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The production of peroxynitrite by human spermatozoa may affect sperm motility through the formation of protein nitrotyrosine
The production of peroxynitrite by human spermatozoa may affect sperm motility through the formation of protein nitrotyrosine Arianna Vignini, M.Sc.,a Laura Nanetti, Ph.D.,a Eddi Buldreghini, B.Sc.,b Cinzia Moroni, B.Sc.,a Giuseppe Ricciardo-Lamonica, Ph.D.,c Franco Mantero, M.D.,d Marco Boscaro, M.D.,b Laura Mazzanti, Ph.D.,a and Giancarlo Balercia, M.D.b a
Institute of Biochemistry, School of Medicine, Polytechnic University of Marche, Ancona; b Andrology Unit, Endocrinology Division, Department of Internal Medicine, Umberto I Hospital, School of Medicine, Polytechnic University of Marche, Ancona; c Department of Economy, School of Economy, Polytechnic University of Marche, Ancona; and d Endocrinology Division, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy
Objective: To detect peroxynitrite and 3-nitrotyrosine production in human spermatozoa of asthenozoospermic infertile patients and normospermic donors, and evaluate any influence on kinetic sperm features. Design: Basic study. Setting: Andrology Unit, Dept of Internal Medicine and Biochemistry Institute, Polytechnic University of Marche, Italy. Patient(s): Sixry-nine infertile patients affected by idiopathic asthenozoospermia and 29 normal fertile donors. Intervention(s): No therapeutic intervention was performed on patients. Main Outcome Measure(s): Production of peroxynitrite (ONOO⫺) and 3-nitrotyrosine by human spermatozoa; kinetic sperm cells parameters. Result(s): Normospermic fertile donors exhibited ONOO⫺ concentrations significantly lower than those of asthenozoospermic infertile men (9.11 ⫾ 3.37 vs. 27.46 ⫾ 5.77 nmol/106 cells); confocal microscopy showed that ONOO⫺ was more evident in spermatozoa of patients than in healthy donors. Moreover, a significant negative correlation was evident between ONOO⫺ concentration and total sperm motility, curvilinear velocity (VCL), straight progressive velocity (VSL), and linearity coefficient. Finally, an increase was found in the nitration of the tyrosine residues in asthenozoospermic samples compared to controls. Conclusion(s): Spontaneous tyrosine nitration occurs in human spermatozoa. This post-translational protein modification is enhanced by an overproduction of peroxynitrite, which is more evident in asthenozoospermic infertile patients when compared with normospermic fertile donors. Motility parameters are negatively affected, suggesting that ONOO⫺ may be involved in defective sperm function. (Fertil Steril威 2006;85:947–53. ©2006 by American Society for Reproductive Medicine.) Key Words: Peroxynitrite, 3-nitrotyrosine, male infertility, sperm cells motility
Defective sperm function is known as one of the common causes of human infertility. There are many studies suggesting a relationship between reactive oxygen species and sperm function impairment (1). In 1943, John MacLeod (2) noted that spermatozoa incubated under high oxygen tensions rapidly lost their motility, and 3 years later it was demonstrated, for the first time, that reactive oxygen species were actively generated in mammalian spermatozoa (3). Several independent investigators have further confirmed these data (4 – 6). Human spermatozoa are extremely sensitive to oxidative attack because they contain high amounts of polyunsaturated fatty acids of phospholipids, particularly C22:6 and are constantly exposed to reactive oxygen species generated by contaminating neutrophils or by the spermatozoa themselves. Spermatozoa are also susceptible to oxida-
Received January 30, 2005; revised and accepted September 19, 2005. Reprint requests: G. Balercia, M.D., Endocrinology, Department of Internal Medicine, Umberto I Hospital, Torrette, Via Conca, 60100 Ancona, Italy (FAX: 39-071-887-300; E-mail: [email protected]).
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tive stress because, due to the paucity of cytoplasm, they lack an adequate reserve of defensive enzymes such as superoxide dismutase, which protect most somatic cells from oxidative attack (7). It has been speculated that peroxynitrite (ONOO⫺) formation is a primary pathway of nitric oxide (NO · ) metabolism (8). Because we have previously shown that human spermatozoa produce NO˙ as well as O⫺ 2 (9), it is reasonable to predict that formation of peroxynitrite can occur in vivo. The unusual stability of peroxynitrite as an anion contributes to its toxicity by allowing it to diffuse far from its site of formation while being selectively reactive with cellular targets (10). In fact, peroxynitrite reacts rapidly with proteins, lipids, and nucleic acids. Moreover, the nitration (addition of an NO2 group) of protein residues gives rise to 3-nitrotyrosine, which represents a protein modification specific for peroxynitrite formation, in vivo, and a widely used marker of its determination. The occurrence of protein tyrosine nitration under pathological conditions is now firmly established and repre-
Fertility and Sterility姞 Vol. 85, No. 4, April 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
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sents a shift from the signal transducing physiological actions of NO to oxidative and potentially pathogenic pathways (11). The precise interplay between NO, oxidants, and the identification of the intermediates responsible for nitration in vivo is still under controversy (12). A recent study indicates that peroxynitrite might cause sperm dysfunction by increasing lipid peroxidation and total sulfydryl groups depletion (13). In addition, Herrero et al. (14) have provided evidence that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of peroxynitrite modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process. In the present study we determined peroxynitrite production in semen and its correlation with kinetic features in spermatozoa, and set out to determine whether protein tyrosine nitration takes place in the same sample to elucidate any pathogenic involvement in sperm cells motility. MATERIALS AND METHODS Patient Selection We evaluated 29 normozoospermic fertile healthy men (control group, aged 27–38 years) and 69 patients (aged 25–37 years) affected by idiopathic asthenozoospermia (sperm concentration ⬎20 ⫻ 106/mL; forward motility grade a ⫹ b ⬍50%, according to World Health Organization [WHO] 1999 criteria) (15) referred to the Andrology Unit of the Division of Endocrinology, Umberto I Hospital, Politechnic University of Marche, Italy, for infertility (of at least 24 months). All subjects in the study showed a normal sperm morphology greater than 30% and a seminal white blood cell count less than 1 ⫻ 106/mL. Sperm culture was negative, including Chlamydia and Mycoplasma ureoliticum detection, as well as the Mar-test. Hormonal serum profile (gonadotropins, T, E2, and PRL) was normal. There was no evidence of anatomical abnormalities of the genital tract, including varicocele (after Doppler sonography), nor was there a history of chriptorchidism, testicular torsion or genital tract infection. The absence of systemic disease or treatment with other drugs within 3 months before enrolment in the present study, the absence of smoking, alcohol, and drug addiction, and the absence of occupational chemical exposure were also verified. Because no causes for motility reduction have been discovered, a conclusive diagnosis of idiopathic asthenozoospermia was reached. This study was approved by the Istitutional Review Board of the Politechnical Marche University, Umberto I Hospital. All patients provided their written informed consent. Semen Analysis Semen samples were collected after 3 days of sexual abstinence. After liquification at room temperature for 30 minutes, standard seminal parameters were analyzed according to WHO 1999 (15) guidelines. Computer-assisted sperm 948
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analysis (CASA) for sperm motility assay was additionally performed as previously reported (16). One semen aliquot (3 L) was placed in a 20-m-deep cell UV chamber (Conception Technologies, La Jolla, CA). Two chambers were loaded, six different fields per chambers were randomly examined, and at least 200 spermatozoa for each field of chambers were scored. Movement characteristics were analyzed using an automated analyser (CellTrack VP110, Motion Analysis Corp, Palo Alto, CA). Kinetic characteristics were evaluated only for motile sperm and expressed as mean values considering total sperm motility (percent), curvilinear velocity (VCL, in m/s), straight progressive velocity (VSL, in m/s), and linearity coefficient (LIN ⫽ VSL/ VCL ⫻ 100). Determination of Peroxynitrite Levels in Semen For peroxynitrite level evaluation, individual sperm samples were diluted to 5 ⫻ 106/mL with Dulbecco phosphatebuffered saline (PBS) (20 mM, pH 7.4), aliquoted to form two replicates and stored at ⫺80°C in sterile tubes until peroxynitrite measurements were performed within 15 days. Peroxynitrite production was determined by the 2,7dichlorofluorescein (DCF) fluorimetric assay as previously described by Kooy et al. (17) and by Tannous et al. (18). DCFDA is a membrane-permeable nonfluorescent dye that is hydrolyzed by esterases, within the sperm cytoplasm, into a free acid, DCFH. ONOO⫺ oxidizes DCFH to a strongly fluorescent dye (17). A DCFDA-free base was prepared by mixing 0.05 mL of 10 mmol/L DCFDA with 2 mL of 0.01 N NaOH at room temperature for 30 minutes. The mixture was neutralized with 18.0 mL of 25 mmol/L PBS at pH 7.4. This solution was maintained on ice in the dark until use. The DCFDAtreated samples were incubated in NO buffer with 100 M L-arginine for 15 minutes at 37°C in a dark room. The mixture was washed with PBS at pH 7.4 then centrifuged for 2 minutes at 214 ⫻ g. Supernatant fluorescence was measured in a Perkin-Elmer MPF-66 spectrofluorometer (Beaconsfield, UK) at an excitation wavelength of 475 nm and emission wavelength of 520 nm. DAF-FM Fluorescence DAF-FM acetate is a membrane-permanent probe that is capable of detecting ONOO⫺ with high sensitivity. Although this probe has also been reported to detect NO, the levels of this radical required to activate DAF fluorescence are beyond the limits found in most biological tissues (⬎7.7 M) (19). The probe was made up as 1-mM stock solution in dimethyl sulfoxide (DMSO) and added to sperm suspensions to give a final concentration of 10 M. The cells were then incubated for 60 minutes at 37°C before being centrifuged at 300 ⫻ g and resuspended in PBS. Confocal microscopy was conducted by using BioRad, MRC 1024 UV confocal laser scanning microscope (Nikon Diaphot inverted microVol. 85, No. 4, April 2006
TABLE 1 The t test results for asthenozoospermic patients and control group.
% Mot Tot. VCL VSL LIN
Normospermic
Asthenozoospermic
P value
63.85 ⫾ 7.38 59.55 ⫾ 9.15 22 ⫾ 1.61 43.29 ⫾ 5.86
37.52 ⫾ 16.65 56.11 ⫾ 9.69 15.42 ⫾ 2.71 33.40 ⫾ 10.57
⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
Results are reported as means ⫾ standard deviation. Note: Abbreviations: see text. Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
scope) equipped with a 75-mW krypton/argon mixed gas laser (BioRad, Hercules, CA), using an excitation wavelength of 488 nm and a 522/35 nm emission filter. To avoid spontaneous photoactivation of the probe, low intensity (5%) laser light excitation was used, as recommended by Roychowdhury et al. (19). Control incubations incorporated cells that had not been incubated with DAF-FM to ensure that neither the cells nor the treatments to which they were exposed resulted in spontaneous fluorescence at the excitation of emission wavelengths used with this probe. Western Blot Analysis Washed spermatozoa were lysed in RIPA lysis buffer containing 1⫻ PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 mg/mL phenylmethylsulfonyl fluoride (PMSF), aprotinin, 100 mM sodium orthovanadate, and 4% protease inhibitor cocktails by microcentrifugation at 10,000 ⫻ g for 10 minutes at 4°C. The supernatants were collected and treated with an equal volume of sample application buffer (125 mmol/L Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, 1% -mercaptoethanol). The mixture (approximately 40 g of protein) was boiled for 5 minutes; 15 L of each sample was applied to each well of an 8% SDS–polycrylamide gel and electrophoresed for 1 hour at 130 V along with a set of molecular weight markers (Broad Range, Sigma Chemical Co, St Louis, MO). The resolved protein bands were then transferred onto PVDF membranes at 100 V for 60 minutes using a transfer buffer of 25 mmol/L Tris base, 192 mmol/L glycine, and 20% methanol. The blots were blocked overnight at 4°C with blocking buffer (5% nonfat milk in 10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20). The blocking buffer was decanted and blots were incubated for 1 hour at room temperature with an antityrosine rabbit polyclonal antibody (Sigma) diluted 1:200 in 5% milk PBS/ Tween. Positive controls were included in all experiments as provided by the manufacturer to confirm antibody specificity. As an internal control, blots were reprobed with an anti--actin antibody (Sigma). Blots were then washed using TTBS (10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20) and incubated with horseradish peroxidaseFertility and Sterility姞
conjugated antirabbit IgG (1:5,000; Sigma) for 1 hour at room temperature following washes in TTBS. Peroxidase activity was revealed using 3,3=-diaminobenzidine (Sigma) as a substrate. Statistical Analysis Statistical analysis was performed using the SAS statistical package (Statistical Analysis System Institute, Cary, NC). Results are reported as mean ⫾ standard deviation. Differences among the groups evaluated by t test and the KolmogorovSmirnov test (K-S test) was used to determine whether the data were random samples from a normal distribution. Finally, the linear dependence was measured using Pearson’s correlation coefficient. RESULTS Although semen features of both normospermic control donors and asthenozoospermic patients were determined according WHO 1999 criteria (15), it is interesting to note that kinetic parameters of the two populations determined by CASA differed significantly (Table 1). The mean values of the seminal peroxynitrite concentration of all subjects enrolled in the study was 22.30 ⫾ 9.78
TABLE 2 Peroxynitrite (ONOOⴚ) seminal concentrations of patients considered on the basis of sperm motility. Patients Normospermic Asthenozoospermic
ONOOⴚ concentration (nmol/106 cells) 9.11 ⫾ 3.37 27.46 ⫾ 5.77a
Note: Results are reported as means ⫾ standard deviation. a P⬍.0001. Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
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FIGURE 1 Two-class plot showing single peroxynitrite (ONOO⫺) seminal level in each donor.
Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
nmol/106 cells. The Kolmogorov-Smirnov test allowed the acceptance of the null hypothesis concerning the normal distribution of the values in the two groups, and the subsequent statistical analysis showed that normospermic fertile donors exhibited peroxynitrite concentrations significantly lower than those of asthenozoospermic infertile men (9.11 ⫾ 3.37 vs. 27.46 ⫾ 5.77 nmol/106 cells; P⬍.0001) (Table 2). Single peroxynitrite seminal levels of each donor are shown in a two-class plot in Figure 1. To further delineate the presence of peroxynitrite in sperms samples, DAF was used to show the presence of this radical using a confocal microscopy imaging technique. Figure 2
FIGURE 2 Confocal images of DAF-labeled spermatozoa from fertile control donors and infertile asthenozoospermic patients. (A and C) Negative controls (without the probe) from normospermic and asthenozoospermic men, respectively. (B and D) Labeled spermatozoa from fertile donors and infertile asthenozoospermic patients, respectively.
shows that peroxynitrite was more evident in spermatozoa of asthenozoospermic patients than in normospermic donors. When all the enrolled subjects were considered, a significant negative correlation was found between peroxynitrite concentration and percentage of total sperm motility ( ⫽ ⫺0.448; P⬍.0001) (Fig. 3A). Furthermore, the CASA evaluation of sperm kinetic features revealed a significant linear negative correlation between peroxynitrite concentration and VCL ( ⫽ ⫺0.598; P⬍.0001) (Fig. 3B), VSL ( ⫽ ⫺0.579; P⬍.0001) (Fig. 3C), and LIN ( ⫽ ⫺0.399; P⬍.0001) (Fig. 3D). It is noteworthy that in all scatter plots of Figure 3, normospermic donors (white squares) exhibited consistently lower levels of peroxynitrite and higher values of kinetics parameters when compared to asthenozoospermic infertile patients (black circles). This situation is so evident that it reproduces, from a statistical point of view, the phenomenon of the spurious correlation. Tyrosine nitration of sperm protein was also investigated by Western Immuno Blot analysis. An increase in the nitration of the tyrosine residues in the asthenozoospermic samples was found compared to controls (Fig. 4) (prestained standard size markers and -actin control; data not shown). DISCUSSION Peroxynitrite formed in vivo from O⫺ 2 and NO can mediate oxidation, nitration, or nitrosation reaction, leading to an impaired function, toxicity, and alterations in signaling pathways (12). Oxidation reactions of peroxynitrite include DNA damage, thiyl radical formation, depletion of thiol pools, as well as lipid peroxidation and hydroxylation of phenols. Nitration reactions are predominantly nitration of phenols, such as nitration of tyrosine residues in proteins (20).
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Proteins tyrosine nitration may interfere with phosphorylation/dephosphorylation signaling or alter protein function (21). Peroxynitrite can also cause nitrosation reactions (22). Although at the level of the whole organism, the reactive chemistry of peroxynitrite can be considered beneficial, because of its cytotoxicity to bacteria or other invading organisms, the formation of protein 3-nitrotyrosine, in vivo, has been shown in a number of inflammatory conditions in human and experimental animals (23). Vol. 85, No. 4, April 2006
FIGURE 3 Negative linear correlations between peroxynitrite (ONOO⫺) concentration and (A) total sperm motility, (B) curvilinear velocity (VCL), (C) straight progressive velocity (VSL), and (D) linearity coefficient (LIN) in asthenozoospermic and normospermic donors.
Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
In a previous work we showed that NO concentrations in semen samples of infertile patients affected by asthenozoospermia are significant higher than those in normozoospermic fertile subjects, and that the overproduction of this free radical and the consequent excessive exposure to oxidative Fertility and Sterility姞
conditions have a potential pathogenic role in the reduction of sperm motility (9). Because peroxynitrite is formed in a rapid reaction from NO and O⫺ 2 , we can speculate that an increase in NO 951
FIGURE 4 Western blot analysis of nitrotyrosine protein expression in both asthenozoospermic (Asteno) and normozoospermic (C ) men. ⫹ ⫽ positive nitrotyrosine control.
Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
production will lead to a subsequent increase of peroxynitrite. At physiological pH, the relative stability of peroxynitrite allows it to diffuse for a considerable distance on a cellular scale and to even cross cell membrane (10). In the present study, peroxynitrite has been detected by two separate methods, in the semen of both studied groups. In the first method, nonfluorescent DCFDA has been shown to be a useful tool because it is nonpolar, may easily cross the cell membrane, and, once inside the cell, DCFDA is hydrolyzed by cytosolic esterases to the hydrophilic fluorescent DCF. Our results show that peroxynitrite is significantly higher in the idiopathic asthenozoospermic infertile patients than in the control group of normozoospermic fertile men. Similar to DCFH-DA, DAF-2-DA can permeate rapidly into the cells where it is hydrolyzed to DAF-2 by intracellular esterases (24). The confocal microscopy imaging evidence of the generation of peroxynitrite by DAF-mediated fluorescence is in full accordance with the hypothesis that sperm cells can generate the oxidant when NO · or O⫺ 2 dominates. Because the leukocyte concentration was less than 1 ⫻ 106/mL in each sample, it is unlikely that it is the main source of peroxynitrite in the semen. The significant negative linear correlation between peroxynitrite concentration and spermatozoa kinetic features of sperm cells (total motility, VCL, VSL, and LIN) is the most striking features of the present study, as it strongly suggests a deleterious effect of high peroxynitrite levels on sperm cell function. The detection of 3-nitrotyrosine, a covalent post-translational protein modification, by analytical and immunological techniques has established that tyrosine nitration occurs under physiological conditions and levels increase in most disease states. The nitration of free tyrosine or protein tyrosine residues has been used as a footprint for the in vivo formation of peroxynitrite (25). Numerous reports have shown that nitration is a biological process derived from the biochemical interaction of NO or NO-derived secondary products with reactive oxygen spe952
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cies (23). It has been demonstrated that tyrosine nitration of proteins occurs in normal muscle fibers and it is dependent mainly on the activity of the inducible Nitric Oxide Synthase (iNOS) isoform (26). In our previous study (9) we showed, by Western immunoblot, the presence in semen samples of iNOS, which was more pronounced in the asthenozoospermic infertile patients. Although the constitutive NO isoforms (which produce small amounts of NO) are beneficial, activation of the inducible isoform of NO (which produces much more NO) causes injury, also because of its toxicity is greatly enhanced when it combines with O⫺ 2 to generate peroxynitrite. Detection of nitrotyrosine at inflammatory sites serves as a biochemical marker for peroxynitrite formation. The present data show an increase of the nitrotyrosine band in the asthenozoospermic sample, and are in full accordance with the data reported here. In fact, the presence of iNOS is in agreement with the notion that NO generated by iNOS may contribute to the production of nitrotyrosine by peroxynitrite. Because the present work shows that an excess of peroxynitrite produced by spermatozoa can negatively modulate cell function, we hypothesize that peroxynitrite can interfere in the post-translational modifications of proteins through either phosphorylation or nitration. These modifications 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. In this regard, it has been demonstrated that peroxynitrite-promoted nitration may exert physiological effects by altering kinase reactions and subsequent signaling pathways (27). In conclusion, this is the first report underlying a possible association between protein tyrosine nitration and the impairment of sperm function. We provide evidence that higher levels of peroxynitrite are produced in sperm cells of idiopathic asthenozoospermic infertile when compared to normospermic fertile donors and peroxynitrite concentration is inversely related to sperm cells motility. As a consequence of high peroxynitrite levels, an excess of spontaneous tyrosine nitration can occur in human spermatozoa proteins and it could be a co-factor in affecting sperm cell function. Such a biochemical mechanism may play a pathogenic role in infertile men when asthenozoospermia is the main problem. REFERENCES 1. Aitken RJ, Buckingham DW, Carreras A, Irvine DS. Superoxide dismutase in human sperm suspensions: relationship with cellular composition, oxidative stress, and sperm function. Free Radic Biol Med 1996;21:495–504. 2. MacLeod J. The role of oxygen in the metabolism and motility of human spermatozoa. Am J Physiol 1943;138:512– 8. 3. Tosic J, Walton A. Formation of hydrogen peroxyde by spermatozoa and its inhibitory effect on respiration. Nature 1946;58:485. 4. Aitken RJ, Harkiss D, Buckingham DW. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 1993;35:302–15. 5. de Lamirande E, Gagnon C. A positive role for the superoxide anion in
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triggering hyperactivation and capacitation of human spermatozoa. Int J Androl 1993;16:21–5. Oehninger S, Blackmore P, Mahony M, Hodgen G. Effects of hydrogen peroxide on human spermatozoa. J Assist Reprod Genet 1995;12:41–7. Zalata AA, Christophe AB, Depuydt CE, Schoonjans F, Comhaire FH. The fatty acid composition of phospholipids of spermatozoa from infertile patients. Mol Hum Reprod 1998;4:111– 8. Reiter CD, Teng RJ, Beckman JS. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. J Biol Chem 2000;275:32460 – 6. Balercia G, Moretti S, Vignini A, Magagnini M, Mantero F, Boscaro M, et al. Role of nitric oxide concentrations on human sperm motility. J Androl 2004;25:245–9. Beckman JS, Chen J, Ischiropoulos H, Crow JP. Oxidative chemistry of peroxynitrite. Methods Enzymol 1994;233:229 – 40. Ischiropoulos H. Biological selectivity and functional aspects of protein tyrosine nitration. Biochem Biophys Res Commun 2003;305:776 – 83. Radi R. Nitric oxide, oxidants, and protein tyrosine nitration. Proc Natl Acad Sci USA 2004;101:4003– 8. Oztezcan S, Turkoglu UM, Kervancioglu E, Kocak T, Kocak-Toker N, Aykac-Toker G. In vitro effects of peroxynitrite on human spermatozoa. Andrologia 1999;31:195– 8. Herrero MB, de Lamirande E, Gagnon C. Tyrosine nitration in human spermatozoa: a physiological function of peroxynitrite, the reaction product of nitric oxide and superoxide. Mol Hum Reprod 2001;7:913–21. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge: Cambridge University Press, 1999. Balercia G, Mosca F, Mantero F, Boscaro M, Mancini A, RicciardoLamonica G, Littarru G. Coenzyme Q(10) supplementation in infertile men with idiopathic asthenozoospermia: an open, uncontrolled pilot study. Fertil Steril 2004;81:93– 8.
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17. Kooy NW, Royall JA, Ischiropoulos H. Oxidation of 2=,7=dichlorofluorescin by peroxynitrite. Free Radic Res 1997;27:245–54. 18. Tannous M, Rabini RA, Vignini A, Moretti N, Fumelli P, Zielinski B, et al. Evidence for iNOS-dependent peroxynitrite production in diabetic platelets. Diabetologia 1999;42:539 – 44. 19. Roychowdhury S, Luthe A, Keilhoff G, Wolf G, Horn TF. Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide. Glia 2002;38:103–14. 20. Aulak KS, Koeck T, Crabb JW, Stuehr DJ. Dynamics of protein nitration in cells and mitochondria. Am J Physiol Heart Circ Physiol 2004;286:H30 – 8. 21. Monteiro HP. Signal transduction by protein tyrosine nitration: competition or cooperation with tyrosine phosphorylation-dependent signaling events? Free Radic Biol Med 2002;33:765–73. 22. Arteel GE, Briviba K, Sies H. Protection against peroxynitrite. FEBS Lett 1999;445:226 –30. 23. Ischiropoulos H. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998;356:1–11. 24. Silveira LR, Pereira-Da-Silva L, Juel C, Hellsten Y. Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions. Free Radic Biol Med 2003;35:455– 64. 25. Greenacre SA, Ischiropoulos H. Tyrosine nitration: localisation, quantification, consequences for protein function and signal transduction. Free Radic Res 2001;34:541– 81. 26. Fries DM, Paxinou E, Themistocleous M, Swanberg E, Griendling KK, Salvemini D, et al. Expression of inducible nitric-oxide synthase and intracellular protein tyrosine nitration in vascular smooth muscle cells: role of reactive oxygen species. J Biol Chem 2003;278:22901–7. 27. Kong SK, Yim MB, Stadtman ER, Chock PB. Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide. Proc Natl Acad Sci USA 1996;93:3377– 82.
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Institute of Biochemistry, School of Medicine, Polytechnic University of Marche, Ancona; b Andrology Unit, Endocrinology Division, Department of Internal Medicine, Umberto I Hospital, School of Medicine, Polytechnic University of Marche, Ancona; c Department of Economy, School of Economy, Polytechnic University of Marche, Ancona; and d Endocrinology Division, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy
Objective: To detect peroxynitrite and 3-nitrotyrosine production in human spermatozoa of asthenozoospermic infertile patients and normospermic donors, and evaluate any influence on kinetic sperm features. Design: Basic study. Setting: Andrology Unit, Dept of Internal Medicine and Biochemistry Institute, Polytechnic University of Marche, Italy. Patient(s): Sixry-nine infertile patients affected by idiopathic asthenozoospermia and 29 normal fertile donors. Intervention(s): No therapeutic intervention was performed on patients. Main Outcome Measure(s): Production of peroxynitrite (ONOO⫺) and 3-nitrotyrosine by human spermatozoa; kinetic sperm cells parameters. Result(s): Normospermic fertile donors exhibited ONOO⫺ concentrations significantly lower than those of asthenozoospermic infertile men (9.11 ⫾ 3.37 vs. 27.46 ⫾ 5.77 nmol/106 cells); confocal microscopy showed that ONOO⫺ was more evident in spermatozoa of patients than in healthy donors. Moreover, a significant negative correlation was evident between ONOO⫺ concentration and total sperm motility, curvilinear velocity (VCL), straight progressive velocity (VSL), and linearity coefficient. Finally, an increase was found in the nitration of the tyrosine residues in asthenozoospermic samples compared to controls. Conclusion(s): Spontaneous tyrosine nitration occurs in human spermatozoa. This post-translational protein modification is enhanced by an overproduction of peroxynitrite, which is more evident in asthenozoospermic infertile patients when compared with normospermic fertile donors. Motility parameters are negatively affected, suggesting that ONOO⫺ may be involved in defective sperm function. (Fertil Steril威 2006;85:947–53. ©2006 by American Society for Reproductive Medicine.) Key Words: Peroxynitrite, 3-nitrotyrosine, male infertility, sperm cells motility
Defective sperm function is known as one of the common causes of human infertility. There are many studies suggesting a relationship between reactive oxygen species and sperm function impairment (1). In 1943, John MacLeod (2) noted that spermatozoa incubated under high oxygen tensions rapidly lost their motility, and 3 years later it was demonstrated, for the first time, that reactive oxygen species were actively generated in mammalian spermatozoa (3). Several independent investigators have further confirmed these data (4 – 6). Human spermatozoa are extremely sensitive to oxidative attack because they contain high amounts of polyunsaturated fatty acids of phospholipids, particularly C22:6 and are constantly exposed to reactive oxygen species generated by contaminating neutrophils or by the spermatozoa themselves. Spermatozoa are also susceptible to oxida-
Received January 30, 2005; revised and accepted September 19, 2005. Reprint requests: G. Balercia, M.D., Endocrinology, Department of Internal Medicine, Umberto I Hospital, Torrette, Via Conca, 60100 Ancona, Italy (FAX: 39-071-887-300; E-mail: [email protected]).
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tive stress because, due to the paucity of cytoplasm, they lack an adequate reserve of defensive enzymes such as superoxide dismutase, which protect most somatic cells from oxidative attack (7). It has been speculated that peroxynitrite (ONOO⫺) formation is a primary pathway of nitric oxide (NO · ) metabolism (8). Because we have previously shown that human spermatozoa produce NO˙ as well as O⫺ 2 (9), it is reasonable to predict that formation of peroxynitrite can occur in vivo. The unusual stability of peroxynitrite as an anion contributes to its toxicity by allowing it to diffuse far from its site of formation while being selectively reactive with cellular targets (10). In fact, peroxynitrite reacts rapidly with proteins, lipids, and nucleic acids. Moreover, the nitration (addition of an NO2 group) of protein residues gives rise to 3-nitrotyrosine, which represents a protein modification specific for peroxynitrite formation, in vivo, and a widely used marker of its determination. The occurrence of protein tyrosine nitration under pathological conditions is now firmly established and repre-
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sents a shift from the signal transducing physiological actions of NO to oxidative and potentially pathogenic pathways (11). The precise interplay between NO, oxidants, and the identification of the intermediates responsible for nitration in vivo is still under controversy (12). A recent study indicates that peroxynitrite might cause sperm dysfunction by increasing lipid peroxidation and total sulfydryl groups depletion (13). In addition, Herrero et al. (14) have provided evidence that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of peroxynitrite modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process. In the present study we determined peroxynitrite production in semen and its correlation with kinetic features in spermatozoa, and set out to determine whether protein tyrosine nitration takes place in the same sample to elucidate any pathogenic involvement in sperm cells motility. MATERIALS AND METHODS Patient Selection We evaluated 29 normozoospermic fertile healthy men (control group, aged 27–38 years) and 69 patients (aged 25–37 years) affected by idiopathic asthenozoospermia (sperm concentration ⬎20 ⫻ 106/mL; forward motility grade a ⫹ b ⬍50%, according to World Health Organization [WHO] 1999 criteria) (15) referred to the Andrology Unit of the Division of Endocrinology, Umberto I Hospital, Politechnic University of Marche, Italy, for infertility (of at least 24 months). All subjects in the study showed a normal sperm morphology greater than 30% and a seminal white blood cell count less than 1 ⫻ 106/mL. Sperm culture was negative, including Chlamydia and Mycoplasma ureoliticum detection, as well as the Mar-test. Hormonal serum profile (gonadotropins, T, E2, and PRL) was normal. There was no evidence of anatomical abnormalities of the genital tract, including varicocele (after Doppler sonography), nor was there a history of chriptorchidism, testicular torsion or genital tract infection. The absence of systemic disease or treatment with other drugs within 3 months before enrolment in the present study, the absence of smoking, alcohol, and drug addiction, and the absence of occupational chemical exposure were also verified. Because no causes for motility reduction have been discovered, a conclusive diagnosis of idiopathic asthenozoospermia was reached. This study was approved by the Istitutional Review Board of the Politechnical Marche University, Umberto I Hospital. All patients provided their written informed consent. Semen Analysis Semen samples were collected after 3 days of sexual abstinence. After liquification at room temperature for 30 minutes, standard seminal parameters were analyzed according to WHO 1999 (15) guidelines. Computer-assisted sperm 948
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analysis (CASA) for sperm motility assay was additionally performed as previously reported (16). One semen aliquot (3 L) was placed in a 20-m-deep cell UV chamber (Conception Technologies, La Jolla, CA). Two chambers were loaded, six different fields per chambers were randomly examined, and at least 200 spermatozoa for each field of chambers were scored. Movement characteristics were analyzed using an automated analyser (CellTrack VP110, Motion Analysis Corp, Palo Alto, CA). Kinetic characteristics were evaluated only for motile sperm and expressed as mean values considering total sperm motility (percent), curvilinear velocity (VCL, in m/s), straight progressive velocity (VSL, in m/s), and linearity coefficient (LIN ⫽ VSL/ VCL ⫻ 100). Determination of Peroxynitrite Levels in Semen For peroxynitrite level evaluation, individual sperm samples were diluted to 5 ⫻ 106/mL with Dulbecco phosphatebuffered saline (PBS) (20 mM, pH 7.4), aliquoted to form two replicates and stored at ⫺80°C in sterile tubes until peroxynitrite measurements were performed within 15 days. Peroxynitrite production was determined by the 2,7dichlorofluorescein (DCF) fluorimetric assay as previously described by Kooy et al. (17) and by Tannous et al. (18). DCFDA is a membrane-permeable nonfluorescent dye that is hydrolyzed by esterases, within the sperm cytoplasm, into a free acid, DCFH. ONOO⫺ oxidizes DCFH to a strongly fluorescent dye (17). A DCFDA-free base was prepared by mixing 0.05 mL of 10 mmol/L DCFDA with 2 mL of 0.01 N NaOH at room temperature for 30 minutes. The mixture was neutralized with 18.0 mL of 25 mmol/L PBS at pH 7.4. This solution was maintained on ice in the dark until use. The DCFDAtreated samples were incubated in NO buffer with 100 M L-arginine for 15 minutes at 37°C in a dark room. The mixture was washed with PBS at pH 7.4 then centrifuged for 2 minutes at 214 ⫻ g. Supernatant fluorescence was measured in a Perkin-Elmer MPF-66 spectrofluorometer (Beaconsfield, UK) at an excitation wavelength of 475 nm and emission wavelength of 520 nm. DAF-FM Fluorescence DAF-FM acetate is a membrane-permanent probe that is capable of detecting ONOO⫺ with high sensitivity. Although this probe has also been reported to detect NO, the levels of this radical required to activate DAF fluorescence are beyond the limits found in most biological tissues (⬎7.7 M) (19). The probe was made up as 1-mM stock solution in dimethyl sulfoxide (DMSO) and added to sperm suspensions to give a final concentration of 10 M. The cells were then incubated for 60 minutes at 37°C before being centrifuged at 300 ⫻ g and resuspended in PBS. Confocal microscopy was conducted by using BioRad, MRC 1024 UV confocal laser scanning microscope (Nikon Diaphot inverted microVol. 85, No. 4, April 2006
TABLE 1 The t test results for asthenozoospermic patients and control group.
% Mot Tot. VCL VSL LIN
Normospermic
Asthenozoospermic
P value
63.85 ⫾ 7.38 59.55 ⫾ 9.15 22 ⫾ 1.61 43.29 ⫾ 5.86
37.52 ⫾ 16.65 56.11 ⫾ 9.69 15.42 ⫾ 2.71 33.40 ⫾ 10.57
⬍.0001 ⬍.0001 ⬍.0001 ⬍.0001
Results are reported as means ⫾ standard deviation. Note: Abbreviations: see text. Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
scope) equipped with a 75-mW krypton/argon mixed gas laser (BioRad, Hercules, CA), using an excitation wavelength of 488 nm and a 522/35 nm emission filter. To avoid spontaneous photoactivation of the probe, low intensity (5%) laser light excitation was used, as recommended by Roychowdhury et al. (19). Control incubations incorporated cells that had not been incubated with DAF-FM to ensure that neither the cells nor the treatments to which they were exposed resulted in spontaneous fluorescence at the excitation of emission wavelengths used with this probe. Western Blot Analysis Washed spermatozoa were lysed in RIPA lysis buffer containing 1⫻ PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 10 mg/mL phenylmethylsulfonyl fluoride (PMSF), aprotinin, 100 mM sodium orthovanadate, and 4% protease inhibitor cocktails by microcentrifugation at 10,000 ⫻ g for 10 minutes at 4°C. The supernatants were collected and treated with an equal volume of sample application buffer (125 mmol/L Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 0.003% bromophenol blue, 1% -mercaptoethanol). The mixture (approximately 40 g of protein) was boiled for 5 minutes; 15 L of each sample was applied to each well of an 8% SDS–polycrylamide gel and electrophoresed for 1 hour at 130 V along with a set of molecular weight markers (Broad Range, Sigma Chemical Co, St Louis, MO). The resolved protein bands were then transferred onto PVDF membranes at 100 V for 60 minutes using a transfer buffer of 25 mmol/L Tris base, 192 mmol/L glycine, and 20% methanol. The blots were blocked overnight at 4°C with blocking buffer (5% nonfat milk in 10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20). The blocking buffer was decanted and blots were incubated for 1 hour at room temperature with an antityrosine rabbit polyclonal antibody (Sigma) diluted 1:200 in 5% milk PBS/ Tween. Positive controls were included in all experiments as provided by the manufacturer to confirm antibody specificity. As an internal control, blots were reprobed with an anti--actin antibody (Sigma). Blots were then washed using TTBS (10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween 20) and incubated with horseradish peroxidaseFertility and Sterility姞
conjugated antirabbit IgG (1:5,000; Sigma) for 1 hour at room temperature following washes in TTBS. Peroxidase activity was revealed using 3,3=-diaminobenzidine (Sigma) as a substrate. Statistical Analysis Statistical analysis was performed using the SAS statistical package (Statistical Analysis System Institute, Cary, NC). Results are reported as mean ⫾ standard deviation. Differences among the groups evaluated by t test and the KolmogorovSmirnov test (K-S test) was used to determine whether the data were random samples from a normal distribution. Finally, the linear dependence was measured using Pearson’s correlation coefficient. RESULTS Although semen features of both normospermic control donors and asthenozoospermic patients were determined according WHO 1999 criteria (15), it is interesting to note that kinetic parameters of the two populations determined by CASA differed significantly (Table 1). The mean values of the seminal peroxynitrite concentration of all subjects enrolled in the study was 22.30 ⫾ 9.78
TABLE 2 Peroxynitrite (ONOOⴚ) seminal concentrations of patients considered on the basis of sperm motility. Patients Normospermic Asthenozoospermic
ONOOⴚ concentration (nmol/106 cells) 9.11 ⫾ 3.37 27.46 ⫾ 5.77a
Note: Results are reported as means ⫾ standard deviation. a P⬍.0001. Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
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FIGURE 1 Two-class plot showing single peroxynitrite (ONOO⫺) seminal level in each donor.
Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
nmol/106 cells. The Kolmogorov-Smirnov test allowed the acceptance of the null hypothesis concerning the normal distribution of the values in the two groups, and the subsequent statistical analysis showed that normospermic fertile donors exhibited peroxynitrite concentrations significantly lower than those of asthenozoospermic infertile men (9.11 ⫾ 3.37 vs. 27.46 ⫾ 5.77 nmol/106 cells; P⬍.0001) (Table 2). Single peroxynitrite seminal levels of each donor are shown in a two-class plot in Figure 1. To further delineate the presence of peroxynitrite in sperms samples, DAF was used to show the presence of this radical using a confocal microscopy imaging technique. Figure 2
FIGURE 2 Confocal images of DAF-labeled spermatozoa from fertile control donors and infertile asthenozoospermic patients. (A and C) Negative controls (without the probe) from normospermic and asthenozoospermic men, respectively. (B and D) Labeled spermatozoa from fertile donors and infertile asthenozoospermic patients, respectively.
shows that peroxynitrite was more evident in spermatozoa of asthenozoospermic patients than in normospermic donors. When all the enrolled subjects were considered, a significant negative correlation was found between peroxynitrite concentration and percentage of total sperm motility ( ⫽ ⫺0.448; P⬍.0001) (Fig. 3A). Furthermore, the CASA evaluation of sperm kinetic features revealed a significant linear negative correlation between peroxynitrite concentration and VCL ( ⫽ ⫺0.598; P⬍.0001) (Fig. 3B), VSL ( ⫽ ⫺0.579; P⬍.0001) (Fig. 3C), and LIN ( ⫽ ⫺0.399; P⬍.0001) (Fig. 3D). It is noteworthy that in all scatter plots of Figure 3, normospermic donors (white squares) exhibited consistently lower levels of peroxynitrite and higher values of kinetics parameters when compared to asthenozoospermic infertile patients (black circles). This situation is so evident that it reproduces, from a statistical point of view, the phenomenon of the spurious correlation. Tyrosine nitration of sperm protein was also investigated by Western Immuno Blot analysis. An increase in the nitration of the tyrosine residues in the asthenozoospermic samples was found compared to controls (Fig. 4) (prestained standard size markers and -actin control; data not shown). DISCUSSION Peroxynitrite formed in vivo from O⫺ 2 and NO can mediate oxidation, nitration, or nitrosation reaction, leading to an impaired function, toxicity, and alterations in signaling pathways (12). Oxidation reactions of peroxynitrite include DNA damage, thiyl radical formation, depletion of thiol pools, as well as lipid peroxidation and hydroxylation of phenols. Nitration reactions are predominantly nitration of phenols, such as nitration of tyrosine residues in proteins (20).
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Proteins tyrosine nitration may interfere with phosphorylation/dephosphorylation signaling or alter protein function (21). Peroxynitrite can also cause nitrosation reactions (22). Although at the level of the whole organism, the reactive chemistry of peroxynitrite can be considered beneficial, because of its cytotoxicity to bacteria or other invading organisms, the formation of protein 3-nitrotyrosine, in vivo, has been shown in a number of inflammatory conditions in human and experimental animals (23). Vol. 85, No. 4, April 2006
FIGURE 3 Negative linear correlations between peroxynitrite (ONOO⫺) concentration and (A) total sperm motility, (B) curvilinear velocity (VCL), (C) straight progressive velocity (VSL), and (D) linearity coefficient (LIN) in asthenozoospermic and normospermic donors.
Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
In a previous work we showed that NO concentrations in semen samples of infertile patients affected by asthenozoospermia are significant higher than those in normozoospermic fertile subjects, and that the overproduction of this free radical and the consequent excessive exposure to oxidative Fertility and Sterility姞
conditions have a potential pathogenic role in the reduction of sperm motility (9). Because peroxynitrite is formed in a rapid reaction from NO and O⫺ 2 , we can speculate that an increase in NO 951
FIGURE 4 Western blot analysis of nitrotyrosine protein expression in both asthenozoospermic (Asteno) and normozoospermic (C ) men. ⫹ ⫽ positive nitrotyrosine control.
Vignini. Peroxynitrite may affect sperm motility. Fertil Steril 2006.
production will lead to a subsequent increase of peroxynitrite. At physiological pH, the relative stability of peroxynitrite allows it to diffuse for a considerable distance on a cellular scale and to even cross cell membrane (10). In the present study, peroxynitrite has been detected by two separate methods, in the semen of both studied groups. In the first method, nonfluorescent DCFDA has been shown to be a useful tool because it is nonpolar, may easily cross the cell membrane, and, once inside the cell, DCFDA is hydrolyzed by cytosolic esterases to the hydrophilic fluorescent DCF. Our results show that peroxynitrite is significantly higher in the idiopathic asthenozoospermic infertile patients than in the control group of normozoospermic fertile men. Similar to DCFH-DA, DAF-2-DA can permeate rapidly into the cells where it is hydrolyzed to DAF-2 by intracellular esterases (24). The confocal microscopy imaging evidence of the generation of peroxynitrite by DAF-mediated fluorescence is in full accordance with the hypothesis that sperm cells can generate the oxidant when NO · or O⫺ 2 dominates. Because the leukocyte concentration was less than 1 ⫻ 106/mL in each sample, it is unlikely that it is the main source of peroxynitrite in the semen. The significant negative linear correlation between peroxynitrite concentration and spermatozoa kinetic features of sperm cells (total motility, VCL, VSL, and LIN) is the most striking features of the present study, as it strongly suggests a deleterious effect of high peroxynitrite levels on sperm cell function. The detection of 3-nitrotyrosine, a covalent post-translational protein modification, by analytical and immunological techniques has established that tyrosine nitration occurs under physiological conditions and levels increase in most disease states. The nitration of free tyrosine or protein tyrosine residues has been used as a footprint for the in vivo formation of peroxynitrite (25). Numerous reports have shown that nitration is a biological process derived from the biochemical interaction of NO or NO-derived secondary products with reactive oxygen spe952
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cies (23). It has been demonstrated that tyrosine nitration of proteins occurs in normal muscle fibers and it is dependent mainly on the activity of the inducible Nitric Oxide Synthase (iNOS) isoform (26). In our previous study (9) we showed, by Western immunoblot, the presence in semen samples of iNOS, which was more pronounced in the asthenozoospermic infertile patients. Although the constitutive NO isoforms (which produce small amounts of NO) are beneficial, activation of the inducible isoform of NO (which produces much more NO) causes injury, also because of its toxicity is greatly enhanced when it combines with O⫺ 2 to generate peroxynitrite. Detection of nitrotyrosine at inflammatory sites serves as a biochemical marker for peroxynitrite formation. The present data show an increase of the nitrotyrosine band in the asthenozoospermic sample, and are in full accordance with the data reported here. In fact, the presence of iNOS is in agreement with the notion that NO generated by iNOS may contribute to the production of nitrotyrosine by peroxynitrite. Because the present work shows that an excess of peroxynitrite produced by spermatozoa can negatively modulate cell function, we hypothesize that peroxynitrite can interfere in the post-translational modifications of proteins through either phosphorylation or nitration. These modifications 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. In this regard, it has been demonstrated that peroxynitrite-promoted nitration may exert physiological effects by altering kinase reactions and subsequent signaling pathways (27). In conclusion, this is the first report underlying a possible association between protein tyrosine nitration and the impairment of sperm function. We provide evidence that higher levels of peroxynitrite are produced in sperm cells of idiopathic asthenozoospermic infertile when compared to normospermic fertile donors and peroxynitrite concentration is inversely related to sperm cells motility. As a consequence of high peroxynitrite levels, an excess of spontaneous tyrosine nitration can occur in human spermatozoa proteins and it could be a co-factor in affecting sperm cell function. Such a biochemical mechanism may play a pathogenic role in infertile men when asthenozoospermia is the main problem. REFERENCES 1. Aitken RJ, Buckingham DW, Carreras A, Irvine DS. Superoxide dismutase in human sperm suspensions: relationship with cellular composition, oxidative stress, and sperm function. Free Radic Biol Med 1996;21:495–504. 2. MacLeod J. The role of oxygen in the metabolism and motility of human spermatozoa. Am J Physiol 1943;138:512– 8. 3. Tosic J, Walton A. Formation of hydrogen peroxyde by spermatozoa and its inhibitory effect on respiration. Nature 1946;58:485. 4. Aitken RJ, Harkiss D, Buckingham DW. Analysis of lipid peroxidation mechanisms in human spermatozoa. Mol Reprod Dev 1993;35:302–15. 5. de Lamirande E, Gagnon C. A positive role for the superoxide anion in
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17. Kooy NW, Royall JA, Ischiropoulos H. Oxidation of 2=,7=dichlorofluorescin by peroxynitrite. Free Radic Res 1997;27:245–54. 18. Tannous M, Rabini RA, Vignini A, Moretti N, Fumelli P, Zielinski B, et al. Evidence for iNOS-dependent peroxynitrite production in diabetic platelets. Diabetologia 1999;42:539 – 44. 19. Roychowdhury S, Luthe A, Keilhoff G, Wolf G, Horn TF. Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide. Glia 2002;38:103–14. 20. Aulak KS, Koeck T, Crabb JW, Stuehr DJ. Dynamics of protein nitration in cells and mitochondria. Am J Physiol Heart Circ Physiol 2004;286:H30 – 8. 21. Monteiro HP. Signal transduction by protein tyrosine nitration: competition or cooperation with tyrosine phosphorylation-dependent signaling events? Free Radic Biol Med 2002;33:765–73. 22. Arteel GE, Briviba K, Sies H. Protection against peroxynitrite. FEBS Lett 1999;445:226 –30. 23. Ischiropoulos H. Biological tyrosine nitration: a pathophysiological function of nitric oxide and reactive oxygen species. Arch Biochem Biophys 1998;356:1–11. 24. Silveira LR, Pereira-Da-Silva L, Juel C, Hellsten Y. Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions. Free Radic Biol Med 2003;35:455– 64. 25. Greenacre SA, Ischiropoulos H. Tyrosine nitration: localisation, quantification, consequences for protein function and signal transduction. Free Radic Res 2001;34:541– 81. 26. Fries DM, Paxinou E, Themistocleous M, Swanberg E, Griendling KK, Salvemini D, et al. Expression of inducible nitric-oxide synthase and intracellular protein tyrosine nitration in vascular smooth muscle cells: role of reactive oxygen species. J Biol Chem 2003;278:22901–7. 27. Kong SK, Yim MB, Stadtman ER, Chock PB. Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide. Proc Natl Acad Sci USA 1996;93:3377– 82.
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