Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print March 17, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027748
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BIOLOGY OF REPRODUCTION 71, 307–318 (2004)
DOI: 10.1095/biolreprod.104.027748
© 2004 by the Society for the Study of Reproduction, Inc.

Male Reproductive Tract

Identification of Cytochrome P450-Reductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations1

Mark A. Baker, Anton Krutskikh3, Benjamin J. Curry, Eileen A. McLaughlin, and R. John Aitken2

ARC Centre of Excellence in Biotechnology and Development, Reproductive Science Group, School of Environmental and Life Sciences, and Hunter Medical Research Institute, University of Newcastle, Callaghan, New South Wales 2308, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes—such as MCLA and luminol—that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

male reproductive tract, sperm, sperm capacitation, sperm maturation, stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Second only to the mitochondrial electron transport chain, the most notable enzyme involved in reactive oxygen species (ROS) production is the NADPH oxidase of phagocytic leukocytes [1]. It belongs to a family of enzymes, which include NOX1 through to NOX5, that uses NADPH as an electron acceptor and catalyzes the one electron reduction of molecular oxygen to produce superoxide anion (O). Originally described as being involved in pathogen defense [1], ROS appears to play pivotal roles in many forms of cell signaling. For example, overexpression of NOX-1 within NIH 3T3 cells leads to an increase in the proliferation and invasiveness of these cells [2]. Moreover, this phenotype can be rescued by coexpressing catalase within these same cells, demonstrating a role for hydrogen peroxide as a signaling molecule. Similarly, ROS are also involved in other physiological processes, including apoptosis [3], capacitation of mammalian spermatozoa [4–7], and cell proliferation [2].

Due to the sensitive nature of the probe, lucigenin has been frequently used for the detection of ROS, especially O [8, 9]. For example, lucigenin chemiluminescence has been used to detect NOX2-dependent O production in phagocytic cells [10]. More convincingly, lucigenin chemiluminescence has been reported in cell free systems generating superoxide such as xanthine oxidase plus xanthine [9]. To understand the role of NADPH oxidases and ROS within cells, investigators have extensively used a simplified assay system for the analysis of these constituents. Such an assay includes the addition of exogenous NADPH together with lucigenin to cell suspensions. Consequently, NADPH-dependent lucigenin chemiluminescence has been reported in many cell and tissue types, including human polymorphonuclear leukocytes [11], rat cardiac muscle [12] rabbit basilar arteries and cerebral arterioles [13], human endothelial cells [14], rabbit smooth muscle [15], calf pulmonary artery smooth muscle [16], human platelet cells [17], vascular tissue [18], bone marrow-derived macrophages [19], spermatozoa [20], and many plant cells [21, 22]. In many of the aforementioned reports, the observation that diphenylene iodonium (DPI; an inhibitor of flavoproteins and NOX isozymes) inhibits the chemiluminescent signal has led to the proposal that NADPH oxidases are responsible for the observed rate of lucigenin reduction.

However, the suggestion that the ability of a cell type to generate an NADPH-dependent chemiluminescent signal is due to oxidases is quite controversial [23]. To investigate this contentious phenomenon further, we have focused our attention on our model system, mammalian spermatozoa.

Spermatozoa were the first cell type demonstrated to produce ROS [24], and current evidence suggests that oxygen metabolites play a crucial role in many physiological and pathological processes in these cells [20, 25–29]. Nevertheless, the mechanism(s) of ROS generation by mammalian spermatozoa remain unclear. Upon addition of NADPH to suspensions of human spermatozoa, an increase in lucigenin-dependent chemiluminescence has been documented [20]. Moreover, the majority of this signal could be inhibited by the addition of superoxide dimutaste (SOD), a scavenger of O [20]. NADPH-dependent lucigenin chemiluminescence in human spermatozoa was shown to be nonmitochondrial in origin because the signal was insensitive to rotenone, antimycin A, carbonyl cyanide m-chlorophenylhydrazone, and sodium azide [20]. The enzyme responsible for this signal appeared to be similar to the NADPH oxidase found in phagocytes because it could be inhibited with quinacrine and the flavoprotein inhibitor DPI. Furthermore, subcellular fractionation studies using sucrose gradients have localized the NADPH-dependent lucigenin signal to a crude membranous fraction [20, 30]. The generation of similar NADPH-dependent signals by mouse [31], rat [30], and equine [32] spermatozoa suggests the presence of a ubiquitous NADPH-dependent enzyme system. However, the addition of NADPH to human spermatozoa failed to stimulate these cells to produce a chemiluminescent signal using the superoxide-dependent probe 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazine-3-one (MCLA), whereas addition of fetal cord serum ultrafiltrates or progesterone did produce a chemiluminescent signal in this system [23]. Furthermore, electron spin measurements failed to detect superoxide production upon addition of NADPH [33]. Therefore, to demonstrate whether NADPH-dependent lucigenin chemiluminescence was indicative of actual enzymatic ROS production, our aim was to purify and identify the biochemical entity responsible for this activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

All chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) with the exception of catalase (specific activity 30 000 U/mg; Calbiochem, Melbourne, Australia), WST-1 (AusPep, Parkville, Victoria, Australia), MCLA (Molecular Probes, Castle Hill, NSW, Australia), purified cytochrome P450-reductase and anti-cytochrome P450-reductase (Research diagnostics, Flanders, NJ), FITC-conjugated anti-mouse secondary antibody (Santa Cruz, CA), and the goat serum (Hunter Antisera, Jesmond, NSW, Australia).

Animals

Laboratory Wister rats were bred and housed at the Central Animal House in the University of Newcastle. Between the ages of 8–12 wk, they were transferred to the Medical Animal Housing Facility, where they were held in pairs in cages under conditions of controlled temperature and light. Food and water were available ad libitum. All experimental animal use was approved by the University of Newcastle Animal Ethics committee and conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals.

Preparation of Spermatozoa

Epididymides from sexually mature male Wistar rats between 12–24 mo old were prepared as previously described [30]. Caput or caudal spermatozoa were obtained by pricking the epididymis with a 20-gauge needle and, in the case of caudal sperm, allowing the sperm to swim into Hanks buffered salt solution (HBSS) surrounded by water-saturated mineral oil. Caput preparations were agitated for at least 1 min following pricking in HBSS surrounded by water-saturated mineral oil. The spermatozoa were pelleted at 750 x g for 5 min, the supernatant discarded, and the pellet resuspended in HBSS. Cell density was determined using a hemocytometer. The spermatozoa from either the cauda or caput epididymides were then diluted in HBSS to the appropriate cell concentration.

Determination of WST-1 Reduction

Approximately 100 µl of epididymal spermatozoa preparation (1 x 106/ml) was added to a 96-well plate. Inhibitors were added at the final concentration indicated in the text and allowed to incubate for 5–10 min at 37°C. 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium, monosodium salt (WST-1) (500 µM), and NADPH (125 µM) were then coadministered. At the appropriate time, the increase in absorbance at 415 nm (WST-1 reduction) was measured in an Ultramark Microplate Imaging System (Bio-Rad, Castle Hill, NSW, Australia).

Nitroblue Tetrazolium Reduction

An isosmotic solution containing 250 µM nitroblue tetrazolium (NBT) and 250 µM NADPH was layered over cells. The mixture was left at 37°C for 1 h before the reduced formazan (blue deposit) was visualized under light microscopy. As a positive control, a solution containing NBT only was used.

Chemiluminescence Measurement

Approximately 400 µl of sperm suspension (8 x 106/ml) from either the cauda or caput epididymis were added to 5-ml luminometer tubes (Sarstedt, Ingle Farm, SA, Australia). Lucigenin (500 µM), luminol (1 mM), or MCLA (1 mM) together with inhibitors, and the vehicle control were added to final concentration indicated. A baseline rate of chemiluminescence was then established for 10 min. NADPH was then added to the tubes and chemiluminescence was measured using an AutoLumat luminometer (Berthold, Bundoora, Victoria, Australia).

Measurement of NADPH Fluorescence

NADPH fluorescence was measured using a spectrofluorophotometer (Shimadzu model RF-5301P1; Rydalmere, NSW, Australia). Briefly, 2 x 106 cells were added to 3 ml fluorometer cuvettes (Starstedt, Ingle Farm, SA, Australia). After normalizing against the tube, 50 µM NADPH was added. Oxidation of the pyridine nucleotides was measured using excitation at 340 nm and emission at 460 nm. Rates of NADPH oxidation were measured for 5–10 min.

Purification of Cytochrome P450-Reductase from Rat Epididymal Preparations

Epididymal sperm preparations were pelleted (500 x g, 5 min) and lysed for 30 min on ice (50 mM Tris, pH 7.4, 2% Triton X-100 [v/v]). The samples were centrifuged (10 000 x g, 30 min) and the supernatant taken. Approximately 100 µg of solubilized protein were loaded onto a 5 cm x 1 cm 2'-5' ADP Sepharose column that had been preequilibrated with Buffer A (1% Triton x-100 [v/v], 50 mM Tris, pH 7.4). The column was washed with 10 ml buffer A, then eluted with a linear gradient of 0– 1 M NADPH (dissolved in buffer A) over 10 ml. One-milliliter fractions were collected and assayed for NADPH-lucigenin or WST-1 activity.

Protein Identification

Protein identification by MADLI-TOF analysis was performed by the Australia Proteome Analysis Facility (Sydney, Australia).

RT-PCR of Cytochrome P450-Reductase

Total RNA was extracted from rat testis based on a procedure described previously [34]. Five micrograms of total RNA from rat testis was reverse transcribed with oligo(dT)15 primer (Promega Corporation, Annandale, NSW, Australia) and M-MLV Reverse Transcriptase RNase H Minus (Promega). Full-length cytochrome P450-reductase was amplified using the Advantage 2 Polymerase Mix (BD Biosciences Clontech, North Ryde, NSW, Australia), combining hot start PCR with proofreading capability. PCR was performed with oligonucleotide primers based on the published sequence, (GenBank accession number NM_031576) [35], with the forward primer designed to facilitate directional TOPO cloning. The forward primer sequence was 5'-CACCATGGGGGACTCTCACGAAGAC-3.' The reverse primer sequence was 5'-GCTCCACACATCTAGTGAGTAGC-3'. The PCR reaction conditions were as follows: 1 cycle of 94°C for 5 min; 35 cycles of 95°C for 30 sec, 66°C for 30 sec, 72°C for 2 min; 1 cycle of 72°C for 10 min.

TOPO Cloning of Cytochrome P45-Reductase

The cytochrome P450-reductase PCR product was cloned into a Gateway entry vector pENTR/D-TOPO (Invitrogen Corporation, Mulgrave, VIC, Australia), according to the manufacturer's instructions, using One Shot TOP10 competent Escherichia coli cells (Invitrogen). Recombinants were confirmed via NotI digestion of plasmid preparations. The insert was then transferred into the Gateway destination vector pcDNA-DEST47 (Invitrogen), using Clonase Enzyme Mix (Invitrogen). Recombinants were confirmed via NdeI digestion of plasmid preparations. Constructs were sequenced with the BigDye Terminator cycle sequencing method on an automated ABI Prism 377 DNA Sequencer (Applied Biosystems, Scoresby, VIC, Australia), performed by the Biomolecular Research Facility at the University of Newcastle. The full-length sequence of cytochrome P450-reductase was shown to be in frame with the green fluorescent protein (GFP) tag of pcDNA-DEST47. This construct was named P450RpcDNA-DEST47.

Cell Culture and Transfection of DNA into COS-7 Cells

COS-7 cells were maintained in Dulbecco modified Eagle medium (Invitrogen) at 37°C in a 5% CO2 atmosphere, supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM L-glutamine. Plasmid DNA was prepared for P450RpcDNA-DEST47 and pcDNA-DEST47 by the plasmid midi-prep protocol according to the manufacturer's instruction (Qiagen, Clifton Hill, VIC, Australia) using a Qiagen-tip 500. The final DNA pellets were resuspended in TE buffer, pH 8.0, and stored in aliquots of 1 µg/µl at 4°C. COS-7 cells were transfected with P450RpcDNA-DEST47 and pcDNA-DEST47 using the Superfect transfection reagent (Qiagen). Briefly, 2 x 105 cells were seeded into six-well plates 24 h before transfection. Five micrograms of DNA was incubated with 25 µl of superfect in serum-free medium at room temperature for 15 min. Six hundred microliters of complete medium were added and the whole mixture incubated with washed COS-7 cells for 3 h at 37°C in a 5% CO2 atmosphere. The cells were then washed and incubated in complete medium for 48 h and assayed for GFP expression by fluorescence.

Immunofluorescence

The epididymides from killed rats were removed, fixed (71% [v/v], picric acid, 24% [v/v], formalin, 5% [v/v] acetic acid), paraffin embedded, sectioned (7 µM), and mounted on glass slides. Individual sections were dewaxed and blocked using TBS (137 mM NaCl, 20 mM Tris-HCl, pH 7.6) containing 1% (w/v) BSA and 5% (v/v) goat serum. For indirect immunofluorescence staining, slides were incubated with anti-rabbit cytochrome P450-reductase antibodies at 1/1000 dilution for 1 h at room temperature. Slides were washed (3 x 5 min) in TBS, then incubated for 1 h with FITC-labeled goat anti-rabbit IgG secondary antibody in the dark. The slides were then washed again (3 x 5 min), mounted with antifade reagent and stored in the dark until analysis. Fluorescence staining was detected using a Zeiss LSM510 laser scanning confocal microscope equipped with an argon and a helium neon laser using excitation 488-nm and emission 500–530-nm wavelengths.

Western Blot Analysis

All Western Blot analyses were essentially performed as previously described [6]. SDS PAGE was carried out on protein extracts (2 µg) from spermatozoa on 10% gels. Following electrophoresis at 30 mA constant current, the proteins were transferred for 1 h onto nitrocellulose hybond super-C membrane (Amersham, Bucks., UK) at 100-V constant voltage in wet blot buffer (0.1 M Tris; 0.767 M glycine; 20% [v/v] methanol). The membrane was blocked for 1 h in Tris buffered saline (TBS), which consisted of 20 mM Tris, 150 mM NaCl, pH 7.6, containing either 3% (w/v) BSA (phosphotyrosine/{alpha}-tubulin antibodies). Labeling with primary antibody was accomplished by incubation for 1 h at 22°C under the following conditions: anti-phosphotyrosine (Py20; Affiniti, Exeter, UK) was incubated at 1/1000 dilution in TBS + 0.1% Tween 20 (TBS-T), and anti-{alpha} tubulin (Clone B-5-1-2; Sigma, NSW, Australia) was incubated at 1/4000 dilution in TBS-T + 0.01% (w/v) BSA. Following four washes with wash buffer (1 x 15 min, 3 x 5 min TBS + 0.01% Tween 20), horseradish-peroxidase-labeled secondary antibody was added for 1 h at 22°C under the following conditions: For phosphotyrosine labeling, goat anti-mouse IgG (Affinity, Exeter, UK) was added at 1/6000 dilution in TBS-T + 1% BSA. In other experiments, goat anti-mouse IgG (Selby Biolab, VIC, Australia) was added at 1/3000 dilution in TBS-T for {alpha}-tubulin. The washes were repeated and detection of proteins was performed using the enhanced chemiluminescence (ECL) kit (Amersham Life Sciences, Bucks, UK) according to the manufacturer's instructions.

Sypro Ruby Staining

Following SDS PAGE, the gel was left in sypro-ruby (Molecualr Probes, Mulgrave, Australia) overnight according to the manufacturer's instructions. Following a brief wash in distilled water, the gel was imaged on the typhoon image system (Amersham Bioscience, Castle Hill, Australia) using the excitation at 532 nm and emission at 610 nm, with a bandpass of 30 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of an NADPH-Dependent Lucigenin Chemiluminescent Signal in Rat Epididymal Spermatozoa

Caput (Fig. 1a) or caudal (Fig. 1b) spermatozoa preparations, incubated with NADPH, demonstrated a cell concentration-dependent increase in lucigenin chemiluminescence, confirming previous reports [30]. Chemiluminescence was higher with sperm preparations originating from the caput epididymis compared with those taken from the cauda, supporting claims that the enzyme(s) responsible for NADPH-lucigenin chemiluminescence become less active as the spermatozoa mature in the epididymal lumen [30]. To determine whether molecular oxygen could serve as an electron acceptor for this system, SOD was added to the incubations. This enzyme was found to inhibit at least 80% of the total signal, indicating the involvement of superoxide (Fig. 1c). To further test for the generation of ROS by spermatozoa on addition of NADPH, the tetrazolium salt WST-1 was used.



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  		FIG. 1. NADPH-induced lucigenin-chemiluminescence in caput and caudal epididymal preparations. Lucigenin was added to increasing numbers of spermatozoa as indicated from either the caput (a) or caudal (b) epididymis. After establishing a background rate for 10 min, NADPH was added as indicated and the increase in lucigenin-dependent chemiluminescence was measured. As a positive control, 1 x 107 spermatozoa were preincubated with 300 U SOD or the vehicle control and NADPH-dependent lucigenin chemiluminescence was measured (c)

Reduction of WST-1 in Rat Epididymal Spermatozoa upon Addition of NADPH

WST-1 has been previously used to detect the presence of an NADH oxidase in cancer cells [36, 37] as well as ROS production by phagocytes [37]. Formation of the reduced formazan was observed in the presence of either caput (Fig. 2a, filled bars) or caudal (Fig. 2a, open bars) spermatozoal preparations and was shown to be cell concentration dependent (Fig. 2). Moreover, reduction of the probe could not be inhibited with the mitochondrial complex I inhibitor, rotenone (Fig. 2b), suggesting a nonmitochondrial source for the reduction of the probe. For further investigation of ROS generation, two additional ROS-detecting probes, luminol and MCLA, were used [23, 38].



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  		FIG. 2. NADPH-dependent WST-1 reduction in caput and caudal epididymal preparations. a) Approximately 500 µM WST-1 and 125 µM NADPH were coadministered to increasing numbers of cells from the caput (filled bars) or caudal (open bars) epididymis. The increase in absorbance was measured at 415 nm. b) Caput (filled bars) or caudal (open bars) cell preparations (0.5 x 106) were incubated with either the vehicle (lane 1), 1 µM rotenone (lane 2), or 10 µM rotenone (lane 3), as indicated. Following a 5-min incubation, WST-1 and NADPH were added according to the Materials and Methods. After 1 h, the increase in absorbance at 415 nm was measured

Luminol and MCLA Chemiluminescence

No chemiluminescent signal was detected when either luminol (Fig. 3a) or MCLA (Fig. 3c) were used as probes for ROS generation in response to NADPH. As a positive control, the addition of 0.05 U xanthine oxidase together with xanthine generated large chemiluminescent signals detectable with both luminol (Fig. 3b) and MCLA (Fig. 3d). Interestingly, the MCLA signal could be inhibited with SOD, suggesting this probe detects superoxide. However, SOD increased the luminol signal, indicating that this probe was detecting hydrogen peroxide as suggested by Nakamura and Nakamura [39]. These data suggest that spermatozoal preparations do not generate significant quantities of ROS upon addition of NADPH to cells incubated in a simple defined culture medium.



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  		FIG. 3. No detectable NADPH-dependent chemiluminescence using luminol or MCLA. Approximately 107 spermatozoa obtained from the caput (squares) or caudal (circles) epididymal region were incubated with (a) luminol (c) MCLA. After establishing a baseline rate, 125 µM NADPH was added (arrow) and the increase in chemiluminescence formation was monitored. The solid line represents the medium control. As a positive control, 0.05 U xanthine oxidase plus xanthine were added and both (b) luminol and (d) MCLA chemiluminescence was measured. The addition of 300 U SOD, as indicated, was added 5 min before chemiluminescence was measured

No NADPH Oxidation by Rat Epididymal Spermatozoa

No NADPH oxidation above background rate could be detected using 2 x 106 spermatozoa (lane 1 versus 2) obtained from the caput (Fig. 4a) or cauda (Fig. 4b) epididymis. However, because the final concentration of NADPH in the spectrofluorometer could not exceed 50 µM (because the emission spectrum generated by doses <50 µM were beyond the detection limit of the flurospectrophotometer), it might be argued that NADPH was not present at an adequate concentration sufficient to ensure penetration across the plasma membrane to fuel the putative oxidoreductase system [38]. To address this potential problem, cells were permeabilized by freeze-thawing (lane 3). This strategy did not result in a further increase in NADPH-oxidation, suggesting that the NADPH-lucigenin and WST-1 signals seen in Figures 1 and 2 were due to the direct enzymatic reduction of these compounds rather than production of reactive oxygen intermediates. As a positive control, cytochrome c was added to measure NADPH-cytochrome c reduction (Fig. 4b, lane 4).



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  		FIG. 4. No NADPH oxidation in the presence of whole or permeabilized spermatozoa. Approximately 1 x 106 viable (horizontal continuous line) or freeze fractured (vertical dashed line) cells from either the caput (a) or cauda (b) were incubated with 50 µM NADPH. NADPH fluorescence was measured in a spectrofluorometer using 340 and 460 nm for excitation and emission, respectively. Medium with NADPH (vertical continuous line) served as a baseline control. The addition of 20 µM oxidized cytochrome c and 50 µM NADPH (horizontal dashed line) to measure NADPH cytochrome c reductase activity was used as a positive control

Pharmacological Profile of NADPH-Dependent Reduction

The presence of an NADPH oxidase in spermatozoa, not dissimilar to the NADPH oxidase (NOX2) present in leukocytes, has been supported on the basis that the flavoprotein inhibitor DPI could inhibit the NADPH-dependent lucigenin chemiluminescence. Similarly, in our hands, the addition of DPI to either the caput (Fig. 5a) or caudal (Fig. 5b) epididymal sperm preparations inhibited lucigenin (Fig. 5) and WST-1 (data not shown) reduction. These data suggest that the enzyme involved contains a flavoprotein group in the active site [36]. Furthermore, the addition of pCMBS, a cysteine chelator, to either caput (Fig. 5c) or caudal (Fig. 5d) epididymal preparations completely inhibited both lucigenin (Fig. 5) and WST-1 signals (data not shown), suggesting that the enzyme system involved contains a cysteine at or near the active site. Furthermore, as both DPI and pCMBS could inhibit both lucigenin and WST-1 reduction, this suggested that the same or a very similar family of enzymes was involved in the oxidoreduction reactions. Having obtained these pharmacological data, we then proceeded to purify and identify the enzyme(s) responsible for NADPH-dependent lucigenin/WST-1 reduction.



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  		FIG. 5. NADPH-dependent lucigenin chemiluminescence is inhibited by DPI and pCMBS. Approximately 107 spermatozoa from the caput (a, c) or caudal (b, d) epididymis region were incubated with 20 µM DPI (a, b), 50 µM pCMBS (c, d), or the vehicle control as indicated. Following a 5-min preincubation, 500 µM lucigenin was added and a baseline rate was established. At the time shown, 125 µM of NADPH was added and the increase in lucigenin-dependent chemiluminescence was measured. Control represents the addition of just lucigenin (500 µM) to cells

Purification of the NADPH-Dependent WST-1/Lucigenin Reductase

Rat epididymal spermatozoa preparations were washed and solubilized in 5% Triton X-100. The soluble proteins were then applied to a 2'-5'-ADP affinity column. Following washing with 10 column volumes, elution of the enzyme was achieved with a 13-ml gradient of 0–500 µM NADPH. One-milliliter fractions were collected and assayed for enzyme activity (Fig. 6a). Fractions either containing or flanking those that demonstrated NADPH-WST-1 activity were then precipitated and separated in an SDS-PAGE (Fig. 6b). A 75-kDa band correlated with enzyme activity (Fig. 6b, arrow), suggesting it might be the enzyme responsible for NADPH-dependent WST-1 reduction. These same fractions also displayed NADPH-dependent lucigenin chemiluminescence, suggesting that the same enzyme may be responsible for both activities (data not shown).



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  		FIG. 6. Purification of cytochrome P450-reductase. a) Approximately 100 µg of solubilized protein was loaded onto a 2'-5' ADP Sepharose column. The column was washed, then eluted with a linear gradient of 0–500 µM NADPH over 13 ml. One-milliliter fractions were collected and assayed for NADPH-WST-1 activity. b) Active fractions eluting from the 2'-5' ADP-Sepharose column were precipitated, resuspended in loading dye, subject to 12% SDS-polyacrylamide gel electrophoresis and sypro ruby stained as described in Materials and Methods. The positions of the molecular mass markers are shown. The arrow indicates the 75-kDa protein coeluting with enzyme activity

The 75-kDa band was excised from the gel and subjected to a matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) analysis. Six resulting peptides with their predicted amino acid sequences were used to search the BLAST and TREMBL databases for sequence homology to known proteins. This search revealed the 75-kDa band to be identical to rat cytochrome P450-reductase (Fig. 7).



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  		FIG. 7. MALDI-TOF analysis reveals cytochrome P450-reductase. a) The 75-kDa protein coeluting with enzyme activity was excised and subject to MALDI-TOF analysis. The resulting seven peptides and their predicted amino acid sequence matched cytochrome P450-reductase. b) The position of the predicted amino acid sequence in cytochrome P450-reductase is shown in bold. Overall, a 10% coverage of the entire protein was found

Homogenous Cytochrome P450-Reductase Can Reduce Lucigenin and WST-1 In Vitro

Coelution of cytochrome P450-reductase with enzyme activity from the 2'-5'-ADP affinity column suggested that this enzyme is likely responsible for the observed lucigenin chemiluminescence and tetrazolium salt reduction. However, because the eluant from the affinity column was not homogeneous (see Fig. 6), further evidence was sought. A commercially available preparation of homogenous cytochrome P450-reductase from Research Diagnostics was obtained. In a 10% SDS-PAGE analysis, the preparation is present as a single band at 75 kDa (Fig. 8, inset) consistent with molecular mass of cytochrome P450-reductase. The addition of this preparation to lucigenin (Fig. 8) and WST-1 (data not shown) in the presence of NADPH demonstrated a marked increase in all signals that was shown to be dose dependent. To ensure that the purified enzyme demonstrated the same pharmacological profile as that seen in whole cells, we tested the ability of pCMBS, DPI, and SOD to inhibit the NADPH-dependent lucigenin (Fig. 9) and WST-1 (data not shown) reduction. In all cases, the activity was suppressed. Furthermore, the cytochrome P450-reductase was shown to be NADPH specific, as NADH could not support either lucigenin (Fig. 9) or WST-1 reduction (data not shown). These data further support the concept that NADPH-dependent redox activity is driven by cytochrome P450-reductase alone and not by another protein coeluting with the activity.



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  		FIG. 8. Purified cytochrome P450-reductase displays NADPH-dependent lucigenin chemiluminescence. Either 564 (solid line), 376 (triangles), or 188 (circles) U of purified enzyme were incubated with lucigenin. After establishing a background rate, NADPH was added and the increase in chemiluminescence was measured. Insert: 5 µl of enzyme extract were loaded onto a 15% SDS polyacrylamide gel and stained with Sypro ruby, 1; purified enzyme, 2; molecular mass markers



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  		FIG. 9. Pharmacological profile of purified NADPH cytochrome P450-reductase. Approximately 75 U of homogenous cytochrome P450-reductase were incubated with 100 units SOD (lane 3), 50 µM pCMBS (lane 4), 20 µM DPI (lane 5), or the vehicle control (lane 1). After a 5-min preincubation, lucigenin (500 µM) was added to the solution and chemiluminescence was measured for 10 min. Following this, 125 µM NADPH (lanes 1, 3–5) or NADH (lane 2) were added. The increase in chemiluminescence over 1 min following addition of the pyridine nucleotide was plotted

Cytochrome P450-Reductase Functions as an NADPH Lucigenin/WST-1 Reductase In Vivo

Although cytochrome P450-reductase can clearly reduce both lucigenin and WST-1 in vitro, it is still uncertain whether this occurs in intact cells (in vivo). To establish this, COS-7 cells (2–3 x 106) were transfected with either 5 µg of P450RpcDNA-DEST47 or the vector control pcDNA-DEST47. P450RpcDNA-DEST47 contains full-length rat cytochrome P450-reductase, with GFP fused to the C-terminus. Approximately 48 h posttransfection, the cells were harvested, washed, and subjected to Western blot analysis (Fig. 10a). As shown, ~100-kDa fusion protein can be seen in the P450RpcDNA-DEST47 transfected cells, which is equivalent to the predicted size of the GFP plus cytochrome P450-reductase fusion protein (Fig. 10a). Further evidence to support expression of the fusion protein was obtained when the transfected cells were visualized using confocal microscopy. Cells transfected with GFP only demonstrated typical widespread cytosolic expression (Fig. 10, e–g). However, in cells transfected with P450RpcDNA-DEST47, GFP fluorescence exhibited a punctate extranuclear distribution consistent with localization within the endoplasmic reticulum, the expected site of cytochrome P450-reductase expression (Fig. 10, b–d).



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  		FIG. 10. Establishing the expression of transient transfection of the rat cytochrome P450-reductase into COS-7 cells. a) Twenty-four hours posttransfection, the cells were harvested, lysed, and subject to Western blot analysis using anticytochrome P450-reductase antibodies. e–g) COS-7 cells were transfected with pcDNA-DEST-47 expression construct alone and viewed under (b) phase contrast (x100) and the corresponding (c) fluorescence (x100). GFP expression can be seen throughout the cell cytosol. A high-power view is also shown (d, x200). b–d) COS-7 cells were transfected with full-length rat cytochrome P450-reductase contained in the expression construct pcDNA-DEST47 (Invitrogen), and viewed under (e) phase contrast (x100) and the corresponding (f) fluorescence (x100). A high-power view is also shown for (g) fluorescence (x200)

To determine if the P450RpcDNA-DEST47 gene product could function as a reductase in vivo, transfected COS7 cells were assayed for their ability to reduce lucigenin and WST-1 in the presence of NADPH. Those cells expressing P450RpcDNA-DEST47 demonstrated a 3-fold stimulation in lucigenin chemiluminescence (Fig. 11a) and WST-1 reduction (Fig. 11b) compared with the vector-only transfected cells (see Fig. 11). Moreover, addition of DPI, pCMBS, or SOD inhibited the reduction of the two probes, confirming the involvement of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin chemiluminescence and WST-1 reduction (Fig. 11, a and b).



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  		FIG. 11. Transient transfection of the rat cytochrome P450-reductase into COS-7 cells increases NADPH-dependent lucigenin and WST-1 reduction. Cells expressing GFP only (filled bars) or the fusion protein (open bars) were assayed for NADPH-dependent (a) lucigenin chemiluminescence or (b) WST-1 reduction in the presence of the vehicle (lane 1), 20 µM pCMBS (lane 2), 20 µM DPI (lane 3), or 100 U SOD (lane 4). The results are representative of the experiment that was performed three times in duplicate analyses

Localization of Cytochrome P450-Reductase Within Epididymal Preparations

Spermatozoal preparations from the caput epididymides have been reported to have a higher NADPH-dependent lucigenin response than that obtained from the cauda [30]. Such a phenomenon was explained by silencing of the enzyme system as the spermatozoa traversed the epididymis. To study why such silencing occurs within these preparations, we have analyzed the localization of cytochrome P450-reductase both biochemically and immunohistologically.

Both caudal and caput sperm preparations were subjected to discontinuous Percoll gradient centrifugation. One-milliliter fractions were collected and assayed for lucigenin-dependent chemiluminescence. As shown (see Fig. 12a), most of the enzymatic activity was found in the fraction above the 20% interface. Microscopic analysis suggested that this preparation consisted mainly of epithelial cells with the occasional spermatozoa present (data not shown). To determine if contaminating epithelial cells could account for the majority of the redox activity in epididymal preparations, NADPH-dependent NBT (NADPH-diaphorase) staining was performed (Fig. 12b). We demonstrated that NBT, a tetrazolium salt similar to WST-1, was also reduced by homogenous cytochrome P450-reductase (data not shown). This compound has the advantage over WST-1 in that reduction can be easily visualized. As shown, although the epithelial cell contains a large formazan deposit, the spermatozoa show no such staining. During the course of obtaining epididymal spermatozoa, it became apparent that caput preparations were highly contaminated with epididymal epithelium compared with caudal preparations. To determine the possible contribution of such epithelial cell contamination to the intense cytochrome P450-mediated redox activity observed in caput epididymal sperm preparations, immunohistochemical analyses were conducted on rat epididymal sections using anticytochrome P450-reductase antibodies (Fig. 12, c and e). Confocal analysis demonstrated abundant fluorescence of epididymal epithelium (arrows). Moreover, in the more distal regions of the epididymis, the presence of cytochrome P450-reductase decreased, in concert with the decline in redox activity detected with lucigenin.



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  		FIG. 12. Localization of rat cytochrome P450-reductase in the epididymides. a) Rat caput spermatozoa preparations were separated on a Percoll density gradient. One-milliliter fractions were collected and assayed for NADPH-dependent lucigenin chemiluminescence. Low-density Percoll = material collected at the medium/20% Percoll interface. High-density Percoll = material collected at the 80%/100% Percoll interface. b) Rat caput preparations were incubated with NADPH and NBT for 1 h at 37°C. The picture demonstrates formazan deposit in epithelial cell rather than spermatozoa. Original magnification, x150. c–e) The epididymides from killed rats were fixed (71% [v/v], picric acid 24% [v/v], formalin 5% [v/v] acetic acid), sectioned, and mounted on glass slides. Immunohistochemical analysis was performed on (c) caput, (d) corpus, and (e) cauda sections. In all cases, the left-hand side shows the phase (top) and indirect FITC-labeled immunofluorescence (bottom) demonstrating the localization of cytochrome P450-reductase. The right-hand side shows the phase (top) and secondary only control (bottom). Scale bar = 100µm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The presence of plasma membrane NADPH oxidases and consequent ROS generation has been described in a wide variety of cell types including fibroblasts [40], endothelial cells [41], glomerular mesangial cells [42], B-lymphocytes [43–45], leukocytes [46], and plant cells [22]. In many of these reports, ROS generation was detected using chemiluminescent probes such as lucigenin [25] and luminol [38] or tetrazolium salts such as WST-1 [37] and NBT [47]. In at least one cell type, the phagocyte, an NADPH oxidase, has been identified and characterized at a molecular level [48].

The production of ROS in germ cells plays a fundamental role in physiological processes including sperm capacitation and acrosome reaction as well as the pathophysiology of male infertility [28, 49]. However, the biochemical entity involved in ROS generation has not been characterized in molecular terms. It has been suggested that rat epididymal spermatozoa possess a plasma membrane NADPH oxidoreductase [30]. Notably, due to the uncertainty in interpreting the redox activity detected with chemiluminescent probes, the activity detected in rat epididymal spermatozoa has been ascribed to an NADPH oxidoreductase rather than an oxidase [30]. This activity could be demonstrated by the addition of lucigenin together with NADPH to cells and measuring an increase in chemiluminescence [30]. This enzyme assay has been used on a wide range of cells and tissues and is commonly interpreted to be the result of NADPH oxidase (NOX2) activation [10– 15]. The results presented here support the observation that spermatozoa preparations are capable of generating a NADPH-dependent lucigenin signal. Furthermore, another ROS detecting probe—WST-1—was also reduced by these same preparations. However, the inability of spermatozoa to invoke an NADPH-dependent luminol or MCLA signal or to oxidize NADPH in whole cell extracts argues against the notion that superoxide is being produced by the cells, and clearly demonstrates that NADPH-dependent reduction of lucigenin/WST-1 is probe specific, a phenomenon that would not be seen if ROS were being generated.

It has been shown that lucigenin and another tetrazolium salt similar to WST-1, NBT, can be directly reduced by enzymes in biological systems [50]. However, as recently pointed out by Fridovich [51], it is unknown which enzymes are responsible for this action. Our results are consistent with the notion that cytochrome P450-reductase is responsible for the majority, if not all, of the NADPH-dependent lucigenin/WST signals detected in this study. This was demonstrated by 1) coelution of a 75-kDa band from a 2'-5' ADP-Sepharose column being positively identified as cytochrome P450-reductase; 2) a purified, homogenous preparation of cytochrome P450-reductase displayed NADPH-dependent lucigenin/WST-1 activities, suggesting that it is this enzyme alone, and not another coprecipitating enzyme, that is responsible for the activity; 3) pCMBS, DPI, and SOD inhibited NADPH-dependent lucigenin/ WST-1 activities in whole cells, purified enzyme preparations and transiently transfected overexpressing cells; and 4) transiently transfected cells, overexpressing cytochrome P450-reductase, demonstrated a 3-fold increase in NADPH-dependent activity compared with the mock controls. The ability of this enzyme to directly reduce lucigenin upon addition of NADPH, explains why, in other studies, NADPH-dependent lucigenin chemiluminescence was high in microsome fractions [50, 52] and not plasma membrane fractions, where NOX isozymes reside.

The common misinterpretation that NADPH-dependent lucigenin/WST-1 reduction is due to ROS production occurs through the observation that SOD is able to quench the signal being generated. However, the ability of SOD to inhibit NADPH-dependent lucigenin chemiluminescence in purified cytochrome P450-reductase suggests that superoxide production is due to redox cycling of the probe [33]. Lucigenin and tetrazolium salts require several steps before chemiluminescence (in the case of the former) or formazan deposits (in the case of the latter) is achieved. In the case of the former, lucigenin (Luc2+) must undergo a one-electron reduction to form a highly reactive, unstable radical (Luc·+) (Fig. 13, step 1). In the system described in this paper, cytochrome P450-reductase is responsible for this reduction. Once reduced, the lucigenin radical can then couple with O2 to produce superoxide anion (O) and ground state lucigenin (Fig. 13, step 2). Superoxide anion then reacts with another molecule of reduced lucigenin, to produce a dioxetane, before its decomposition into two molecules of N-methyl acridone (Fig. 13). One of the N-methyl acridone molecules is in an excited state, and upon returning to ground state, light is emitted. The constant coupling of the lucigenin radical with O2 to produce O causes the probe to undergo redox cycling. Addition of SOD inhibits this cycling (as shown), and consequently, a major decrease in light emittance occurs.



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  		FIG. 13. Schematic showing the reduction of lucigenin by cytochrome P450-reductase in the presence of NADPH

In the case of tetrazolium salts, similar chemistry applies. Thus, cytochrome P450-reductase reduces tetrazolium salts like WST-1 into the radical (Eqn. 1). The radical then has two options: either react with another radical to produce the blue formazan WST-1H2 (Eqn. 2) or react with ground state oxygen to produce O (Eqn. 3). Upon addition of SOD, which converts superoxide anion to hydrogen peroxide (Eqn. 4), Equation 3 is shifted to the right. As a consequence, the WST-1 radical preferentially reacts with ground state oxygen (Eqn. 3) to produce more O rather than generate formazan deposit (Eqn. 2). The ultimate result is the formation of oxidized WST-1 and not the reduced, dark-yellow colored, formazan product.

Similar chemistry has been applied to NBT, which upon reduction with glucose oxidase becomes a source of superoxide; in this case, SOD inhibits formazan formation, leading to the incorrect conclusion that glucose oxidase produces superoxide [51].

In conclusion, it appears evident that the NADPH:lucigenin and WST-1 signals generated in rat epididymal sperm preparations are due to the presence of NADPH-dependent cytochrome P450-reductase that originates largely, but not exclusively [30], from contaminating epithelial cells. This ubiquitous enzyme directly reduces lucigenin and the tetrazolium salts, WST-1 and NBT, thus initiating their redox cycling with molecular oxygen and production of superoxide anion.


   FOOTNOTES
 
1 Funded by the Ernst-Schering Trust through the AMPPA network and also by the Australian Research Council. Back

2 Correspondence: R. John Aitken, Discipline of Biological Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia. FAX: 61 2 4921 6308; jaitken{at}mail.newcastle.edu.au Back

3 Current address: Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom Back

Received: 27 January 2004.

First decision: 26 February 2004.

Accepted: 4 May 2004.


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