MARÃA BELE´N HERRERO AND CLAUDE. GAGNON. From the ..... Francavilla F, Santucci R, Macerola B, Ruvolo G, Romano R. Nitric oxide synthase inhibition ...
Journal of Andrology, Vol. 22, No. 3, May/June 2001 Copyright American Society of Andrology
Nitric Oxide: A Novel Mediator of Sperm Function MARI´A BELE´N HERRERO AND CLAUDE GAGNON
Review
stitutive and the inducible NOS (Knowles and Moncada, 1994). Constitutive NOS was first localized in the nervous system (nNOS) and vascular endothelium (eNOS), although it is now known to be expressed in various cells in different organs (Knowles and Moncada, 1994). NO generated by neurons acts as a neurotransmitter, whereas within the vasculature, NO induces vasodilation, inhibits platelet aggregation, and regulates programmed cell death, among other functions. Both constitutive NOSs (nNOS and eNOS) are calcium- and calmodulin-dependent and generate small quantities of NO that carry out a variety of physiological functions. However, under certain conditions, the activity of constitutive NOS is increased due to an up-regulation of the enzyme by sex hormones. An increase in messenger RNA (mRNA) encoding both constitutive NOSs was observed after treatment with estradiol in the uterus, suggesting that this isoform can actually be induced (Weiner et al, 1994). Because the eNOS NH2 terminus lacks a 220 amino acid domain that is present in nNOS, these isoforms differ in molecular mass (130 kilodaltons [kd] for eNOS, 160 kd for nNOS), and eNOS has a consensus Nmyristoylation site that contributes to its membrane localization (Sessa, 1994). Transcriptional induction was first demonstrated for another isoform of NOS that is induced by immunological or inflammatory stimuli and gives rise to the sustained release of NO (Nustler and Billiar, 1993). This enzyme was originally identified in macrophages, where the NO generated contributes to the cytotoxic/cytostatic actions of these cells against tumors and invading microorganisms. Although inducible NOS (iNOS) is also calcium- and calmodulin-dependent, it differs from nNOS and eNOS in that it needs very low levels of calcium to be fully activated, and it appears to bind calmodulin very tightly so that calmodulin forms a constitutive subunit to this isoform (Nathan and Xie, 1994). Originally, iNOS was believed to be induced only in pathological conditions, but now it has been also identified in physiological settings such as ovulation, pregnancy, and labor (Shukorski and Tsafriri, 1994; Buhimschi et al, 1996; Bansal et al, 1997). Studies using antibodies against nNOS, eNOS, and iNOS have revealed that these enzymes are present in nearly every tissue studied, including the reproductive system (Burnett et al, 1995; Suburo et al, 1995; Roselli et al, 1996). In addition, NOS enzymes share a 60% amino acid homology, represent a dispersed gene family, and
From the Urology Research Laboratory, Royal Victoria Hospital, McGill University, Montre´al, Que´bec, Canada.
Nitric oxide (NO) is currently one of the most studied molecules in biomedical sciences because of the multiplicity of roles it plays in various physiological systems and because of the prospect of developing new therapeutic drugs aimed at curing important diseases (Loscalzo and Welch, 1995; Corbin and Francis, 1999). Indeed, NO plays a decisive role in regulating multiple functions within the male reproductive system. In this article we will review the important role played by NO on sperm function. We will first present a brief description of the enzymes that are responsible for NO biosynthesis, followed by the observations obtained on the role of NO in sperm functions that helped lead us and others to our present understanding of the effects of NO in sperm fertilizing ability.
Nitric Oxide Synthases NO is synthesized from L-arginine by the NO-synthase (NOS) enzymes (Palmer et al, 1988). This reaction requires a number of cofactors, namely b-nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide, flavin adenine dinucleotide, and tetrahydrobiopterin, as well as calmodulin and calcium (Moncada and Higgs, 1995). Molecular oxygen is also used in this reaction, which proceeds via synthesis of the intermediate N-hydroxyarginine and results in the formation of L-citrulline in addition to NO. NOSs are heme-containing enzymes that have sequence similarities with cytochrome P-450 reductase and are the only mammalian proteins known to catalyze both a hydroxylation reaction and NADPH reduction (Sessa, 1994). Two types of NOS have been described, the conSupported by a grant of the Medical Research Council (MRC) of Canada to C.G. M.B.H. was supported by a fellowship from the National Institutes of Health Fogarty International Center (D43 TW/HD00671). Correspondence to: Dr Marı´a B. Herrero, CEFYBO-CONICET, Serrano 669, Piso 5, 1414 Buenos Aires, Argentina (e-mail: herrerob@ hotmail.com). Received for publication August 25, 2000; accepted for publication November 13, 2000.
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350 are encoded by at least 3 separate genes; in humans, genes encoding for eNOS, nNOS, and iNOS are located on chromosomes 7, 12, and 17, respectively (Knowles and Moncada, 1994).
Nitric Oxide Synthase Isoforms in Spermatozoa Before 1996, there was no evidence to suggest that spermatozoa could generate NO. In 2 separate reports, Herrero et al (1996a) and Lewis et al (1996) demonstrated that NOS was present in mouse and human spermatozoa. In mouse, NOS was observed on the acrosome and tail of noncapacitating spermatozoa. Localization of NOS to the head disappeared with time when spermatozoa were incubated under capacitating conditions, suggesting a role for NOS in capacitation (Herrero et al, 1996a). In contrast, in human spermatozoa, specific labeling with nNOS and eNOS was observed on postacrosomal and equatorial segments, but not on the flagellum. Immunoreactivities to eNOS and nNOS antibodies were most intense on sperm samples derived from normozoospermic individuals compared with those from asthenozoospermic patients, which had little or no NOS immunoreactivity (Lewis et al, 1996). Furthermore, O’Bryan et al (1998) classified human sperm eNOS immunostaining into 4 patterns: 1) faint postacrosomal and equatorial staining, 2) intense postacrosomal and equatorial staining, 3) diffuse sperm head staining, and 4) sperm midpiece staining with or without diffuse head staining. Patterns of types 1 and 2 were always observed in normal spermatozoa, whereas types 3 and 4 were often associated with morphologically abnormal spermatozoa and with a decrease in sperm motility, suggesting that NO may be associated with normal sperm physiology. The presence of NOS in spermatozoa was subsequently confirmed by Western blot analysis. Under nonreducing conditions, a band corresponding to a human sperm protein of 145 kd was recognized after immunoblotting with the eNOS antiserum (Revelli et al, 1999). A similar molecular mass protein (140 kd) was observed for sea urchin (Kuo et al, 2000) and mouse sperm NOS (Herrero et al, 1997a), although it is intriguing that in the mouse, the 3 NOS antibodies (anti-nNOS, anti-eNOS, and anti-iNOS) tested could recognize a unique band. The high degree of homology that exists between the neuronal, endothelial, and inducible isoforms, and the fact that antibodies used in the study were polyclonal, may explain why sperm NOS reacted positively with all 3 antisera. Nevertheless, at present, it is not known whether spermatozoa possess 1 or more NOS isoforms and only molecular biology techniques will provide conclusive evidence to answer this question.
Nitric Oxide Production by Spermatozoa Several methods have been described in the literature to detect NO production in different tissues. These include
Journal of Andrology · May/June 2001 measuring NOS activity by following the conversion of [14C]L-arginine to [14C]L-citrulline (Bredt and Snyder, 1989), detecting NO end by-products such as nitrates and nitrites, spin-trapping techniques combined with electron paramagnetic resonance (EPR) spectroscopy (Komarov et al, 1993), or fluorescence changes after nitrosation of the NO indicator dye, diaminofluorescein (DAF) (Kojima et al, 1998). In teleosts, no measurable production of NO by spermatozoa could be detected by EPR (Creech et al, 1998). However, in that study, spermatozoa were mixed with seminal plasma, which is known to scavenge reactive oxygen species (ROS) and thus, NO. Recently, it was demonstrated that in sea urchin spermatozoa, NOS is present at high concentration (⬃0.44% ⫾ 0.05% of total NP-40 soluble sperm protein) and is enzymatically active as determined by the citrulline assay, accumulation of nitrites, and by fluorescence changes (DAF; Kuo et al, 2000). In mouse spermatozoa, NOS activity was evidenced by the conversion of [14C]L-arginine to [14C]L-citrulline (Herrero et al, 1997a). Formation of [14C]L-citrulline decreased in a concentration-dependent fashion when spermatozoa were incubated with a specific NOS inhibitor. Furthermore, kinetic assays demonstrated that NO formation increased during the first 120 minutes of incubation and then reached a plateau at 120–180 minutes. This may indicate that mouse sperm NOS could have a physiological significance during capacitation (Herrero et al, 1997a). In human spermatozoa, studies on NO generation have produced different results, some of which appear to directly contradict others. This controversy could be in part due to the small amounts of NO produced by these cells. The first report that suggested NO production by human spermatozoa was based on evidence of the accumulation of nitrites produced by these cells following 2 hours of incubation. However, the level of nitrites (1.6–2.9 M nitrites) produced by 106 spermatozoa was surprisingly high (Lewis et al, 1996). Using an Iso-NO electrode, Donnelly et al (1997) then suggested that spermatozoa synthesized NO, although indirect NO measurement was done in nonliving spermatozoa. Recently, a study by Revelli et al (1999) showed data on NO production by human spermatozoa measured by the conversion of [3H]Larginine to [3H]L-citrulline, but in this case the measurements were also done on human sperm lysates. However, Zini et al (1995) as well as Schaad et al (1996) could not detect NO production in live human spermatozoa. Therefore, the question that arises is whether live human spermatozoa can synthesize NO. To address this question, EPR was used to measure NO (Herrero et al, 2000). EPR is a specific and reliable technique that allows in vivo real-time detection of NO production on motile
Herrero and Gagnon · Nitric Oxide and Sperm Functions spermatozoa. Two relevant findings emerged from the EPR studies. First, the direct measurement of NO conclusively showed that NO is synthesized by the human male gamete. Second, this NO production was associated with capacitation, because spermatozoa incubated under capacitating conditions generated eightfold more NO than those incubated under noncapacitating conditions.
Involvement of Nitric Oxide in Different Events Leading to Fertilization Sperm Motility—The first reports on NO and sperm motility analyzed the effects of NO-releasing compounds on sperm motion and viability. For example, Hellstro¨m and coworkers (1994) demonstrated that low concentrations of a NO-releasing compound, sodium nitroprusside (SNP), was beneficial to the maintenance of post-thaw human sperm motility and viability. In parallel, a study suggested that NO could also act as a stimulator of mouse sperm hyperactivation (Herrero et al, 1994). Moreover, it was recently demonstrated that low concentrations of SNP enhanced sperm motility in hamsters as well as in teleosts (Creech et al, 1998; Yeoman et al, 1998). In contrast, several reports showed that sperm motility is decreased in the presence of different concentrations of SNP (Tomlinson et al, 1992; Herrero et al, 1994; Roselli et al, 1995; Weinberg et al, 1995), although one of the studies demonstrated that sperm viability was not affected (Tomlinson et al, 1992). Inhibition of sperm motility was observed with high concentrations of the NO-releasing compound, and this effect was most likely caused by an inhibition of sperm respiration (Weinberg et al, 1995). Despite the fact that some of these cited reports appear to be contradictory, the results may be completely compatible and the variance attributable to the concentration of NO in the sample as well as the time frame over which the experiments were conducted. Hence, low concentrations of NO enhance sperm motility, whereas high concentrations of NO decrease it. It is interesting that in semen collected from different donors, a negative correlation between the concentrations of NO and the percentage of motile cells was observed, supporting the contention that low concentrations of NO enhance motility (Nobunaga et al, 1996). In addition, Schaad et al (1996) also reported that human seminal plasma inhibited nNOS activity. The inhibitor present in seminal plasma was a heat-stable, noncompetitive inhibitor of nNOS, although the physiological relevance of the presence of such an endogenous inhibitor in the seminal plasma is unclear. It is tempting to speculate that the function of this inhibitor may be to prevent the development of hyperactivation and capacitation or to maintain NO at low concentrations in order to protect spermatozoa from toxic damage. Consistent with this hypothesis, high NO levels have been found in semen of infertile men with
351 decreased sperm motility, and it is in these clinical cases that inhibitors of NOS have succeeded in enhancing motility (Roselli et al, 1995; Nobunaga et al, 1996; Perera et al, 1996). Addition of SNP to spermatozoa of infertile patients further diminished motility, as may be expected because the level of oxidative stress was probably already high. It is therefore conceivable that excessive generation of NO, mainly by leukocytes, could cause a spontaneous hyperactivation that impairs sperm transport along the lower female reproductive tract or leads to a premature capacitation and therefore cause infertility. In summary, the concentration of NO will determine its effects on sperm motility and viability. This bimodal motility response to various concentrations of NO-releasing compounds is not unexpected because the dual nature of NO as both a transduction molecule at low concentrations and a cytotoxic effector at high concentrations is well known in other systems. Bearing in mind that NO can be synthesized by spermatozoa, it is reasonable to speculate that endogenous NO produced by spermatozoa regulates sperm motility. The addition of the NOS inhibitor N6-nitro-L-arginine-methyl ester (L-NAME) to the incubation medium depressed human and hamster sperm motility (Donelly et al, 1997; Yeoman et al, 1998). Moreover, concentrations of nitrite produced by spermatozoa were observed to be lower in samples from asthenozoospermic men than in those from normozoospermic donors, suggesting that the decrease of endogenous NO may influence sperm motility and thus fertilization (Lewis et al, 1996). Sperm Capacitation—The first experimental evidence for the involvement of NO in capacitation came from the observation that human spermatozoa incubated with low concentrations of NO-releasing compounds increased the percentage of capacitation measured by the lysophosphatidylcholine-induced acrosome reaction (Zini et al, 1995). In addition, the presence of NO-releasing compounds (SNP and diethylamine-NONOate), accelerated capacitation and thus the ability of spermatozoa to undergo the acrosome reaction (Herrero et al, 1999). The ability to accelerate the capacitation process varied among NO-releasing compounds. This difference in potency was probably related to the kinetics of NO formation, because SNP generated NO instantaneously (3–10 seconds) whereas diethylamine-NONOate released NO over a longer period of time (15–30 minutes). Thus, it is possible that NO required only a short period of time to exert its action, perhaps by initiating a cascade of events that would lead to capacitation in a manner similar to that observed with cyclic adenosine monophosphate (cAMP) in many other systems. Moreover, the fact that the addition of NOS inhibitors at the onset of the incubation period caused an important decrease of capacitation (measured by the human follicular fluid or calcium ionophore-induced acro-
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Journal of Andrology · May/June 2001
Figure 1. Nitric oxide regulates sperm functions.
some reaction) indicated that endogenous NO was necessary for spermatozoa to display their full fertilizing ability (Herrero et al, 1999). The importance of NO on sperm capacitation was also reflected on the levels of tyrosine phosphorylation of 2 sperm proteins (p81 and p105). When capacitation was accelerated by a NO-releasing compound, there was an increase in tyrosine phosphorylation, whereas when sperm capacitation was inhibited by L-NAME, there was an attenuation in the tyrosine phosphorylation of these 2 sperm proteins (Herrero et al, 1999). Therefore, tyrosine phosphorylation of sperm proteins appears to be regulated by NO. It is interesting that these 2 proteins are also modulated by superoxide anion and hydrogen peroxide (Leclerc et al, 1997). Thus, the fact that tyrosine phosphorylation of at least these 2 proteins are regulated by 3 different ROS support the concept that sperm capacitation is a redox regulated event. Zona Pellucida Binding, Acrosome Reaction, Fusion, and Activation—The involvement of NO in the acquisition of fertilizing ability is not limited to sperm motility or capacitation. NO seems to participate in the zona pellucida binding as well as in the acrosome reaction and fusion events. Treatment of human spermatozoa with low concentrations of SNP (10⫺7–10⫺8 M) in the capacitating medium increased the number of spermatozoa bound to the hemizona (Sengoku et al, 1998). In addition, mild oxidative conditions were previously shown to improve by 50% the zona pellucida binding ability of mouse spermatozoa (Kodama et al, 1996). These observations could suggest that modifications of phospholipid integration within the sperm membrane caused by limited lipid peroxidation induced by low concentrations of NO may change the membrane fluidity and trigger rearrangements of membrane components, possibly promoting zona pellucida binding. In contrast, a recent study demonstrated that the addition of various concentrations of L-NAME to capacitated spermatozoa did not affect zona pellucida binding, although a 50% inhibition was observed in the fusion event (Francavilla et al, 2000). Differences in the experimental design and the methods used to capacitate spermatozoa could explain these discrepancies. NO is also involved in the process of acrosome reac-
tion. A recent study in sea urchin established that 2 inducers of the acrosome reaction (the egg jelly and ionomycin) increased NO levels in spermatozoa, although the latter was less effective than the egg jelly coat (Kuo et al, 2000). In human spermatozoa, NO-releasing compounds induced the acrosome reaction in a concentration-dependent manner, whereas hemoglobin, a NO scavenger, prevented the human follicular fluid-induced acrosome reaction of capacitated spermatozoa. (Revelli et al, 1999). Therefore, NO appears to participate in the human follicular fluidinduced acrosome reaction. Similarly, in the mouse, the addition of 2 NOS inhibitors (L-NAME and nitro-arginine) completely blocked the ability of progesterone to stimulate acrosomal exocytosis, whereas spermine-NONOate, a NO-releasing compound, directly elicited this event (Herrero et al, 1997b). It is interesting that 100 M spermine-NONOate triggered the acrosome reaction in 45% of mouse spermatozoa, as was observed with progesterone. However, at a higher concentration (1000 M), spermine-NONOate failed to induce the acrosome reaction. This bell-shaped, dose-response curve is not surprising and indicates once more that high concentrations of NO have deleterious effects on cellular functions, a response now well established for ROS. In vitro fertilization studies demonstrated that the percentage of oocytes with 2 pronuclei was decreased by 80% when mouse spermatozoa were pretreated with 1 mM of L-NAME, suggesting once more that sperm NOS activity is required for spermatozoa to display their full fertilizing ability (Herrero et al, 1996b). Finally, a recent study effectively demonstrated that microinjection of NO-releasing compounds or recombinant NOS to sea urchin eggs recapitulated events of egg activation, suggesting that NO synthesized by spermatozoa may be an universal activator of eggs (Kuo et al, 2000). In summary, NO is associated with most processes leading to fertilization (Figure 1), emphasizing the concept that ROS are involved in sperm acquisition of fertilizing ability.
Herrero and Gagnon · Nitric Oxide and Sperm Functions
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Possible targets of nitric oxide on spermatozoa during capacitation and acrosome reaction* Effect Observed Increased Increased Increased Increased
cGMP cAMP PGE2 5-HETE
Possible Target Guanylyl cyclase Adenylyl cyclase Cyclooxygenase Lipoxygenase
Sperm Function Acrosome reaction Capacitation Capacitation–Acrosome reaction Capacitation
Species Bovine Human Mouse Mouse
* cGMP indicates cyclic guanosine monophosphate; cAMP, cyclic adenosine monophosphate; PGE2, prostaglandin E2; 5-HETE, 5-hydroxyeicosatetraenoic acid.
Nitric Oxide Metabolism and its Possible Mechanisms of Action in Spermatozoa Evidence presented above support the concept that NO plays an important role in regulating sperm functions. However, many questions about its mechanism of action remain to be clarified. Although initially the effects of NO were believed to be solely mediated via activation of soluble guanylyl cyclase (GC; Murad, 1994), recent studies indicated that NO can also induce its biological effects via non-cyclic guanosine monophosphate (cGMP)–dependent pathways (Stamler, 1994; Pfeiffer et al, 1999). Be-
cause of its short half-life (⬍30 seconds) and high reactivity, NO was shown to react with oxygen (O2), superoxide anion (O2•⫺), sulfhydryl (⫺SH) groups, and transition metals (heme complexes of iron and copper, iron-sulfur centers of proteins; Stamler et al, 1992; Mohr et al, 1994). In mammalian spermatozoa, few studies suggested possible targets of NO during capacitation and acrosome reaction (Table; Figure 2). In bovine spermatozoa, SNP caused a 50% increase in cGMP levels during the acrosome reaction (Zamir et al, 1995), although in mouse
Figure 2. Current signal transduction pathways that could be involved in NO-induced capacitation. In mammalian spermatozoa, there is a stimulation of NO production through the action of a constitutive nitric oxide synthase (cNOS) during capacitation. Calcium influx, which occurs immediately at the beginning of capacitation, could possibly activate cNOS. An increase in NO could then activate, directly or indirectly, sperm adenylyl cyclase (AC) with a subsequent increase in levels of cAMP. In addition, NO can activate COX, which results in an increase in prostaglandin production. Although there is no evidence yet, it is possible that during capacitation, NO transforms lipids or modulates enzymes such as cyclic nucleotide phosphodiesterases (PDEs), protein tyrosine kinases (PTKs), phosphatases (PTPs), or a combination of these, because these enzymes are very sensitive to oxidation. (⫹) Indicates positive regulation; (⫺) indicates negative regulation. Bicarbonate (HCO3⫺); protein kinase A (PKA).
354 spermatozoa, cGMP levels were not affected by treatment with the NOS inhibitor, L-NAME (Herrero et al, 1998). It is interesting that in this last study, the activity of cyclooxygenase (COX), another heme-containing enzyme, appeared to be modulated by NO during the progesteroneinduced acrosome reaction, because the presence of the NOS inhibitor, L-NAME completely blocked the increase in prostaglandin E2 (PGE2) synthesis induced by progesterone. Conversely, treatment of mouse spermatozoa with SNP stimulated both COX and lipoxygenase (LOX) activities during capacitation (Herrero et al, 1995). In other tissues such as hypothalamus and uterus (Rettori et al, 1993; Salvemini et al, 1993), the activation of heme-containing proteins (ie, GC, COX, LOX) appears to result from a structural change upon NO binding to the heme group, similar to that induced by O2 binding to hemoglobin. Thus, it is possible that such an NO-induced conformational change in GC, COX, and LOX occurs in spermatozoa during capacitation, the acrosome reaction, or both. During the progesterone-induced acrosome reaction, mouse sperm NOS activity is increased by about 70% (Herrero et al, 1998), raising the question of how this steroid can modulate sperm NO production. It is tempting to speculate that progesterone, which promotes the influx of extracellular calcium, activates a calcium-dependent isoform of sperm NOS. This would lead to an increase in NO synthesis, which in turn would activate enzymes (ie, COX) involved in different signal transduction pathways that finally result in the acrosome reaction. Another relevant mechanism of NO action is the Snitrosylation of thiols, which has been implicated in the regulation of key enzymes, including protein kinase C (PKC; Gopalakrishna et al, 1993) and glyceraldehyde-3phosphate dehydrogenase (Clancy et al, 1994). We recently demonstrated that NO participates in human sperm capacitation and tyrosine phosphorylation of proteins by interacting with a cAMP pathway (Herrero et al, 2000). Indirect evidence suggests that low concentrations of NO increased sperm cAMP levels by stimulating adenylyl cyclase activity (Herrero et al, 2000). These results are in agreement with those obtained in cardiac myocytes, in which low concentrations of an NO-releasing compound also increased cAMP levels (Vila-Petroff et al, 1999). Moreover, it was recently demonstrated that in neuroblastoma cells, adenylyl cyclase activity is regulated by NO, because high concentrations of SNP (1–3 mM) inhibited its activity. This inhibition is reversible if an appropriate reducing agent is present in the incubation medium (McVey et al, 1999), suggesting S-nitrosylation as a possible mechanism for NO modulation of adenylyl cyclase activity. However, it remains to be established whether sperm adenylyl cyclase could also be regulated in a similar manner.
Journal of Andrology · May/June 2001 Finally, an important reaction of NO in biological media is a direct bimolecular reaction with O2•⫺, yielding peroxynitrite (ONOO⫺; Beckman and Koppenol, 1996). Peroxynitrite is a potent oxidant and has been shown to react with virtually all classes of biomolecules in vitro, such as myeloperoxidase, glutathione peroxidase, cytochrome c, ascorbate, etc (Pryor and Squadritto, 1995). In human platelets (Mondoro et al, 1997) as well as in synaptic proteins (Di Stasi et al, 1999), peroxynitrite can induce nitrosylation of several tyrosine molecules that regulate enzyme function as well as lipid peroxidation. Because spermatozoa produce both O2•⫺ (de Lamirande and Gagnon, 1995) and NO, for¨ ztezmation of ONOO⫺ is conceivable. In a recent study, O can et al (1999) showed that the addition of 100 M ONOO⫺ to human spermatozoa decreases sperm motility and the total sulfhydryl content by about 20%–25%, whereas lipid peroxidation is increased 2.5-fold. The authors proposed that ONOO⫺ may cause sperm dysfunction through lipid peroxidation stimulation and sulfhydryl depletion, although it remains to be determined if low concentrations of ONOO⫺ can play a physiological role in sperm function. The defense of cells against NO, which in turn produces ONOO⫺, largely depends on decreasing the O2•⫺ concentration, controlled by the superoxide dismutase enzyme. It is therefore feasible that the effects of NO will depend on the chemistry it undergoes in a given biological milieu. Reactions of NO with targets such as O2•⫺ may be toxic or protective, depending on the nature and extent of the chemical insult.
Conclusions Experimental evidence demonstrates that although excessive NO concentrations can cause defective sperm function, low and controlled concentrations of NO play an important role in the control of sperm physiology. Spermatozoa are the source of NO and a constitutive NOS appears to be involved in sperm motility, capacitation, and acrosome reaction, suggesting that more than 1 component of the signal transduction pathway could be modified by NO. Therefore, the type and extent of the modifications induced by NO depend not only on the amount of NO, but also on the moment and duration of NO exposure as well as the cell’s redox potential. The challenge will be to determine the precise targets for NO in spermatozoa if we are to comprehend how NO exerts its biological effects on sperm fertilizing ability.
Acknowledgment We thank Dr Eve de Lamirande for a critical reading of the manuscript.
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