Molecular Human Reproduction, Vol. 7, No. 10, 913-921,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Tyrosine nitration in human spermatozoa: a physiological function of peroxynitrite, the reaction product of nitric oxide and superoxide
1 Centro de Estudios Farmacológicos y Botánicos (CEFYBO-CONICET), Serrano 669, 1414, Buenos Aires, Argentina and 2 Urology Research Laboratory, Royal Victoria Hospital, McGill University, 687 Pine Avenue West, H3A 1A1 Montréal, Canada
Abstract
Tyrosine nitration is a widely used marker of peroxynitrite (ONOO–) produced from the reaction of nitric oxide (NO.) with superoxide (O2.–). Since human spermatozoa are able to produce both NO. and O2.– during capacitation in vitro, we investigated whether spontaneous tyrosine nitration of proteins occurs in human spermatozoa and evaluated the physiological effects of peroxynitrite on sperm function. We report here that human spermatozoa, incubated for 8 h under conditions conducive to capacitation, display a reproducible pattern of protein tyrosine nitration. Several proteins with mol. wt of 105–14 kDa become increasingly tyrosine-nitrated after 15 min incubation and then minimal changes are observed. Treatment of capacitated spermatozoa with human follicular fluid or calcium ionophore causes an increase of the nitrotyrosine content of proteins at the mol. wt of 85 kDa. Moreover, exposure of spermatozoa to ONOO– (2.5–50 µmol/l) increases motility and primes spermatozoa to respond earlier to human follicular fluid. ONOO– also increases protein tyrosine nitration and phosphorylation in a concentration-dependent manner. Taken together, these results demonstrate that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of ONOO– modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process.
capacitation/human spermatozoa/nitric oxide/peroxynitrite/protein nitration
Introduction
Sperm capacitation can be defined as a maturational process that occurs in vivo in the female genital tract (Chang, 1951
; Austin, 1952), and it can also be accomplished in defined media in vitro. The main purpose of capacitation is to ensure that spermatozoa will reach the oocyte at the appropriate time and in the appropriate state to fertilize the oocyte.
The molecular basis of sperm capacitation is still poorly understood, although a calcium uptake, an increase in cAMP concentration, a rise in intracellular pH, an efflux of cholesterol from the sperm plasma membrane (Yanagimachi, 1994; Visconti et al., 1998), and tyrosine phosphorylation of specific proteins have been demonstrated to occur during this process (Aitken et al., 1995; Visconti et al., 1995; Leclerc et al., 1996). It has also been reported that reactive oxygen species (ROS), such as superoxide anion (O2.–), hydrogen peroxide (H2O2) and nitric oxide (NO.) can induce sperm capacitation in vitro (Griveau et al., 1994; de Lamirande and Gagnon, 1995; Herrero et al., 1999). Indeed, spermatozoa generate small amounts of O2.– and NO. which are both tightly related to the cAMP pathway in the control of human sperm capacitation and protein tyrosine phosphorylation (Aitken et al., 1998; Herrero et al., 2000).
Under physiological conditions, both NO. and O2.– exist at very low concentrations. However, at loci near cells that produce both O2.– and NO., the local concentrations of the two should be significant and the formation of ONOO– appears likely (Pryor and Squadrito, 1995). A landmark paper (Beckman et al., 1990) suggested that these two radicals could combine to form ONOO– (Equation 1), holding enormous implications for the understanding of free radicals in biological systems.
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This reaction occurs at 6.7x109/mol/l/s and it is essentially irreversible due to its highly exothermic nature (Koppenol, 1998). Peroxynitrite is not a free radical because the unpaired electrons on NO. and O2.– have combined to form a new N–O bond in peroxynitrite, but it is a strong one or two electron oxidant and nitrating agent. Even at physiological pH, the relative stability of ONOO– allows it to diffuse for a considerable distance on a cellular scale, and even to cross cell membranes (Beckman et al., 1994). Peroxynitrite can react rapidly with proteins (Moreno and Pryor, 1992; Ischiropoulos and Al-Mehdi, 1995; Pietraforte and Minetti, 1997; Sies and Arteel, 2000), lipids (Rubbo et al., 1994) and DNA (Epe et al., 1996). Moreover, sulphydryl groups on cell structures are also targets of ONOO–, and either affect the function of signalling systems or result in the production of tissue-derived NO.–-releasing compounds (Radi et al., 1991; Wu et al., 1994; White et al., 1999; Crow, 2000).
Like NO., ONOO– has been associated with both deleterious and beneficial effects (Moro et al., 1994; Vinten-Johansen, 2000). A review of the literature suggests that the effects of ONOO– do show a dependency on the environment in which the anion is present (Ronson et al., 1999; Ma et al., 2000). Moreover, the physiological effects of ONOO– are also dependent on its concentration. For instance, Mallozzi et al. demonstrated that ONOO– at low concentrations (10–100 µmol/l) stimulates a metabolic response in human erythrocytes, leading to a rise of tyrosine phosphorylation proteins and an enhancement of lactate production, whereas ONOO– at high concentrations (200–1000 µmol/l) inhibits tyrosine phosphorylation and glycolysis (Mallozzi et al., 1997). This concentration-dependent effect is in agreement with two other independent studies, in which cardioprotection of isolated rat hearts occurs in the presence of low concentrations of ONOO– (2–4 µmol/l) but not with 40 µmol/l ONOO– (Schulz et al., 1997; Nossuli et al., 1998).
Measuring ONOO– is problematic in that it cannot be measured directly; thus the surrogate measures of nitrotyrosine footprints are frequently used as an assay for ONOO– (Beckman et al., 1994). The nitration (addition of a NO2 group) of protein tyrosine residues gives rise to 3-nitrotyrosine which represents a protein modification specific for ONOO– formation in vivo (Beckman, 1996). Although considerably increased levels of nitrotyrosines have been found in tissues of several pathological conditions (Ischiropoulos, 1998; Reiter et al., 2000), the possible role of tyrosine nitration in cellular function has not been fully explored.
Therefore, in this study, we set out to determine whether protein tyrosine nitration takes place in human spermatozoa during capacitation and to evaluate whether ONOO– has beneficial effects on sperm functions.
Materials and methods
Reagents
The following reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA): water tissue culture grade, bovine serum albumin (BSA; fraction V), 1,4-diazabicyclo-[2.2.2.]octane (DABCO), Pisum sativum agglutinin conjugated to fluorescein isothiocyanate (PSA–FITC), and 3-morpholinosydnonimine (SIN-1). Percoll was obtained from Amersham, Pharmacia Biotech (Baie d'Urfé, Québec, Canada). NG-Nitro-L-arginine methyl ester (L-NAME) was bought from Research Biochemicals International (Natick, MA, USA). Ethanamine, N-ethyl-1,1-diethyl-2-hydroxy-2-nitrosohydrazine (diethylamine-NONOate, DEA) and nitrotyrosine were bought from Cayman Chemical Co. (Ann Arbor, MI, USA). Chemically synthesized ONOO– was obtained from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Tris-(2-carboxyethyl) phosphine (TCEP) was from Molecular Probes (Eugene, OR, USA).
Monoclonal anti-phosphotyrosine antibody (1 mg/ml) (clone 4G10) and polyclonal anti-nitrotyrosine antibody (1 µg/ml), standards (Upstate Biotechnology Inc.), nitrocellulose membranes (0.22 µm pore size) (Micron Separations Inc., Westboro, MA, USA), goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (Life Technologies, Burlington, ON, Canada), goat anti-rabbit IgG (H+L) conjugated to horseradish peroxidase (Zymed, San Francisco, CA, USA), an enhanced chemiluminescence kit (Lumi Light) (Roche Diagnostics, Laval, Que, Canada), and X-ray films (Fuji, Minami-Ashigara, Japan) were used for immunodetection of phospho- and nitrotyrosine-containing proteins. Ready strips, IPG strips and Bio-Lytes 3/10 for isoelectrofocusing were from Biorad. All other chemicals were at least of reagent grade.
The human follicular fluid (HFF) was collected from preovulatory follicles after gonadotrophin stimulation, centrifuged for 10 min at 20 000 g and kept frozen in aliquots at –20°C until used.
Sperm preparation
Semen samples were obtained by masturbation from healthy volunteers after 3 days of sexual abstinence. All ejaculates used in the experiments had 
65% motile cells, 60% normal morphology and cells counts of 15x106/ml. The specimens were allowed to liquefy for 30–60 min at room temperature and then motile spermatozoa were selected by centrifugation (800 g for 20 min) through a four-step (20–40–60–95%) Percoll gradient (de Lamirande and Gagnon, 1991). Spermatozoa from the 95% Percoll layer and the 95–60% Percoll interface were pooled, resuspended in 5 ml modified Tyrode's medium consisting of 117.5 mmol/l NaCl, 0.3 mmol/l NaH2PO4, 8.6 mmol/l KCl, 25 mmol/l NaHCO3, 2.5 mmol/l CaCl2, 0.5 mmol/l MgCl2, 2 mmol/l glucose, 0.25 mmol/l Na pyruvate, 19 mmol/l Na lactate, 70 µg/ml of both streptomycin and penicillin, and centrifuged at 300 g for 10 min. The supernatant was discarded and the remaining soft sperm pellet was resuspended in Tyrode's medium supplemented with 2% BSA.
Measurement of capacitation
Sperm aliquots (200 µl), at 20x106 cells/ml, were incubated in Tyrode's medium supplemented with 2% BSA in the presence or absence of different agents at 37°C in 5% CO2 in air for 4 or 8 h. Spermatozoa were then washed twice in Tyrode's medium with BSA, and challenged with 20% HFF for another 30 min. Sperm capacitation was measured as the ability of spermatozoa to respond to the HFF by undergoing acrosome reaction. Spermatozoa were then incubated in hypo-osmotic swelling medium (HOS) for 1 h at 37°C (Aitken et al., 1993) in order to determine viability, centrifuged, resuspended in 30 µl ice-cold ethanol and stored at 4°C. The acrosome reaction was evaluated by using PSA–fluorescein isothiocyanate (FITC) as previously described (Cross et al., 1986) on at least 100 spermatozoa. Only spermatozoa with curly tails were considered viable and thus scored.
Measurement of sperm motility parameters
The motility parameters of spermatozoa were analysed at 37°C by videomicroscopy using the CellSoftTM computer-assisted digital image analysis system (Cryo Resources, Montgomery, NY, USA) after 8 h treatment. For each treatment, curvilinear velocity, linearity, and amplitude of the lateral head displacement (ALH) from 200 spermatozoa were analysed.
Electrophoresis and immunoblot analyses
For one-dimensional immunoblots, sperm aliquots (200 µl), at 20x106 cells/ml, were incubated in Tyrode's medium supplemented with 2% BSA in the presence or absence of different agents at 37°C in 5% CO2 in air. In some sets of experiments, spermatozoa were incubated for 8 h and then stimulated with A23187 (5 µmol/l) or with 20% HFF for 30 min. Following the incubations, spermatozoa were washed twice in Tyrode's medium without BSA (500 g for 5 min), and resuspended in Laemmli's sample buffer supplemented with sodium vanadate 0.5 mmol/l and heated at 100°C for 5 min. Proteins (from 0.5x106 spermatozoa/lane) were then separated by 12% sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) (Laemmli, 1970) and electrotransferred onto nitrocellulose membranes (Towbin et al., 1979). Non-specific binding sites on the membrane were blocked with 5% (w/v) skimmed milk in Tris-buffered saline (0.9% NaCl, 20 mmol/l Tris–HCl, pH 7.8) supplemented with Tween 20 (0.1%) (TTBS). The nitrocellulose membrane was incubated for 1 h at room temperature with the anti-phosphotyrosine antibody (1/10 000) or for 2 h at room temperature with the anti-nitrotyrosine antibody (1/300). The membrane was extensively washed in TTBS, and goat anti-mouse IgG (1/5000) or goat anti-rabbit (1/5000), both conjugated with horseradish peroxidase, was added. After a 45 min incubation period at room temperature, the membrane was extensively washed and positive immunoreactive bands were detected by chemiluminescence using Lumi Light according to the manufacturer's instructions.
For two-dimensional (2-D) electrophoresis, isoelectrofocusing was performed prior to SDS–PAGE. Spermatozoa (8x106 cells/200 µl) submitted to various treatments were washed twice in Tyrode's medium without BSA (500 g for 5 min) and once in sucrose 0.3 mol/l. The pellet (100 µl) was resuspended in 100 µl sucrose 0.3 mol/l and sperm proteins were extracted in 400 µl rehydration sample buffer (8 mol/l urea, 4% CHAPS, 2 mmol/l TCEP, 0.2% Bio-Lytes 3/10) for 15 min. Samples were then centrifuged at 19 000 g for 5 min and a 150 µl portion of the supernatant, used to rehydrate Ready Strip immobilized pH gradient strips, was separated with a pH 3.0–10.0 ampholine range. Proteins were then separated by isoelectrofocusing (5 h at 400 v/h) and then SDS–PAGE (12% polyacrylamide) as previously described. The continuation of the immunoblotting procedure was the same as that described above.
Statistical analysis
The data were analysed by Fisher's protected least-significant difference test following a one-way analysis of variance on paired observations.
Results
Tyrosine nitration of sperm proteins
It has been speculated that ONOO– formation is a primary pathway of NO. metabolism (Pryor and Squadrito, 1995). Because we have previously shown that human spermatozoa produce NO. as well as O2.– during capacitation (de Lamirande and Gagnon, 1995; Herrero et al., 2000), it was reasonable to predict that formation of ONOO– can occur in vivo. Since tyrosine nitration is a widely used marker of ONOO–, we evaluated the presence of 3-nitrotyrosine on sperm proteins.
When spermatozoa were incubated for various time intervals under conditions conducive to capacitation, there was an increase in the nitration of tyrosine residues, with detectable levels in the first 15 min of incubation and a maximum reached at 2 h. Tyrosine nitration persisted for at least 8 h with minimal changes in the pattern or intensity of the bands. The most prominently nitrated proteins had mol. wts between 105 and 14 kDa (Figure 1). The labelling was specific since it was abolished after incubation of the antibody with nitrotyrosine (5 mmol/l) (Figure 1). Moreover, incubation of the anti-nitrotyrosine antibody with 5 mmol/l phosphotyrosine did not modify the nitrotyrosine pattern of sperm proteins (data not shown).
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Since increased production of NO. (Herrero et al., 1998
; Revelli et al., 1999) and of O2.– (Griveau et al., 1995; de Lamirande et al., 1998) have been demonstrated during the acrosome reaction and since these two ROS react rapidly to form ONOO–, the possibility that nitration of proteins could be increased during the acrosome reaction was then examined. This possibility was confirmed since treatment of capacitated spermatozoa with HFF or the calcium ionophore A-23187 caused an increase of nitrotyrosine content of the proteins at a mol. wt of 85 kDa, indicating that nitration of some proteins occurs within spermatozoa at the time of the acrosome reaction (Figure 2). The percentage of acrosome reaction induced by A23187 was 22 ± 3, whereas for HFF, it was 13 ± 1.8.
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Peroxynitrite induces sperm capacitation and modulates sperm motility
From the results presented above, it appears that formation of ONOO– can occur in spermatozoa. Thus, it was necessary to investigate whether ONOO– could also modulate human sperm functions, such as capacitation and motility.
Spermatozoa incubated in Tyrode's medium supplemented with BSA need at least a 6 h period of capacitation so that they can induce the acrosome reaction upon addition of HFF (Calvo et al., 1993; Herrero et al., 1999). However, the presence of ONOO– or SIN-1 (a product that generates simultaneously NO. and O2.–) (Beckman et al., 1994) accelerated the capacitation process since spermatozoa incubated for only 4 h were responsive to the HFF (Figure 3A, B). However, ONOO– and SIN-1 did not trigger a spontaneous acrosome reaction in spermatozoa (Figure 3A, B). In all cases, the degradation products of ONOO– (preincubated for 1h in Tyrode's + BSA medium before use) were assayed and the results obtained were not significantly different from those obtained with the Tyrode + BSA medium.
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To further analyse the effect of ONOO– on sperm functions, we then investigated the action of ONOO– and SIN-1 on different motility parameters. Both compounds at concentrations capable of inducing capacitation increased ALH and decreased linearity without affecting the percentage of motile spermatozoa. Moreover, ONOO– was also able to increase sperm velocity (Table I
).
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Peroxynitrite induces tyrosine nitration and phosphorylation of sperm proteins
Exposure of spermatozoa to ONOO– (2.5–50 µmol/l) rapidly and dose-dependently increased the nitrotyrosine immunoreactivity (Figure 4A
). This nitrotyrosine protein modification seemed to occur rapidly and to be stable, because nitrotyrosine immunoreactivity was essentially unchanged in spermatozoa incubated for 8 h after ONOO– addition (Figure 4A). It seems that ONOO– nitrated the same bands as those observed in the control. Moreover, these same proteins were nitrated when spermatozoa were exposed to SIN-1 but not when exposed to an NO.-releasing compound alone (DEA, 1 mmol/l) (Figure 4B), suggesting that endogenous ONOO– formation or its degradation products might be responsible for nitration of certain cellular sperm proteins in vivo.
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Since tyrosine phosphorylation of sperm proteins appears to be associated with sperm capacitation and regulated by oxidoreduction reactions (Leclerc et al., 1997
; Herrero et al., 1999), we further delineated the involvement of ONOO– on the tyrosine phosphorylation event. The effect of ONOO– or SIN-1 on tyrosine phosphorylation of sperm proteins was observed only after 8 h, and not at the beginning (30 min) of the incubation (Figure 5A, B). The addition of ONOO– to spermatozoa incubated for 8 h resulted in an increase in the phosphotyrosine content of proteins (Figure 5A). The enhancement primarily involved a complex set of protein bands, within a mol. wt of 116–50 kDa. This phosphorylation pattern was similar to the one observed when spermatozoa were incubated with DEA (Figure 5B). Moreover, SIN-1 also caused a concentration-dependent increase in the tyrosine phosphorylation event, although some other proteins (mol. wt of 50–31 kDa) were modulated by 1 mmol/l SIN-1 (Figure 5B).
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Since ONOO– is the reaction product of NO. and O2.–, we were interested in identifying whether ONOO– and NO. modulate tyrosine phosphorylation of the same set of proteins. Therefore, sperm proteins were separated by 2-D electrophoresis before immunoblotting. Spermatozoa incubated for 8 h in the presence of ONOO– or DEA showed increased immunoreactivity of several protein groups ranging in size from 97 to 45 kDa with isoelectric points (pI) of ~4–8.5 (Figure 6
). However, a set of proteins between 45 and 50 kDa with pI of ~5.3–6.6 and another of 55–60 kDa with pI of ~4.6–5.3 were tyrosine-phosphorylated in the presence of ONOO– but not with DEA. In contrast, proteins at 85 kDa with pI of ~5.2–6.6 were mainly modulated by DEA. These findings demonstrated that NO. and ONOO– might share targets during the tyrosine phosphorylation event, although they might also be able to exert their effects on specific sperm proteins.
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Discussion
Mammalian sperm capacitation is defined as an obligatory maturational process that leads to the development of the fertilization-competent state of spermatozoa. Studies on sperm capacitation have shown that conventional thought regarding ROS as harmful should be carefully reconsidered since it has effectively been demonstrated that small amounts of NO., O2.– and H2O2 act as intracellular signals in spermatozoa during capacitation (de Lamirande et al., 1997; Herrero and Gagnon, 2001). In this regard, two main findings are reported in the present study: (i) the beneficial effects of ONOO– on sperm function; (ii) the occurrence of nitration of proteins on capacitated spermatozoa with potentially significant biological implications.
Our work on motility and capacitation indicates that exogenous ONOO– is able to modulate sperm functions. The addition of ONOO– to spermatozoa decreases sperm linearity and increases sperm velocity and ALH without modifying the percentage of motile cells. These observations are consistent with the notion that ONOO– modulates sperm motility and could be implicated in the regulation of sperm hyperactivation, which is the specific erratic and whiplash-like type of motility that capacitated spermatozoa display at the site of fertilization.
The presence of ONOO– or SIN-1 in the incubation medium accelerates sperm capacitation, indicating that ONOO– may be involved in this process. These results are similar to those obtained with NO.-releasing compounds (Herrero et al., 1999), suggesting that ONOO– and NO. could act on similar cellular targets. In fact, these similarities have also been described in different tissues. These include the induction of blood vessel relaxation by stimulating cyclic GMP synthesis (Tarpey et al., 1995; Davidson et al., 1997), the inhibition of platelet aggregation (Heath Mondoro et al., 1997), and the activation of prostaglandin biosynthesis (Landino et al., 1996).
Post-translational modifications of proteins such as tyrosine phosphorylation or nitration can have dramatic effects on a protein's structure, intracellular compartmentalization, catalytic activity, or rate of degradation and turnover and can thus participate in many regulatory processes (Kamisaki et al., 1998). Whereas several studies have reported the importance and regulation of tyrosine phosphorylation during sperm capacitation and acrosome reaction (O'Toole et al., 1996; Visconti and Kopf, 1998), this is the first report on the association of tyrosine nitration of proteins with sperm function.
We have observed a spontaneous tyrosine nitration of proteins in capacitating spermatozoa. The pattern of protein tyrosine nitration was always the same and differences were observed only in the relative intensity of some bands among donors.
Considering that under our experimental conditions tyrosine residues are nitrated by ONOO– but not by NO. itself, it seems likely that ONOO– is the one involved in the formation of 3-nitrotyrosine in human spermatozoa. Similarly, several laboratories have demonstrated that endogenous nitration of certain proteins, such as prostacyclin synthase (Zou et al., 1998), neurofilament L (Crow et al., 1997) and calcium ATPase (Klebl et al., 1998) occurs when cells are exposed to ONOO– but not when exposed to NO. alone or nitrite. The fact that an increase in tyrosine nitration of proteins takes place only at the beginning of capacitation may indicate that ONOO– requires a short period of time to exert its action. Perhaps ONOO– initiates a cascade of events in a manner similar to that observed with NO. during human sperm capacitation (Herrero et al., 2000).
We previously reported that during the acrosome reaction there was an increase of sperm NO. synthase activity (Herrero et al., 1998). Since ONOO– is formed in a rapid reaction from NO. and O2.–, it is tempting to speculate that an increase in NO. production will lead to a subsequent increase in ONOO–. In agreement with this hypothesis, we observe that the intensity of specific nitrotyrosine proteins from capacitated spermatozoa challenged with HFF or calcium ionophore is higher than that of acrosome non-reacted spermatozoa, suggesting the involvement of nitrated proteins during the sperm acrosome reaction.
Since the primary effect of ONOO– on proteins is nitration of tyrosine residues, we then evaluated the effects of ONOO– and SIN-1 on sperm proteins. A concentration-dependent increase of tyrosine-nitrated proteins occurs in spermatozoa. In addition, both ONOO– and SIN-1 induce a rapid and long-lasting nitration of tyrosine of many sperm proteins. The persistence of nitration in sperm proteins may indicate that a positive tyrosine nitration balance exists in the presence of some nitrotyrosine denitrase activity or that spermatozoa lack the enzymatic ability to remove nitrate from tyrosine residues. However, this issue remains to be determined since the existence of a nitrotyrosine denitrase has been suggested only in rat spleen and lung (Kamisaki et al., 1998).
The involvement of ONOO– on sperm functions is also reflected in the levels of tyrosine phosphorylation of sperm proteins. Examination of this process in capacitated spermatozoa demonstrates that ONOO– increases protein tyrosine phosphorylation. Stimulation of tyrosine phosphorylation by ONOO– is not specific to spermatozoa since it has been demonstrated in other cells and tissues, such as human erythrocytes (Mallozi et al., 1997), platelets (Heath Mondoro et al., 1997) and human neuroblastoma cells (Li et al., 1998).
A similar type of enhancement on sperm tyrosinephosphorylated proteins has also been observed for NO. (Herrero et al., 1999), and confirmed in this study. Despite the fact that NO. and ONOO– seem to modulate the same phosphotyrosine proteins, 2-D immunoblots demonstrate that some tyrosine-phosphorylated proteins are only modulated by ONOO–, raising the question of how ROS can become selective for modifying specific residues on certain proteins in complex biological systems.
Although the identities of phosphotyrosine proteins affected by ONOO– remain to be identified, phosphorylation of tyrosines of some proteins appears to be of the same molecular weights as the substrates for tyrosine nitration. In this regard, it has been shown that in lymphocytes, nitration of a tyrosine residue impairs the subsequent phosphorylation of the same residue, indicating that ONOO–-promoted nitration may exert physiological effects by altering kinase reactions and subsequent signalling pathways (Kong et al., 1996). On the other hand, two separate reports suggest that protein nitration and phosphorylation might be mutually exclusive (Berlett et al., 1996; Gow et al., 1996). Although the present experiments are not intended to address this issue, the simultaneous increase in tyrosine nitration and phosphorylation of sperm proteins observed after 8 h exposure to ONOO– lend support to the latter hypothesis. However, determination of the identities of tyrosine-nitrated and -phosphorylated proteins, as well as site-specific measurements, are clearly needed to resolve this issue.
One additional point deserves comment: a recent study indicates that ONOO– might cause sperm dysfunction through an increase in lipid peroxidation, and total sulphydryl group depletion (Öztezcan et al., 1999). These findings appear to be in disagreement with our results. Because ONOO– effects depend on its concentration and on the environment in which the anion is present (Crow, 2000), we believe that the differences between the two reports could be attributable to the concentration of ONOO– used in the samples, as well as the time frame over which the experiments were conducted. Thus, ONOO– may have physiological functions in addition to pathological ones.
The precise biological targets for ONOO– and the nature of the modification of those targets will vary dramatically depending on their relative concentrations and the rates and duration of ONOO– formation. The rate of formation of ONOO– depends primarily on the concentration of oxygen to provide both precursors (NO. and O2.–). It has been demonstrated that oxygen concentration in fluids of the female reproductive tract remains at low levels except at the time of ovulation (Maas et al., 1976), thus supporting the concept that ONOO– could be generated in vivo. Peroxynitrite can react with various cellular compounds such as sugars or other molecules containing an alcohol or a sulphydryl group and the resulting compounds can act as long-lasting NO.-releasing compounds. Therefore, ONOO– could interact with NO. signalling. For instance, the reaction between ONOO– and glutathione (GSH) forms nitrosoglutathione or a similar nitrosothiol, which in turn will liberate NO.. Hence the presence of glutathione may prevent the accumulation of ONOO– to toxic levels and may convert ONOO– to secondary products with protective properties (Muijsers et al., 1997).
Finally, since ONOO–, NO. and O2.– are cellular messengers involved in cell function, including spermatozoa, the question that arises is how ROS can modulate specific cell signalling pathways. Presumably, absolute specificity in the interactions of the oxidants is not a requirement for cell signalling. What is required is that the signal downstream of the reaction between the oxidant and the target molecule can be effectively amplified. Therefore, although multiple reactions may occur between the oxidant and the various target molecules, only those modifications that are efficiently coupled to activation of downstream events will be able to elicit a biological response.
In summary, spontaneous tyrosine nitration occurs in capacitating spermatozoa. This tyrosine nitration is enhanced by ONOO– treatment that also affects motility parameters and causes an increase in capacitation and tyrosine phosphorylation, suggesting that ONOO– may be involved in sperm function. These results raise critical topics to be addressed, including the significance of nitration of proteins on sperm fertilizing ability, as well as the identification of key molecules affected specifically by ONOO–.
Acknowledgements
C.G. was supported by a grant from the Medical Research Council of Canada. M.B.H. was supported by the National Research Council of Argentina and by a re-entry grant (Pre-032/99) from PLACIRH (Programa Latinoamericano de Capacitación e Investigación en Reproducción Humana). The authors are thankful to the McGill Reproduction Centre for providing human follicular fluid and to all the volunteers who participated in this study.
Notes
3 Present address: Cell Biology Department, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22904, USA 
4 To whom correspondence should be addressed at: Urology Research Laboratory (H6.44), Royal Victoria Hospital, McGill University, 687 Pine Avenue West, H3A 1A1 Montréal, Canada. E-mail: claude.gagnon{at}muhc.mcgill.ca
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Submitted on February 2, 2001; accepted on July 20, 2001.
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