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Biology of Reproduction 65, 1102-1113 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Analysis of Reactive Oxygen Species Generating Systems in Rat Epididymal Spermatozoa1

Patrick Verneta,c, Norma Fultona, Ceanne Wallaceb, and R. John Aitken2,a,b

a MRC Reproductive Biology Unit, Edinburgh EH3 9ET, Scotland b School of Biological and Chemical Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia c Laboratoire Epididyme et Maturation du Gamète Mâle, Université Blaise Pascal, 63177 Aubiere, France

ABSTRACT

Epididymal sperm maturation culminates in the acquisition of functional competence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction cascade that controls the tyrosine phosphorylation status of the spermatozoa during capacitation. Analysis of superoxide anion (O2-·) generation by rat epididymal spermatozoa has revealed a two-component process involving electron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lactate-, and pyruvate-free medium (-GLP), O2-· generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of lactate, malate, or succinate to spermatozoa incubated in medium -GLP stimulated O2-· generation. This increase could be blocked by rotenone and oligomycin (R/O) in the presence of malate or lactate but not succinate. Stimulation with all three substrates, as well as spontaneous O2-· production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodonium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigenin-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in the sperm plasma membrane and its activity was regulated by zinc and uncharacterized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite distinct from the equivalent leukocyte system.

epididymis, gamete biology, male reproductive tract, sperm, sperm maturation

INTRODUCTION

Epididymal sperm maturation is considered an essential process for the transformation of immature testicular spermatozoa to mature gametes capable of fertilization. Many modifications occur during this maturation process in association with the acquisition of functional competence [1, 2]. The sperm plasma membrane undergoes extensive remodeling in terms of its surface glycosylation characteristics, fatty acid composition, and cholesterol:phospholipid ratio. Maturing epididymal spermatozoa also exhibit changes in intracellular calcium concentration, cAMP content, and protein phosphorylation pattern that are pivotal to the development of coordinated movement [3–5]. In addition, maturing epididymal spermatozoa exhibit changes in their capacity for reactive oxygen species (ROS) generation and tyrosine phosphorylation that are thought to be related to the ability of the spermatozoa to engage in the process of sperm capacitation [6–9]. The latter is thought to involve a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade that is dependent on the ability of the spermatozoa to generate reactive oxygen metabolites [6, 10]. Thus, if ROS generation by human spermatozoa is suppressed with flavoprotein inhibitors, scavenged by catalase, or counteracted with membrane-permeant reducing agents, then the spermatozoa will not become capacitated and will not exhibit biological responses to physiological agonists such as progesterone [9–12]. On the other hand, if spermatozoa are exposed to, or generate, excessive levels of ROS then their fertilizing capacity and genetic integrity are compromised [13, 14].

In light of these observations, tight control of the redox status of the intra- and extracellular environments is clearly a central feature of epididymal sperm maturation [9]. Cellular redox status is, in turn, dependent upon the metabolism of ground-state oxygen to ROS including the superoxide anion (O2-·) and H2O2. O2-· is the primary component formed by the one-electron reduction of molecular oxygen. The ability to generate O2-· is linked to the presence of an NAD(P)H oxidase-like activity in the spermatozoa of all mammalian species examined to date [12, 15]. This activity is stimulated by NAD(P)H but inhibited by flavoprotein inhibitors such as diphenylene iodonium (DPI) and quinacrine [15]. In principle, such activity is similar to the well-characterized NAD(P)H oxidases of phagocytic leukoytes [16]. These cells produce copious amounts of O2-· in response to bacterial provocation or artificial secretagogues such as fMLP (formyl methionyl leucyl phenylalanine) or PMA (12-myristate, 13-acetate phorbol ester). The NAD(P)H oxidase in leukocytes is a multicomponent enzyme including a membrane-bound cytochrome b558 (with two components: p22phox and gp91) and cytosolic proteins (p47phox, p67phox, Rac 1 and 2, and p40phox) that are translocated to the membrane when the system is activated during the oxidative burst [17]. However, generation of low levels of O2-· by several nonphagocytic cells apart from spermatozoa has also been reported, including endothelial cells [18], fibroblasts [19], mesangial cells [20], vascular smooth muscle [21], and thyroid tissue [22]. In general the biochemical features of oxygen free radical generation in nonphagocytic cells have not been resolved, although in some cases components of the leukocyte NAD(P)H oxidase system have been identified [23].

It has been known for some time that the leukocytes that frequently contaminate washed sperm preparations are powerful generators of ROS [24]. However, recent results from several independent laboratories have demonstrated that spermatozoa themselves are able to generate O2-· [24–27]. Two potential O2-· sources have been suggested for spermatozoa in independent studies: some authors have focused on the above-mentioned NAD(P)H oxidase while others have emphasized the leakage of electrons from the mitochondrial electron transport chain [12, 28]. The purpose of this study was to investigate the different mechanisms involved in the generation of O2-· by epididymal spermatozoa in an animal model, the rat. By using a combination of different inhibitors and novel techniques for the subcellular fractionation of spermatozoa, we have demonstrated that both the mitochondria and a plasma membrane NAD(P)H oxidase are potential sources of O2-· in mammalian spermatozoa. Moreover, the membrane-bound NAD(P)H oxidase activity was found to be quite distinct from the leukocyte free radical-generating system on the basis of a detailed reverse transcription-polymerase chain reaction (RT-PCR) analysis. Further elucidation of the structure of this putative NAD(P)H oxidase may have important implications for our understanding of the fundamental cellular mechanisms regulating sperm function and the development of novel contraceptive strategies targeting the male gamete.

MATERIALS AND METHODS

Chemicals

All reagents were obtained from Sigma Chemical Company (Poole, Dorset, UK) with the exception of Hepes, penicillin, and streptomycin (Gibco, Paisley, UK) and superoxide dismutase (SOD, 3000 U/mg; Calbiochem, Nottingham, UK).

Epididymal Dissection and Spermatozoa Preparation

Sexually mature male Wistar rats (Charles River, Margate, UK) between 12 and 24 wk were killed by CO2 inhalation followed by cervical dislocation. The epididymides were removed; cleared of fat and connective tissue; rinsed in Biggers, Whitten, and Whittingham medium (BWW) [29]; and dissected into caput and caudal regions. Each piece of tissue was transferred to small petri dishes, each containing 3 ml of BWW prewarmed to 34°C and repeatedly punctured with a 26-gauge needle. The epididymides were incubated for 10 min to allow diffusion of the spermatozoa. The remaining tissues were then removed, and the cells were counted and adjusted to 107 spermatozoa/ml.

Reactive Oxygen Species Detection

The levels of ROS generated by rat spermatozoa were too low to permit their accurate quantification using conventional reagents such as ferricytochrome C or scopoletin, despite repeated attempts. Highly sensitive chemiluminescent techniques were therefore used for this purpose that have been verified for use with mammalian spermatozoa [30]. These probes comprised lucigenin and the combination of luminol and horseradish peroxidase to measure O2-· and H2O2, respectively [30]. Lucigenin was used at a final concentration of 250 µM in a volume of 400 µl containing 4 x 106 spermatozoa or 10 µg of membrane protein. The signals generated with this probe can be fully scavenged by SOD (see this paper) and, in the case of NADPH stimulation, can be replicated by acetylated ferricytochrome C measurements [12], indicating the validity of this probe as an indicator of superoxide anion generation. Measurements were conducted at 37°C using a Berthold 9505C luminometer (Berthold, Wilbad, Germany).

Nitroblue tetrazolium (NBT) reduction was also used to confirm the generation of O2-· by rat spermatozoa. For this assay rat spermatozoa were isolated from the caput and caudal regions of the epididymis, counted, and resuspended in BWW at a final concentration of 20 x 106 /ml. Unless indicated, this sperm suspension was then diluted in BWW to a concentration of 5 x 106/ml for the assay. Twenty-five-microliter aliquots of this sperm suspension were added to the wells of a microtiter plate and allowed to air dry in order to permeabilize the cells, the NBT cation being membrane impermeant. Reagents were added to these cells in a 50 mM Tris (Trizma) buffer, pH 7.0 at 37°C. Superoxide was recorded as a SOD (15 U/well)-inhibitable NBT signal, the latter being present at a final concentration of 0.75 mM. Substrates added to the system were NADP+ (0.75 mM), glucose-6-phosphate (G-6-P; 0.4 mM), and, as a positive control, NADPH (100 µM). The plates were incubated at 37°C for 24 h unless otherwise indicated, and the intensity of the formazan deposit was measured with an ELISA plate reader (Bio-Rad 550; Bio-Rad, Hemel Hempstead, Hertfordshire, UK) at 490 nm.

For H2O2 the sperm samples (8 x 106 cells) were incubated at 37°C in a total volume of 400 µl in the presence of 250 µM luminol and horseradish peroxidase type VI (5 U/assay). After 5 min baseline recording, possible contamination of the sperm suspensions with leukocytes was assessed using an fMLP (100 µM) provocation test, and 5–10 min after the signal returned to a stable baseline, by the addition of 10 µM PMA.

Effect of Energy Substrates on ROS Generation

Rat epididymal spermatozoa were incubated in BWW depleted of glucose, lactate, and pyruvate at 37°C (BWW-GLP). The generation of ROS was recorded for 20 min in the presence of 250 µM lucigenin as described above. Substrate (glucose, pyruvate, lactate, malate, or succinate) was then added to the reaction mixture and the response monitored for the following 8 min. The concentrations of substrate employed corresponded to the concentrations found in normal BWW medium, except for succinate and malate that are not normal constituents of this medium. The final concentrations were: 5.5 mM glucose, 273 µM pyruvate, 15 mM lactate, 10 mM malate, and 10 mM succinate, and throughout these manipulations osmolarity of the medium was in the range 275–300 mosmol/kg.

Effect of Specific Inhibitors on ROS Generation

DPI was added to the sperm or protein extract at a final concentration of 15 µM prior to addition of any ROS inducers; controls were treated with dimethylsulfoxide (DMSO) only.

For the suppression of mitochondrial function, a 10 mM stock solution of rotenone in DMSO and a 25 mM stock solution of oligomycin in ethanol were prepared. Spermatozoa were incubated for 12 h in BWW with 10 µM rotenone and 25 µM oligomycin at 34°C. The motility of the spermatozoa and the level of superoxide anion generation were analyzed every 2 h.

In order to suppress the hexose monophosphate shunt (HMS), spermatozoa from the caput or cauda epididymidis were incubated in BWW in the presence of 200 µM 6-aminonicotinamide (6-AN) and 100 µM dehydroepiandrosterone (DHEA) or in presence of 2% DMSO (v:v) as a control [31]. A 12-h incubation was performed at 34°C. Motility and superoxide anion generation were recorded every 2 h.

Preparation of Sperm Plasma Membranes

Spermatozoa were obtained as described above. After 10 min incubation, the supernatant containing the spermatozoa was centrifuged at 500 x g for 5 min. The pellet was resuspended in hypo-osmotic 10 mM potassium phosphate buffer [32], pH 7.4 at 4°C (75 x 106/ml spermatozoa) and agitated for 5 min on rollers. The medium was centrifuged at 1300 x g for 15 min at 4°C, and the supernatant (S1) was retained. The pellet was resuspended in fresh 10 mM potassium phosphate buffer and the previous step was repeated. Supernatant S2 was collected and mixed with S1. NaCl and MgSO4 were added to the supernatant at a final concentration of 0.2 M and 2 mM, respectively. A further centrifugation was carried out at 11 000 x g for 30 min at 4°C to separate the mitochondria from the plasma membrane fraction. The pellet containing the mitochondria was stored at 4°C. The supernatant containing the plasma membrane was centrifuged at 100 000 x g for 90 min at 4°C. The supernatant corresponding to the sperm cell cytosol was retained. The plasma membrane and mitochondrial pellets were extracted with 1% octyl-beta-D-thioglucopyranoside (OSGP) (w:v), 10% glycerol (v:v), 20 mM Tris, pH 8.6, containing protease inhibitors (Complete Mini, Boehringer Mannheim, Mannheim, Germany) with frequent vortexing for 30 min at 4°C; the soluble protein fraction was recovered and the protein concentration determined using the BCA Protein Assay Kit (Pierce, Rockford, IL).

In order to dissociate high from low molecular weight compounds, the cytosolic fraction was centrifuged through a 10-kDa cutoff membrane (Microcon 10; Amicon, Millipore, Watford, Hertfordshire, UK) at 10 000 x g, 4°C for 15 min. The filtrate (<10-kDa material) was retained, and the retentate (>10-kDa material) was resuspended to the initial volume in 10 mM potassium phosphate buffer, pH 7.4, containing 0.2 M NaCl and 2 mM MgSO4.

Western Blot Analysis of Plasma Membrane Purity

Proteins (20 µg/lane) were separated by SDS-PAGE (12% [w:v] gel). The gels were electroblotted onto polyvinylidene fluoride membranes (Hybond-P, Amersham International, Little Chalfont, Buckinghamshire, UK) overnight at 25 V. The efficiency of the transfer was evaluated using Ponceau red staining according to the manufacturer's conditions. Blots were blocked for 1 h with 3% BSA (w:v) in TBS (20 mM Tris, 150 mM NaCl), then probed with anti-bovine cytochrome oxidase subunit IV (Molecular Probes Europe, Leiden, Netherlands) at 83 ng/ml in TBS containing 1% BSA (w:v) and 0.1% (v:v) Tween 20 for 2 h. After washing in 0.1% Tween 20 in TBS, blots were incubated for 1 h with horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham International). Following a final wash in 0.1% Tween 20 in TBS, detection of bound antibodies was achieved by enhanced chemiluminescence and exposure to Hyperfilm ECL (Amersham International).

Reverse Transcription and PCR Amplification

The RT-PCR experiments were conducted on RNA extracted from pachytene spermatocytes, first-strand cDNA synthesis being performed using random primers. The spermatocytes were prepared by centrifugal elutriation [33] and were a generous gift from Dr. P. Saunders. Rat leukocytes were purified from whole blood using Polymorphoprep (Nycomed Pharma, Asker, Norway) and the RNA extracted using RNAzol B (Biogenesis, Poole, Dorset, England) according to the manufacturer's instructions. RNA concentrations were estimated by absorbance at 260 nm and their purity determined using the OD260:OD280 ratio. In this case first-strand cDNA synthesis was performed on 2 µg RNA previously primed with 50 µM oligo-dT in the presence of 10 mM dithiothreitol, 1 mM dNTP, 0.5 µl RNasin, and 50 U Expand reverse transcriptase (Boehringer Mannheim) for 2 h at 40°C. One tenth of the reaction mixture was used for the PCR reaction in the presence of 200 µM dNTP, 100 pmol of 5' primer, 100 pmol of 3' primer, and 2 U Taq DNA polymerase (Promega, Southampton, Hampshire, England) with manufacturer's buffer. The conditions used for the PCR analysis of p22 and p47 were 30 sec at 94°C, 30 sec at 60°C, and 90 sec at 72°C for 30 cycles followed by 7 min at 72°C. The conditions for p67 were identical except that the cycle temperatures were 94°C, 55°C, and 72°C for 30 cycles followed by 7 min at 72°C.

Statistical Analysis

The data were analyzed by ANOVA for repeated measures using the Statview program (Abacus Concepts, Berkeley, CA). Differences between individual groups were examined with Fisher probable least-square difference (PLSD) test, and all experiments were repeated at least three times on independent samples. All data are expressed as mean ± SEM; probability values of P < 0.05 were considered significant.

RESULTS

Generation of ROS by Rat Spermatozoa

Spermatozoa were incubated in medium BWW at 37°C in the presence of lucigenin, and the chemiluminescence was recorded over a 15-min incubation period. Spontaneous ROS generation was recorded in both caput and caudal epididymal spermatozoa giving chemiluminescence values of similar magnitude (22 225 ± 1904 counts/min and 29 764 ± 3213 counts/min), respectively. Although this difference was statistically significant (Fig. 1), it was not consistently observed in independent data sets (see Fig. 3). By contrast, the addition of 500 µM NADPH invariably led to a highly significant (P < 0.001) burst of chemiluminescence, commensurate with a 30-fold increase in O2-· generation by caput spermatozoa and a 10-fold increase in caudal cells. This difference between caput and caudal cells was again highly significant (Fig. 1; P < 0.01) and clearly emphasized the greater sensitivity of the former to NADPH stimulation. Treatment of these sperm preparations with fMLP in the presence of luminol and horseradish peroxidase did not generate a chemiluminescent response, indicating that leukocyte contamination was negligible (data not shown). Similarly treatment with PMA (100 nM) did not activate O2-· production by rat sperm suspensions, again emphasizing that leukocyte contamination was not a significant factor in the analyses described below.



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  		FIG. 1. Comparison of the lucigenin-dependent signals generated by caput and cauda epididymal rat spermatozoa both at a basal spontaneous level (control) and in response to NADPH (500 µM). (**P < 0.01 compared with caput spermatozoa; n = 5.)



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  		FIG. 3. Impact of substrate availability on lucigenin-dependent chemiluminescence. A) Rate of spontaneous superoxide anion generation by caput and caudal rat spermatozoa in the presence of normal BWW medium (BWW) and BWW depleted of glucose, lactate, pyruvate (-GLP). The results correspond to a variation in the number of counts over a 2-min period (**P < 0.01 in relation to BWW control; n = 3). B) The effect of glucose, lactate, and pyruvate depletion (-GLP) on superoxide anion generated by rat epididymal spermatozoa after NADPH stimulation. The results are expressed as a total number of counts over 2 min (n = 3)

Confirmation that rat spermatozoa could generate O2-· in response to NADPH stimulation was obtained using an NBT assay. Addition of substrates for NADPH generation by the HMS (NADP+ and G-6-P) to permeabilized rat spermatozoa induced a highly significant (P < 0.001) increase in NBT reduction that was inhibited by 15 U SOD (Fig. 2A) and highly dependent on sperm concentration (Fig. 2B). Addition of either substrate in isolation had no such effect. Addition of NADPH (100 µM) directly to the spermatozoa also induced NBT reduction via mechanisms that were inhibitable by SOD (Fig. 2A). Under the conditions of this assay, the levels of NBT reduction observed with NADPH or the combination of NADP+ and G-6-P were not significantly different, presumably because both approaches provided a rich supply of substrate, and the only limiting factor was oxidase availability. Consistent with the lucigenin data was the observation that the level of NADPH-induced NBT reduction detected following exposure to NADP+ and G-6-P was significantly higher with caput than caudal epididymal spermatozoa (Fig. 2B; P < 0.001).



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  		FIG. 2. Analysis of NBT reduction by rat spermatozoa. A) neither NADP+ nor G-6-P stimulated a change in NBT reduction, but the combination of these reagents induced a significant change that was completely inhibited by SOD (15 U). Similarly, direct addition of NADPH (100 µM) to the spermatozoa induced a change in NBT reduction that was inhibited by SOD. ANOVA indicated that the overall significance of the effect of treatment was P < 0.001 (***P < 0.001 and **P < 0.01 compared with control incubations by Fisher PLSD; n = 6). B) NBT reduction in response to NADP+ and G-6-P was highly dependent on the concentration of spermatozoa in the reaction mixture. ANOVA indicated that the overall significance for the sperm concentration effect was P < 0.001 (***P < 0.001; **P < 0.01; *P < 0.05 compared with control by Fisher PLSD). ANOVA also indicated that the caput responses were significantly greater than the caudal; ***P < 0.001

Mitochondria and Spontaneous ROS Generation

Although NADPH was clearly a potential substrate for ROS generation by rat spermatozoa, inhibitors of the HMS (6-AN and DHEA), the major intracellular source of NADPH, had no effect on the spontaneous basal level of free radical generation by these cells (data not shown). If NADPH generation by the HMS were not driving spontaneous basal O2-· generation by rat spermatozoa, an alternative possibility was that this activity reflected electron leakage from the mitochondrial electron transport chain. In order to examine this proposal, spermatozoa were recovered in substrate-free BWW lacking lactate, pyruvate, and glucose (-GLP) and the spontaneous generation of O2-· compared to spermatozoa incubated in normal BWW. As observed in Figure 3A, the spontaneous generation of O2-· was significantly lower in -GLP medium at 37°C (P < 0.01), in the absence of any overt change in sperm motility. In contrast, the NADPH-induced O2-· response was increased in the absence of lactate, pyruvate, and glucose, but this difference did not reach statistical significance (Fig. 3B).

The possible involvement of the mitochondrial electron transport chain in the spontaneous release of O2-· was examined by investigating the impact of rotenone and oligomycin on O2-· generation by rat epididymal spermatozoa. These inhibitors block electron transfer at complex I in the respiratory chain as well as at ATP synthetase. A time course analysis of rat epididymal spermatozoa in a normal BWW medium in the presence of rotenone and oligomycin revealed an instantaneous suppression of ROS generation that was sustained over a 12-h incubation period (Fig. 4). In contrast, the stimulation of O2-· production by NAD(P)H was not influenced by the presence of these inhibitors (data not shown).



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  		FIG. 4. Time course for the effect of rotenone and oligomycin (R/O) on spontaneous lucigenin-dependent chemiluminescence by caput and caudal epididymal rat spermatozoa. The results are expressed as a total number of counts over 2 min (n = 3)

The above results suggested that electron leakage from the mitochondrial respiratory chain was involved in the spontaneous generation of O2-· by rat epididymal spermatozoa. However, it was also possible that the loss of O2-· production in -GLP medium was due to the absence of glucose and a consequential suppression of glycolysis. The latter would reduce cytoplasmic NADH generation, and this coenzyme is known to be an effective substrate for ROS generation by mammalian spermatozoa [12]. In order to explore this possibility, five substrates were examined in sequence for their capacity to promote spontaneous O2-· production by rat spermatozoa in substrate-free medium BWW: glucose, lactate, pyruvate, malate, and succinate. No increase of ROS generation was observed after glucose or pyruvate addition (Table 1). However the addition of lactate, malate, or succinate resulted in a dramatic increase of ROS generation by rat spermatozoa from both the caput and cauda epididymidis (Table 1). The addition of rotenone and oligomycin completely abolished the stimulatory effect observed following supplementation with lactate and malate, consistent with electron leakage from complex I of the respiratory electron transport chain. By contrast, these inhibitors had no effect on the succinate-induced response, indicating that with this substrate, electron leakage was occurring at the level of complex II (Table 1).


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  		TABLE 1. Impact of substrate availability on O2-· generation by epididymal spermatozoa

Influence of DPI

The generation of O2-· was studied in the presence or in the absence of DPI [30], a known inhibitor of flavoproteins (Table 1). In the presence of 15 µM DPI, the level of O2-· spontaneously generated by rat spermatozoa was decreased both in caput and in caudal epididymal spermatozoa. Moreover, the generation of O2-· in response to lactate, malate, and succinate was also suppressed by this inhibitor (Table 1). DPI was also found to decrease the NAD(P)H-induced chemiluminescent signal in intact cells and cell membranes (see below).

Resolving the Sites of O2-· Production

The above data suggested that rat spermatozoa possess two potential sources of production: 1) the mitochondria, which are largely responsible for spontaneous O2-· generation using lactate, malate, or succinate as substrates, via systems that are inhibitable by DPI and, in the case of lactate and malate, rotenone and oligomycin; and 2) an NAD(P)H-induced response that was also inhibitable with DPI but could not be suppressed with mitochondrial inhibitors. In order to confirm that the NAD(P)H response did not involve electron leakage from the mitochondria, plasma membrane preparations free of mitochondrial contamination were generated from spermatozoa treated with low osmolarity buffer as described in Materials and Methods. The plasma membrane preparations created in this way were confirmed to be free of detectable mitochondrial membranes by Western blot analysis using an anti-cytochrome oxidase antibody. The latter gave clear positive signals in lanes corresponding to whole sperm extract, prior to any cellular fractionation, as well as the mitochondrial fraction; however, no signal was observed in the plasma membrane fraction (Fig. 5).



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  		FIG. 5. Western blot analysis of subcellular rat caput and caudal epididymal sperm fractions with anti-cytochrome oxidase subunit IV antibody. W, Whole sperm extract; Mit, mitochondrial fraction; Mem, membrane fraction

This purified plasma membrane fraction was shown to be fully capable of generating O2-· in response to the presence of both NADH and NADPH (Figs. 6 and 7, A and B), the cytosol having no detectable capacity for ROS production (Fig. 7). These responses could be completely inhibited by the presence of SOD, confirming that the major reactive oxygen metabolite being generated by these membrane preparations was O2-· (Fig. 7, A and B).



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  		FIG. 6. Superoxide-generating potential of isolated plasma membrane of caput epididymal spermatozoa in response to NADPH in comparison with detergent extracts of whole cells and cytosol. Additions of membrane, cytosol, and whole cell extract standardized at 10 µg protein per incubation. Representative of three independent analyses



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  		FIG. 7. Free radical generation by isolated rat caput sperm plasma membranes. A) NADPH as the stimulus and effective inhibition of the signal by SOD (300 U). B) NADH as the stimulus and effective inhibition of the signal by SOD (300 U). C) Direct comparison of superoxide anion generation in response to NADH and NADPH. D) Comparison of superoxide anion generation by isolated membranes and OSGP extracts of the same preparation. Additions of membrane standardized at 10 µg protein per incubation. Results are representative of three independent analyses

The pattern of response induced by NADH and NADPH in these membrane preparations was markedly different. NADH induced an extremely high level of O2-· production that peaked after 10 min and was 2 log orders higher than the NADPH response (Fig. 7C). The latter was characterized by a sustained low level response that continued for several hours. The O2-·-generating capacity of these plasma membrane preparations was significantly impaired by the presence of mild detergents such as OSGP (Fig. 7D), suggesting that this activity involves a complex association of components that must be closely linked within the plasma membrane for optimal O2-· generation to occur. The responses of these membrane preparations to NAD(P)H were also significantly curtailed by the presence of low concentrations (<5 µM) of the flavoprotein inhibitor, DPI (Fig. 8, A and B). This inhibition was particularly marked in relation to the substantial NADH-induced responses (Fig. 8A).



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  		FIG. 8. Dose-dependent analysis of the impact of DPI on NAD(P)H-induced, lucigenin-dependent chemiluminescence in purified caput sperm membrane preparations. A) NADH-induced response. B) NADPH-induced response. Results are representative of three independent analyses

Cytosolic Regulation of NAD(P)H Oxidase Activity

Figure 6 also reveals that the levels of ROS generation observed in these purified membrane preparations were severalfold greater than the response observed with intact cells (Fig. 1). Although this difference in activity could be explained by a combination of factors, a key element appeared to be the presence of an inhibitor of NAD(P)H oxidase activity in the cytoplasm. Thus addition of sperm cytosol to the membrane fraction resulted in a decrease in the response to NADPH (Fig. 9) irrespective of whether the cytosol or membrane preparations were prepared from caput or caudal epididymal spermatozoa. To examine the possibility of an O2-·-scavenging compound in the cytosol, the xanthine-xanthine oxidase system [34] was used to produce O2-· chemically. In the presence of the cytosolic fraction, no significant inhibition of the O2-· signal generated by this system was observed (data not shown). Similarly, the cytosolic factor was not SOD. When SOD was added to the membrane preparations at a level equivalent to that found in the cytosol preparations (1.5 x 10-3 U), only a minor reduction in chemiluminescence was observed (Fig. 10A). In keeping with this conclusion, the effects of cytosol and SOD were additive in suppressing the generation of O2-· by rat sperm plasma membranes (Fig. 10, B and C).



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  		FIG. 9. The inhibitory effect of sperm cytosol on superoxide anion generation by rat sperm plasma membranes. A) Inhibitory effect was observed with both caput and caudal epididymal spermatozoa. B) Inhibitory effect of whole cytosol was due to a factor exhibiting a molecular mass of >10 kDa. C) The inhibitory effect was also observed when sperm cytosol was added to OSGP-solubilized sperm plasma membranes that had been activated by NADPH (***P < 0.001). Additions of membrane and cytosol standardized at 10 µg protein per incubation



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  		FIG. 10. The inhibitory effect of sperm cytosol on superoxide anion generation by rat spermatozoa was not due to SOD. A) Addition of SOD to OSGP extracts of sperm plasma membrane at a concentration equivalent to that found in sperm cytosol (1.5 x 10-3 U) did not suppress the O2-· signal to the same degree as cytosol itself. B) Cytosol induced a sustained inhibition of O2-· generation by caudal sperm plasma membranes. C) Addition of SOD (300 U) to such preparations caused an additional suppression of the O2-· signal

To investigate further the nature of the inhibitor(s) present in sperm cytosol, the latter was passed through a 10-kDa molecular cutoff filter. Both filtrate and retentate were tested for their ability to inhibit O2-· generation by different subcellular fractions. As revealed in Figure 9B, the filtrate had no significant inhibitory effect on O2-· generated by sperm plasma membrane. However, addition of the retentate fraction to the plasma membrane fraction resulted in a significant decrease in the levels of O2-· generated in response to NADPH (Fig. 9B).

Regulation of Sperm NAD(P)H Oxidase Activity by Zinc

In light of a recent report indicating that zinc can suppress the generation of O2-· by human spermatozoa [35], the impact of this cation on ROS generation by rat epididymal spermatozoa and plasma membranes was investigated. The incubations were performed in the presence of different doses of zinc from 1 µM to 5 mM, and the spermatozoa were stimulated with NADPH. The presence of zinc in the incubation medium of intact rat epididymal spermatozoa did not interfere with the generation of O2-· at concentrations below 1 mM. However, at the 1 mM dose level, detectable O2-· generation was suppressed by 89.7 ± 4.3% and 95.3 ± 0.9% in caput and caudal epididymal spermatozoa, respectively (Fig. 11A). At the 5 mM dose the suppression of O2-· was virtually complete (Fig. 11A). When the same experiment was performed on purified sperm plasma membranes, the amount of zinc needed to inhibit O2-· generation was dramatically reduced into the nano- to micromolar range (Fig. 11, B and C).



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  		FIG. 11. Impact of zinc on NAD(P)H oxidase activity. A) Effect of different ZnCl2 concentrations on superoxide anion generated by intact rat spermatozoa from the caput and cauda epididymis after induction by NADPH. The results are expressed as a percentage of the activity found in rat epididymal spermatozoa without ZnCl2 (**P < 0.01). B) Inhibitory influence of zinc on O2-· generation by caput epididymal sperm plasma membranes using NADH as substrate. C) Inhibitory influence of Zn on O2-· generation by caput epididymal sperm plasma membranes using NADPH as substrate. Results are representative of three independent analyses

Relationship with Leukocyte NAD(P)H Oxidase

In order to determine the level of homology between the NADPH oxidases from rat leukocytes and germ cells, an RT-PCR analysis was performed using primers capable of amplifying mRNA species involved in the assembly of a functional leukocyte NADPH oxidase. Three components of the leukocyte NADPH oxidase complex were chosen: p22Phox, p47Phox, and p67Phox. In view of the lack of known nucleic sequences for these three components in the rat, primers were designed that targeted regions of these NADPH oxidase constituents that were highly conserved between species (Table 2). A preliminary experiment was carried out on rat leukocyte mRNA to validate the primers. All of these primers gave amplifications of cDNA with the expected size: 540 base pairs (bp) for p22Phox, 430 bp for p47Phox, and 470 bp for p67Phox (Fig. 12). These primers therefore appeared to amplify correctly the different components of the leukocyte NAD(P)H oxidase in the rat. PCR analysis with these primers under identical conditions employing rat pachytene spermatocyte RNA did not generate any detectable signals (Fig. 12).


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  		TABLE 2. Primers used to detect constituents of the leukocyte NADPH oxidase complex in rat spermatozoa



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  		FIG. 12. Comparison of rat NADPH oxidase from leukocyte mRNA (left) and from pachytene spermatocyte mRNA (right). RT-PCR amplifying three different components: p22phox, p47phox, and p67phox were used. Glucose-6-phosphate dehydrogenase is used as a positive control. MW, Molecular weight standards. The 500-bp standard is indicated. Results were visualized after UV detection of ethidium bromide-stained gel

DISCUSSION

Reactive oxygen species generation has been described in a wide variety of different cell types including human glomerular mesangial cells, fibroblasts, thyroid tissue, vascular smooth muscle, endothelial cells, and leukocytes [16, 18–23, 36–39]. The results obtained in this study confirm that mammalian spermatozoa are also capable of generating ROS and indicate that in the rat this activity involves at least two independent mechanisms. These sources of O2-· comprise an enzymatic system located in the sperm plasma membrane that utilizes NAD(P)H as substrate and a second system involving the mitochondrial electron transport chain. While previous reports have identified either electron leakage from sperm mitochondria [28] or NAD(P)H oxidases [12] as sites of ROS generation in mammalian spermatozoa, this is the first report to indicate that both sources of ROS generation may be operative.

When rat spermatozoa are incubated at 37°C in a simple defined culture medium they reveal a spontaneous capacity to generate O2-· that appears to be largely dependent on the mitochondrial electron transport chain. In the presence of substrates such as malate and lactate, the mitochondria spontaneously generated O2-· via mechanisms that could be blocked with rotenone and oligomycin, suggesting the involvement of complex I of the respiratory chain: NADH-CoQ reductase. Succinate was also able to stimulate O2-· generation in intact rat spermatozoa, but in this case the combination of rotenone and oligomycin was unable to suppress O2-· production. These observations are consistent with O2-· generation from complex II (succinate-CoQ reductase complex), which is known to be insensitive to rotenone-oligomycin. This conclusion is also supported by the inhibitory action of DPI, as this reagent is effective against flavoproteins and FAD is an essential component of the succinate dehydrogenase complex. ROS generation as a consequence of electron leakage from complex I and II of the mitochondrial electron transport chain has previously been reported for other cell types, for example bovine heart mitochondria [40, 41], and is clearly a feature of rat sperm biochemistry when these cells are cultured in vitro. Interestingly, rotenone-oligomycin appears to have little effect on spontaneous ROS generation by human spermatozoa, possibly reflecting the reduced dependence of the latter on oxidative phosphorylation as a source of ATP.

Surprisingly, PMA did not initiate any free radical response from rat epididymal spermatozoa in contrast to the powerful effect of this compound on leukocyte O2-· production. These results are in keeping with data obtained with human spermatozoa indicating that normal cells cannot respond to PMA stimulation with enhanced O2-· production, only defective cells can exhibit such responses [42]. The inability of PMA to activate the NAD(P)H oxidase in spermatozoa is not due to a lack of protein kinase C (PKC) activity within these cells. The presence of different PKC isoforms in rat germ cells [43], the localization of two PKC isoforms in the plasma membrane of bovine spermatozoa [44], and the existence of other intermediates linked to the PKC pathway [45] all suggest that this signal transduction pathway is operative in mammalian spermatozoa; however, it is not normally coupled to the activation NAD(P)H oxidase activity.

To date, the only factor that appears to be capable of activating this enzyme complex in spermatozoa is the provision of substrate in the form of NAD(P)H. In this respect the sperm oxidase is quite unlike the leukocyte enzyme that must be activated before it can generate O2-·. The lack of a need for oxidase activation may explain why PMA is not effective in stimulating O2-· production by rat spermatozoa. The difference between sperm and leukocyte oxidases in terms of their need for activation may simply reflect fundamental differences in the structure and function of these disparate cell types. In leukocytes, an activation mechanism is imposed on the oxidase in order to ensure that O2-· is not constantly generated by these cells but is only produced during the oxidative burst when phagocytes are activated by an appropriate stimulus such as a foreign cell or organism. In contrast, spermatozoa have a chronic need for ROS production in order to stimulate the redox-regulated signal transduction cascades that drive capacitation [6, 10], a process that may take many hours to complete. Moreover, the lack of significant cytoplasmic space within the spermatozoon, plus the competing needs of cytoplasmic NADPH to fuel the glutathione cycle [46], mean that under normal cirumstances, the oxidase will only have access to sufficient substrate to maintain the constant low level of activity needed to support capacitation. If the cytoplasmic space is increased (as in cases of male infertility characterized by the presence of spermatozoa that have retained excess residual cytoplasm during spermiogenesis) then substrate availability is elevated and O2-· generation is enhanced to the point where peroxidative damage is induced [47].

At present, the metabolic pathways that contribute substrate to this putative oxidase during sperm capacitation have not yet been resolved. The correlation between G-6-P dehydrogenase activity and free radical generation by human spermatozoa has led to the suggestion that glucose oxidation through the HMS may represent an important source of substrate for the oxidase [48]. This proposal is supported by recent studies on mouse spermatozoa indicating that these cells have a functional HMS and have an obligatory requirement for glucose to support capacitation [49]. The fact that this glucose requirement for capacitation can be replaced by NADPH strongly suggests that the generation of this coenzyme through the HMS is an important attribute of capacitation in the mouse [49]. In the present study, addition of substrates for the HMS, NADP+ and G-6-P, to permeabilized rat spermatozoa resulted in the generation of O2-·, as judged by SOD-inhibitable NBT reduction. Although such results do not prove the importance of the shunt as a means of supplying electrons to the spermatozoon's free radical-generating system in vivo, they are consistent with such a hypothesis. However, at this stage we cannot exclude the possibility that other enzyme systems capable of generating NADPH, including mitochondrial dehydrogenases linked to malate and isocitrate, could also contribute to the supply of reducing equivalents during capacitation [50, 51]. Alternatively, NADH generated via glycolysis may provide the link between glucose metabolism and capacitation given the preferential way in which this coenzyme was oxidized by rat sperm plasma membranes. Notwithstanding such possibilities, the evidence obtained in the present study, as well as the mouse [49], strongly suggest that NADPH generated via the HMS is involved in the redox control of capacitation. The inability of known HMS inhibitors to modulate the spontaneous basal levels of O2-· production by rat spermatozoa may simply reflect the fact that these cells are not capacitated as readily as mouse spermatozoa in vitro. Similarly, the poor ability of rat spermatozoa to undergo capacitation in vitro may also explain why the only detectable spontaneous ROS signal generated by cultured rat spermatozoa is of mitochondrial origin. In vivo, estrous uterine fluids may well contain physiological factors that activate the redox systems controlling sperm capacitation. However, this hypothesis has yet to be tested.

Apart from substrate availability, one of the other controlling influences on sperm NAD(P)H oxidase activity is the availability of zinc. Zinc is known to suppress the leukocyte NADPH oxidase following stimulation with PMA [52]. This cation appears to be effective in the inhibition of leukocyte NADPH oxidase activity by disrupting an electrogenic proton-transporting pathway that plays a central role in the regulation of intracellular pH during the oxidative burst [53]. In the case of spermatozoa, zinc must possess some other mechanism of action because it can suppress ROS generation by isolated sperm plasma membranes at nM to µM levels. Zinc is a major element in seminal fluid and spermatozoa where it is present in abundance, with ejaculated rat spermatozoa containing 1.055 µg zinc/million cells according to a recent report [54]. Moreover there is evidence to suggest that there is secretion of zinc into the lumen of the rat cauda epididymis [55] where it might play an important physiological role in silencing the sperm NAD(P)H oxidase. The fact that several independent analyses have indicated that zinc will suppress sperm capacitation [56–59] is in keeping with the ability of this cation to suppress the oxidase and thereby disrupt the major signal transduction pathway involved in the attainment of a capacitated state. Our results indicate that the site of zinc action is intracellular with the result that high concentrations (>1 mM) of this cation are required to exert an impact on ROS generation in intact cells. At lower levels of extracellular zinc, other effects of this cation predominate. For example, rat spermatozoa incubated with micromolar amounts of extracellular zinc have been found to exhibit increased levels of tyrosine phosphorylation through inhibitory effects of this metal on tyrosine phosphatase activity (unpublished observations). In light of these results, it would appear that the intracellular levels of zinc could have a key regulatory role in the control of sperm function. Elucidating the mechanisms by which the intracellular concentration of this cation is regulated in different sperm compartments could shed new light on the biochemistry of sperm capacitation.

A second inhibitory influence on the sperm NAD(P)H oxidase appears to be localized in the cytosol but remains uncharacterized (Fig. 10). The information gained in this study shows clearly that this component has a molecular mass greater than 10 kDa, is not a free radical scavenger, and in particular, is unlikely to be SOD. Elucidating the identity of this factor will not only be significant in resolving redox regulation of sperm function but may also contribute to our understanding of the origins of oxidative stress in the male germ line.

While some progress has been made on the cellular mechanisms involved in the regulation of NAD(P)H oxidase activity in leukocytes [60], the sperm oxidase appears to be quite distinct from its counterpart in leukocytes according to the RT-PCR analysis described in this paper. In view of this uncertainty over the identity of this free radical-generating system in spermatozoa, it may be more appropriate to refer to the activity recorded in this study as a function of a plasma membrane NAD(P)H oxidoreductase system rather than a specialized oxidase. Although oxygen can serve as an electron acceptor for this system resulting in the generation of O2-·, this may not be the only or even the preferred electron acceptor for this complex in vivo. Further studies are clearly needed to characterize this plasma membrane redox system and its relationship with known NAD(P)H oxidases and oxidoreductases thought to be important in the regulation of many disparate cell types [60].

FOOTNOTES

First decision: 26 February 2001.

1 This study was largely funded by a grant from the Ernst Schering Foundation and Rockefeller Foundation to promote research on post-testicular methods of contraception. Back

2 Correspondence: John Aitken, School of Biological and Chemical Sciences, Centre for Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia. FAX: 61 2 4921 6923; jaitken{at}mail.newcastle.edu.au Back

Accepted: May 22, 2001.

Received: January 25, 2001.

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