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Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.

The investigation of biomarkers is constantly evolving. New molecules and molecular assemblies, such as soluble and particulate complexes, emerged as biomarkers from basic research and investigation of different proteomes, genomes, and glycomes. Extracellular vesicles (EVs), and glycans, complex carbohydrates are ubiquitous in nature. The composition and structure of both reflect physiological state of paternal cells and are strikingly changed in diseases. The EV-associated glycans, alone or in combination with soluble glycans in related biological fluids, used as analytes, aim to capture full complex biomarker picture, enabling its use in different clinical settings. Bringing together EVs and glycans can help to extract meaningful data from their extreme and distinct heterogeneities for use in the real-time diagnostics. The glycans on the surface of EVs could mark their subpopulations and establish the glycosignature, the solubilisation signature and molecular patterns. They all contribute to a new way of looking at and looking for composite biomarkers. [ABSTRACT FROM AUTHOR]

Résumé Les Vésicules Extracellulaires (VE) sont des constituants d’origine membranaire contenant des protéines solubles ou membranaires, des lipides, des métabolites ou des acides nucléiques. Ces vésicules sont très hétérogènes en taille et en contenu. Trois catégories de VE ont été décrites dans le tractus génital mâle: les microvésicules, les myélinosomes et les exosomes. Les types de VE sont différents selon le lieu de production testiculaire, prostatique ou encore épididymaire. Il a été montré que les VE peuvent fusionner et délivrer une information à la cellule réceptrice, en l’occurrence le spermatozoïde dans le tractus génital mâle. La cryo-microscopie électronique reste la technique de référence pour déterminer la morphologie des VE mais la quantification de la concentration absolue de ces VE dans les liquides biologiques reste un challenge dans le cadre d’une approche clinique. Le domaine de la biodétection s’est. considérablement développé avec l’introduction des nanomatériaux dans les biocapteurs et va permettre de mieux comprendre l’impact de ces VE. Or les modifications fonctionnelles des gamètes mâles résultent d’interactions avec les composants du liquide intraluminal tout le long du tractus génital et dépendent de la sécrétion et de l’absorption de protéines et de lipides du microenvironnement local. On ne peut donc pas exclure la possibilité d’une modulation épigénétique des informations qui seront transmises à l’embryon donc à la génération suivante via les VE.

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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.

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.

Exosomes are nano-sized membrane vesicles, secreted by different types of cells into the body's biological fluids. They are found in abundance in semen as compared to other fluids. Exosomes contain a cargo of lipid molecules, proteins, phospholipids, cholesterol, mRNAs, and miRNAs. Each molecule of seminal exosomes (SE) has a potential role in male reproduction for childbirth. Many potential candidates are available within the seminal exosomes that can be used as diagnostic markers for various diseases or syndromes associated with male reproduction. Also these seminal exospmes play a major role in female reproductive tract for effective fertilization. The aim of this review is to focus on the advancement of human seminal exosomal research and its various properties. We used many databases like Scopus, Google scholar, NCBI-NLM and other sources to filter the articles of interest published in exosomes. We used phrases like "Exosomes in human semen", "Composition of exosomes in human semen" and other relevant words to filter the best articles. Seminal exosomes play a major role in sperm functions like cell-to-cell communication, motility of the sperm cells, maintaining survival capacity for the sperm in the female reproductive tract and spermatogenesis. Also, seminal exosomes are used as a carrier for many regulatory elements using small RNA molecules. miRNAs of the seminal exosomes can be used as a diagnostic marker for prostate cancer instead of prostate specific antigen (PSA). Epididymosomes can be used as a biomarker for reproductive diseases and male infertility. Seminal exosomes could be used as biological markers for various reproductive disorders, male infertility diagnosis, and it can be used in anti-retroviral research for the identification of novel therapeutics for HIV-1 infection and transmission. [ABSTRACT FROM AUTHOR]

Prostate cancer is the second most diagnosed disease in men worldwide, with an increasing mortality rate in recent years. Currently, there are two early detection tests, the measurement of blood levels of prostate-specific antigen and digital rectal examination of the prostate. However, these tests do not present optimal specificity and sensitivity for their detection. Although different studies have looked for new biomarkers by means of the implementation of technologies, such as new generation sequencing, mass spectrometry, among others, the same disadvantages persist, therefore, they have not allowed their use in clinical practice; The discovery of new biomarkers for the diagnosis of prostate cancer is a challenge for the scientific community. Prostasomes correspond to extracellular vesicles secreted by normal prostate or tumor tissue that can be detected in different fluids. Structurally, prostasomes differ from other exosomes, by their size, membrane composition and specific protein content, which makes them a potential and novel source of clinical biomarkers. In this context, this review presents an overview of protein biomarkers, isolated from prostasomes present in different biological fluids, for the possible diagnosis of prostate cancer. For this, a systematic PubMed search was carried out for studies in proteomics for prostate cancer, with criteria such as extracellular vesicles, exosomes and prostasomes, as well as blood, urine, seminal fluid, among other biological samples.

Abstract 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.

Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR−/−: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFβ: transforming growth factor β; uNK: uterine natural killer

Extracellular vesicles are highly abundant in seminal fluids and have a known role enhancing sperm function. Clinical pregnancy rates after IVF treatment are improved after female exposure to seminal fluid. Seminal fluid extracellular vesicles (SF-EVs) are candidate enhancers, however, whether SF-EVs interact with cells from the endometrium and modulate the implantation processes is unknown. Here, we investigated whether SF-EVs interact with endometrial stromal cells (ESCs) and enhance decidualisation, a requisite for implantation. SF-EVs, isolated from human seminal fluid (n = 11) by ultracentrifugation, were characterised by nanoparticle tracking analysis and Western blotting, and purified using size exclusion chromatography. Non-decidualised and decidualised primary ESCs (n = 5) were then treated with SF-EVs. Binding of bio-maleimide-labelled SF-EVs was detected by flow cytometry and fluorescence microscopy. Prolactin and IGFBP-1 protein levels in culture media were also analysed after single and multiple SF-EV exposure. SF-EVs size ranged from 50 to 300 nm, and they expressed exosomal markers (ALIX, SYNTENIN-1, CD9 and CD81). SF-EVs bound to non-decidualised and decidualised ESCs at similar levels. ESCs prolactin secretion was increased after single (p = 0.0044) and multiple (p = 0.0021) SF-EV exposure. No differences were found in IGFBP-1 protein levels. In conclusion, SF-EVs enhance in vitro ESC decidualisation and increase secretion of prolactin, an essential hormone in implantation. This elucidates a novel role of SF-EVs on endometrial receptivity. Abbreviations: ECACC: European Collection of Authenticated Cell Cultures; ESCs: endometrial stromal cells; EVs: extracellular vesicles; FCS: foetal calf serum; HRP: horse-radish peroxidase; IFNγ: interferon-gamma; IGF: insulin-like growth factor; IGFBP-1: insulin-like growth factor binding protein 1; IVF: in vitro fertilisation; MVB: multivesicular bodies; NTA: nanoparticle tracking analysis; PRLR−/−: homozygous prolactin receptor knockout; RT: room temperature; SF-EVs: seminal fluid extracellular vesicles; STR: short tandem repeat; TGFβ: transforming growth factor β; uNK: uterine natural killer [ABSTRACT FROM AUTHOR]

Prostasomes are extracellular vesicles that fuse with sperms thereby improving its functional parameters. Present study aimed to isolate and characterise prostasomes from semen of KF bulls, and to investigate prostasomes effects on functional parameters of KF bull spermatozoa. Isolated prostasomes were characterized with respect to the binding of FITC-conjugated CD 26 antibodies, as well as protein, cholesterol and phospholipids content. Subsequently, effects of prostasomes supplementation (1mg/ml) were investigated on ROS production, Ca2+ signalling, mitochondrial membrane potential and acrosome integrity of fresh and cryopreserved-thawed spermatozoa. Isolated prostasomes were immunostained positively. Prostasomes showed higher proportion of both protein and cholesterol as compared to phospholipids. When sperm samples were supplemented with prostasomes, ROS production decreased, while all other functional parameters improved. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process.

Extracellular vesicles (EV) are small membrane-bound vesicles enriched in a selective repertoire of mRNA, miRNA, proteins and cell surface receptors from parental cells and are actively involved in the transmission of inter and intracellular signals. Cancer cells produce EV that contain cargo including DNA, mRNA, miRNA and proteins that allow EV to create epigenetic changes in target cells both locally and systemically. Cancer-derived EV play critical roles in tumorigenesis, cancer cell migration, metastasis, evasion of host immune defense, chemoresistance, and they promote a premetastatic niche favourable to micrometastatic seeding. Their unique molecular profiles acquired from originator cells and their presence in numerous body fluids, including blood and urine, make them promising candidates as biomarkers for prostate, renal and bladder cancers. EV may ultimately serve as targets for therapy and as platforms for personalized medicine in urology. As urologic malignancy comprises 28% of new solid tumour diagnoses and 15% of cancer-related deaths, EV-related research is rapidly emerging and providing unique insights into disease progression. In this report, we review the current literature on EV in the setting of genitourinary fertility and malignancy.

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

Summary: 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. [ABSTRACT FROM AUTHOR]

The prostate of the koala (Phascolarctos cinereus), and of marsupials more generally, is the primary contributor of seminal fluid, yet comparatively little is known about its microanatomy or biochemistry. This study explored evidence of parenchymal segmentation of the koala prostate. The prostate of three sexually mature koalas were processed for histopathology, histochemistry (Masson's trichrome, Alcian Blue, periodic acid Schiff staining), and immunohistochemistry using basal (tumor protein 63, cytokeratin 14) and luminal (cytokeratin 8/18, prostate specific antigen, androgen receptor) markers. Results confirmed clear segmentation of the koala prostate into three zones, anterior, central, and posterior, characterized by differences in the proportion of glandular tissue, as well as the thickness of collagen fibers; there were also distinct differences in the secretions produced in each zone. Based on immunohistochemistry, the koala prostate showed evidence of both basal proliferative and luminal secretory cells. The ratio of cell types varied across the three segments, with the central segment housing the highest density of basal cells. Globular bodies produced in the anterior zone were shown to possess the same markers as those described for human prostasomes. This study is the first to comprehensively document the marsupial prostate in terms of microanatomy and corresponding immunohistochemistry. While further biochemical analysis, such as proteomics of each segment will better define the relative functions of each tissue, the data presented here are consistent with the hypothesis that the koala prostate potentially represents an example of an ontological stage in the evolutionary differentiation of male eutherian accessory glands., (© The Author(s) 2023. Published by Oxford University Press behalf of Society for the Study of Reproduction.)

We have demonstrated the applicability of cyclic voltammetry and rotating disk electrode techniques to study adsorption, aggregation, and self-fusion of prostasomes, nanoparticles present in human semen. Prior to the electrochemical experiments, we have characterized prostasomes through nanoparticle tracking analysis, dynamic light scattering, and zeta potential measurements at various pH. One of our main findings in this work has been the prostasome concentration in human semen, determined to be 4.8 × 10 11 cm −3 , which is 20000 times higher than the sperm cell concentration. We have found the hydrodynamic diameter of the prostasome to range from 35 to 475 nm, with the mean value of 151 nm, in agreement with the literature. Then, we have adsorbed a monolayer of prostasomes at the surface of gold rotating disk electrode and studied its effect on the limiting diffusion current at the electrode. We have conducted our measurements in a standard three-electrode cell. We have described the monolayer effect in terms of the equivalent layer thickness of stagnant solution layer. Next, we have added a small amount of prostasomes to the three-electrode cell and decreased pH to 4.0 to allow their aggregation at the surface of rotating disk electrode. We have used the chronoamperometry method to monitor multilayer adsorption of the prostasome for 180 min. Then, we have again characterized the adsorbed layer of prostasomes in terms of the equivalent layer thickness. Through a comparison of the equivalent layer thickness determined for the mono- and multilayer, we have found the amount of prostasomes adsorbed during chronoamperometry. We have also compared the number of prostasomes adsorbed at pH 4.0 with the number of prostasomes transported to the rotating disk electrode during this experiment. From that comparison, we have estimated the two-prostasome aggregation probability and the surface area fractions corresponding to the positive and negative surface charges of the prostasome. We believe that our results can be useful for better understanding of prostasome surface properties and, consequently, processes such as prostasome-sperm cell fusion or prostasome-bacterium interactions. [ABSTRACT FROM AUTHOR]

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process. [ABSTRACT FROM AUTHOR]

Membrane vesicles (MV) have been identified in seminal plasma from various species and they are thought to have a significant impact on semen quality and fertilisation. Although recently presence of MV has been also described in the canine ejaculate, detailed knowledge on their morphology is missing by now. This is, however, needed to provide a basis for detailed biochemical and functional studies as it is generally assumed that different MV populations are responsible for distinct tasks. MV were prepared for light (LM) and transmission electron microscopy (TEM) analysis using samples from normospermic dogs ( n = 15), hypokinozoospermic dogs ( n = 2, h) and one castrated azoospermic dog (a). For TEM, a new preparation protocol was used resulting in a higher MV retrieval rate. Using fractionated semen samples, most MV were identified in the second (sperm-rich) fraction in LM. Using pooled ejaculates, three different MV types could be identified in LM: (1) large MV with a marginal accumulation of opaque, granulated material, (2) medium- to small size MV with dense, opaque content and (3) small MV with no further defined contents. No direct contact between sperm and MV could be visualised. In TEM, 11 different MV types were identified based on diameter, structure, contents and electron density of contents as well as presence, number and size of smaller MV inside the MV itself. In normospermic males, secondary vesicles (type i, H, K1/2) included smaller vesicles and had a weighted mean diameter of 409.46 nm; hereof types i, H and K1 were smaller (mean: 287.55 nm, range: 51.25–994.86 nm) and type K2 was larger (mean: 1746.43 nm, range: 1003.66–3289.34 nm). Primary vesicles (mean diameter: 135.29 nm) – without vesicles inside – were differentiated into larger MV (A, B, C1/2) with a mean diameter of 219.63 nm (range: 39.08–1300.13 nm) and small primary MV (F, G) with a mean diameter of 66.12 nm (range: 24.62–99.84 nm). Whereas all mentioned MV were round to oval and mostly double-, rarely multiple-membrane surrounded, one longish primary MV type (L) was identified. In general, small primary vesicles were most common independent of semen quality, but distribution frequency of vesicle types differed between normospermic, pathospermic dogs and the castrated male. Mean weighted diameter of MV was 195.14 nm (range: 24.62–3289.34 nm) in normospermic males with the maximum diameter being smaller in the other dogs (h: 2096.78 nm; a: 1314.06 nm). Our results provide new information about ultrastructure and distribution frequency of canine MV in normospermic males and point to possible differences in MVs depending on semen quality. They provide the basis for further detailed functional analysis of MV subpopulations. Furthermore, the presence of MV in the castrated azoospermic male confirms an at least partly prostatic origin of canine MV. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EV) are small membrane-bound vesicles enriched in a selective repertoire of mRNA, miRNA, proteins and cell surface receptors from parental cells and are actively involved in the transmission of inter and intracellular signals. Cancer cells produce EV that contain cargo including DNA, mRNA, miRNA and proteins that allow EV to create epigenetic changes in target cells both locally and systemically. Cancer-derived EV play critical roles in tumorigenesis, cancer cell migration, metastasis, evasion of host immune defense, chemoresistance, and they promote a premetastatic niche favourable to micrometastatic seeding. Their unique molecular profiles acquired from originator cells and their presence in numerous body fluids, including blood and urine, make them promising candidates as biomarkers for prostate, renal and bladder cancers. EV may ultimately serve as targets for therapy and as platforms for personalized medicine in urology. As urologic malignancy comprises 28% of new solid tumour diagnoses and 15% of cancer-related deaths, EV-related research is rapidly emerging and providing unique insights into disease progression. In this report, we review the current literature on EV in the setting of genitourinary fertility and malignancy. [ABSTRACT FROM AUTHOR]

Background: One of the problems in prostate cancer (CaP) treatment is the appearance of the multidrug resistance phenotype, in which ATP-binding cassette transporters such as multidrug resistance protein 1 (MRP1) play a role. Different localizations of the transporter have been reported, some of them related to the chemoresistant phenotype. Aim: This study aimed to compare the localization of MRP1 in three prostate cell lines (normal, androgen-sensitive, and androgen-independent) in order to understand its possible role in CaP chemoresistance. Methods: MRP1 and caveolae protein markers were detected using confocal microscopy, performing colocalization techniques. Lipid raft isolation made it possible to detect these proteins by Western blot analysis. Caveolae and prostasomes were identified by electron microscopy. Results: We show that MRP1 is found in lipid raft fractions of tumor cells and that the number of caveolae increases with malignancy acquisition. MRP1 is found not only in the plasma membrane associated with lipid rafts but also in cytoplasmic accumulations colocalizing with the prostasome markers Caveolin-1 and CD59, suggesting that in CaP cells, MRP1 is localized in prostasomes. Conclusion: We hypothesize that the presence of MRP1 in prostasomes could serve as a reservoir of MRP1; thus, taking advantage of the release of their content, MRP1 could be translocated to the plasma membrane contributing to the chemoresistant phenotype. The presence of MRP1 in prostasomes could serve as a predictor of malignancy in CaP. [ABSTRACT FROM AUTHOR]

Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in human prostasomes) were identified via peptide mass fingerprinting, several of which may be different isoforms. Out of the three high molecular weight bands characteristic for human prostasomes (CD10, CD13, and CD26), CD10 and CD13 were retrieved in equine prostasomes. We present some new proteins of horse prostasomes not found in their human counterparts. Further studies are warranted to reveal the function of these proteins. [ABSTRACT FROM AUTHOR]

Abstract: 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 10–150kDa. 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Prostasomes are prostate-derived, exosome-like microvesicles that transmit signaling complexes between the acinar epithelial cells of the prostate and sperm cells. The vast majority of prostasomes have a diameter of 30-200 nm, and they are generally surrounded by a classical membrane bilayer. Using a selected proteomic approach, it became increasingly clear that prostasomes harbor distinct subsets of proteins that may be linked to adenosine triphosphate (ATP) metabolic turnover that in turn might be of importance in the role of prostasomes as auxiliary instruments in the fertilization process. Among the 21 proteins identified, most of the enzymes of anaerobic glycolysis were represented, and three of the glycolytic enzymes present are among the top 10 proteins found in most exosomes, once again linking prostasomes to the exosome family. Other prostasomal enzymes involved in ATP turnover were adenylate kinase, ATPase, 5=-nucleotidase, and hexose transporters. The identified enzymes in their prostasomal context were operational for ATP formation when supplied with substrates. The net ATP production was low due to a high prostasomal ATPase activity that could be partially inhibited by vanadate that was utilized to profile the ATP-forming ability of prostasomes. Glucose and fructose were equivalent as glycolytic substrates for prostasomal ATP formation, and the enzymes involved were apparently surface located on prostasomes, since an alternative substrate not being membrane permeable (glyceraldehyde 3-phosphate) was operative, too. There is no clear-cut function linked to this subset of prostasomal proteins, but some possible roles are discussed. [ABSTRACT FROM AUTHOR]