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Keywords:

  • exosomes;
  • intercellular communication;
  • microvesicles;
  • prostasomes;
  • prostate cancer;
  • reproduction;
  • spermatozoa

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

Ronquist G (University Hospital, Uppsala, Sweden). Prostasomes are mediators of intercellular communication: from basic research to clinical implications (Review). J Intern Med 2012; 271: 400–413

Abstract.  Prostasomes are nanosized microvesicles secreted by acinar epithelial cells of the prostate gland. Furthermore, they are intracellular microvesicles inside another larger vesicle, a so-called storage vesicle, equivalent to multivesicular bodies of late endosomal origin. Prostasomes are thought to play an important role in intercellular communication by direct interaction primarily between the immobile acinar cells of the prostate gland and the mobile spermatozoa. Prostasomes transfer not only membrane components but also genetic material to spermatozoa. They are rich in various transferable bioactive molecules (e.g. receptors and enzymes) that promote the fertilizing ability of spermatozoa. In this review, the pleiotropic biological effects of prostasomes that are relevant for successful fertilization will be discussed. The ability to synthesize and export prostasomes to the extracellular space is observed not only in normal prostate epithelial cells but also in malignant prostate cells. Release of prostasomes by prostate cancer cells suggests a role in malignant cell growth and proliferation. These findings may provide new therapeutic and diagnostic strategies.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

More than 30 years ago, we reported the extracellular presence of nanosized, membrane-surrounded microvesicles in prostatic fluid and seminal plasma [1–3]. Later, the biological concept of prostasomes was broadened, as similar extracellular microvesicles termed exosomes were described in other cell systems grown in vitro [4–10]. Hence, although initially met with much scepticism, the existence of extracellularly occurring microvesicles is now well established. Our electron microscopy studies of acinar cells from surgically removed specimens of human prostatic tissue revealed that the extracellular microvesicles, which we termed prostasomes, corresponded to intracellular microvesicles inside another larger vesicle, a so-called storage vesicle, equivalent to multivesicular bodies (MVBs) of late endosomal origin [11, 12] (Fig. 1). Accordingly, prostasomes are produced by the endosome-containing epithelial cells lining the acinar ducts of the prostate gland, and their release into the prostate ducts (and from there into prostatic fluid and seminal plasma) is the result of a fusion process between the membrane surrounding the storage vesicle and the plasma membrane of the prostate acinar cell (exocytosis) [11, 12]. We produced monoclonal antibodies against purified seminal prostasomes, and immunohistochemistry using one monoclonal antibody (mAb78) demonstrated positive staining of all secretory cells in the prostate gland epithelium with most intense staining in the apical area of the cells establishing these cells as the production sites of prostasomes [13].

image

Figure 1. Schematic illustration of endosomal membrane invagination giving rise to a multivesicular body or storage vesicle containing prostasomes. The size of the storage vesicles is 1000–2000 nm, and prostasomes have a mean diameter of about 150 nm.

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Morphological and biochemical definition of prostasomes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

The identification of microvesicles in prostatic fluid and seminal plasma as prostasomes was based on both morphological and biochemical principles of selection. Given their small size, prostasomes can only be visualized by electron microscopy. Secretory epithelial cells of the prostate contain storage vesicles that in turn are almost filled with prostasomes displaying some heterogeneity with regard to size and appearance [11, 12, 14]. A similar ultrastructure of the prostasomes was also found when sectioning prostasome pellets obtained after preparative ultracentrifugation from prostatic fluid and seminal plasma (Fig. 2) [2, 3, 15, 16]. We measured the diameter of prostasomes in three different locations: an intracellular location (within storage vesicles), extracellularly in the acinar lumen (within the prostate gland) and another extracellular location after isolation from prostatic fluid and seminal plasma (outside the prostate gland). We found very similar mean values of about 150 nm (range 30–500 nm) for all three locations [12]. This mean diameter is in good agreement with recent measurements of seminal prostasomes using nanoparticle tracking analysis (by courtesy of Dr. Mark Ware, NanoSight, Amesbury, UK; mean value of 142 nm) (Fig. 3, right). Further support for the idea of a tight relationship between the intracellular and extracellular bodies is based on findings that the concentrations of calcium, magnesium and zinc in cytoplasmic ‘dense bodies’ (i.e. prostasomes) of the prostate cells [17] correlated with the concentrations of the same ions in the ‘dense bodies’ of the semen (i.e. in the seminal prostasomes) [18]. Immunocytochemical analysis carried out on human prostate cancer cells in culture (PC-3 cells [19]) demonstrated that the same proteins present in the storage vesicles and therefore in the prostasomes could be found in the prostasomes recovered from the external growth medium [20].

image

Figure 2. Thin-section transmission electron microscopy image of human prostasomes isolated from seminal plasma. An electron lucid prostasome surrounded by a bilayered membrane containing a secondary vesicle is shown [16] (long arrow). Another electron-dense prostasome is shown (short arrow). Bar represents 200 nm.

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image

Figure 3. Nanoparticle tracking analysis (Nanosight Ltd, Amesbury, UK) of prostasomes in liquid suspension. Right: size distribution of purified seminal prostasomes; mean diameter 142 nm. The vast majority of prostasomes are distributed within the size range of 30–200 nm. Left: size distribution of purified seminal prostasomes subjected to flotation on sucrose gradient centrifugation (main fraction) followed by filtration through a 0.2-μm filter; mean diameter 117 nm.

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Colloidal silica gradient centrifugation revealed a main band at a density of 1.03 as well as minor bands [2, 3], and this was confirmed by Laurell et al. [21] again suggesting some degree of heterogeneity of prostasomes. The banding of the main component at this unusually low density may be explained by a steric exclusion effect and is consistent with the position of, for example, lysosomes [22] (Table 1). Later, we subjected prostasomes first to flotation on sucrose gradients and then to passage through a 0.2-μm filter in accordance with a reported protocol for preparation of other exosomes from different types of cell growth medium [23]. A main fraction was obtained with a density of 1.13–1.19 g mL−1 in sucrose in addition to smaller fractions, consistent with previous findings from colloidal silica gradient centrifugation. The mean prostasomal diameter of the main fraction was 117 nm using nanoparticle tracking analysis (Fig. 3, left). It thus appears that prostasomes isolated from body fluid (seminal plasma) in accordance with the protocol for isolation of exosomes from in vitro cellular growth medium are somewhat larger than exosomes, which have a diameter of 30–100 nm [23].

Table 1. Key features of human prostasomes
Features References
Size range (mean value)30–500 nm (approximately 150 nm)[12]
Site of generationStorage vesicles inside prostate acinar cells (both acinar and neoplastic cells)[11, 12, 14]
Mechanism of dischargeExocytosis of storage vesicles[11, 12, 14]
IsolationDifferential centrifugation steps, preparative ultracentrifugation, Sephadex G 200 chromatography[2, 3, 12, 28]
DetectionElectron microscopy, mass spectrometry, flow cytometry[2, 3, 11, 12, 15, 16, 30, 31]

The justification for the presence of prostasomes in semen

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

The notion that prostasomes are important components of semen raised the well-founded question of their relevance to reproductive health. A starting point for this was that the release of prostasomes was part of a cellular mechanism of mediating one or more biological functions, the target cells of which could be the spermatozoa. Hence, the physiological relevance of prostasomes was supported by our observation that prostasomes have the ability to interact with spermatozoa, albeit that both prostasomes and spermatozoa display a net negative surface charge favouring repulsive forces [24]. The binding forces between prostasomes and spermatozoa are most probably hydrophobic in nature [24]. This important extracellular reaction between a cell (spermatozoon) and an organelle (prostasome) was subsequently confirmed [25–29].

Prostasomes can carry information from one cell (the immobile prostate acinar cell) to another (the mobile spermatozoon). Transfer of a message to distant cells could occur by three possible mechanisms: (i) by direct contact between the prostasomal membrane and the sperm cell plasma membrane [24, 27]; (ii) by fusion of the two membranes [25, 26]; or (iii) by sperm cell internalization of the prostasome [28, 29]. Accordingly, the prostate acinar cell is able to mediate different abilities at a distance to several spermatozoa, which is important for their survival in the female genital tract and for their ability to reach and penetrate the zona pellucida for fertilization of the ovum [29]. In this context, it is noteworthy that on a stoichiometric basis, the number of prostasomes in an ejaculate is much greater than that of spermatozoa [30].

Prostasome composition

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

Proteins

The protein composition of human prostasomes is varied and has been comprehensively examined. Utleg et al. [31] used microcapillary high-performance liquid chromatography–electrospray ionization tandem mass spectrometry coupled with an iterative gas-phase fractionation to identify a minimum of 139 proteins. These proteins were subdivided into six different categories: enzymes (35% of total); transport/structural proteins (19%); guanosine triphosphate GTP-binding proteins (14%); chaperone proteins (6%); signal transduction proteins (17%); and annotated proteins (9%) [31]. A more recent study by Poliakov et al. [16] using trypsin in-gel digestion and liquid chromatography/mass spectrometry led to the identification of 440 proteins in human prostasomes.

Several of the prostasomal enzymes are involved in redox reactions [32, 33]. The enzyme dipeptidyl peptidase 4 (CD26) has an extremely high specific activity in prostasomes [34] and was identified as the antigen for one of our monoclonal antibodies against human prostasomes [35]. The first membrane-bound enzyme (in a lipoprotein complex) to be identified in prostasomes was ATPase; the activity of this enzyme is dependent on divalent cations [1–3, 36, 37]. Other hydrolysing enzymes were found to be associated with the prostasome membrane via a glycosylphosphatidylinositol (GPI) anchor [38]. In comparison with the surrounding medium (cytosol and seminal plasma, respectively), an enrichment of metals (magnesium, calcium and zinc) was observed in prostasomes [17, 18]. The aforementioned ATPase system of the prostasome membrane [36, 37] may be the molecular basis for the vectorial transport of these divalent cations. Therefore, prostasomes may exert a regulatory function on spermatozoa by modulating the concentration of divalent cations in the microenvironment necessary for, amongst other things, sperm motility. We studied the influences of prostasomes in the presence of divalent cations (and sometimes chelators) on different types of sperm motility. Our data indicated that magnesium ions were essential for forward motility of sperm. There appeared to be a safety margin concerning calcium ions, and zinc ions did not seem to be involved in forward motility in the presence of prostasomes [39].

Lipids

Quite unexpectedly, we found that sphingomyelin was the predominant phospholipid class amongst prostasomal phospholipids, representing almost half of the total amount measured [40]. The saturated fatty acids were quantitatively dominant [40]. Cholesterol is known to be particularly abundant (for physical/chemical reasons) in biomembranes that contain saturated phospholipid acyl chains and have a high content of sphingomyelin. We observed a remarkably high cholesterol/phospholipid ratio of almost 2 [40]. These results were subsequently confirmed by others [41]. Such a high cholesterol/phospholipid ratio renders the membrane highly ordered and stable [40] as reflected by resistance to detergents [38] (Table 2). The finding of sphingomyelin phosphodiesterase in prostasomes [16] suggests the possibility of release of ceramide by removal of the phosphocholine moiety from sphingomyelin. Ceramide might be involved in the inward budding and scission of the limiting late endosomal (storage vesicle) membrane of secretory epithelial cells of the prostate gland giving rise to prostasome formation (see [42]). It should be kept in mind in this context that ceramide was found to be highly represented in oligodendroglial exosomes [42] but, to date, the presence of ceramide has not been reported in prostasomes.

Table 2. Summary of signs of recognition and main characteristics of human prostasomes
CharacteristicsReferences
Gradient centrifugation yields a characteristic banding pattern[2, 3, 21]
Highly reproducible sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) pattern[28]
Membrane-associated enzymes: Mg2+/Ca2+-ATPase (transport ATPase), dipeptidylpeptidase 4 (extremely high specific activity), aminopeptidase N (zinc dependent), protein kinases, ecto-diadenosine polyphosphate hydrolase, arachidonic acid 15-lipoxygenase, 5′-nucleotidase[1–3, 34–38, 79, 80, 115–117]
Membrane composition: proteins are highly varied; sphingomyelin is the predominant phospholipid class; an extremely high cholesterol/phospholipid ratio rendering the membrane very stable[16, 31, 40, 41]

The lipid composition of the plasma membrane of spermatozoa is very different from that of the prostasomal membrane, the cholesterol/phospholipids ratio of the sperm plasma membrane is 0.83 [43], which is similar to most other biological membranes. The very high prostasomal concentration of sphingomyelin and the high cholesterol/phospholipid ratio, that initially surprised us, were subsequently explained by the introduction of the concept of ‘lipid rafts’ [44]. Although the exact mechanisms involved in prostasome biogenesis remain unknown, we believe that the inward budding of the limiting late endosomal membrane mentioned above (Fig. 1) has its starting point in separate membrane microdomains, the so-called lipid rafts [45, 46].

Nucleic acids

In 1990, we first reported the presence of DNA in prostasomes [47]. The prostasomal occurrence of DNA was independent of spermatozoa, as the same amount of DNA was obtained from seminal plasma lacking spermatozoa as a result of vasectomy [47]. Subsequently, fragments of human chromosomal DNA were identified in purified prostasomes, and treatment with DNase demonstrated that the prostasome-shielded DNA was protected from enzyme attack, corroborating the idea that the DNA was present inside the prostasome [48]. This was also in line with the finding in a flow cytometric analysis of prostasomes that membrane-permeable DNA fluorescence staining was increased, whereas membrane-impermeable staining was not [48]. A genome-wide DNA copy number analysis revealed that human prostasomes contained fragments of DNA randomly selected from the entire genome [28]. Furthermore, transfer of genetic material from prostasomes to spermatozoa was demonstrated [28].

Elevated levels of DNA damage in sperm have been associated with early pregnancy loss [49]. It has been claimed that environmental factors and lifestyle behaviours, such as smoking, may be related to DNA damage in the spermatozoon and thereby increased risk of childhood cancer in the offspring [50]. Also, elevated levels of reactive oxygen species (ROS) lead to oxidative stress (the spermatozoon is unusually sensitive to oxidative stress because of its configuration and constitution), which can in turn lead to decreased sperm motility, viability and fertilizing potential, and increased sperm DNA fragmentation rates [51]. Hence, the integrity of the paternal genome seems to be important for fertility, embryonic and foetal development and the long-term health of the offspring. It also follows that there is a need for DNA repair mechanisms in vulnerable spermatozoa. However, there are no clear data available to corroborate the view of a direct prostasomal involvement in sperm DNA repair.

In terms of different types of RNA, there have been no reports to date on their presence in prostasomes. By contrast, the first study regarding RNA in exosomes was carried out in 2007 [52], although whether or not DNA is present in exosomes is unclear. Using microarray technology, it was possible to show that exosomes derived from mouse bone marrow cells contain messenger RNA (mRNA) and microRNA (miRNA). RNA from mast cell exosomes was transferable to other mouse and human mast cells [52]. On the other hand, cells in culture predominantly export miRNA in exosome-independent form [53, 54], reflecting the nonmicrovesicular origin of extracellular miRNA.

Prostasome function

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

Once released from the secretory epithelial cells of the prostate gland and becoming a constituent of semen, it is very likely that the prostasome interacts and possibly fuses with the membrane of spermatozoa [24–29], thus transferring molecules from one cell (the prostatic secretory cell) to another (the spermatozoon).

Sperm motility

Sperm motility is a critical factor in judging semen quality, and the motility pattern influences the fertilizing capability of spermatozoa. In the lower female reproductive tract, motility is important to penetrate the cervical mucus, whilst vigorous beating of the sperm tail is necessary for the penetration of the zona pellucida of the ovum in the upper tract [55]. The motility pattern of spermatozoa evoked by prostasomes [39, 56–58] supports oocyte fertilization. We have suggested that prostasomes may have the ability to regulate the divalent cation concentrations in the microenvironment of the spermatozoa to promote motility [36, 39, 57].

The detailed mechanisms by which prostasomes promote sperm motility and male fertility were recently elucidated by Park et al. [29] in an elegant study. The authors found that prostasome fusion with the spermatozoon was a prerequisite for the transfer from prostasomes of a range of calcium ion signalling tools (including receptors and enzymes) for regulating sperm flagella. In other words, spermatozoa with their tiny configuration do not have to manufacture or maintain all the important signalling proteins; instead, they acquire them from their ‘rucksacks’, that is, the prostasomes, on their way to the target, that is, the ovum [29].

Immunosuppressive and complement inhibitory activity

Spermatozoa are considered as ‘foreign invaders’ in the female genital tract, which is accordingly a potentially hostile environment. To survive, the spermatozoa need to evade potent female immune effectors. Indeed, prostasomes have been identified as inhibitors in lymphoproliferation assays [59, 60]. This ability accounts for a significant proportion of the immunosuppressive activity of human seminal plasma [61]. Because prostasomes have the capacity to interact with spermatozoa [24, 27], it is likely that the immunosuppressive effect associated with prostasomes can be carried up the female genital tract with the spermatozoa [61].

Prostasomes contain in their membrane the membrane attack complex (MAC) inhibitory protein CD59, which is also known as membrane inhibitor of reactive lysis [62]. The CD59 protein is localized on the prostasome surface in a GPI anchor, and CD59 is probably transferred from prostasomes to spermatozoa [62]. Thus, prostasomes may represent a pool of CD59 from which protein lost from spermatozoa, possibly as a result of normal membrane turnover or of low-level complement attack, may be replenished. This would ensure that spermatozoa advance along the female reproductive tract and are guarded against attack by the female complement system. In this context, it is noteworthy that prostasomal CD59 can be transferred in vitro with preserved functionality to erythrocytes lacking CD59 [63]. Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired chronic clonal disorder characterized by increased susceptibility of erythrocytes to complement-mediated lysis; the principal protein that is deficient in this disorder is CD59. A transfer of functionally active CD59 from human prostasomes to CD59-deficient erythrocytes from patients with PNH was observed during short-term incubation, resulting in almost complete abrogation of complement-mediated haemolysis [63].

Antioxidant capacity

Reactive oxygen species are a major cause of idiopathic male infertility. An abnormally high level of production of ROS has been found in 40% of semen samples from infertile individuals, whereas very few samples with high levels of ROS were reported in fertile donors [64]. As mentioned earlier, human spermatozoa are very sensitive to oxidative stress, resulting in peroxidative damage. The origin of ROS in semen is controversial, but leucocytes infiltrating the semen, especially the polymorphonuclear neutrophils, seem to be the major source of ROS generation, and it has been proposed that prostasomes may have the ability to reduce ROS production by sperm preparations containing these leucocytes [65]. Subsequent work suggested that prostasomes inhibit the NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) oxidase activity of polymorphonuclear neutrophils by lipid transfer from prostasomes to the plasma membrane of these cells [66].

Antibacterial properties

We described the presence of prostasomal chromogranin B in abundance over chromogranin A, that is unusual [15]. What is more, the C-terminal fragment (secretolytin) of chromogranin B was found to have potent antibacterial activity [67]. Even other parts of the chromogranin B molecule demonstrated antibacterial properties [68]. These observations prompted us to test the antibacterial properties of whole prostasomes; we found that the antibacterial activity was associated with bacterial membrane deformation [69]. Hence, this process involves the creation of membrane cavities resulting in bacterial cell death at low concentrations of prostasomes [69]. The bactericidal effect of prostasomes differs mechanistically from that of neutrophil granulocytes, as the mechanism involves the generation of ROS in the latter cells. Therefore, it appears appropriate that prostasomes rather than neutrophil granulocytes serve as antibacterial agents in semen because of the extreme sensitivity of human spermatozoa to oxidative stress.

Capacitation and the acrosome reaction

Spermatozoa must undergo capacitation and the acrosome reaction before being capable of fertilization. Mammalian sperm capacitation is defined as the period of time during which the spermatozoa must reside in the female reproductive tract before acquiring the ability to fertilize the ovum. Capacitation is characterized by a complex of structural and functional changes in spermatozoa in the seminal plasma but proceeds throughout the transit of spermatozoa through the female reproductive tract and is considered to be complete when the spermatozoa are able to respond to ligands in the zona pellucida by undergoing the acrosome reaction [70]. It therefore follows that capacitation and the acrosome reaction should not occur prematurely. Cholesterol is one factor in the seminal plasma that has clearly been identified as having an inhibitory influence on capacitation [71], and most of the inhibitory activity is contained in prostasomes [72, 73].

Progesterone released by cumulus cells surrounding the ovum is a potent stimulator of the acrosome reaction [74]. Human spermatozoa are extremely sensitive to progesterone, showing a chemotactic response to picomolar concentration of the hormone [75]. Park et al. [29] in their detailed study found that picomolar levels of progesterone induced a well-adapted, high-amplitude, calcium ion signal in spermatozoa, provided they had been subjected to fusion with prostasomes, and it appeared that this effect of progesterone was dose dependent. Other data also support the view that prostasome–sperm fusion can stimulate the acrosome reaction making spermatozoa more sensitive to the effect of progesterone [76]. This viewpoint was supported by the results of a study of porcine seminal prostasomes and spermatozoa [77] (Table 3).

Table 3. Putative functions of human prostasomes that support successful fertilization
FunctionsCommentsReferences
Promotion of sperm forward motility and hyperactivation with vigorous beating of the sperm tailProstasomes regulate Ca2+ homeostasis in the microenvironment of spermatozoa. Sperm motility depends on the acquisition of Ca2+ signalling tools from prostasomes[29, 36, 39, 57]
Immunosuppressive and complement inhibitory activitySpermatozoa are considered to be ‘foreign invaders’ in the female genital tract. Prostasomes are inhibitory in lymphoproliferation assays, and this ability accounts for the significant immunosuppressive activity. CD59 is transferred from prostasomes to spermatozoa, thus ensuring that they will be guarded against attack by the female complement system[59–62]
Antioxidant capacityReactive oxygen species (ROS) produced by leucocytes are a major cause of idiopathic male infertility. This production of ROS can be inhibited by prostasomes[64–66]
Antibacterial propertiesShort-term incubation of growing bacteria with low concentrations of prostasomes results in total bacterial cell death because of bacterial membrane deformation and creation of membrane cavities[69]
Effects on capacitation and the acrosome reactionCapacitation and the acrosome reaction are essential, multistep reactions that enable the spermatozoon to fertilize the ovum. Prostasomes have a regulatory influence on capacitation exerted via cholesterol transfer. Progesterone released by cumulus cells surrounding the ovum is crucial for the acrosome reaction to occur. However, the progesterone effect can only be exerted after prior interaction between spermatozoa and prostasomes[29, 71–77]

Prostasomes could also influence the acrosome reaction via their hydrolases. Hydrolytic enzymes, such as ecto-diadenosine polyphosphate hydrolase, present on sperm acrosomal membranes, are essential for the acrosome reaction to take place [78]. This ecto-enzyme is bound to the prostasome membrane via a GPI anchor but, similar to CD59 as described earlier, is still transferable [79]. Accordingly, this enzyme is primarily not a constituent of the spermatozoon but can be acquired after interaction between the spermatozoon and the prostasome [79]. Moreover, prostasomes contain arachidonic acid 15-lipoxygenase [80], and this enzyme has been implicated in the acrosome reaction of bull spermatozoa [81]. Thus, prostasome–sperm interactions of various types seem to be essential for capacitation and the acrosome reaction, which in turn are vital for fertilization of the ovum.

Clinical views implicating prostasomes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

Immunological infertility

Spermatozoa are hidden from the immune system by the presence of the blood–testis barrier, which prevents immunoglobulins and immunocompetent cells from entering the lumen of the seminiferous tubules. The protection provided by the blood–testis barrier is, however, incomplete. It has been known for a long time that antisperm antibodies (ASAs) against human spermatozoa from immunologically infertile men can spontaneously appear [82, 83]. The targets of ASAs have been antigens on spermatozoa, and the antibodies can be detected in semen either bound to spermatozoa [84] or free in the seminal plasma [85]. Additionally, these antibodies can be present in blood serum of both men and women [86]. The prevalence of ASAs in infertile couples (both men and women) has been reported to vary between 9% and 36% depending on the study centre [87, 88]. It appears that, in vital spermatozoa, only those ASAs that bind to the sperm membrane will be of functional relevance and their binding sites will be crucial for fertility and viability [89].

Antisperm antibodies may decrease fertility by inhibiting sperm transport and/or gamete interactions [90]. ASAs can cause the agglutination of spermatozoa and thereby prevent sperm motility [91], impair penetration of the cervical mucus [92], block capacitation, the acrosome reaction [93] and binding to the zona pellucida [94] and obstruct sperm–oocyte fusion [95]. We found somewhat unexpectedly that ASAs recognize not only sperm membrane proteins but also prostasomes [96]. We also found very high frequency of recognition of prostasomes as antigens by circulating human ASAs from immunoinfertile men and women [97]. Also, polyclonal antibodies raised against prostasomes resulted in the agglutination of a high percentage of human spermatozoa displaying several types of formation in a similar fashion to that seen in immunologically infertile patients with demonstrable ASAs [96]. As a result of these findings, prostasomes provide a new category of sperm antigens with interesting implications for studies of the mechanism(s) involved in ASA-mediated infertility and immunological control of fertility.

Several prostasome antigens serving as targets for ASAs have been identified, and unexpectedly, they mostly differed from those identified in spermatozoa [98, 99]. The most frequently occurring prostasomal antigens were prolactin-inducible protein (PIP) and clusterin [100]. The observations that prostasomes can act as sperm surface antigens and that most immunoinfertile men have sperm-agglutinating ASAs recognizing prostasomes add further complexity to the problem of immunological infertility. Still, the uneven allocation of prostasomal antigens as targets for ASAs (predominantly PIP and clusterin) could be of interest with regard to fertility regulation. Both antigens are glycoproteins, meaning that they may display different post-translational modification patterns, perhaps giving rise to different isoforms and antibody responses. This in turn increases the possibility of finding unique antibodies against these prostasome glycoproteins relevant to infertility. The recognition of such antigens may also be important for potential immunocontraceptive efforts.

Prostate cancer

Prostate cancer is the most common cancer amongst men older than 50 years in western societies [101], and the incidence is increasing steadily in countries in general. The causes of prostate cancer are essentially unknown. Nevertheless, several factors have been connected with a higher risk of this disease. These factors include increasing age, family history of prostate cancer, living in western countries and race (prostate cancer is especially common in African-American men) [102]. In addition, the increasing incidence of prostate cancer affects those who have migrated from low-risk to high-risk countries [103]. Androgen stimulation may be a carcinogenic factor, as testosterone promotes proliferation of prostate epithelial cells and prevents apoptosis [104]. Prostate cancer is extremely rare in men castrated before puberty [105].

Primary cancer in the seminal vesicles is extremely rare [106], in contrast to the very high incidence of cancer in the prostate gland. This deserves attention because the two types of glands represent neighbouring anatomical locations, and both are exocrine accessory genital glands under similar hormonal control. This discrepancy prompted us to think in terms of the prostasome being an accessory to the development of prostate cancer, because the secretion of seminal vesicles is devoid of prostasomes and prostasome-like structures [107]. It is noteworthy that not only normal prostate acinar secretory cells but also neoplastic prostate cells and even poorly differentiated prostate cancer metastases are able to synthesize and export prostasomes to the extracellular space [14, 108, 109].

It has long been known that tumour cells tend to exploit opportunistically the host′s physiological system to obtain support in terms of nutrition, growth and metastasis. We proposed some years ago that several attributes of the prostasome, primarily developed to sustain the fertilizing spermatozoa, can also promote the transition from a normal to a neoplastic prostatic cell and help the prostasome-producing, poorly differentiated cancer cell to survive and proliferate in a primarily hostile environment [110]. The control of cell proliferation, differentiation and signal transduction pathways is generally mediated by protein kinases and phosphatases [111–113], whose actions are modified by hormones, growth factors and mitogens [112, 114]. Hence, the phosphorylation of a protein is carried out by protein kinases, which can be subdivided into two main categories depending on the acceptor amino acid of the transferred phosphoryl group: hydroxy (OH) groups on serine/threonine residues and phenolic groups on tyrosine residues of their respective proteins. The serine/threonine protein kinases are generally second messenger (e.g. cyclic AMP) dependent. Protein kinases that phosphorylate the tyrosine-containing protein residue include hormone receptor-associated kinases. Protein phosphorylation is controlled reversibly by phosphoprotein phosphatases that cleave the phosphoryl group from the acceptor amino acid facilitating a phosphorylation/dephosphorylation cycle. Approximately half of the protein kinase activity in prostatic fluid is associated with prostasomes [115, 116], and co-incubation of spermatozoa and prostasomes was shown to result in a 10-fold increase in total protein phosphorylation compared to the level of phosphorylation achieved through incubation with either component alone [115]. This finding supports the interactive relationship between prostasomes and spermatozoa, even though they have different origins in the genital apparatus.

Our studies on prostasomes derived from different types of prostate cancer cells revealed distinctly upregulated protein kinase (A and C) and casein kinase activities compared to normal seminal prostasomes [117]. This enhanced protein kinase activity of cancer cell-derived prostasomes may participate in the self-defence programme of prostate cancer cells against attack by the complement system. The main event in the activation of complement is proteolytic cleavage of C3, producing C3a and C3b. Two enzyme complexes (convertases), which are assembled by three different activation pathways, accomplish the cleavage. These converge in a common pathway, forming the MAC (C5b–C9), which elicits cell lysis by insertion into the lipid bilayer of plasma membranes. Complement activation on autologous cells is controlled by several soluble and membrane-bound regulators. The role of complement activation as a mechanism for destruction of malignant cells is not yet well understood. However, it is highly probable that the complement system is involved in control of malignant tumours, as these cells are extremely sensitive to altered self- and nonself-structures on cell surfaces [118]. It is therefore assumed that cells that are unable to protect themselves against complement attack will be eliminated early in the process of cancer development. It was reported that phosphorylation of complement component C3 made it inaccessible to physiological activation [119]. Our findings demonstrated that upregulated protein kinase A of cancer-derived prostasomes was indeed able to phosphorylate complement component C3 [117]. We therefore believe that these cancer cell-derived prostasomes have the ability to disarm complement activation by regulatory phosphorylation. Additionally, CD59 expression was higher in prostasomes from cancer cells than in those from normal cells [120]. CD59 could be transferred, functionally active, from prostasomes to other cells including prostate cancer cells, thereby inhibiting complement-mediated lysis [120]. Thus, prostate cancer cells with the support of their prostasomes have two mechanisms of survival against the host complement system.

It has been suggested that fibrinogen plays a role to induce migration of tumour cells and is associated with an infiltrative histological phenotype in bladder cancer [121]. We found that fibrinogen was phosphorylated by all three types of protein kinases (A, C and casein kinase) of malignant cell-derived prostasomes [117]. This is interesting in the light of the findings that phosphorylated fibrinogen is more resistant to cleavage [122] and that suppression of fibrinolysis is important for metastatic prostate cancer cells.

Patients with prostate cancer as well as other types of cancer experience a much higher than expected incidence of thromboembolic events; this is commonly referred to as Trousseau’s syndrome. Although this association is well documented, the aetiology of the hypercoagulable state has remained obscure. Tissue factor (CD142), serving as a receptor and essential cofactor for factors VII and VIIa of the coagulation cascade, is the principal initiator of both coagulation and thrombosis [123]. It is present in huge amounts in prostasomes [124]. Prostasomes derived from different human prostate cancer cell lines overexpress and are able to phosphorylate tissue factor [125, 126]. Tissue factor is also known to support cancer growth and proliferation, including the promotion of tumour angiogenesis [123, 127], cell adhesion [128], cell migration [129] and tumour cell invasion [130]. In addition, it binds with high affinity to plasminogen [131], an effector that may be involved in the enhancement of tumour growth and metastasis [132]. Therefore, prostasomes by virtue of their high levels of tissue factor may play an active role in prostate cancer proliferation.

The clotting ability of prostasomes, seemingly in a dose-dependent fashion, may be related to their level of expression of tissue factor [126, 133]. However, no difference in plasma level of soluble tissue factor between patients with prostate cancer and controls has also been reported [134]. Recent data support the view that prostate cancer patients with aggressive disease do have circulating prostasomes with membrane-bound tissue factor in their peripheral blood [135] and, therefore, circulating prostasomes may be the underlying cause of Trousseau’s syndrome in aggressive prostate cancer.

Prostasomes as diagnostic markers

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

Prostate-specific antigen (PSA; also known as human glandular kallikrein 3) belongs to the traditional human kallikrein family, which is a subgroup of the serine protease family [136]. Serum PSA analysis along with digital rectal examination has been the standard methods for prostate cancer screening for the past two decades [137], despite the fact that PSA is not a cancer-specific marker. The clinically used tumour marker PSA shows high organ specificity but does not indicate the proliferation and metastatic potential of prostate cancer cells [137, 138]. More precise biomarkers are needed to differentiate between aggressive and indolent cancers. We sought to determine whether prostasomes could exist extracellularly and thereby appear in peripheral blood during the development of prostate cancer. This possibility was based on the fact that there is a transition, both morphological and functional, of the highly polarized, tall, columnar cells of the prostate into more cuboidal cells (with loss of polarity) that can appear in small masses during cancer development [110]. Loss of polarity means that the strictly vectorial transport of prostasomes into the acinar ducts and then into prostatic fluid and finally seminal fluid essentially ceases. Instead, it is possible that prostasomes can be released into the interstitial fluid (see [109]) which is connected with peripheral blood. Blood samples from patients with prostate cancer did show traces of prostasomes in the form of autoantibodies with, however, limited discriminatory power between malignant and benign prostate disease [139, 140]. We therefore aimed to develop a method for direct measurements of prostasomes in blood samples from patients with prostate cancer. By using an extremely sensitive and specific assay, we were able to demonstrate that prostasomes can be detected at elevated levels in blood plasma from patients with prostate cancer whereas levels of prostasomes are not elevated in plasma from patients with benign prostatic hyperplasia, prostatitis or indolent prostate cancer [135]. Accordingly, the assay appeared to distinguish between patients with aggressive prostate cancer and those with indolent prostate cancer or benign disease [135]. This investigation was, however, based on a limited number of patients (Table 4), and the data must therefore be confirmed with a larger, well-characterized patient cohort.

Table 4. Blood plasma levels of prostasomes (ng protein per mL) in samples from patients with prostate cancer (PCa) classified into three groups according to Gleason score. Each patient group was analysed in a separate experiment together with the same control group, which consisted of age-matched urology patients with benign results from transrectal ultrasound-guided biopsy. The plasma levels of prostasomes in patients with Gleason score 7 (medium) and 8/9 (high; aggressive cancers) were both significantly elevated compared to those with Gleason score of 6 or lower (indolent cancers) and those of controls (P = 0.001). There was no difference in the levels of prostasomes between patients with Gleason scores of 6 and lower and controls (P = 0.94) [135]
Patients/controlsnMedianRange
PCa, Gleason <6201.00.9–2.3
Controls201.00.9–4.5
PCa, Gleason 7192.20.5–17.3
Controls200.90.5–3.5
PCa, Gleason 8/9202.90.3–7.2
Controls201.21.2–3.0

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References

Prostasomes represent a subclass of secreted membrane vesicles. A wide range of regulatory functions related to reproduction has been attributed to these microvesicles with diverse applications in cell physiology and pathology. Therefore, prostasomes have appealing properties that can be exploited for therapeutic intervention and as biomarkers in blood plasma and other biological fluids in prostate cancer and other pathologies. However, many questions remain concerning the physiological relevance of prostasomes. The current expansion of the scientific area of microvesicle research is promising and will address these issues and lead to a growing understanding of the distinctive character and functions of prostasomes.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Morphological and biochemical definition of prostasomes
  5. The justification for the presence of prostasomes in semen
  6. Prostasome composition
  7. Prostasome function
  8. Clinical views implicating prostasomes
  9. Prostasomes as diagnostic markers
  10. Concluding remarks
  11. Acknowledgements
  12. Conflict of interest statement
  13. References