Molecular Human Reproduction, Vol. 7, No. 3, 237-244,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
A critical investigation of NADPH oxidase activity in human spermatozoa
University of Bristol, Division of Obstetrics & Gynaecology, St Michael's Hospital, Southwell Street, Bristol BS2 8EG, UK
Abstract
It has been suggested that human spermatozoa contain an NADPH oxidase that could generate reactive oxygen species involved in signalling pathways to promote fertility. The proposal depends on observations that the addition of NADPH to purified human spermatozoa stimulates chemiluminescence by the superoxide (O2–) probe, lucigenin. We confirmed these observations, but demonstrated that lucigenin increases NADPH consumption by spermatozoa and stimulates artefactual O2– production via a diphenyleneiodonium (DPI) sensitive flavoprotein. In the absence of cytochrome c, DPI-inhibitable NADPH oxidation by permeabilized spermatozoa was 8 times too small to account for the rate of NADPH-stimulated cytochrome c reduction. Thus NADPH can directly reduce cytochrome c by a flavoprotein dependent mechanism making this O2– assay also unreliable in sperm suspensions. We were unable to observe O2– production by 40 x 106 spermatozoa/ml using electron paramagnetic resonance spectroscopy but could identify O2– generation from 2000 4ß-phorbol-12-myristate-13-actetate (PMA)-stimulated leukocytes. Using spectrophotometry, we did not detect the reduced cytochrome b558 component of the neutrophil NADPH oxidase in human spermatozoa. No hydrogen peroxide generation was observed using a sensitive Amplex Red assay. We conclude that human spermatozoa do not possess significant NADPH oxidase activity and that the mechanism by which NADPH promotes capacitation must be re-evaluated.
human spermatozoa/lucigenin/NADPH oxidase/spin trapping/superoxide
Introduction
Reactive oxygen species (ROS) can act as signalling molecules (e.g. Suzuki et al., 1997) and there is now much evidence to suggest that sperm capacitation, hyperactivation and acrosome reaction can be enhanced by ROS including the superoxide anion (O2–), hydrogen peroxide (H2O2) and nitric oxide (Bize et al., 1991
; de Lamirande and Gagnon, 1993a,b, 1995; Griveau et al., 1994, 1995; Aitken et al., 1995, 1996, 1998b; Zini et al., 1995; Leclerc et al., 1997; de Lamirande et al., 1998a,b). Contaminating leukocytes are the primary source of ROS in unpurified sperm suspensions (Aitken and West, 1990; Kessopoulou et al., 1992; Whittington and Ford, 1999) and ROS generated by these leukocytes are responsible for the deleterious effects of O2 on human spermatozoa (Whittington and Ford, 1998). However, spermatozoa themselves may produce low levels of ROS sufficient for a signalling role.
The mechanism of ROS production by spermatozoa is a matter of much controversy. It has been suggested that human spermatozoa contain an NADPH oxidase similar to that found in phagocytic leukocytes (Babior et al., 1997). This proposal depends on observations using a lucigenin-based chemiluminescence assay. Addition of NADPH to suspensions of highly purified human spermatozoa induced a rapid increase in lucigenin-dependent chemiluminescence (LDCL) that was taken to reflect increased O2– production. NADPH-induced chemiluminescence was inhibited by superoxide dismutase (SOD), a scavenger of O2– LDCL was unaffected by mitochondrial inhibitors but was decreased by diphenyleneiodonium (DPI) and quinacrine, suggesting the involvement of a flavoprotein. Permeabilizing the spermatozoa by repeated freeze–thawing greatly increased O2– production and oxidase activity was localized to the plasma membrane (Aitken et al., 1997). It was also shown that the addition of NADPH or micromolar values of H2O2 promoted capacitation of human spermatozoa and that both treatments induced similar changes in protein tyrosine phosphorylation (Aitken et al., 1995, 1998b). Hence, it was suggested that the putative oxidase could generate O2– to promote capacitation. Under physiological conditions, NADPH would be produced by glucose metabolism through the pentose phosphate pathway. Higher concentrations of NADPH were found to mimic pathological changes characteristic of oxidative stress (Aitken et al., 1998a; Twigg et al., 1998).
In contrast to the above observations, NADPH was shown not to stimulate extracellular O2– production as detected by 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA) chemiluminescence, whereas addition of progesterone or ultrafiltrates of fetal chord serum, follicular fluid or seminal plasma to human spermatozoa did so (de Lamirande et al., 1998a). NADPH promoted capacitation but unlike the effects of progesterone and ultrafiltrates of biological fluids, this was not blocked by SOD. Recently, the absence of NADPH oxidase activity in human spermatozoa has been reported by another group using a highly sensitive spin trapping technique (Armstrong et al., 1999).
Our study had two main aims: (i) to confirm the presence of an NADPH oxidase in human spermatozoa; and (ii) to investigate the regulation of its activity to ensure that the capacitation-promoting signal was produced at the appropriate time. However, after the project was underway, we became aware that lucigenin is not a reliable probe for O2–. Before reacting with O2–, lucigenin must be reduced to form the lucigenin cation anion, Luc+. Luc+ may then react with O2– to form an unstable dioxetane intermediate which decays by a light emitting process (Faulkner and Fridovitch, 1993). However, Luc+ can also react with oxygen to produce O2– and the redox potential favours this reaction (Spasojevic et al., 2000). Therefore, lucigenin can both initiate and signal O2– generation in the absence of any physiological O2–-producing system. Moreover, LDCL due to artefactual O2– generation can be suppressed with SOD, a control widely accepted to demonstrate genuine O2– production. Since the reduction of lucigenin to Luc+ may be catalysed by numerous enzymatic systems including flavoprotein containing enzymes, this probe should not be used to detect biological O2– production (Liochev and Fridovitch, 1997,1998; Vasquez-Vivar et al., 1997; Spasojevic et al., 2000). In view of these considerations, we have tried to establish whether the putative NADPH oxidase activity of human spermatozoa is a genuine phenomenon or an artefact of the lucigenin assay method of O2– detection.
Materials and methods
Digitonin (high purity), D (+) glucose and sodium hydrogen carbonate were purchased from BDH Laboratory Supplies, Poole, UK. Dynabeads (M-450, anti-CD45 and CD15) were obtained from Dynal A.S (Oslo, Norway). All other reagents were purchased from Sigma (Poole, UK). Unless otherwise stated, reagents were dissolved in Biggers–Whitten–Whittingham (BWW) medium (Biggers et al., 1971) buffered with HEPES (20 mmol/l) and supplemented with polyvinyl alcohol (1 mg/ml) in place of albumin (Aitken et al., 1997). The pH of the medium was adjusted to pH 7.45 with sodium hydroxide.
Sperm preparation
Semen samples were produced by healthy donors and collected via masturbation into sterile containers. All ejaculates exceeded the WHO criteria of normality (World Health Organization, 1992). After liquefaction for up to 45 min at 37°C, spermatozoa were purified from seminal plasma by centrifugation on 40/80% Percoll gradients (Ford et al., 1992). Briefly, 1 ml semen was layered on each gradient and centrifuged (at 330 g for 25 min). The supernatant was removed and pellets washed twice in 2 ml BWW (330 g for 10 min). The final pellet was suspended in 1 ml BWW and the round cell concentration estimated using a Makler chamber. Leukocytes were removed using magnetic Dynabeads coated with monoclonal antibodies against CD15 and the common leukocyte antigen, CD45. The beads were added in accordance with the manufacturer's guidelines and incubated for 1 h at room temperature with gentle agitation. Beads and bound leukocytes were removed with a magnetic separator and the purified spermatozoa pelleted by centrifugation (600 g, 10 min) before being resuspended to the appropriate concentration in BWW medium. All preparations were challenged with 50 µmol N-formyl-methionyl-leucyl-phenylalanine (N-FMLP)/l as described by Whittington and Ford (1999) except that luminesence was measured in a `Bertholdt Microlumat LB96P' chemiluminesence plate reader. The absence of a detectable increase in chemiluminesence was taken to confirm successful removal of leukocyte contamination (Krausz et al., 1992).
Measurement of NADPH-induced ROS production by LDCL
This method was based on a protocol described by Aitken et al (1997) but adapted so that the assays could be performed in 96-well (200 µl) plates. Chemiluminescence was monitored at 37°C using a Berthold luminometer (Microlumat LB96P) in repeated measurement mode. Two µl of 25 mmol/l lucigenin in dimethyl sulphoxide (DMSO) were added to 100 µl of sperm suspension (30–60x106/ml) and the basal signal recorded for 5 min. Then, 100 µl of NAD(P)H (final concentrations 0–10 mmol/l) were injected and chemiluminescence recorded for a further 10–30 min. The background chemiluminescence from cell-free controls was subtracted from all measurements before data analysis. Figures for basal and induced LDCL were calculated from the mean signal recorded over a 5 min period of maximum light output. Chemiluminescence was expressed as relative light units (RLU)/s/108 spermatozoa.
The effect of membrane permeabilization on NADPH-induced ROS production was investigated by treatment of spermatozoa with 1.25–160 µmol digitonin/l for 5 min at 37°C prior to addition of NAD(P)H 0–10 mmol/l. The hypo-osmotic swelling test (HOST) (Jeyendran et al., 1984) was used to assess the integrity of the plasma membrane at each detergent concentration used.
ROS production by both intact and permeabilized cells (10 µmol digitonin/l) induced by NADPH 1 mmol/l was also monitored in the presence of SOD 0.1 mg/ml (S2515, Sigma), catalase 0.2 mg/ml (C-9322, Sigma) or diphenyleneiodonium chloride (DPI) 25 µmol/l (10 mmol/l stock in DMSO, diluted in BWW medium).
Measurement of O2– by reduction of cytochrome c
Cytochrome c reduction was monitored on a dual-wavelength optical plate reader (Tecan Spectra thermo) by following the increased absorbance of the 
band at 550 nm using 540 nm as a reference wavelength (Jones and Hancock, 1994). Assays were performed at 37°C in 96-well (200 µl) plates with shaking between cycles. Plates were prepared with all reagents and pre-warmed before addition of ~4x106 spermatozoa to initiate the reaction. Sperm-free controls were prepared for each test. The signals from these were subtracted from those of the corresponding experimental well so that only sperm-associated reduction of cytochrome c was used for calculation of O2– production. The extinction coefficient (
E550–540) was taken as 19.1 cm/mmol/l.
Intact and permeabilized cells were incubated with NADPH 2mmol/l and the effects of the addition of SOD 0.1mg/ml or DPI 25 µmol/l were also observed.
Measurement of NADPH consumption by spermatozoa
In the first series of experiments, NADPH concentration was monitored by following the decline in absorption at 340 nm using 410 nm as a reference wavelength. The maximum concentration of NADPH that could be used in this protocol was 0.3 mmol/l. To measure the oxidation of NADPH at concentrations comparable with those used in chemiluminescence experiments, the production of NADP+ was determined. Intact or permeabilized spermatozoa (20x106/ml) were incubated in a total volume of 200 µl with NADPH 1 mmol/l for 2 h in darkness at 37°C. Lucigenin 250 µmol/l, digitonin 10 µmol/l and/ or DPI 25 µmol/l were added to selected tubes. At the end of the incubation, 100 µl of 1 mol perchloric acid/l were added to stop the reaction and destroy remaining NADPH before transferring the tubes to ice. Control preparations were acidified at zero time and kept in darkness on ice. Each tube was centrifuged (10 000 g, 1 min) and 200 µl of supernatant transferred to a clean tube and mixed with 20 µl of 2.3 mol K2CO3 + morpholinoethanesulphonic acid (MES) 0.7 mol/l. The tubes were centrifuged a second time (10000 g, 1 min) and 150 µl of the supernatant frozen for assay the following day. NADP+ was measured with a fluorimetric enzymatic assay using glucose 6-phosphate dehydrogenase to catalyse its reduction to NADPH (Williamson and Corkey, 1969). The assay was performed on a Perkin Elmer LS50B luminescence spectrometer (excitation wavelength 340nm, emission 460nm) and calibrated using internal standards.
Measurement of O2– production by electron paramagnetic resonance (EPR) spectroscopy
The superoxide-specific and highly sensitive spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), was used to detect O2– produced by spermatozoa stimulated with NADPH 1 mmol/l. Measurements were performed at room temperature (23°C) using a Bruker ESP 310 X-band EPR spectrometer. The instrument settings were; modulation frequency, 100 kHz; modulation amplitude, 0.403 G (leukocyte measurements), or 1.429 G (sperm measurements); time constant, 10.24 ms; sweep time, 41.943 s; microwave frequency, 9.75 GHz; and microwave power 2e+01 mw. Reaction mixtures were placed in a 1 ml flat cell and serial scans performed in the presence or absence of SOD 0.1 mg/ml.
The EPR spectra of 4ß-phorbol-12-myristate-13-actetate (PMA)-stimulated leukocytes were used as a positive control for O2– production (Roubaud et al., 1998). Leukocytes were prepared from peripheral blood of human volunteers (Markert et al., 1984). After purification, cells were suspended at various concentrations (2000–1.6x106/ml) in BWW medium supplemented with diethylenetriaminepentaacetic acid (DTPA) 0.1 mmol/l and DEPMPO 10 mmol/l in phosphate-buffered saline (PBS) plus DTPA 2 mmol/l. Stimulated cells were treated with PMA 100 nmol/l immediately prior to recording. Control incubations were prepared with BWW media replacing cells.
Equivalent experiments were performed with 40x106 spermatozoa/ml replacing leukocytes and NADPH 1 mmol/l as the stimulus for O2– production.
Spectrophotometric detection of cytochrome b558
Absorption spectra of 0.6 ml samples containing ~60x106/ml spermatozoa were recorded in a rapid scanning spectrophotometer (Cross et al., 1985). Spectra were also examined for cells treated with 1 mmol NADPH/l, 100 nmol PMA/l or a few crystals of sodium dithionite.
Measurement of H2O2 production with Amplex Red reagent
Determination of H2O2 produced by oxidase-mediated reactions can be accomplished with high sensitivity and specificity using N-acetyl-3, 7-dihydroxyphenoxazine (Amplex Red) (Mohanty et al., 1997; Zhou et al., 1997). Fluorescence due to resorufin formation from 50 µmol Amplex Red reagent/l in the presence of 1 IU horse-radish peroxidase/ml was followed on a spectrofluorimeter (excitation 563 nm, emission 587 nm) at 37°C. The reaction mixture (1.8 ml) was pre-warmed for 10 min in darkness. Then, 20x106 spermatozoa were added and background fluorescence monitored for 5 min. NADPH (1 mmol/l) or a buffer blank was added to the cuvette and the reaction returned to the incubator. Fluorescence was monitored for a further 5 min at 10, 20, 30 and 60 min intervals. Parallel incubations were performed with BWW medium replacing spermatozoa.
Results
Stimulation of LDCL by NADPH
The addition of 1 mmol NADPH/l rapidly stimulated LDCL in human sperm suspensions. Chemiluminescence (RLU/s/108 spermatozoa) increased from a basal rate of 92 ± 29.0 to a plateau of 720 ± 92 (mean ± SEM, n = 7) after 5 min. NADPH-stimulated chemiluminesence was decreased by 26 ± 7.9% (n = 3) by 0.1 mg SOD/ml. A combination of SOD and 0.2 mg CAT/ml decreased chemiluminescence by 11 ± 18.5% (n = 3). Addition of 25 µmol DPI/l had a progressive inhibitory effect and decreased NADPH induced chemiluminesence by 86 ± 3.2 % (n = 4) after 15–20 min (Figure 1a,b).
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Permeabilization of the plasma membrane with
5 µmol digitonin/l caused a large increase in NADPH-induced chemiluminesence. Treatment with 10 µmol digitonin/l increased peak chemiluminescence to 4300 ± 540 RLU/s/108 spermatozoa (mean ± SEM, n = 6) or ~6-fold compared with intact cells. NADPH-dependent chemiluminesence produced by permeabilized cells was partially inhibited by SOD 0.1 mg/ml or by 0.1 mg SOD plus CAT 0.2 mg/ml and suppressed to basal levels by DPI 25 µmol/l. Chemiluminescence (mean ± SEM, n = 3) was 2400 ± 330, 1500 ± 260 and 280 ± 89 RLU/s/108 spermatozoa for each of these treatments respectively (Figure 2a,b).
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Both NADPH and NADH increased LDCL by spermatozoa permeabilized with digitonin 10 µmol/l in a dose-dependent manner. Maximal chemiluminescence was similar with a saturating dose of either nucleotide, although at lower concentrations, NADPH was a more effective stimulus. The nucleotide concentration required for half-maximal chemiluminesence was 35 ± 4.6 µmol NADPH/l compared with 180 ± 46 µmol/l NADH (mean ± SEM, n = 3).
Cytochrome c reduction
NADPH-dependent cytochrome c reduction by intact spermatozoa was difficult to demonstrate consistently, because the changes in absorbance were very small and approached the sensitivity limit of the assay. There was a variation between donors in both background and NADPH-induced rates of cytochrome c reduction by spermatozoa. Hence, only some samples, with a low background rate and good response to NADPH, demonstrated a SOD-inhibitable rate of NADPH-induced cytochrome c reduction that was detectably different from the background level in the absence of NADPH. In three such samples, NADPH 2 mmol/l increased the rate of cytochrome c reduction (nmoles/min/108 spermatozoa, mean ± SEM) from 0.06 ± 0.017 to 0.34 ± 0.029. Cytochrome c reduction was unaffected by DPI 25 µmol/l but could be inhibited to near basal levels by SOD 0.1 mg/ml giving a SOD-inhibitable rate of 0.24 ± 0.017.
When spermatozoa were permeabilized with 10µmol digitonin/l, NADPH-dependent cytochrome c reduction could be demonstrated consistently. Permeabilization increased both basal and stimulated levels of cytochrome c reduction (nmoles/min/108 spermatozoa, mean ± SEM, n = 7) to 0.21 ± 0.044 and 1.7 ± 0.28 respectively. DPI (25 µmol/l) inhibited NADPH-enhanced cytochrome c reduction by permeabilized spermatozoa by ~70%. SOD (0.1 mg/ml) had no effect (Table I).
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Consumption of NADPH by sperm suspensions
Permeabilized spermatozoa incubated with 0.3 mmol NADPH/l oxidized 0.55 ± 0.091 nmoles NADPH/min/108 spermatozoa (mean ± SEM, n = 3) averaged over a 2 h incubation period. However, the DPI-inhibitable `oxidase-associated rate' of NADPH oxidation by permeabilized spermatozoa was only 0.09 ± 0.046. Intact spermatozoa did not consume 0.3 mmol/l NADPH at a measurable rate.
When intact spermatozoa were incubated with 1 mmol/l NADPH, addition of 250 µmol/l lucigenin increased the basal rate of NADPH oxidation by 440 ± 76% (mean ± SEM, n = 4). Addition of DPI 25 µmol/l reduced the effect of lucigenin to 220 ± 61 % (n = 3) but did not inhibit the basal level of NADPH consumption (Figure 3).
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Treatment of spermatozoa with digitonin 10 µmol/l dramatically increased NADPH oxidation to ~1400 ± 220 % (n = 4) of the basal rate in intact spermatozoa. NADPH oxidation by permeabilized cells was further increased to 3500 ± 320% (n = 3) by the addition of 250 µmol/l lucigenin. The stimulatory effect of lucigenin on NADPH consumption by permeabilized cells was strongly inhibited by DPI 25 µmol/l (Figure 3
).
EPR spectroscopy for O2– production
Superoxide production by activated leukocytes was reflected by formation of the characteristic EPR-detectable adduct (DEPMPO-OOH). This assay was very sensitive and O2– generation could be detected from 6000 but not 1000 leuko-cytes/ml. Calculated detection limits, based on the observed peak amplitudes and assuming a signal-to-noise ratio of 2:1, suggest that O2– production could be detected from just 2000 activated leukocytes/ml. A strong signal was generated by 1.6x106 leukocytes/ml but was abolished by the addition of 0.1 mg/ml SOD (Figure 4a,b). In contrast, no O2– production could be detected from 40x106 spermatozoa/ml (Figure 4c). Incubation of spermatozoa for 2 h with 10 mmol/l DEPMPO did not affect motility, compared with control cells (data not shown).
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Spectrophotometry for cytochrome b558
The characteristic absorption spectra of human neutrophil cytochrome b558 (reduced state) was not observed in suspensions of human spermatozoa treated with NADPH, PMA or sodium dithionite to induce complete reduction of the cell.
Measurement of H2O2 production with Amplex Red reagent
This assay could detect the addition of 5 pmoles H2O2/ml to intact sperm suspensions. Addition of 1 mmol NADPH/l caused an immediate increase in fluorescence but this increase was also observed in the absence of spermatozoa. Incubation of spermatozoa for up to 1 h with Amplex Red reagent, in the absence or presence of NADPH, caused no increase in fluorescence above cell free background levels.
Discussion
The proposal that human spermatozoa contain an NADPH oxidase is largely based on two observations. Firstly, that the addition of NADPH to sperm suspensions increases chemiluminescence from the O2– probe lucigenin and secondly, that this increase in chemiluminescence can be inhibited by SOD or DPI (Aitken et al., 1997; Fulton et al., 1997). Our early results (Richer et al., 1998), agreed closely with these data and the observation that NADPH supported cytochrome c reduction by human spermatozoa, seemed to support further the theory of NADPH oxidase activity. However, several factors gave us cause for concern: (i) NADPH-induced LDCL from intact or permeabilized spermatozoa was only partially inhibited by SOD; (ii) The rate of NADPH oxidation by permeabilized spermatozoa showed very little sensitivity to DPI and was too low to account for the rate of O2– production calculated from cytochrome c reduction. The cytochrome c assay suggested a DPIinhibitable rate of O2– production by permeabilized cells that was 1.54 nmoles/min/108 spermatozoa. This rate would require 0.77 nmoles/min NADPH on the basis of NADPH oxidase stoichemistry. However, in the absence of cytochrome c, the DPI-inhibitable rate of NADPH oxidation was only 0.09 nmoles/min/108 spermatozoa, over 8 times less than predicted. We consider that most sources of error, e.g. diaphorase activity, would lead to an overestimate of NADPH consumption. Furthermore, NADPH-induced cytochrome c reduction by permeabilized cells was insensitive to inhibition by SOD; (iii) We were unable to detect cytochrome b558. This observation has since been reported by others (Armstrong et al., 1999).
At about this time, as discussed above, we became aware of publications drawing attention to possible artefacts in the measurement of O2– with lucigenin or cytochrome c. Such artefactual O2– production in the presence of lucigenin has been observed with glucose/glucose oxidase (Liochev and Fridovitch, 1997) and the endothelial nitric oxide synthase (eNOS) (Vasquez-Vivar et al., 1997). In the latter system, addition of lucigenin to eNOS caused a rapid DPI sensitive oxidation of NADPH demonstrating that lucigenin could accept electrons from the flavin component of the eNOS reductase domain. Our observations that lucigenin increases NADPH oxidation by human spermatozoa and that this increase can be inhibited by DPI indicate that a similar mechanism is operating in human spermatozoa. However, we consider it unlikely that the flavoprotein involved is part of an eNOS complex since in preliminary experiments neither L-argenine or NG-nitro-L-argenine methyl ester affected NADPH-induced LDCL. Whatever the mechanism, it is clear that lucigenin is not a reliable probe for O2– production by spermatozoa and that the majority if not all of the chemiluminesence promoted by NADPH is derived from redox cycling independent of O2– production by spermatozoa themselves.
It is also possible for NADPH to reduce cytochrome c independently of O2– (Vasquez-Vivar et al., 1999). The discrepancy between rates of cytochrome c reduction and NADPH oxidation in the absence of cytochrome c coupled with the lack of sensitivity of NADPH oxidation to DPI strongly suggest that NADPH-induced cytochrome c reduction by human spermatozoa does not involve an NADPH oxidase. The results with intact spermatozoa indicate that cells from some ejaculates may be able to produce O2– by an NADPH oxidase-independent mechanism, e.g. leakage of electrons from the mitochondrial electron transport chain. On the other hand, in preliminary experiments, it was not possible to demonstrate the reduction of acetylated cytochrome c by human spermatozoa. We were unable to utilize the fluorimetric assay to confirm directly that cytochrome c increased NADPH oxidation as cytochrome c caused a high background rate of NADPH oxidation in the absence of spermatozoa. Nevertheless, the inhibition of NADPH-induced cytochrome c reduction by permeabilized spermatozoa with DPI and the lack of sensitivity to SOD suggest direct reduction of cytochrome c by a sperm flavoprotein. This phenomenon has been observed for several flavoenzymes with reductase activity including cytochrome P450 reductase, sulphite reductase and NOS (Vasquez-Vivar et al., 1999).
EPR spin-trapping, although not totally free of experimental artefacts, is reported to be the only viable technique for detecting and quantifying O2– (Vasquez-Vivar et al., 1999). We therefore tried to detect O2– production by EPR spectroscopy using the spin trap DEPMPO (Frejaville et al., 1995; Roubaud et al., 1997, 1998, Vasquez-Vivar et al., 1999). Superoxide production by leukocytes could be easily identified and the EPR-detectable adduct was completely blocked by SOD. Sensitivity calculations showed that we could detect O2– production by as few as 2000 PMA-stimulated leukocytes; this is similar to the figure reported by Roubaud et al. (1998). However, we were unable to observe any O2– production from 40x106 spermatozoa, even after prolonged incubation with NADPH. These results also exclude genuine O2– generation by routes independent of NADPH oxidase activity as suggested by our observations for intact spermatozoa with cytochrome c. We are unable to say whether this discrepancy is due to a variation in spermatozoa between different donors or to an artefact arising from use of the cytochrome c assay close to its limit of sensitivity.
It is possible that endogenous SOD rapidly converts O2– to H2O2 or that this ROS is formed directly by spermatozoa (Holland and Storey, 1981). However, we were unable to observe any H2O2 production by intact spermatozoa using an Amplex Red assay that was sensitive to 5 pmoles/ml H2O2.
We cannot exclude the possibility that human spermatozoa produce ROS at minute levels below the detection limit of the assays available. However, the concentration of exogenous H2O2 reported to influence sperm capacitation is in the micromolar range (Griveau et al., 1994; Aitken et al., 1995) and if present, would have been easy to detect. We are confident that human spermatozoa do not produce sufficient O2– by an NADPH oxidase to match these levels, although it remains possible that local ROS production could be an effective regulatory signal without raising the concentration of ROS in the extracellular medium so high. Alternatively, sperm-derived ROS may be scavenged with tremendous speed and efficiency before they can diffuse out of the cell. These conclusions agree with the failure of others to detect extracellular O2– production by human spermatozoa (de Lamirande et al., 1998a; Armstrong et al., 1999) and imply that the mechanism by which NADPH affects sperm function should be re-examined.
Acknowledgments
The authors are grateful to John Maher, University of Bristol for his kind assistance with the EPR spectroscopy. We would also like to thank John Hancock, University of the West of England for his help and advice on the cytochrome b558 specrophotometry experiments and the University of Bristol for the award of a scholarship to S.C.R.
Notes
1 To whom correspondence should be addressed. E-mail: chris.ford{at}bristol.ac.uk 
References
Aitken, R.J. and West, K.M. (1990) Analysis of the relationship between reactive oxygen species production and leukocyte infiltration in fractions of human semen separated on Percoll gradients. Int. J. Androl., 13, 433–451.[ISI][Medline]
Aitken, R.J., Paterson, M., Fisher, H. et al. (1995) Redox regulation of tyrosine phosphorylation in human spermatozoa and its role in the control of human sperm function. J. Cell Sci., 108, 2017–2025.[Abstract]
Aitken, R.J., Buckingham, D.W., Harkiss, D. et al. (1996) The extragenomic action of progesterone on human spermatozoa is influenced by redox regulated changes in tyrosine phosphorylation during capacitation. Mol. Cell. Endocrinol., 117, 83–93.[ISI][Medline]
Aitken, R.J., Fisher, H.M., Fulton, N. et al. (1997) Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Mol. Reprod. Dev., 47, 468–482.[ISI][Medline]
Aitken, R.J., Gordon, E., Harkiss, D. et al. (1998a) Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol. Reprod., 59, 1037–1046.
Aitken, R.J., Harkiss, D., Knox, W. et al. (1998b) A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. J. Cell Sci., 111, 645–656.[Abstract]
Armstrong, J.S., Rajasekeran, M., Hellmstom, W. et al. (1999) Lack of involvement of a membrane NADPH oxidase in superoxide anion generation by human spermatozoa. J. Androl., 20 (Suppl. 1), 33.
Babior, B.M., El Benna, J., Chanock, S.J. et al. (1997) The NADPH oxidase of leukocytes: The respirtory burst oxidase. In Scandalios, J.G. (ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, New York, USA, pp. 737–784.
Biggers, J.S., Whitten, W.K. and Whittingham, D.G. (1971) The culture of mouse embryos in vitro. In Daniels, J.C. (ed.), Methods in Mammalian Embryology. Freeman, San Francisco, USA, pp. 86–116.
Bize, I., Santander, G., Cabello, P. et al. (1991) Hydrogen peroxide is involved in hamster sperm capacitation in vitro. Biol. Reprod., 44, 398–403.[Abstract]
Cross, A.R., Parkinson, J.F. and Jones, O.T.G. (1985) Mechanism of the superoxide-producing oxidase of neutrophils. Biochem. J., 226, 881–884.[ISI][Medline]
de Lamirande, E. and Gagnon, C. (1993a) Human sperm hyperactivation and capacitation as parts of an oxidative process. Free Rad. Biol. Med., 14, 157–166.[ISI][Medline]
de Lamirande, E. and Gagnon, C. (1993b) A positive role for the superoxide anion in triggering hyperactivation and capacitation of human spermatozoa. Int. J. Androl., 16, 21–25.[ISI][Medline]
de Lamirande, E. and Gagnon, C. (1995) Capacitation-associated production of superoxide anion by human spermatozoa. Free Rad. Biol. Med., 18, 487–495.[ISI][Medline]
de Lamirande, E., Harakat, A. and Gagnon, C. (1998a) Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion. J. Androl., 19, 215–225.
de Lamirande, E., Tsai, C., Harakat, A. et al. (1998b) Involvement of reactive oxygen species in human sperm acrosome reaction induced by A23187, lysophosphatidylcholine, and biological fluid ultrafiltrates. J. Androl., 19, 585–594.
Faulkner, K. and Fridovitch, I. (1993) Luminol and lucigenin as detectors for O2–. Free Rad. Biol. Med., 15, 447–451.[ISI][Medline]
Ford, W.C.L., McLaughlin, E.A., Prior, S.M. et al. (1992) The yield, motility and performance in the hamster egg test of human spermatozoa prepared from cryopreserved semen by four different methods. Hum. Reprod., 7, 654–659.
Frejaville, C., Karoui, H., Tuccio, B. et al. (1995) 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide: A new efficient phosphorylated nitrone for the in vitro and in vivo spin trapping of oxygen centred radicals. J. Med. Chem., 38, 258–265.[ISI][Medline]
Fulton, N., Fisher, H., Paterson, M. et al. (1997) Purification and characterization of NADPH oxidase in human spermatozoa. [Abstr. no. P-165] Hum. Reprod., 12 (Abstract Book), 198.[ISI][Medline]
Griveau, J.F., Renard, P. and Le Lannou, D. (1994) An in vitro promoting role for hydrogen peroxide in human sperm capacitation. Int. J. Androl., 17, 300–307.[ISI][Medline]
Griveau, J.F., Renard, P. and Le Lannou, D. (1995) Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process. Int. J. Androl., 18, 67–74.
Holland, M.K. and Storey, B.T. (1981) Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa. Biochem. J., 198, 273–280.[ISI][Medline]
Jeyendran, R.S., Van der Ven, H.H., Perez-Pelaez, M. et al. (1984) Development of an assay to assess the functional integrity ofthe human sperm plasma membrane and its relationships to other semen characteristics. J. Reprod. Fertil., 70, 219–228.[Abstract]
Jones, O.T.G. and Hancock, J.T. (1994) Assays of plasma membrane NADPH oxidase. Methods Enzymol., 233, 222–229.[ISI][Medline]
Kessopoulou, E., Tomlinson, M.J., Barratt, C.L.R. et al. (1992) Origin of reactive oxygen species in human semen – spermatozoa or leukocytes? J. Reprod. Fertil., 94, 463–470.[Abstract]
Krausz, C., West, K., Buckingham, D. et al. (1992) Development of a technique for monitoring the contamination of human semen samples with leukocytes. Fertil. Steril., 57, 1317–1325.[ISI][Medline]
Leclerc, P., de Lamirande, E. and Gagnon, C. (1997) Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives. Free Rad. Biol. Med., 22, 643–656.[ISI][Medline]
Liochev, S.I. and Fridovitch, I. (1997) Lucigenin (bis-N-methylacridinium) as a mediator of superoxide anion production. Arch. Biochem. Biophys., 337, 115–120.[ISI][Medline]
Liochev, S.I. and Fridovich, I. (1998) Lucigenin as mediator of superoxide production: revisited. Free Rad. Biol. Med., 25, 926–928.[ISI][Medline]
Markert, M., Andrews, P.C. and Babior, B.M. (1984) Measurement of O2– production by human neutrophils. The preparation and assay of NADPH oxidase-containing particles from human neutrophils. Methods Enzymol., 105, 358–369.[ISI][Medline]
Mohanty, J.G., Jaffe, J.S., Schulman, E.S. et al. (1997) A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydrophenoxazine derivative. J. Immunol. Methods, 202, 133–141.[ISI][Medline]
Richer, S., Whittington, K. and Ford, W.C.L. (1998) Confirmation of NADPH oxidase activity in human sperm. J. Reprod. Fertil. (Abstr. Ser.), 21, 46.
Roubaud, V., Sankarapandi, S., Kuppusamy, P. et al. (1997) Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide. Anal. Biochem., 247, 404–411.[ISI][Medline]
Roubaud, V., Sankarapandi, S., Kuppusamy et al. (1998) Quantitative measurement of superoxide generation and oxygen consumption from leukocytes using electron paramagnetic resonance spectroscopy. Anal. Biochem., 257, 210–217.[ISI][Medline]
Spasojevic, I., Liochev, S.I. and Fridovich, I. (2000) Lucigenin: redox potential in aqueous media and redox cycling with O2- production. Arch. Biochem. Biophys., 373, 447–450.[ISI][Medline]
Suzuki, Y.J., Forman, H.J. and Sevanian, A. (1997) Oxidants as stimulators of signal transduction. Free Rad. Biol. Med., 22, 269–285.[ISI][Medline]
Twigg, J., Fulton, N., Gomez, E. et al. (1998) Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum. Reprod., 13, 1429–1436.
Vasquez-Vivar, J., Hogg, N., Pritchard, K.A. et al. (1997) Suproxide formation from lucigenin: an electron spin resonance spin trapping study. FEBS Letters, 403, 127–130.[ISI][Medline]
Vasquez-Vivar, J., Martasek, P., Hogg, N. et al. (1999) Electron spin resonance spin-trapping detection of superoxide generated by neuronal nitric oxide synthase. Methods Enzymol., 301, 169–177.[ISI][Medline]
Whittington, K. and Ford, W.C.L. (1998) The effect of incubation periods under 95% oxygen on the stimulated acrosome reaction and motility of human spermatozoa. Mol. Hum. Reprod., 4, 1053–1057.
Whittington, K. and Ford, W.C.L. (1999) Relative contribution of leukocytes and of spermatozoa to reactive oxygen species production in human sperm suspensions. Int. J. Androl., 22, 229–235.[ISI][Medline]
Williamson, J.R. and Corkey, B.E. (1969) Assays of intermediates of the citric acid cycle and related compounds by fluorimetric enzyme methods. Methods Enzymol., 80, 324–332.
World Health Organization (1992) WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge University Press, Cambridge, UK.
Zhou, M.J., Diwu, Z.J., Panchuk Voloshina, N. et al. (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem., 253, 162–168.[ISI][Medline]
Zini, A., Delamirande, E. and Gagnon, C. (1995) Low levels of nitric oxide promote human sperm capacitation in vitro. J. Androl., 16, 424–431.
Submitted on September 18, 2000; accepted on December 15, 2000.
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