Progesterone-induced increase of sperm cytosolic calcium is enhanced by previous fusion of spermatozoa to prostasomes

Ejaculated spermatozoa must undergo a number of modifications before becoming able to fertilize the oocyte. The interaction of sperm with other semen components may influence these phenomena; human semen contains vesicles of prostatic origin, called prostasomes that may fuse to sperm at slightly acidic to neutral pH values.Prostasomes contain calcium and it has been demonstrated that their fusion with spermatozoa produces a transient increase (wave) of [Ca(2+)](i) in these cells. The fusion process also transfers protein and lipid to spermatozoa. These phenomena may induce long-lasting changes of sperm properties. We test the hypothesis that spermatozoa, as modified by fusion, change their ability to undergo the progesterone-induced increase of [Ca(2+)](i) and we find that the increase of [Ca(2+)](i) produced by the fusion with prostasomes and by the stimulation with progesterone are independent and additive phenomena. We also find that spermatozoa present a stronger response to the progesterone-induced increase of [Ca(2+)](i) if they are previously made to fuse with prostasomes. This effect does not depend directly on the [Ca(2+)](i) increase due to fusion, since it is still present after the [Ca(2+)](i) has returned to resting values.     

Prostasomes, dérivés actifs de l’oxygène et sperme humain

Prostasomes are particular lipid vesicles secreted by the human prostate and found in the semen. No specific action has yet been attributed to prostasomes, but they appear to act at various levels. For example, prostasomes enhance sperm motility in vitro and participate in the immunomodulation properties of seminal plasma. Excessive production of reactive oxygen species (ROS) in human semen has a negative influence on the functional capacities of spermatozoa. The presence of leukocytes in semen is associated with increased production of ROS that can be harmful to sperm cells, under certain conditions. Previous results tend to suggest a possible role of prostasomes on ROS production in human semen. After reviewing the literature concerning the structural and functional characteristics of prostasomes and the role of ROS in human semen, we report our results concerning the influence of prostasomes on ROS production and the consequences on semen. We have demonstrated that prostasomes exert an antioxidant function in human semen. This function is effective both on polymorphonuclear neutrophils and on sperm cells. The mechanism of action of prostasomes is unusual, as they act on ROS production mainly on the plasma membranes of neutrophils. They induce a decrease of NADPH-oxidase activity associated with rigidification of the plasma membrane. Prostasomes protect the functional capacities of spermatozoa during an oxidative stress created by the presence of NADPH in the incubation medium.     

Fusion of Human Sperm to Prostasomes at Acidic pH

Prostasomes are membranous vesicles (150–200 nm diameter) present in human semen. They are secreted by the prostate and contain large amounts of cholesterol, sphingomyelin and Ca²⁺. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated spermatozoa and are involved in a number of additional biological functions. The possibility that they may fuse to sperm has never been proved. In this work, we studied the fusion of sperm to prostasomes by using various methods (relief of octadecyl Rhodamine B fluorescence self-quenching, fluorescence microscopy and flow cytometry) and we found that it occurs at acidic pH (4–5), but not at pH 7.5 pH-dependent fusion relies on the integrity of one or more proteins and is different from the Ca²⁺-stimulated fusion between rat liver liposomes and spermatozoa that does not require any protein and occurs at neutral pH. We think that the H⁺-dependent fusion of prostasomes to sperm may have physiological importance by modifying the lipid and protein pattern of sperm membranes. Received: 19 June 1996/Revised: 4 September 1996     

Role of human prostasomes in the activation of spermatozoa

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

Hamster sperm antigen P26h is a phosphatidylinositol‐anchored protein

We have previously identified a hamster sperm protein, P26h, proposed to be involved in the interaction between spermatozoa and the egg's zona pellucida. In this study we investigated the mechanism of P26h accumulation on hamster spermatozoa during epididymal maturation. Immunocytochemical studies showed an accumulation of P26h on the acrosomal cap of hamster spermatozoa during epididymal transit. To document the anchoring mechanism of P26h, cauda epididymal spermatozoa were exposed to different treatments. High‐salt buffered solutions were unable to remove P26h from the surface of intact spermatozoa. P26h was released in a dose‐dependent manner when live spermatozoa were treated with a solution of phospholipase C specific to phosphatidylinositol. In contrast, the P26h remained associated to the sperm surface following treatment with trypsin. To document the transfer mechanisms of P26h on the maturing spermatozoa, prostasomes were isolated from the epididymal fluid and subjected to immunodetection. Western blots and immunogold studies showed that P26h was associated to epididymal prostasomes. Phospholipase C treatment performed on epididymal prostasomes, indicated that P26h also is anchored to these vesicles via a phosphatidylinositol. These results suggest that epididymal sperm maturation involves a cell to cell transfer of a phosphaditylinositol‐anchored protein and that prostasomes may be implicated in this process. Mol. Reprod. Dev. 52:225–233, 1999. © 1999 Wiley‐Liss, Inc.     

The roles of the epididymis and prostasomes in the attainment of fertilizing capacity by stallion sperm

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.     

The motility of human spermatozoa as influenced by prostasomes at various pH levels.

Human semen contains several components among which spermatozoa, membranous vesicles called 'prostasomes', secreted by the prostate gland and unorganized material. Prostasomes possess an unusual lipid composition, contain a number of proteins and small molecules and have been claimed to take a part in the immune response, in seminal fluid liquefaction and in sperm motility. Since sperm may come in contact with an acidic environment in the vagina, it may be of some interest to know whether prostasomes may affect spermatozoon motility or may protect spermatozoa upon the exposure to an acidic milieu. Human semen was supplied by donors. From whole semen we collected spermatozoa by centrifugation and used the supernatant to prepare prostasomes (centrifugation at 105,000 g for 120 min, followed by purification step on Sephadex G 200); spermatozoa were then collected by a swim-up procedure and exposed to an acidic pH medium (from 5 to 7) in the presence or absence of prostasomes. Spermatozoa motility was subsequently assessed with a superimposed image analysis system (SIAS). Results indicate that the motility of spermatozoa was affected by the pH value of the medium. Acidic media reduced the percentage of motile cells and decreased the straight line velocity of spermatozoa (VLS). Prostasomes had a protective effect and increased the percentage of motile cells. However, they did not change the characteristics of motility (curvilinear and straight). Prostasomes may be considered as a system for counteracting the negative effects of acidic pH values that may be present in the vagina after coitus.     

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.     

Selenoprotein P in seminal fluid is a novel biomarker of sperm quality

Hepatically-derived selenoprotein P (SePP) transports selenium (Se) via blood to other tissues including the testes. Male Sepp-knockout mice are infertile. SePP-mediated Se transport to Sertoli cells is needed for supporting biosynthesis of the selenoenzyme glutathione peroxidase-4 (GPX4) in spermatozoa. GPX4 becomes a structural component of sperm midpiece during sperm maturation, and its expression correlates to semen quality. We tested whether SePP is also present in seminal plasma, potentially correlating to fertility parameters. Semen quality was assessed by sperm density, morphology and motility. SePP was measured by an immunoluminometric assay, and trace elements were determined by X-ray fluorescence spectroscopy. SePP levels were considerably lower in seminal plasma as compared to serum (0.4 ± 0.1 mg/l vs. 3.5 ± 1.0 mg/l); Se concentrations showed a similar but less pronounced difference (48.9 ± 20.7 μg/l vs. 106.7 ± 17.3 μg/l). Se and Zn correlated positively in seminal fluid but not in serum. Seminal plasma SePP concentrations were independent of serum SePP concentrations, but correlated positively to sperm density and fraction of vital sperm. SePP concentrations in seminal plasma of vasectomized men were similar to controls indicating that accessory sex glands are a testes-independent source of SePP. This notion was corroborated by histochemical analyses localizing SePP in epithelial cells of seminal vesicles. We conclude that SePP is not only involved in Se transport to testes supporting GPX4 biosynthesis but it also becomes secreted into seminal plasma, likely important to protect sperm during storage, genital tract passage and final journey.     

Prostasome-like vesicles stimulate acrosome reaction of pig spermatozoa

Background  

The presence of small membranous particles characterizes the male genital fluids of different mammalian species. The influence of semen vesicles, denominated prostasomes, on sperm functional properties has been well documented in humans, but their biological activity is scarcely known in other species. The present work investigated prostasome-like vesicles in pig semen for their ability to interact with spermatozoa and to affect acrosome reaction.     

Interactions between prostasomes and leukocytes

Prostasomes are membranous vesicles (150–200 nm diameter) present in human semen. They are secreted by the prostate gland and contain large amounts of cholesterol, sphingomyelin and Ca²⁺. In addition, some of their proteins are enzymes. Prostasomes enhance the motility of ejaculated sperm and are involved in a number of biological functions. In a previous work, we found that prostasome can fuse to spermatozoa at slightly acidic pH values, as demonstrated by the transfer of the lipophilic octadecylrhodamine probe. In this paper, we study the interactions of two leukocyte populations (polymorphonuclear and mononuclear) with prostasomes and find a pH-dependent adhesion (revealed by microscopic observation), but no fusion. These phenomena may be relevant for the functions of leukocytes in human reproduction.     

Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse.

To analyse the effect of the state of the sperm plasma membrane on oocyte activation rate following intracytoplasmic sperm injection (ICSI), three types of human and mouse spermatozoa (intact, immobilised and Triton X-100 treated) were individually injected into mouse oocytes. At 30, 60 and 120 min after injection, maternal chromosomes and sperm nuclei within oocytes were examined. Following human sperm injection, the fastest and the most efficient oocyte activation and sperm head decondensation occurred when the spermatozoa were treated with Triton X-100. Intact spermatozoa were the least effective in activating oocytes. Thus, the rate of mouse oocyte activation following human sperm injection is greatly influenced by the state of the sperm plasma membrane during injection. When mouse spermatozoa were injected into mouse oocytes, the rates of oocyte activation and sperm head decondensation within activated oocytes were the same irrespective of the type of sperm treatment prior to injection. We witnessed that live human spermatozoa injected into moue oocytes often kept moving very actively within the ooplasm for more than 60 min, whereas motile mouse spermatozoa usually became immotile within 20 min after injection into the ooplasm. In 0.002% Triton X-100 solution, mouse spermatozoa are immobilised faster than human spermatozoa. These facts seem to suggest that human sperm plasma membranes are physically and biochemically more stable than those of mouse spermatozoa. Perhaps the physical and chemical properties of the sperm plasma membrane vary from species to species. For those species whose spermatozoa have 'stable' plasma membranes, prior removal or 'damage' of sperm plasma membranes would increase the success rate of ICSI.     

CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa.

Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, which showed the localization of CD26 at the postacrosomal region and at the midpiece of the spermatozoa after incubation with vesicles. Moreover, a similar localization of CD26 and ecto-ADA on the spermatozoa was evidenced after fusion. Our results suggest that the interaction CD26/ecto-ADA might be responsible for fusion. Since A(1)ARs are said to be second receptors for ecto-ADA to form ecto-ADA/A(1)AR complexes, and since horse spermatozoa have A(1)ARs at their surface, the interaction CD26/ecto-ADA/A(1)AR during the fusion process cannot be ruled out.     

Role of Cholesterol, DOTAP, and DPPC in Prostasome/Spermatozoa Interaction and Fusion

Prostasomes are membranous vesicles present in ejaculated human semen. They are very rich in cholesterol and can interact with spermatozoa. Their physiological roles are still under study. Prostasomes were mixed with liposomes prepared from various lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), DOTAP/1,2-dipalmytoyl-sn-glycero-3-phosphorylcholine (DPPC, 4:1 molar ratio) and DOTAP/cholesterol (4:1, molar ratio) at different pH values (5–8). The mixing of the lipid phases (fusion) was determined by the relief of octadecyl rhodamine B chloride (R₁₈) self-quenching and the radii of the vesicles, by light scattering measurements. The mixing of lipids and the radii of prostasomes were both influenced by the addition of liposome, although in a different manner. The ability of prostasomes (modified by previous treatment with liposomes) to transfer lipid to spermatozoa was also measured. Pretreatment with DOTAP decreased the phenomenon and addition of DPPC abolished it. On the other hand, pretreatment of prostasomes with DOTAP/cholesterol liposomes did not affect the transfer of lipid between prostasome and spermatozoa. Therefore, the ability of vesicles to fuse (or, at least, to exchange the lipid component) was affected by the enrichment in either natural or artificial lipid. This may open new possibilities for the modulation of spermatozoa capacitation and acrosome reaction.     

Feasibility of sex-sorting sperm from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis)

The objective of these studies was to investigate the practicality of flow cytometric sex-sorting for spermatozoa from the white and the black rhinoceros (Ceratotherium simum, Diceros bicornis). In Experiment 1, four semen extenders were tested regarding their suitability for liquid preservation of spermatozoa before sorting. Dilution in MES-HEPES-based semen extender followed by incubation generated best sperm quality parameters (motility, viability, and acrosome integrity). In Experiment 2, the effect of staining method (15 °C for 4 to 6 h during transport or 37 °C for 1 to 1.5 h) on sort efficiency and sperm quality was investigated. Staining at 15 °C during transport resulted in a higher percentage of sperm samples showing a resolution of X- and Y-chromosome-bearing populations (60%) compared with that for staining at 37 °C after transport (33%) and resulted in superior sperm integrity after staining (43.8 ± 11.3% vs. 19.6 ± 12.1%). Sort rate was 300 to 700 cells/sec and sort purity, determined for one sorted sample, was 94% for X-chromosome-bearing spermatozoa. In Experiment 3, the highly viscous component of rhinoceros seminal plasma, which complicates the process of sperm sorting, was examined by gel electrophoresis and mass spectrometry. Results suggested a 250-kDa glycoprotein (most likely originating from the bulbourethral gland) to be responsible for the characteristic viscosity of ejaculates. In Experiment 4, viscosity of seminal plasma, as measured by electron spin resonance spectroscopy, was significantly decreased after addition of α-amylase or collagenase (0.5 and 3 IU per 100 μL seminal plasma, respectively) by 28% and 21%, respectively, with no negative effect on sperm characteristics. The results of this study demonstrate for the first time that rhinoceros spermatozoa can be successfully sorted into high-purity X- and Y-chromosome-bearing populations. Furthermore, the successful liquefaction of viscous ejaculates provides the means to greatly improve sort-efficiency in this species.     

In vitro evaluation of fresh sperm quality in tomcats: A comparison of two collection techniques

The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P < 0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P < 0.01), while CT sperm contained more spermatozoa with tail abnormalities (P < 0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P > 0.05) between CT and EP sperm. Nevertheless, no difference (P > 0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.     

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.)

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 μm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 μm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg⁻¹ (average: 283.88 ± 33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P < 0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg⁻¹. Osmolality above 375 mOsmol kg⁻¹ inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.     

Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig

The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 °C for up to 48 h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Cζ (PLCζ) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCζ in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18 h at 24 °C (78%, 62%, and 35%, respectively) and for 48 h at 14 °C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCζ in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.     

Identification of distinct populations of prostasomes that differentially express prostate stem cell antigen, annexin A1, and GLIPR2 in humans

In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 ± 13 nm vs. 105 ± 25 nm) but similar equilibrium buoyant density (～1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostate-specific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals.     

A possible role for the pentose phosphate pathway of spermatozoa in gamete fusion in the mouse

Glucose metabolism is essential for successful gamete fusion in the mouse. Although the metabolic activity of the oocyte does not appear to play a significant role in the fusion step, the metabolic role of the spermatozoon is not known. The aim of this study was therefore to characterize the role of glucose metabolism in mouse spermatozoa. Initially, the high-affinity glucose transporter GLUT3 was identified in mouse sperm. In characterizing the glucose metabolism of mouse sperm, we have shown 1) that mouse epididymal spermatozoa have a functional pentose phosphate pathway (PPP), implying that they produce NADPH, which is required for reducing reactions, and ribose 5-phosphate, which is required for nucleic acid synthesis; and 2) that sperm are able to fuse with the oocyte when NADPH is substituted for glucose, suggesting that sperm need to produce NADPH via the PPP in order to be able to achieve fertilization. The existence of an NADPH-regulated event that influences the ability of the sperm to fuse with the oocyte is envisaged.