult clinical enigma. Therefore, the development of novel strategies for diagnosis and treatment (e.g. immunotherapy) of prostate cancer is essential. We tried to identify the prostasome-derived proteins that were immunogenic in prostate cancer patients. Prostate cancer patients' sera (n = 44) with high enzyme-linked immunosorbent assay (ELISA) titers against prostasomes were selected for immunoblotting against purified seminal prostasomes. The SDS-PAGE and immunoblotting experiments were performed with Bio-Rad systems. Twenty-five of the recognized proteins were isolated and analyzed by means of mass spectrometry. Out of 44 patients' sera, 31 (70%) demonstrated in immunoblotting experiments reactivity against several prostasomal protein bands in the molecular weight range of 10-200 kDa. Some of the bands (55, 70 and 170 kDa) were more frequently recognized by the patients' sera. Concomitantly run control sera generated only very weak or no bands at all. The most frequently occurring prostasomal proteins were identified as heat shock proteins (HSP 70, 71) and clusterin. This study identified the most important molecular targets of autoantibodies against prostasomes generated in connection with the development of prostate cancer in man. These immunogenic prostasomal proteins could be appropriate target molecules for specific immunotherapy of prostate cancer patients.