Molecular Human Reproduction, Vol. 6, No. 10, 883-891,
October 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Prostasomes inhibit the NADPH oxidase activity of human neutrophils
1 Laboratoire de Biologie de la Reproduction – CECOS, and 2 Laboratoire de Biochimie, CHU Hôtel Dieu, 63003 Clermont-Ferrand, France
Abstract
Prostasomes are particular lipid vesicles secreted by the prostate in human semen and involved in several physiological functions such as the improvement of sperm motility or immunomodulation. We have previously shown that they reduced the overall reactive oxygen species (ROS) production of seminal polymorphonuclear neutrophils (PMN). The present study was conducted to define the mechanism by which prostasomes inhibit the ROS production of blood and seminal PMN. The luminol chemiluminescence measuring total ROS production of blood PMN stimulated by either a phorbol ester (PMA) or a chemoattractant peptide, formyl-Met-Leu-Phe (fMLP) was significantly inhibited by prostasomes. The NADPH oxidase activity of the PMN was measured by 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA) chemiluminescence. Prostasomes inhibited the NADPH oxidase activity of blood or seminal PMN and increased the lag-phase of the enzyme after PMA stimulation. Prostasomes also inhibited significantly the NADPH oxidase activity of fMLP stimulated blood PMN, but the inhibition was not significant for seminal PMN. The lipid composition of blood PMN was analysed and compared to the lipid composition of prostasomes. This showed that prostasomes had a high cholesterol:phospholipid molar ratio and a high proportion of sphingomyelin. Together with the fact that prostasomes can rigidify the plasma membrane of blood PMN, these results led us to postulate that prostasomes inhibit the NADPH oxidase activity of PMN by lipid transfer from the prostasomes to the plasma membrane of the PMN.
NADPH oxidase/polymorphonuclear neutrophils/prostasomes/reactive oxygen species/semen
Introduction
The importance of oxidative stress as a cause of male infertility has been a growing idea for the last 20 years. It is now known that the presence of a high amount of reactive oxygen species (ROS) in human semen can be deleterious for the functions of spermatozoa (Griveau and Le Lannou, 1997
; Aitken, 1999). On the other hand, low doses of ROS and particularly of the superoxide anion (O2·–) are also implicated in the initiation of the capacitation process and the acrosome reaction, both of which are prerequisites for fertilization (DeLamirande and Gagnon, 1995; Griveau et al., 1995). Spermatozoa have been long considered as the major ROS producing cells in semen but it now appears that the white blood cells sometimes infiltrating the semen are the main producers of ROS (Aitken and West, 1990; Wolff, 1995; Whittington and Ford, 1999).
The seminal plasma possesses antioxidant properties to protect spermatozoa against the ROS-induced damage, but these capacities can sometimes be overwhelmed (Aitken et al., 1995). Indeed, ROS can cause lipoperoxidation of the sperm plasma membrane due to its high content of polyunsaturated fatty acids (Jones et al., 1979), as well as attacking DNA and provoking fragmentation (Twigg et al., 1998).
Leukocytospermia (>1x106 PMN/ml) is usually a consequence of the inflammation of one or more of the male accessory sex glands (Wolff, 1995). However, it is not always associated with a decrease in sperm function in vivo (Tomlinson et al., 1993). The important capacity of polymorphonuclear neutrophils (PMN) to produce ROS arise from a specialized enzyme, NADPH oxidase, which catalyses the production of O2·– by the reduction of oxygen, using NADPH as the electron donor: 2 O2 + NADPH 
2O2·– + NADP+ + H+.
This enzyme is in an inactive conformation in the resting cells, with separate cytoplasmic and membrane subunits, and is assembled into an active form upon activation with stimuli, e.g. interleukin-8 (IL-8), and the C5a fraction of complement or formylated peptides (for review see Babior, 1999). In semen, the infiltrating PMN seem to be at least in part in an activated state (Aitken et al., 1995), supporting the fact that they probably come from a site of inflammation, and thus represent an oxidative stress towards sperm cells.
Prostasomes are human-specific extracellular organelles secreted by the prostate into seminal plasma; they are implicated in a number of physiological functions, e.g. the improvement of sperm motility (Fabiani et al., 1994), seminal fluid liquefaction, and immunomodulation (Kelly, 1999). Prostasomes are characterized mainly by their particular structure and composition: lipid vesicles ranging in size from 30 to 500 nm, surrounded by a multi-lamellar membrane, and containing a high proportion of cholesterol and sphingomyelin, with a cholesterol:phospholipid molar ratio of ~2:1 giving rise to a high ordering of their phospholipid bilayers (Arvidson et al., 1989). In a previous work (Saez et al., 1998), we showed that prostasomes caused a decrease in ROS production by seminal PMN. This effect was not due to a scavenging capacity of the prostasomes. Prostasomes also provoked rigidity in the plasma membrane of blood PMN, which was used as a model.
The aim of this study was to further investigate the mechanism by which prostasomes inhibit the production of ROS by PMN. We first evaluated the effect of prostasomes on luminol chemiluminescence of blood PMN stimulated with either 12-myristate-13-acetate phorbol ester (PMA) or formyl-Met-Leu-Phe (fMLP). The NADPH oxidase activity of blood and seminal PMN was then measured in the presence or absence of different concentrations of prostasomes, using a chemiluminescence method with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA), a Cypridina luciferin analogue, as a probe. We show that prostasomes inhibit NADPH oxidase activity of both blood and seminal PMN with different characteristics, depending upon whether the cells were stimulated by PMA or fMLP. We propose that the mechanism of action of prostasomes could be the inhibition of the enzyme assembly in its active form at the cell surface. The particular lipid composition and structure of the prostasomes is probably responsible for their inhibitory properties.
Materials and methods
Semen samples
Semen samples were obtained from men referred to our laboratory for semen analysis screening. The ejaculates were collected by masturbation after 2–3 days of sexual abstinence and allowed to liquefy at 37°C for ~30 min before use. Sperm concentration, motility, vitality, morphology and round cells count were assessed according to World Health Organization guidelines (WHO, 1992). The characterization of PMN was carried out using the benzidin–cyanosin coloration technique yielding a brown-coloured PMN. A semen sample was considered normozoospermic if the following parameters were verified: >20x106 spermatozoa/ml; progressive motility >50%; vitality >75%; normal morphology >30% and PMN <1x106 ml. For the experiments of O2·– production by seminal PMN, 16 different semen samples were used. All of these samples were characterized by a strong leukocytospermia and some were associated with teratozoospermia (n = 7), oligoasthenoteratozoospermia (n = 3), oligoteratozoospermia (n = 2), asthenoteratozoospermia (n = 1), oligozoospermia (n = 1) or azoospermia (n = 1); one sample had isolated leukocytospermia. In addition, all these patients had negative bacteriospermia according to described previously criteria (Grizard et al., 1985). In certain cases, the O2·– production was measured for the same sample after PMA stimulation and also after fMLP stimulation. Semen characteristics are summarized in Table I.
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Preparation of prostasomes
The semen samples used for the preparation of prostasomes were normozoospermic. Prostasomes were obtained as described previously (Ronquist and Brody, 1985
). Briefly, seminal plasma (pooled from 14 different samples) was centrifuged at 105 000 g for 2 h at 4°C. The prostasomes were separated from an amorphous material, by chromatography on Sephadex G-200. Finally, purified prostasomes were harvested by centrifugation at 105 000 g for 2 h at 4°C, resuspended in phosphate-buffered saline (PBS) pH 7.4 (Sigma-Aldrich, Saint Quentin Fallavier, France) and stored at –20°C. The quantities of prostasomes used for experiments are expressed in terms of cholesterol and the physiological concentration (i.e. 1x is ~130 nmol/ml; Sion et al., 1994). The concentrations used in this study were prostasomes 0.05x, prostasomes 0.1x and prostasomes 0.2x, corresponding to 6.5, 13 and 26 nmol/ml respectively of cholesterol. In a series of experiments, the prostasomes were subjected to a 5 min boiling step before use, in order to investigate the influence of their protein content on their action towards ROS production by the PMN.
Preparation of blood PMN
The purification of PMN from whole blood collected in heparin–lithium tubes was performed by gradient centrifugation using Histopaque (Sigma) (Saez et al., 1998). The final PMN suspension was resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma) and counted. PMN vitality determined by Trypan Blue exclusion was always >95%.
Preparation of seminal PMN
To obtain the cellular components of the selected leukocytospermic semen (PMN >1x106/ml), whole semen was washed with 10 ml RPMI 1640 medium and centrifuged at 500 g for 10 min at room temperature. The pellet was resuspended in 500 µl of RPMI 1640 and the cells were counted again. The seminal PMN were not isolated from the other cells contained in the original semen (spermatozoa and immature germinal cells).
Assay of individual phospholipid molecular species by high performance liquid chromatography (HPLC)
Prostasomes and PMN were characterized by measuring their lipid content. Total cholesterol and phospholipids were estimated after lipid extraction according to a previously described method (Folch et al., 1957). The cholesterol:phospholipid molar ratio was calculated from these values.
The phospholipid patterns of prostasomes and PMN were determined from the lipid extract using HPLC, performed on a Kontron System (Milan, Italy) equipped with a programmable ternary gradient pump (system 325), an automatic injector with a 100 µl loop (autosampler 360) and coupled with a light scattering detector (DDL 21; Eurosep Instruments, Cergy Pontoise, France). The assays were carried out on a 250x3 mm column packed with Inerstil 5 µm silica and a ternary mobile phase composed of solvent A (hexan), solvent B (chloroform:methanol, 100:8, v/v) and solvent C (methanol:formic acid 0.05 mol/l in water, 100:4 v/v). Elution was performed at room temperature at a flow rate of 0.35 ml/min (Grizard et al., 2000). This method enabled evaluation of the relative proportions of five major membrane phospholipids: sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylserine (PS).
Chemiluminescence
Luminol chemiluminescence
The total production of ROS (intra- and extra-cellular) by blood PMN was measured by luminol chemiluminescence as previously described (Saez et al., 1998). Briefly, 200 000 blood PMN were placed in a measuring cuvette in a final volume of 500 µl in RPMI 1640 medium and, when needed, prostasomes were added at a final concentration varying between prostasomes 0.05x and 0.2x. From a stock solution of 25 mmol/l luminol in dimethylsulphoxide (DMSO), 4 µl was added to each sample and the luminescence of individual samples was measured at a chamber temperature of 37°C in a Bio-Orbit 1251 luminometer (Kontron, Paris, France).
After 330 s corresponding to the basal state, the cells were stimulated by 80 nmol/l of PMA (Sigma) and monitored for the following 30 min. PMA is a phorbol ester, a structural analogue of the diacyl glycerol, and has the ability to pass through the plasma membrane and directly activates protein kinase C, leading to activation of the respiratory burst in PMN. The resulting curve enabled several values to be calculated: the lag phase of the NADPH oxidase, corresponding to the time required for its assembly in an active conformation on the cell plasma membrane and measured on the curve as the time between the injection of PMA and the increase of the signal (expressed in seconds, see Figure 1), the maximum net chemiluminescence (Net CL expressed in mV), and the integral under the curve 10–20 min after the lag phase corresponding to the enzyme activity (Int 10 min and Int 20 min, expressed in mV/s/104 PMN, see Table II and Figure 1). Luminol reacts with both intra- and extra-cellular ROS produced by the activated PMN but is not O2·–-specific. Consequently, the chemiluminescence signal obtained is related to the NADPH oxidase activity, but is not specific.
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In another set of experiments, the same protocol was used except that the cells were stimulated by 1 µmol/l of the chemotactic peptide fMLP (Sigma) and 500 000 blood PMN per sample were used for sensitivity reasons. fMLP possesses membrane receptors coupled to the heterotrimeric G proteins, also leading to the activation of the protein kinase C and the subsequent respiratory burst. Here, no lag phase was observed and the values measured were the maximum net chemiluminescence (Net CL in mV) and the integral under the curve after stimulation (Instim) during the rest of the measurement (570 s), expressed in mV/s/104 PMN.
MCLA chemiluminescence: measurement of the NADPH oxidase activity
The production of O2·– secreted by the PMN in the extracellular milieu was monitored by chemiluminescence with MCLA (Interchim, Montlucion, France) as the luminescent probe. This molecule is a Cypridina luciferin analogue and is specific for the detection of extracellular O2·–. The samples were prepared as described above and 1 µl from a stock solution of 10 mmol/l MCLA in DMSO was used. After 330 s corresponding to the basal state, the cells (200 000 blood or seminal PMN) were stimulated by either 80 nmol/l PMA or 1 µmol/l fMLP and the chemiluminescent signal was measured for individual samples for the following 30 min or 270 s respectively. The same values as for the luminol chemiluminescence were measured, except that the integral under the curve after fMLP stimulation (Intstim) was measured for only 170 s (expressed in mV/s/104 PMN). Due to the high auto-oxidation of the probe MCLA giving a strong background signal, the values of the integrals under the curves were always corrected by the values obtained with a blank (same sample with no stimulation) in order to only measure the signal depending on the production of O2·– by the cellular NADPH oxidase. The specificity of the signal was also confirmed by the use of superoxide dismutase (SOD, an O2·– scavenger, 500 IU/ml; Sigma) which inhibited the chemiluminescence to the blank level, and by catalase (hydrogen peroxide scavenger, 0.2 mg/ml, Sigma) which had no effect. The specificity for NADPH oxidase activity was confirmed by the inhibition of the chemiluminescence by 25 µmol/l diphenylene–iodonium (Sigma), a specific inhibitor of NADPH oxidase (data not shown).
Statistical analysis
Wilcoxon-signed Rank test (non-parametric test) for paired data was applied to evaluate the influence of the different concentrations of prostasomes; P < 0.05 was considered to be statistically significant.
Results
Luminol chemiluminescence on blood PMN
Luminol is a permeant probe which reveals both intracellular and extracellular produced ROS, with a superior affinity towards hydrogen peroxide and O2·–. When 200 000 blood PMN were stimulated by 80 nmol/l PMA (see Table II
), prostasomes had a significant inhibitory action on all the parameters of ROS production, from prostasome concentration 0.05x to 0.2x (except on Int 20 min for prostasomes 0.05x). In the same time, the lag-phase of the enzyme was significantly increased and this effect was more pronounced with prostasome concentration 0.2x, as indicated by the significant difference between prostasomes concentration 0.2x and 0.05x.
When 500 000 blood PMN were stimulated by 1 µmol/l fMLP (Table III), the net chemiluminescence was significantly inhibited for all the prostasome concentrations, and the total ROS production (represented as the integral under the curve after stimulation, Intstim) was significantly inhibited for the prostasomes concentrations 0.1x and 0.2x.
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Thus, prostasomes inhibited the overall production of ROS by PMA or fMLP-stimulated blood PMN. In semen, oxidative stress for spermatozoa is mainly related to extracellular released ROS and the primary enzyme responsible for this synthesis is membrane NADPH oxidase. We therefore investigated the effect of prostasomes on the kinetics of this enzyme in blood and seminal PMN.
MCLA chemiluminescence on blood PMN
In contrast to luminol, MCLA is not cell permeable and thus stays in the extracellular compartment. After the kinetics of individual samples had been determined, the same values for luminol chemiluminescence were measured and the results are presented in Figure 1 and Table IV. The action of prostasomes appears on the kinetic curves of Figure 1, showing a reduction in net chemiluminescence and enzyme activities, as well as an increase in the lag phase. All the parameters were significantly different (P < 0.05) between the prostasome concentrations of 0.2x and 0.05x (Table IV). The lag-phase of NADPH oxidase is the most representative parameter, showing a significant increase for prostasome concentration 0.05x compared with no prostasomes and also between prostasome concentrations 0.2x and 0.1x. The lag-phase is probably the most specific parameter of the NADPH oxidase activation, indicating a high sensitivity to the presence of prostasomes.
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Another set of experiments was carried out on blood PMN stimulated by 1 µmol/l fMLP and the results are shown in Figure 2
and Table V. Whatever the concentration of prostasomes used, there was a significant inhibition of the net chemiluminescence (except for prostasome concentration 0.1x) and Intstim.
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MCLA chemiluminescence on seminal PMN
The action of prostasomes on the NADPH oxidase activity of seminal PMN was evaluated based on the results obtained with blood PMN. The measurements were performed on cell suspensions obtained from semen containing >1x106 PMN/ml (see Table I
). The results of PMA-stimulated PMN, presented in Table IV, show that when no prostasomes were added, the kinetic of the luminescence was very similar to that obtained for blood PMN. Prostasomes also had a significant inhibitory action on all the parameters of the NADPH oxidase of seminal PMN (except on Int 20 min for prostasomes 0.05x). The lag-phase of the enzyme was very sensitive to the presence of prostasomes as there was a significant difference between all the different concentrations of prostasomes. We also noticed higher SEM values for the lag-phase of seminal PMN compared with blood PMN, indicating important inter-individual differences concerning the activation of the NADPH oxidase after PMA stimulation. For the three concentrations of prostasomes with seminal PMN, in contrast to blood PMN, the other parameters are not significantly different between each others, except Int 10 min between prostasomes 0.2x and 0.1x.
The results obtained for the fMLP stimulated seminal PMN (Table V) do not show any significant difference for any parameter in the presence of prostasomes. This emphasized the differences between blood and seminal PMN, and raised the importance of the inter-individual discrepancies; the enzyme was inhibited in almost all individual cases, but the comparison of the means did not lead to any significant differences.
Effect of the temperature on the inhibition capacity of the prostasomes
To test if the numerous enzymes contained by the prostasomes could play a role in the NADPH oxidase inhibition, the prostasomes were submitted to a 5 min boiling step prior to their addition to the chemiluminescence reaction medium. The results obtained are summarized in Figure 3. There is no difference in the inhibition whether the prostasomes were boiled or not, indicating that their enzymatic content does not play a role in the inhibition of the NADPH oxidase activity.
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Phospholipid composition of prostasomes and of blood PMN
The results presented in Table VI
reveal the very particular composition of the prostasomes, with a very important proportion of SM and a high PE/PC ratio. These are two characteristics of the rigid biological membranes. The mean cholesterol: phospholipid ratio of 1.7 in prostasomes is also very high and is another feature of a rigid membrane. In comparison with the PMN phospholipid composition, the main differences are the cholesterol:phospholipid ratio and the proportions of SM, PE and PC.
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Discussion
The white blood cells, particularly the PMN, infiltrating human semen are now recognized as the major source of ROS, and are potentially hazardous to the male gamete's functional activity. We previously demonstrated that prostasomes could decrease the overall ROS production in cell suspensions isolated from PMN containing semen, in the basal state or stimulated by PMA (Saez et al., 1998).
In this study, the inhibitory capacity of prostasomes on luminol chemiluminescence was tested on blood PMN stimulated by PMA (80 nmol/l) or the chemoattractant peptide fMLP (1 µmol/l). The inhibition was confirmed when cells were stimulated by PMA and we found that the ROS production was also significantly decreased by prostasomes when fMLP was the stimulant. The overall ROS production triggered by both activation pathways was thus inhibited by the presence of prostasomes. These results are in agreement with previous results (Skibinski et al., 1992) who found that blood PMN pre-incubated with prostasomes were less responsive to PMA and fMLP, as revealed by the measurement of O2·– with the reduction of ferricytochrome c to ferrocytochrome c. However, this method does not allow a kinetic study (the authors used an end-point method to measure O2·–) and is much less sensitive than the chemiluminescence method (Aitken et al., 1992). In addition, these authors did not suggest a mechanism of action for the prostasomes, except that they probably do not act as scavengers, a fact that we can also verify.
The enzyme responsible for the synthesis of ROS in PMN is NADPH oxidase, which produces O2·– from O2 and NADPH. This enzyme is activated by PMA or fMLP and the secretion of O2·– in the extracellular space as well as in the phagosomes is the final step of the activation. The NADPH oxidase activity, measured by MCLA chemiluminescence, was first evaluated on blood and seminal PMN stimulated by PMA. In absence of prostasomes, we noticed that the chemiluminescence profiles as well as the values calculated from the curves are very similar for blood and seminal PMN. This result is in accordance with the fact that PMN are the major ROS producing cells in the ejaculate. Furthermore, it emphasizes the fact that spermatozoa in the cell suspensions do not secrete O2·– in the extracellular medium in a sufficient amount, after PMA stimulation, to be detected by this method. If spermatozoa possess an NADPH oxidase, it is probably not a PMN-like enzyme, as already mentioned (Aitken et al., 1997). This is supported by the fact that spermatozoa isolated from semen containing no PMN, and stimulated by PMA, do not produce a chemiluminescence signal higher than the blank in our experimental conditions (data not shown). The small contribution of spermatozoa to O2·– production in semen had already been highlighted (Alvarez et al., 1987) who also showed strong inter-individual differences in the levels of production.
One of the main features of the inhibition by the prostasomes is the increase of the lag-phase of the enzyme. Normally, the secretion of O2·– is detectable ~30 s after stimulation by PMA, but the presence of even low doses of prostasomes (0.05x) caused a 4–5-fold increase of this lag-phase, whether in blood or seminal PMN.
Previous studies (Kobayashi et al., 1998; Vaissiere et al., 1999) both showed that upon stimulation, the NADPH oxidase was first assembled in an active conformation in intracellular granules, and that O2·– was secreted in these granules which were then exocytosed, secreting O2·– in the extracellular space. It also seems that some active NADPH oxidase would be assembled on the plasma membrane itself. The increase in the lag-phase and the fact that the presence of prostasomes caused a rigidification of the PMN plasmic membrane (Saez et al., 1998) allow us to propose that prostasomes may well interfere with the important vesicle trafficking and membrane modifications following cell stimulation by PMA.
The O2·– production of blood PMN stimulated by fMLP was significantly inhibited by prostasomes. Thus, the prostasome-dependent inhibition of O2·– production also showed an inhibition of this pathway. It confirms that the steps downstream of protein kinase C activation, particularly the exocytosis of the vesicles of secretion, are probably inhibited. It is also possible that the activity of the fMLP receptor, a member of the seven transmembrane spanning domain receptor family, is impaired by the presence of prostasomes and the subsequent membrane rigidification. This hypothesis could account for the fact that seminal PMN are less responsive to fMLP than to PMA, as compared to blood PMN. Indeed, the plasma membrane of seminal PMN might already be impaired by previous contact with prostasomes in the semen or even in the prostate. The fact that prostasomes cause a reduction of the NADPH oxidase activity measured after the lag-phase following PMA stimulation, favours this hypothesis. Thus, the rigidification of the PMN plasma membrane provoked by the prostasomes might have consequences at two levels of the NADPH oxidase activation: the activity of the membrane receptor (initial step) and the secretion of the oxidant products (final step), accompanied by a decrease in the overall activity. This hypothesis is supported by our experiments with `boiled' prostasomes which were shown to have the same inhibitory activity as native prostasomes, thus indicating that the different proteins of the prostasomes do not seem to be implicated in the phenomenon. Also, the phospholipid composition of the prostasomes, compared to the blood PMN, reveals much more sphingomyelin and cholesterol, two rigidifying components of the biological membranes. A previous study (Arienti et al., 1998) showed that prostasomes did not fuse with PMN but only adhered to the cells in a pH-dependent fashion, the adhesion being more efficient at pH values corresponding to our experimental conditions (pH 7.4). This observation is in accordance with a possible lipid exchange between these two components. Another study (Day et al., 1997) showed that, after treatment of hypercholesterolaemic patients with simvastatin, an inhibitor of the HMGCoA reductase, there was a strong correlation between cell cholesterol content and NADPH oxidase lag phase of isolated blood PMN, a conclusion that further supports our results and hypotheses.
With regard to seminal PMN, the important inter-individual variations might come from the point that before the experiments and the separation of the cells from the seminal plasma, the PMN were already in contact with prostasomes in the ejaculate, as mentioned previously. Furthermore, prostatitis is one of the common inflammations of the male genital tract, which means that the contact might have occurred in certain cases in the prostate, before ejaculation. It was shown (Vicari, 1999) that white blood cells from the 45% Percoll fraction isolated from patients with prostatitis had a significantly reduced capacity to produce ROS, when compared with samples from patients with prostato-vesiculitis or prostato-vesiculo-epididymitis. This result might reflect the in-vivo inhibition of the enzyme from PMN present in the prostate. Depending on these parameters and considering our experimental procedure, the influence of prostasomes on seminal PMN might differ from case to case.
In cases of leukocytospermia, the physiological importance of the inhibition of NADPH oxidase in PMN by prostasomes may also be relevant when the cells are separated from the seminal plasma for sperm preparation in assisted reproduction techniques. Indeed, the high antioxidant capacities of seminal plasma and prostasomes are not available in this situation.
In conclusion, we demonstrate that the antioxidant action of the human prostasomes comes from the inhibition of NADPH oxidase in PMN, probably due to the particular lipid composition and structure of the prostasomes. These results are original because they represent, to our knowledge, the only physiological inhibition of the NADPH oxidase not acting via a chemical pathway. In our model, the properties of the prostasomes could lead to an interference with the important vesicle trafficking and membrane modifications following the PMN stimulation, thus reducing the secretion of O2·– in the extracellular compartment by the activated NADPH oxidase.
Acknowledgments
The authors thank Mr Benoit Sion and Mrs Claudine Nouailles for carrying out the high performance liquid chromatography.
Notes
3 To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction CHU Hôtel Dieu, 63003 Clermont-Ferrand, France. E-mail: fabrice.saez{at}u-clermont1.fr 
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Submitted on May 8, 2000; accepted on July 11, 2000.
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