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BOR - Papers in Press, published online ahead of print April 27, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037960
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BIOLOGY OF REPRODUCTION 73, 334–342 (2005)
DOI: 10.1095/biolreprod.104.037960
© 2005 by the Society for the Study of Reproduction, Inc.

Identification of Cytochrome-b5 Reductase as the Enzyme Responsible for NADH-Dependent Lucigenin Chemiluminescence in Human Spermatozoa1

Mark A. Baker , Anton Krutskikh 3 , Benjamin J. Curry , Louise Hetherington , and R. John Aitken 2 

The 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 together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

cytochrome-b5 reductase, female reproductive tract, lucigenin, reactive oxygen species, sperm, sperm capacitation, sperm maturation, spermatozoa, WST-1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS) are now accepted as playing pivotal roles in many forms of cell signaling [1]. For example, overexpression of NADPH-oxidase 1 within NIH 3T3 cells leads to an increase in proliferation and invasiveness via mechanisms that can be reversed by the concomitant overexpression of catalase [2]. Reactive oxygen species are also involved in mediating the bacteriocidal functions of phagocytic leukocytes, the biosynthesis of key hormones, and the induction of apoptosis [3]. Within the reproductive field, ROS have been implicated in male infertility and the redox regulation of tyrosine phosphorylation during sperm capacitation in a variety of species, including humans [4–7].

Because of the sensitive nature of the probe, lucigenin has been frequently used for the detection of ROS, especially the superoxide anion () [8, 9]. For example, lucigenin chemiluminescence has been used to detect NOX 2-dependent production by both phagocytic leukocytes and endothelial cell [10, 11]. Furthermore, lucigenin chemiluminescence has been reported in cell-free systems generating superoxide such as xanthine oxidase plus xanthine [9]. To understand the role of free radical-generating systems within cells, investigators have made extensive use of model systems incorporating exogenous NADH or NADPH as a source of electrons and either lucigenin or tetrazolium salts such as WST-1 or nitroblue tetrazolium (NBT) to detect the generated. Using this approach, NADH-dependent redox activity has been reported in many cells and tissue types including rabbit basilar arteries and cerebral arterioles [12], human endothelial cells [13], calf pulmonary artery smooth muscle [14], human platelet cells [15], vascular tissue [16], spermatozoa [17, 18], and plant cells [19, 20]. In many of the previously mentioned reports, the observation that diphenylene iodonium (DPI; an inhibitor of flavoproteins and NOX isozymes) inhibits the chemiluminescent signal has led to the proposal that NADH-oxidases are responsible for the observed rate of probe activation.

Whether the addition of NAD(P)H to suspensions of human spermatozoa results in the generation of by oxidases is quite controversial [21]. Addition of NAD(P)H to suspensions of human spermatozoa induces a clear increase in both lucigenin-dependent chemiluminescence and WST-1 reduction [17, 18]. Moreover, these signals can be effectively inhibited by the addition of copper, zinc superoxide dismutase (SOD1), a specific scavenger of , suggesting the production of this ROS in this system. However, the addition of NADPH to human spermatozoa failed to stimulate these cells to produce a chemiluminescent signal using another 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 [21]. Furthermore, electron spin measurements failed to detect production on addition of NADPH [22]. To clarify this paradox, we have analyzed the mechanisms responsible for NADPH-dependent lucigenin chemiluminescence in rat epididymal sperm suspensions and identified cytochrome-p450 reductase (CP450R) as the enzyme responsible [23]. Thus, this enzyme was found to coelute with NADPH-dependent lucigenin chemiluminescence from a 2–5 ADP Sepharose affinity column. Second, NADPH-dependent lucigenin chemiluminescence activity could be immunoprecipitated with anti-CP450R antibodies. Third, a homogenous, commercially available preparation of CP450R displayed NADPH-dependent lucigenin chemiluminescence. Finally, overexpression of recombinant CP450R led to a 3-fold increase in whole cell NADPH-dependent lucigenin chemiluminescence. Importantly, although CP450R could reduce lucigenin and tetrazolium salts including WST-1, this was due not to production but rather to the direct one-electron reduction of the previously mentioned probes [23].

Although CP450R is involved in the NADPH-dependent redox activity associated with mammalian sperm suspensions, NADH appears to act through an alternative pathway since this activity is much less susceptible to DPI suppression [18]. In this report, we describe the identification of cytochrome-b5 reductase (CYB5R2) as a key enzyme involved in the mediation of NADH-induced redox activity in human spermatozoa.


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

All chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) with the exception of catalase (Calbiochem, Melbourne, Australia; specific activity 30 000 U/mg), WST-1 (AusPep, Parkville, VIC, Australia), MCLA (Molecular Probes, Castle Hill, NSW, Australia), and anti-GFP (green fluorescent protein) antibodies (GE Healthcare, Castle Hill, NSW, Australia).

Preparation of Human Spermatozoa

The study population comprised a population of unselected normozoospermic donors [24] who had been counseled to exclude individuals exhibiting a high risk for sexually transmitted diseases such as HIV that might have influenced the quality or cellular composition of their semen. Institutional and state government ethical approval was secured for the use of human semen samples for the purposes of this research. The semen samples were produced by masturbation and collected into sterile containers for immediate transportation to the laboratory. After allowing at least 30 min for liquefaction to occur, the spermatozoa were fractionated on a discontinuous two-step Percoll gradient. For this procedure, an isotonic solution was prepared by adding 90 ml of Percoll (Pharmacia LKB, Uppsala, Sweden) to 10 ml of 10 x Ham F10 (Flow Laboratories, Irvine, UK) supplemented with 100 mg polyvinyl alcohol (PVA) to give a preparation that was designated 100% Percoll [24]. This solution was diluted 1:1 with HEPES (20 mM) buffered medium BWW [25] each 100 ml of which was supplemented with 1 mg/ml PVA, 3 mg of sodium pyruvate, 0.37 ml of a 60% sodium lactate syrup (Sigma Chemical Company, St. Louis, MO) and 200 mg of sodium hydrogen carbonate. Discontinuous gradients were then created by layering this low-density Percoll preparation above 3 ml of isotonic Percoll. Liquefied semen was then pipetted onto the gradient and centrifuged for 20 min at 500 x g. Spermatozoa were recovered from the base of the gradient and the low-density/high-density Percoll interface was washed with 7 ml of medium BWW and pelleted (5 min at 500 x g) for further analysis.

Chemiluminescence Measurement

Approximately 400 µl of sperm suspension (5 x 106/ml) or 50 µl of lysate were added to 5-ml luminometer tubes (Starstedt, Ingle Farm, SA, Australia). Stock solutions of lucigenin (500 µM), luminol (1 mM), or MCLA (1 mM) were dissolved in DMSO and added to the tubes together with inhibitors or vehicle control preparations at the final concentrations indicated in Results. A baseline rate of chemiluminescence was then established for 10 min. NADH was then added to the tubes, and chemiluminescence was measured using an AutoLumat luminometer (Berthold, Bundoora, VIC, Australia). The measurements for each time were taken 30 sec following addition of NADH.

Determination of WST-1 Reduction

Approximately 100 µl of epididymal spermatozoa preparation (5 x 106/ml) or cell lysate were 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. WST-1 (500 µM) and NADH (125 µM) were then coadministered. At the appropriate time, the increase in absorbance at 415 nm was measured in an Ultramark Microplate Imaging System (Bio-Rad, Castle Hill, NSW, Australia).

Measurement of NADH Fluorescence

NADH fluorescence was measured using a spectrofluorophotometer (Shimadzu model RF-5301P1; Rydalmere, NSW, Australia). Briefly, 2 x 106 cells were added to 1-ml fluorometer cuvettes (Starstedt). After normalizing against the tube, 50 µM NADH were added. Oxidation of the pyridine nucleotides was measured using excitation and emission wavelengths of 340 and 460 nm, respectively. Rates of NADH oxidation were measured for 5–10 min.

Purification of Cytochrome-b5 Reductase from Human Spermatozoa

Percoll purified sperm preparations were pelleted (500 x g, 5 min) and lysed for 30 min on ice (2% [v/v] Triton X-100 in 50 mM Tris, pH 8.2; 100 µl buffer for 5 x 106 spermatozoa). The samples were centrifuged (10 000 x g, 30 min), and the supernatant was taken. Approximately 100 µg of solubilized protein were loaded onto a 5-cm mono Q column that had been pre-equilibrated with buffer A (1% Triton X-100 [v/v], 50 mM Tris, pH 8.2). The column was washed with 10 ml buffer A, then eluted with a linear gradient of buffer A containing 200 mM NaCl over 10 ml. One-milliliter fractions were collected and assayed for NADH-lucigenin or WST-1 activity.

Protein Identification

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

Cloning of Human Cytochrome-b5 Reductase

Total RNA was extracted from human spermatozoa using the Trizol reagent (Invitrogen Corporation, Mulgrave, VIC, Australia), based on the manufacturer's instructions, except that before isopropanol precipitation, 5 µl of 2 mg/ml glycogen (Ambion, Austin, TX) were used to facilitate RNA precipitation. FirstChoice PCR-Ready cDNA was the source of human testis cDNA (Ambion). Five micrograms of total RNA was reverse transcribed with oligo(dT)15 primers (Promega Corporation, Annandale, NSW, Australia) and M-MLV Reverse Transcriptase RNase H Minus (Promega). Full-length CYB5R2 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 no. NM_016229) [26], with the forward primer designed to facilitate directional TOPO cloning. The forward primer sequence was 5'-CACCATGGACTCCAGGAGGAGAGAG-3'. The reverse primer sequence was 5'-GTAGGTGAAAATCATGTCCTGGG-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. The PCR product was gel purified with the Wizard SV Gel Purification kit (Promega).

TOPO Cloning of Cytochrome-b5 Reductase

The CYB5R2 PCR product was cloned into a Gateway entry vector pENTR/D-TOPO (Invitrogen), 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 CYB5R2 was shown to be in frame with the GFP tag of pcDNA-DEST47. This construct was named b5RpcDNA-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 b5RpcDNA-DEST47 and pcDNA-DEST47 by the plasmid midi-prep protocol according to the manufacturer's instructions (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 b5RpcDNA-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 were 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 was 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.

Western Blot Analysis

All Western blot analyses were essentially performed as previously described [6]. Anti-GFP antibody was used at 1/1000 dilution, and the goat anti-rabbit FITC conjugated secondary antibody was used at 1/2000 dilution.

Measurement of NADPH Fluorescence

NADPH fluorescence was measured using a spectrofluorophotometer (Shimadzu model RF-5301P1; Rydalmere). Briefly, 30 x 106 cells were added to 3 ml fluorometer cuvettes (Starstedt). After normalizing against the tube, 50 µM NADH were added. Oxidation of the pyridine nucleotides was measured using excitation at 340 nm and emission at 460 nm. Rates of NADH oxidation were measured for 5–10 min. Ferricyanide (100 µM) was used as the electron acceptor for CYB5R2 [27, 28].

Statistics

The overall statistical significance of any differences due to treatment was determined by analysis of variance (ANOVA). If significance was observed, then the Student t-test, assuming equal variance, was used to test for differences between group means. A value of P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of an NADH-Dependent Lucigenin Chemiluminescant Signal in Human Spermatozoa

Human sperm preparations demonstrated an NADH-dependent lucigenin chemiluminescence signal that was cell number dependent (Fig. 1A; P < 0.001). Addition of superoxide dismutase (SOD1; Fig. 1B) to the system inhibited the redox activity (P < 0.01), suggesting the involvement of . At this dose of NADH, para-chloromercuribenzenesulfonate (pCMBS), a membrane-impermeant sulfydryl reagent, also inhibited NADH-induced chemiluminescence (P < 0.01), suggesting the involvement of surface thiols in this form of redox activity (Fig. 1B). However, addition of the flavoprotein inhibitor DPI had only a minor impact, suppressing the chemiluminescence by around 25% of the control value (Fig. 1B; P < 0.05). The recent observation that CP450R activity (an enzyme capable of mediating NADPH-dependent lucigenin chemiluminescence [23]) is completely inhibited by DPI suggested that it was not the same enzyme using both electron sources. To further elucidate the mechanisms of NADH-dependent ROS generation, the tetrazolium salt WST-1 was used.



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  		FIG. 1. NADH-induced lucigenin chemiluminescence in human spermatozoa. A) Lucigenin (500 µM) was added to increasing numbers of human spermatozoa cells as indicated. After establishing a background rate for 10 min, 125 µM NADH were added, and lucigenin-dependent chemiluminescence was measured. B) diphenylene iodonium (DPI), zinc superoxide dismutase (SOD1), or pCMBS was added 5 min before the addition of lucigenin and NADH to cells. The level of chemiluminescence was measured and compared to the vehicle control; * P < 0.05, ** P < 0.01

NADPH oxidation by human spermatozoa Very small amounts of NAPH oxidation above background rate could be detected using 30 x 106 human spermatozoa (Fig. 2, lane 1 vs. 2; P < 0.01). On addition of the cell-impermeant compound ferricyanide, no further increase was seen (lane 3). To determine whether an intracellular oxidoreductase existed, cells were permeabilized by freeze-thawing (Fig. 2, lanes 4–5; P > 0.05). This strategy did not result in a further increase in NADH-oxidation. As a positive control, ferricyanide was again added to measure NADH-cytochrome-c reduction (Fig. 2, lane 5; P > 0.01).



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  		FIG. 2. No NADPH oxidation in the presence of whole or permeabilized spermatozoa. Approximately 10 x 106 viable (lanes 2–3) or freeze-fractured (lanes 4–5) cells were incubated with 50 µM NADH. Fluorescence was measured in a spectrofluorometer using 340 and 460 nm for excitation and emission, respectively. Medium with NADH (lane 1) served as a baseline control. The addition of 100 µM ferricyanide and 50 µM NADH (lanes 3 and 5) to measure NADH-dependent ferricyanide reductase activity was used as a positive control

Reduction of WST-1 in Spermatozoa upon Addition of NADH

WST-1 has been previously used to detect the presence of an NADH oxidase in cancer cells [29, 30] as well as ROS production by professional phagocytes [30]. Formation of the reduced formazan was observed with populations of human spermatozoa and was shown to be cell-concentration (data not shown) and time dependent (Fig. 3A; P < 0.001). Much like lucigenin-dependent chemiluminescence, NADH-dependent WST-1 reduction was inhibitable with pCMBS (Fig. 3B; P < 0.01) and SOD1 (Fig. 3B; P < 0.01) but again DPI had only a slight inhibitory effect (Fig 3B; P < 0.05). The recorded activity was dependent on NADH since without this cofactor, no reduction of WST-1 was seen (Fig. 3A). Although one interpretation of these data is that populations of human spermatozoa are capable of generating ROS, we have previously emphasized that the signals generated by both WST-1 and lucigenin may not necessarily reflect the primary production of [21, 23, 31–34]. This point was further emphasized by the absence of a detectable NADH-dependent chemiluminescent signal with two additional probes (MCLA and luminol), one of which (MCLA) is held to be specific for (data not shown). To resolve the biochemical basis of the redox activity detected by WST-1 and lucigenin with suspensions of human spermatozoa exposed to NADH, fractions exhibiting these activities were isolated and characterized by mass spectrometry.



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  		FIG. 3. NADH-dependent WST-1 reduction. A) WST-1 (500 µM), either alone (open symbols) or together with 125 µM NADH (filled symbols), was added to 0.5 x 106 human spermatozoa, and the rate of absorbance at 415 nm was measured and plotted over time. B) Human sperm preparations (0.5 x 106) were incubated with either the vehicle (lane 1), 20 µM diphenylene iodonium (DPI) (lane 2) or 50 µM pCMBS (lane 3) or 300 U zinc superoxide dismutase (SOD1) (lane 4) as indicated. Following 5 min of incubation, WST-1 and NADH were added according to Materials and Methods. After 1 h, the increase in absorbance at 415 nm was measured; * P < 0.05, ** P < 0.01

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

Suspensions of purified human spermatozoa, isolated from the high-density region of Percoll gradients, were solubilized (2% Triton X-100, 50 mM Tris, pH 8.2) and centrifuged to remove cell debris. The soluble proteins were then applied to a mono Q column that was pre-equilibrated with lysis buffer. Following washing with 10 column volumes, elution of the enzyme was achieved with a 13-ml gradient of 0–200 mM NaCl. One-milliliter fractions were collected and assayed for enzyme activity (Fig. 4A). Fractions either containing or flanking those that demonstrated both NADH-WST-1 (Fig. 4A) or lucigenin (Fig. 5) activity were then precipitated and separated by SDS-PAGE (Fig. 4B). A ~32-kDa band correlated with enzyme activity (Fig. 4B, arrow), suggesting that it might be the enzyme responsible for NADH-dependent WST-1/lucigenin reduction.



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  		FIG. 4. Coelution of cytochrome-b5 reductase with NADH-dependent WST-1 reduction. A) Approximately 500 µg of solubilized protein were loaded onto a mono Q column. The column was washed, then eluted with a linear gradient of 0–200 mM NaCl over 10 ml. One-milliliter fractions were collected and assayed for NADH-WST-1 activity. B) Active fractions eluting from the mono Q column were precipitated, resuspended in loading dye, subjected to 12% SDS-PAGE, and sypro ruby stained as described in Materials and Methods. The positions of the molecular mass markers are shown. The arrow indicates the 32-kDa protein coeluting with enzyme activity



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  		FIG. 5. Coelution of cytochrome-b5 reductase with NADH-dependent lucigenin chemiluminescence. Those fractions eluting from the mono Q column with NADH-dependent WST-1 reduction (Fig. 3) were also assayed for NADH-dependent lucigenin chemiluminescence as described in Materials and Methods

The 32-kDa band was excised from the gel and subjected to a MALDI-TOF analysis. Seven resulting peptides with their predicted amino acid sequences (Fig. 6, underlined) were used to search the BLAST and TREMBL databases for sequence homology to known proteins. This search revealed the 30-kDa band to be identical to human CYB5R2. The presence of a cysteine group and an NADH-docking site (Fig. 6, boldface), similar to that of VDAC1 [27], was further evidence to suggest that CYB5R2 was the enzyme responsible for NADH-dependent WST-1/lucigenin activity.



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  		FIG. 6. MALDI-TOF analysis reveals cytochrome-b5 reductase. The 32-kDa protein coeluting with enzyme activity was excised and subjected to MALDI-TOF analysis. The resulting seven peptides and their predicted amino acid sequence (underlined) matched cytochrome-b5 reductase. The position of the NADH-binding domain for CYB5R2 is shown in bold. Overall, a 35% coverage of the entire protein was found

To assess this enzyme's pharmacological profile, we added SOD1, pCMBS (data not shown), and DPI (Fig. 7) to fraction 15 from the mono Q column. All three inhibitors, including DPI (Fig. 7), were able to completely inhibit enzyme activity.



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  		FIG. 7. Pharmacological profile of NADH cytochrome-b5 reductase fractions. Active (fraction 15) and inactive (fraction 10) fractions eluting from the mono Q column were taken, and NADH-dependent lucigenin chemiluminescence was measured in the presence or absence of 50 µM diphenylene iodonium (DPI)

Redox Activity in Cytochrome-b5 Reductase-Transfected Cells

Coelution of CYB5R2 with enzyme activity from the mono Q affinity column suggested that this enzyme is likely responsible for the lucigenin chemiluminescence and tetrazolium salt reduction observed in the presence of NADH. To confirm this hypothesis, recombinant CYB5R2 was prepared and assessed for enzyme activity following transfection into COS cells.

The cDNA from human spermatozoa was prepared, and primers were selected specifically for CYB5R2 mRNA. The putative reductase band was excised, cloned into the pcDNA-DEST47 vector, sequenced, and shown to be identical to human CYB5R2 (GenBank accession no. AY665398). The final construct was CYB5R2 fused in frame to GFP at the C-terminus. This construct was named b5RpcDNA-DEST47. To confirm the production and correct folding of the fusion protein, COS-7 cells (2–3 x 106) were transfected with either 5 µg of b5RpcDNA-DEST47 or the vector control pcDNA-DEST47. Approximately 48 h posttransfection, the cells were harvested, washed, and subjected to confocal microscopy. Cells transfected with both GFP only and CYB5R2 demonstrated typical, predicted widespread cytosolic expression (Fig. 8).



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  		FIG. 8. Establishing the expression of transiently transfected COS 7 cells with cytochrome-b5 reductase. A–C) COS-7 cells were transfected with pcDNA-DEST47 (Invitrogen) expression construct alone and viewed by (A) phase contrast and (B) fluorescence microscopy. Green fluorescent protein (GFP) expression can be seen throughout the cell cytosol. A high-power view is also shown (C). D–F) COS-7 cells were transfected with b5RpcDNA-DEST47 containing full-length human CYB5R2 and viewed by (D) phase contrast and (E) fluorescence microscopy. F) A high-power view is also shown for fluorescence. Original magnification A, B, D, E x100; C, F x200

To confirm the production of the fusion protein, b5RpcDNA-DEST47-transfected cells were lysed and subjected to anti-GFP Western blot analysis as described in Materials and Methods. As shown (Fig. 9A), a ~60-kDa fusion protein can be seen in the transfected cells, which is equivalent to the predicted size of the GFP (27 kDa) plus CYB5R2 (32 kDa) fusion protein.



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  		FIG. 9. Transient transfection of human cytochrome-b5 reductase into COS-7 cells increases NADH-dependent lucigenin reduction. A) Twenty-four hours posttransfection, the cells were harvested, lysed, and subject to Western blot analysis using anti-GFP (green fluorescent protein) antibodies. The Western blot shows the full-length fusion product. B) Cells expressing GFP only (Control) or the fusion protein (Trans) were assayed for NADH-dependent lucigenin chemiluminescence (125 µM NADH). A duplicate analysis is shown. The results are representative of an experiment that was performed three times in duplicate

To determine if the b5RpcDNA-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 NADH. Those cells expressing b5RpcDNA-DEST47 demonstrated a 3-fold stimulation in lucigenin chemiluminescence (Fig. 9B). Moreover, addition of pCMBS and SOD1 very effectively inhibited the lucigenin-induced chemiluminescence (P < 0.01), just as observed with the signal generated by intact human spermatozoa (Fig. 1B and Fig. 10, lane 3 vs. lanes 8 and 9). Addition of the flavoprotein inhibitor DPI also reduced NADH-induced lucigenin chemiluminescence to a limited extent (P < 0.05), again reflecting the results obtained with intact spermatozoa (Fig. 1B and Fig. 10, lane 3 vs. lanes 4–7). In contrast to the lucigenin results, b5RpcDNA-DEST47-transfected cells did not show an increase in NADH-dependent WST-1 reduction over the mock-transfected controls (control 0.85 ± 0.5; transfected 0.87 ± 0.3 absorption units at 415 nm). Thus, while CYB5R2 can reduce WST-1 in the presence of NADH in vitro, this enzyme is not completely responsible for the WST-1 signal generated by intact spermatozoa.



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  		FIG. 10. Transient transfection of the human cytochrome-b5 reductase into COS7 cells. Cells expressing green fluorescent protein (GFP) only (open bars) or the fusion protein (filled bars) were assayed for NADH-dependent lucigenin chemiluminescence in the presence of the vehicle (Con) and the inhibitors diphenylene iodonium (DPI), pCMBS, and zinc superoxide dismutase (SOD1), as described in Materials and Methods. The difference between the GFP- and GFP- CYB5R2 fusion proteins was statistically significant, as were the differences between the controls and the presence of inhibitors; * P < 0.05, ** P < 0.01


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The presence of plasma membrane NADH-oxidases capable of generating ROS has been described in a wide variety of cell types, including fibroblasts [35], glomerular mesangial cells [36], B-lymphocytes [37–39], leukocytes [40], endothelial cells [41], and plant cells [20]. In many of these reports, ROS generation was detected using chemiluminescent probes such as lucigenin [42] and luminol [32] or tetrazolium salts such as WST-1 [30] and NBT [43]. To date, the plasma membrane tNOX [44, 45] is the only nonmitochondrial NADH-dependent oxidase that has been identified and cloned at a molecular level, which may form part of a plasma membrane redox complex [5, 39, 46].

The production of ROS in germ cells has been documented to play a role in physiological processes, including sperm capacitation and acrosomal exocytosis, as well as the pathophysiology of male infertility [25, 47, 48]. Nevertheless, 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 NADH oxidoreductase [49]. This activity could be demonstrated by the addition of lucigenin together with NADH to cells and measuring an increase in chemiluminescence [49]. The results presented here support the observation that mammalian sperm preparations are capable of generating an NADH-dependent lucigenin signal [33]. Furthermore, another putative ROS detecting probe, WST-1, was also reduced by these same preparations. However, the inability of spermatozoa to invoke an NADH-dependent luminol or MCLA signal or to oxidize NADH directly does not support the concept that either or other species of ROS are being produced by these cells in response to this nucleotide [34]. These seemingly paradoxical results come together with the identity of CYB5R2 as an enzyme capable of NADH-dependent reduction of both lucigenin and WST-1 but not luminol or MCLA.

The generation of lucigenin-dependent chemiluminescence in the absence of ROS generation can be explained by a biochemical process initiated by the one-electron reduction of the probe (L2+) to generate a lucigenin radical (LH•+). The latter is unstable and will revert back to the parent compound with the release of an electron to oxygen, thereby generating . LH•+ then combines with to generate the oxygenated dioxetane that subsequently decomposes with the emission of light [23]. The identification of CYB5R2 as an enzyme responsible for NADH-induced, lucigenin-dependent chemiluminescence in human spermatozoa is perfectly in keeping with this proposed reaction mechanism since it would account for the initial one-electron reduction of the probe. CYB5R2 was identified as the 32-kDa enzyme coeluting with NADH-lucigenin/WST-1 activity from a mono Q ion exchange column. Furthermore, COS7 cells overexpressing CYB5R2 demonstrated a 3-fold increase in NAPH-dependent lucigenin activity compared to the mock-transfected controls. The incomplete suppression observed with DPI is difficult to understand because this compound is a recognized flavoprotein inhibitor, and CYB5R2 is a flavoprotein. It is possible that in intact COS 7 cells and spermatozoa (Fig. 10), there are additional pathways mediating NADH-dependent lucigenin chemiluminescence. Alternatively, DPI penetration to the sites of lucigenin action may be limited in intact cells. This would explain why DPI was so much more effective in suppressing the lucigenin chemiluminescence observed in active mono Q fractions (Fig. 7) compared with intact cells (Fig. 1).

Although the addition of SOD1 inhibited redox activity, this should not be interpreted as evidence for the primary production of ROS, as pointed out more than a decade ago [31]. In the case of lucigenin, is an essential component of the chemistry leading to chemiluminescence since it is responsible for the formation of the unstable dioxetane that decomposes to acridone with the generation of light. It is for this reason that SOD1 is such an effective inhibitor of the chemiluminescence recorded in the presence of human spermatozoa. However, the generated in the presence of NADH is a consequence rather than a cause of lucigenin activation. The initiating event appears to be the CYB5R2-mediated reduction of the probe rather than the presence of generated by an NADH oxidase. Similarly, the fact that SOD1 inhibits the signal generated in the presence of WST-1 and NADH cannot be taken as evidence for the primary production of by human spermatozoa. In this case, the one-electron reduction of WST-1 by the combination of NADH and CYB5R2 leads to the formation of a WST-1 radical (WST-1H•) that by dismutation generates the reduced soluble formazan product (WST-1H2) detected in the spectrophotometric assay:

However, the WST-1H• radical can also combine with oxygen to generate :

In the presence of SOD1, the previously mentioned reaction is driven to the right, reducing the availability of WST-1H• and hence suppressing the production of formazan (WST-1H2). Thus, as with lucigenin, the inhibitory action of SOD1 is not evidence for the primary production of but rather an artifact created as a consequence of the redox cycling of the probe.

A similar logic applies to the formazan generated as a consequence of the reaction between NBT and glucose oxidase. In this case also, SOD1 inhibits formazan formation, leading to the false conclusion that glucose oxidase produces [50]. It appears that both lucigenin and tetrazolium salts, including WST-1, fit into the active site of CYB5R2 and are directly reduced by this enzyme. In the case of one of the other ROS probes, MCLA, there does not appear to be a conformational fit between the probe and the reductase, and as a consequence no chemiluminescant signal is generated by human spermatozoa in the presence of NADH and this compound. No signal was observed in the presence of luminol because this probe requires a one-electron oxidation rather than a reduction to become sensitized to the presence of ROS.

Although CYB5R2 reduced WST-1 in the isolated FPLC fractions, there was no change in WST-1 reduction in the presence of cells transfected with this enzyme. Since CYB5R2 reduced lucigenin in intact spermatozoa, isolated mono Q fractions, and transfected cells, the lack of WST-1 reduction in the latter may reflect differences in the bioavailability of these probes following transfection. There are large physicochemical differences between lucigenin and WST-1. The former can be dissolved only in organic-based solvents, whereas the latter is readily dissolved in water. This difference in hydrophobicity would be expected to reflect the degree of cellular penetrance associated with these probes, the hydrophilic nature of WST-1 being reflected in a relative lack of membrane permeability. This lack of cell penetration may explain why transfected cells did not increase their WST-1 response, even though the isolated CYB5R2 can clearly reduce this compound. This explanation cannot apply to human spermatozoa because in this case the combination of WST-1 and NADH did elicit a powerful redox response from these cells. These data suggest that in the case of intact human spermatozoa, WST-1 must be reduced by an alternative pathway, such as the surface NADH oxidase reported by Berridge and Tan [29]. The NADH-elicited oxidoreductase activities recorded in human sperm suspensions in the presence of lucigenin and WST-1 are certainly pharmacologically distinct, the WST-1 signal being significantly more susceptible to inhibition by capsaicin, pCMBS, and DPI [31]. Furthermore, the signals generated by these two probes have quite different biological implications, for while the NADH/lucigenin signal is significantly enhanced in defective human sperm populations, this is not true of the NADH/WST-1 response [31].

Interestingly, the addition of ferricyanide to intact spermatozoa did not induce further NADH-oxidation above background. This cell-impermeable probe is very sensitive for the measurement of CYB5R2. Permeabilization of the cells through freeze thawing enabled access of the probe to CYB5R2 and led to a consequent enhanced of NADH-oxidation.

In conclusion, it appears that CYB5R2 is capable of reducing both lucigenin and WST-1 in an NADH-dependent manner. The reduction of these probes is not due to production of ROS since both MCLA and luminol do not respond in this same system. In intact human spermatozoa, CYB5R2 appears to be particularly associated with the lucigen response, which is, in turn, negatively associated with defective sperm function [31]. It is possible that this association exists because the CYB5R2 detected by the NADH/ lucigenin system is a cytosolic enzyme. The magnitude of the response elicited by this redox system should therefore be correlated with the degree of cytoplasmic retention associated with a given sperm population. Given the negative correlation that exists between the retention of excess residual cytoplasm and sperm function [51], NADH-induced lucigenin-dependent chemiluminescence may be a very simple, effective means of monitoring the degree of cytoplasmic retention in human sperm samples and hence the quality of the underlying spermatogenic process.


   ACKNOWLEDGMENTS
 
The authors would like to thank Ceanne Wallace for help with preparation of human spermatozoa and FPLC.


   FOOTNOTES
 
1 Supported by the ARC Centre of Excellence in Biotechnology and Development, Ernst-Schering Trust through the AMPPA network, and the Australian Research Council. Back

2 Correspondence: R. John Aitken, Discipline of Biological Sciences, University of Newcastle, Callaghan, New South Wales 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: 10 November 2004.

First decision: 13 December 2004.

Accepted: 15 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Babior BM, Kipnes RS, Curnutte JT. Biological defense mechanisms: the production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 1973 52:741-744
  2. Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD. Cell transformation by the superoxide-generating oxidase Mox1. Nature 1999 401:79-82[CrossRef][Medline]
  3. Domenicotti C, Marengo B, Verzola D, Garibotto G, Traverso N, Patriarca S, Maloberti G, Cottalasso D, Poli G, Passalacqua M, Melloni E, Pronzato MA, Marinari UM. Role of PKC-delta activity in glutathione-depleted neuroblastoma cells. Free Radic Biol Med 2003 35:504-516[CrossRef][Medline]
  4. Aitken RJ, Baker MA, Sawyer D. Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease. Reprod Biomed Online 2003 7:65-70[Medline]
  5. Baker MA, Krutskikh A, Aitken RJ. Biochemical entities involved in reactive oxygen species generation by human spermatozoa. Protoplasma 2003 221:145-151[CrossRef][Medline]
  6. Baker MA, Lewis B, Hetherington L, Aitken RJ. Development of the signalling pathways associated with sperm capacitation during epididymal maturation. Mol Reprod Dev 2003 64:446-457[CrossRef][Medline]
  7. Aitken RJ, Baker MA. Reactive oxygen species generation by human spermatozoa: a continuing enigma. Int J Androl 2002 25:191-194[Medline]
  8. Li Y, Stansbury KH, Zhu H, Trush MA. Biochemical characterization of lucigenin (Bis-N-methylacridinium) as a chemiluminescent probe for detecting intramitochondrial superoxide anion radical production. Biochem Biophys Res Commun 1999 262:80-87[CrossRef][Medline]
  9. Li Y, Zhu H, Kuppusamy P, Roubaud V, Zweier JL, Trush MA. Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J Biol Chem 1998 273:2015-2023[Abstract/Free Full Text]
  10. Trush VD, Efremova TM. Orienting reflex and spectral characteristics of cortical biopotentials in rabbits. Neurosci Behav Physiol 1972 5:347-354[CrossRef][Medline]
  11. Simonet S, Rupin A, Badier-Commander C, Coumailleau S, Behr-Roussel D, Verbeuren TJ. Evidence for superoxide anion generation in aortas of cholesterol-fed rabbits treated with L-arginine. Eur J Pharmacol 2004 492:211-216[CrossRef][Medline]
  12. Didion SP, Faraci FM. Effects of NADH and NADPH on superoxide levels and cerebral vascular tone. Am J Physiol Heart Circ Physiol 2002 282:H688-H695[Abstract/Free Full Text]
  13. Jones SA, O'Donnell VB, Wood JD, Broughton JP, Hughes EJ, Jones OT. Expression of phagocyte NADPH oxidase components in human endothelial cells. Am J Physiol 1996 271:H1626-H1634
  14. Mohazzab KM, Wolin MS. Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle. Am J Physiol 1994 267:L815-L822
  15. Seno T, Inoue N, Gao D, Okuda M, Sumi Y, Matsui K, Yamada S, Hirata KI, Kawashima S, Tawa R, Imajoh-Ohmi S, Sakurai H, Yokoyama M. Involvement of NADH/NADPH oxidase in human platelet ROS production. Thromb Res 2001 103:399-409[CrossRef][Medline]
  16. Souza HP, Liu X, Samouilov A, Kuppusamy P, Laurindo FR, Zweier JL. Quantitation of superoxide generation and substrate utilization by vascular NAD(P)H oxidase. Am J Physiol Heart Circ Physiol 2002 282:H466-H474[Abstract/Free Full Text]
  17. Aitken RJ, Fisher HM, Fulton N, Gomez E, Knox W, Lewis B, Irvine S. Reactive oxygen species generation by human spermatozoa is induced by exogenous NADPH and inhibited by the flavoprotein inhibitors diphenylene iodonium and quinacrine. Mol Reprod Dev 1997 47:468-482[CrossRef][Medline]
  18. Aitken RJ, Ryan AL, Baker MA, McLaughlin EA. Redox activity associated with the maturation and capacitation of mammalian spermatozoa. Free Radic Biol Med 2004 36:994-1010[CrossRef][Medline]
  19. Wojtaszek P. Oxidative burst: an early plant response to pathogen infection. Biochem J 1997 322:pt 3681-692
  20. Auh CK, Murphy TM. Plasma membrane redox enzyme is involved in the synthesis of O2– and H2O2 by phytophthora elicitor-stimulated rose cells. Plant Physiol 1995 107:1241-1247[Abstract]
  21. de Lamirande E, Harakat A, Gagnon C. Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion. J Androl 1998 19:215-225[Abstract/Free Full Text]
  22. Richer SC, Ford WC. A critical investigation of NADPH oxidase activity in human spermatozoa. Mol Hum Reprod 2001 7:237-244[Abstract/Free Full Text]
  23. Baker MA, Krutskikh A, Curry BJ, McLaughlin EA, Aitken RJ. Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations. Biol Reprod 2004 71:307-318[Abstract/Free Full Text]
  24. Lessley BA, Garner DL. Isolation of motile spermatozoa by density gradient centrifugation in Percoll. Gamete Research 1983 7:49-61[CrossRef]
  25. Baker MA, Hetherington L, Ekroyd H, Roman SD, Aitken RJ. Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation. J Cell Sci 2004 117:211-222[Abstract/Free Full Text]
  26. Zhu H, Qiu H, Yoon HW, Huang S, Bunn HF. Identification of a cytochrome-btype NAD(P)H oxidoreductase ubiquitously expressed in human cells. Proc Natl Acad Sci USA 1999 96:14742-14747[Abstract/Free Full Text]
  27. Baker MA, Lane DJ, Ly JD, De Pinto V, Lawen A. VDAC1 is a transplasma membrane NADH:ferricyanide reductase. J Biol Chem 2004 279:4811-4819[Abstract/Free Full Text]
  28. Baker MA, Ly JD, Lawen A. Characterization of VDAC1 as a plasma membrane NADH-oxidoreductase. Biofactors 2004 21:215-221[Medline]
  29. Berridge MV, Tan AS. High-capacity redox control at the plasma membrane of mammalian cells: trans-membrane, cell surface, and serum NADH-oxidases. Antioxid Redox Signal 2000 2:231-242[Medline]
  30. Tan AS, Berridge MV. Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J Immunol Methods 2000 238:59-68[CrossRef][Medline]
  31. Faulkner K, Fridovich I. Luminol and lucigenin as detectors for O2. Free Radic Biol Med 1993 15:447-451[CrossRef][Medline]
  32. Aitken RJ, Buckingham DW, West KM. Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in luminol- and lucigenin-dependent chemiluminescence. J Cell Physiol 1992 151:466-477[CrossRef][Medline]
  33. Aitken RJ, Baker MA, O'Bryan M. Shedding light on chemiluminescence: the application of chemiluminescence in diagnostic andrology. J Androl 2004 25:455-465[Free Full Text]
  34. Aitken RJ, Ryan AL, Curry BJ, Baker MA. Multiple forms of redox activity in populations of human spermatozoa. Mol Hum Reprod 2003 9:645-661[Abstract/Free Full Text]
  35. Meier B, Radeke HH, Selle S, Younes M, Sies H, Resch K, Habermehl GG. Human fibroblasts release reactive oxygen species in response to interleukin-1 or tumour necrosis factor-alpha. Biochem J 1989 263:539-545[Medline]
  36. Radeke HH, Meier B, Topley N, Floge J, Habermehl GG, Resch K. Interleukin 1-alpha and tumor necrosis factor-alpha induce oxygen radical production in mesangial cells. Kidney Int 1990 37:767-775[Medline]
  37. Vaillant F, Larm JA, McMullen GL, Wolvetang EJ, Lawen A. Effectors of the mammalian plasma membrane NADH-oxidoreductase system: short-chain ubiquinone analogues as potent stimulators. J Bioenerg Biomembr 1996 28:531-540[CrossRef][Medline]
  38. Wolvetang EJ, Larm JA, Moutsoulas P, Lawen A. Apoptosis induced by inhibitors of the plasma membrane NADH-oxidase involves Bcl-2 and calcineurin. Cell Growth Differ 1996 7:1315-1325[Abstract]
  39. Baker MA, Lawen A. Plasma membrane NADH-oxidoreductase system: a critical review of the structural and functional data. Antioxid Redox Signal 2000 2:197-212[Medline]
  40. Henderson LM, Chappel JB. NADPH oxidase of neutrophils. Biochim Biophys Acta 1996 1273:87-107[Medline]
  41. Matsubara T, Ziff M. Increased superoxide anion release from human endothelial cells in response to cytokines. J Immunol 1986 137:3295-3298[Abstract]
  42. Aitken RJ, Harkiss D, Knox W, Paterson M, Irvine DS. A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. J Cell Sci 1998 111:pt 5645-656[Abstract]
  43. Ginsburg I, Borinski R, Sadovnic M, Eilam Y, Rainsford K. Poly L-histidine: a potent stimulator of superoxide generation in human blood leukocytes. Inflammation 1987 11:253-277[CrossRef][Medline]
  44. Wilkinson F, Kim C, Cho N, Chueh PJ, Leslie S, Moya-Camarena S, Wu LY, Morre DM, Morre DJ. Isolation and identification of a protein with capsaicin-inhibited NADH oxidase activity from culture media conditioned by growth of HeLa cells. Arch Biochem Biophys 1996 336:275-282[CrossRef][Medline]
  45. Chueh PJ, Morre DJ, Wilkinson FE, Gibson J, Morre DM. A 33.5-kDa heat- and protease-resistant NADH oxidase inhibited by capsaicin from sera of cancer patients. Arch Biochem Biophys 1997 342:38-47[CrossRef][Medline]
  46. Lawen A, Baker MA, Malik S. The function of the plasma membrane oxidoreductase: a critical review of the structural and functional data as described to date. Antioxid Redox Signal 2000 2:197-212
  47. Griveau JF, Renard P, Le Lannou D. An in vitro promoting role for hydrogen peroxide in human sperm capacitation. Int J Androl 1994 17:300-307[Medline]
  48. Griveau JF, Renard P, Le Lannou D. Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process. Int J Androl 1995 18:67-74
  49. Vernet P, Fulton N, Wallace C, Aitken RJ. Analysis of reactive oxygen species generating systems in rat epididymal spermatozoa. Biol Reprod 2001 65:1102-1113[Abstract/Free Full Text]
  50. Fridovich I. Editorial commentary on "superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide" by H. Zhao et al. Free Radic Biol Med 2003 34:1357-1358[CrossRef][Medline]
  51. Gomez E, Buckingham DW, Brindle J, Lanzafame F, Irvine DS, Aitken RJ. Development of an image analysis system to monitor the retention of residual cytoplasm by human spermatozoa: correlation with biochemical markers of the cytoplasmic space, oxidative stress, and sperm function. J Androl 1996 17:276-287[Abstract/Free Full Text]



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