Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways

Animal Reproduction Science 85 (2005) 231–242

Nitric oxide-induced capacitation of cryopreserved bull spermatozoa and assessment of participating regulatory pathways P.C. Rodriguez, C.M. O’Flaherty, M.T. Beconi, N.B. Beorlegui∗ Biological Chemistry, School of Veterinary Sciences, University of Buenos Aires, Chorroar´ın 280, Buenos Aires C1427 CWO, Argentina Received 13 December 2003; received in revised form 29 April 2004; accepted 17 May 2004

Abstract The effect of nitric oxide (NO• ) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from four bulls were incubated in TALP medium in the presence of heparin (10 IU/ml) or sodium nitroprusside (SNP, 0.05–100 ␮M), a NO• donor. The participation of NO• was confirmed by the use of scavengers, i.e. methylene blue (50 100 ␮M) and hemoglobin (20–40 ␮g/ml). The role of nitric oxide synthase in heparin-induced capacitation was evaluated using enzyme inhibitors N␻-nitro-l-arginine methyl ester (l-NAME) and N␻-nitro-l-arginine (l-NA) in concentrations ranging from 1 to 500 ␮M. The effects of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK), on NO• -induced capacitation were evaluated by incubation with specific inhibitors of these enzymes (H-89, 50 ␮M; bisindolylmaleimide I, 0.1 ␮M and genistein, 3 ␮M). The role of hydrogen peroxide or superoxide anion in NO• -induced capacitation was evaluated by incubation with catalase (20–100 ␮g/ml) or superoxide dismutase (SOD, 0.05–0.5 mg/ml), respectively. Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC). SNP concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of the 0.05 ␮M SNP treatment (31 ± 5.15%) were similar to those of heparin treated samples (33 ± 4.27%). Inhibitors of nitric oxide synthase (NOS) diminished capacitation percentages in a dose-dependent manner as did the addition of NO• scavengers (P < 0.05). The presence of PKA, PKC and PTK inhibitors likewise decreased capacitation percentages (6.25 ± 0.71, 12.75 ± 1.41, 9.00 ± 1.41%, respectively). The presence of catalase or SOD in the incubation medium had no effect on capacitation percentages. These results indicate that NO• may be generated by a sperm NOS during heparin-induced capacitation and that exogenous NO• ∗ Corresponding author. Present address: Area de Qu´ımica Biol´ ogica, Facultad de Ciencias Veterinarias, University of Buenos Aires, Chorroar´ın 280, Buenos Aires C1427 CWO, Argentina. Tel.: +54-11-4524-8452; fax: +54-11-4524-8452. E-mail address: [email protected] (N.B. Beorlegui).

0378-4320/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2004.05.018

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acts as a capacitation inducer and involves the participation of PKA, PKC and PTK as part of the intracellular mechanisms that lead to capacitation in cryopreserved bull spermatozoa. © 2004 Elsevier B.V. All rights reserved. Keywords: Cryopreserved bull spermatozoa; Nitric oxide; Sperm capacitation; Protein kinases

1. Introduction Mammalian spermatozoa require a period of preparation defined as capacitation to acquire the capacity to fertilize mature oocytes. During this process, changes take place in the fluidity of the plasma membrane, intracellular ionic concentration, energy metabolism, phosphorylation of proteins in tyrosine and modifications in the activity of several enzymes such as protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK). This process is followed by an exocytotic event termed the acrosome reaction that is an absolute prerequisite for the achievement of fertilization (Yanagimachi, 1994). There is evidence that capacitation is part of an oxidative process and that the superoxide anion (O2 •− ) is required to induce capacitation in human (de Lamirande and Gagnon, 1993) and bull spermatozoa (O’Flaherty et al., 1997), while H2 O2 is involved in the capacitation in hamster (Bize et al., 1991) and human spermatozoa (Griveau et al., 1994). In diverse cell types, the intracellular redox state is important in regulation of enzymes presumably involved in signal transduction mechanisms (Aitken et al., 1995; Hancok et al., 2001). At physiological concentrations, O2 •− , H2 O2 and nitric oxide (NO• ) are considered the major reactive species that participate in redox reactions in diverse biological processes (Hancok et al., 2001). Nitric oxide is a biologically active gas generated by almost all the cells of the organism (Nathan, 1992) that, due to its low molecular weight and lipophilic nature, diffuses quickly through lipid membranes and is implicated in a variety of physiological cellular signalling mechanisms in many tissues by activating guanylyl cyclase (Lowenstein et al., 1994). NO• is synthesized in vivo during the conversion of l-arginine to l-citruline by the enzyme nitric oxide synthase (NOS) using O2 and NADPH as substrates (Moncada and Higgs, 1993). Three isoforms of the enzyme have been described, two constituent and calcium dependent (i.e. nNOS expressed mainly in CNS neuronal and peripheral cells and eNOS identified in the vascular endothelium) and an inducible, calcium-independent isoform (iNOS, mainly synthesized in macrophages; Marletta, 1993). NOS has been detected in the human epididymis, testicle (Zini et al., 1996) and uterus (Telfer et al., 1995) suggesting that it could be involved in sperm maturation and capacitation. The presence of NOS has also been shown in human, mouse and ram spermatozoa (Lewis et al., 1996; Herrero et al., 1996; Funahashi, 2002). NOS inhibitors have proven essential to evaluate the role of NO• in physiological processes. Most l-arginine analogs, such as N␻-nitro-l-arginine (l-NA) or N␻-nitro-l-arginine methyl ester (l-NAME) are specific inhibitors of constituent and inducible NOS forms, whereas aminoguanidine (AG) selectively inhibits inducible NOS (Joly et al., 1994). In experimental systems, hemoglobin functions as a NO• scavenger when

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binding to the heme group (Herrero et al., 1994) and methylene blue blocks its effects by acting on guanylyl cyclase (Donnelly et al., 1997). The magnitude and duration of nitric oxide synthesis by cells determine whether its action is pathological or physiological. High concentrations of NO• lead to decreased motility and viability in human spermatozoa (Weinberg et al., 1995), attributable to NO• capacity to interact with the Fe–S center of aconitase and of some enzymes of the mitochondrial electron transport chain, such as NADH dehydrogenase or succinate oxidoreductase, altering energy metabolism and cellular respiration (Castro et al., 1994). At low NO• concentrations, a significant increase in capacitation was observed in human spermatozoa (Herrero et al., 1999) that was inhibited by catalase, indicating that H2 O2 would be necessary for the action of NO• (Zini et al., 1995). Mechanisms that control sperm capacitation have not been completely clarified but the process is regulated by several signal transduction elements that involve PKA (Leclerc et al., 1996), PKC (Thundathil et al., 2002) and PTK (Leclerc et al., 1997; Herrero et al., 1997). A better understanding of the effects of nitric oxide on motility and viability and its participation in the capacitation process will contribute to clarify intracellular mechanisms in order to improve the conditions required to optimize in vitro fertilization procedures. The objective of the present research was to determine the effect of NO• in the capacitation of cryopreserved bull spermatozoa, and whether the presence of other reactive oxygen species and the participation of protein kinases are necessary in this process.

2. Materials and methods 2.1. Semen freezing Semen was collected by artificial vagina from four Holstein bulls (4–5 years old) of proven fertility. The bulls belonged to a controlled program of artificial insemination and were maintained under uniform nutritional conditions and management during the period the research was conducted. For all ejaculates, progressive motility was greater than 70% and the percentage of abnormal spermatozoa was less than 20%. Two ejaculates from each bull were obtained once a week for 12 weeks. Ejaculates from all the bulls were pooled and diluted to obtain a final concentration of 3.0–4.5 × 107 sperm/ml, in a buffer containing Tris (0.20 mM), citric acid (0.06 mM), glycine (0.12 mM), fructose (0.06 mM), 20% egg yolk and 7% glycerol. A slow cooling curve to 5 ◦ C (1 ◦ C/min) was performed, and the semen was then equilibrated at 5 ◦ C for a further 90 min. It was frozen at −76 ◦ C on dry ice, and pellets were preserved at −196 ◦ C in liquid nitrogen. 2.2. Evaluation of sperm motility and viability Progressive motility was evaluated by light microscopy (400× magnification) with a thermal stage (37 ◦ C) three times by the same observer, in the following cases: 10 min after thawing, after 60 min of incubation and after each treatment (45 min). The percentage of live spermatozoa was determined by the supravital technique with trypan blue 0.25%. At least 200 spermatozoa were counted in each sample.

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2.3. Determination of sperm concentration Assessment of sperm concentration in semen or in sperm suspensions was conducted in a Neubauer chamber. 2.4. Preparation of the sperm suspension for capacitation Samples of frozen semen were thawed for 10 min in TALP medium, pH 7.4 at 36 ◦ C, without calcium and without bovine serum albumin (BSA, Parrish et al., 1988) in a 1:3 ratio. Samples were washed twice by cetrifugation at 600 × g for 5 min to separate the seminal plasma and the freezing buffer. The pellets were re-suspended to a final concentration of 1.5 × 107 spermatozoa/ml, in TALP with the addition of CaCl2 (2 mM) and BSA (6 mg/ml), used as incubation medium (completed TALP) for all the experiments. Sperm suspensions corresponding to each experience were incubated for 45 min at 38 ◦ C under 5% CO2 in humidified air in the presence or absence of a capacitation inducer (Fukui et al., 1990; O’Flaherty et al., 1997). The sperm concentration and the percentage of progressive motility were evaluated. 2.5. Determination of capacitation The chlortetracycline (CTC) fluorescent technique was used to detect changes in the plasma membrane of the bovine spermatozoon (Fraser et al., 1995). Three patterns were observed: F (fluorescent), intact non-capacitated sperm displaying fluorescence throughout their surface; C (capacitated), intact capacitated spermatozoa that lost fluorescence in the post-acrosomal region; AR (acrosome reacted), spermatozoa with a reacted acrosome that lost fluorescence in the post-acrosomal and acrosomal regions, expressing fluorescence only in the midpiece. CTC (500 ␮l) was added to an equal volume of the medium containing the spermatozoa. Glutaraldehyde was then added to the mixture reaching a final concentration of 0.1%. Slides were examined at 400× magnification under epifluorescence exitation at 410 nm using a Carl Zeiss Jena Jenamed 2 epifluorescence microscope. The percentage of capacitated spermatozoa determined at zero time was subtracted from the values obtained in control and capacitated samples to rule out cells damaged during freezing– thawing. 2.6. Experiment 1: effect of NO• on capacitation The effects of NO• on capacitation were evaluated in sperm suspensions (n = 5) incubated for 45 min at 38 ◦ C, in the presence of different concentrations of sodium nitroprusside (0.05–200 ␮M), a well-known generator of NO• in vitro (Herrero et al., 1994). In these samples, the percentage of capacitated spermatozoa was determined by the CTC technique. 2.7. Experiment 2: effect of NOS and NO• scavenger on capacitation In order to discern the possible participation of NOS during capacitation, sperm suspensions (n = 5) were incubated for 45 min at 38 ◦ C, in the presence of heparin (10 IU/ml)

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and different concentrations of NOS inhibitors l-NA (1–500 ␮M) or l-NAME (1–500 ␮M; Donnelly et al., 1997). The effect of NO• scavengers was studied in sperm suspensions (n = 5) incubated for 45 min at 38 ◦ C, in the presence of heparin (10 IU/ml) or SNP (0.05 ␮M) as capacitation inducers. In both cases, hemoglobin (20–40 ␮g/ml; Herrero et al., 1994) and methylene blue (50–100 ␮M; Donnelly et al., 1997) were added as NO• scavengers. 2.8. Experiment 3: effect of protein kinases on NO• -induced capacitation To study the effect of NO• on the activity of protein kinases involved in capacitation, sperm suspensions were incubated for 45 min at 38 ◦ C, with sodium nitroprusside (0.05 ␮M) in the presence or absence of H-89 50 ␮M (Galantino-Homer et al., 1997), a specific PKA inhibitor, bisindolylmaleimide I (BM) 0.1 ␮M (O’Flaherty et al., 2001), a specific PKC inhibitor and genistein 3 ␮M, a specific PTK inhibitor. The percentage of capacitated spermatozoa was determined by CTC. 2.9. Experiment 4: effect of ROS on NO• -induced capacitation To study the requirement of superoxide anion and hydrogen peroxide on NO• -induced capacitation, sperm suspensions (n = 5) were incubated 45 min at 38 ◦ C, with sodium nitroprusside (0.05 ␮M) in the presence or absence of SOD (0.05–0.5 mg/ml) and catalase (20–100 ␮g/ml). 2.10. Additional experiment In order to evaluate whether SNP 0.05 ␮M was able to inhibit catalase in the experimental model, catalase activity (20, 40 and 100 ␮g/ml) was determined in the presence or absence of SNP 0.05 ␮M, measuring spectrophotometrically the breaking down of H2 O2 (10 mM) at 240 nm during 30 s at 25 ◦ C (Aebi, 1984). The slope for each catalase concentration used was determined with and without SNP 0.05 ␮M and the inhibition percentages were calculated. 2.11. Statistical analysis Percentages of capacitated spermatozoa are given as mean ± S.D. Treatments in the different experiences were analyzed by ANOVA and the Bonferroni test was used for means comparison. A P < 0.05 value was considered as statistically significant.

3. Results Progressive motility was not affected by SNP concentrations ranging from 0.05 to 200 ␮M, however, a dose-dependent decrease was observed at concentrations ≥400 ␮M (Fig. 1). Sperm viability (56 ± 2%) was not affected by SNP (P > 0.05).

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Progressive Motility (%)

236 70 60 50 40

a a

a

a b

b,c

b,c

c

30 20 10 0 Time 0

C

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400

600 SNP

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Fig. 1. Effect of sodium nitroprusside (NP) on progressive motility of bull spermatozoa. Time 0, sample at time 0: C, control; SNP, sodium nitroprusside (␮M). Samples were incubated for 1 h at 38 ◦ C in complete TALP medium. Different letters indicate significant differences (P < 0.05), n = 5.

Capacitation (%)

All concentrations of SNP increased (P < 0.05) capacitation rates compared with values from the control group with maximal effects observed at concentrations of 0.05 and 0.1 ␮M (Fig. 2). Peak capacitation percentages did not differ from those of heparin treated samples. With concentrations between 1 and 100 ␮M of SNP, capacitation amounts achieved was 14% (data not shown). The addition of l-NAME or l-NA produced a dose-dependent inhibition (P < 0.05) of heparin-induced capacitation, reaching amounts for the control group at concentrations ≥100 ␮M. At 1 ␮M concentrations, inhibition percentage with l-NAME (60%) was significantly greater than with l-NA (46%; Fig. 3). NO• scavengers were employed to confirm the participation of NO• as capacitation inducer in the experimental model and in spermatozoa capacitated with heparin. Both hemoglobin and methylene blue lowered the amount of capacitation significantly (Figs. 4 and 5). With 40 ␮g/ml of hemoglobin, capacitation was entirely blocked, in both cases, without modifying sperm motility and viability. Methylene blue not only inhibited capacitation to a lesser degree than hemoglobin but it also negatively affected sperm motility at all concentrations. Sperm viability (56 ± 2%) was not influenced by methylene blue.

40 35 30 25 20 15 10 5 0

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Fig. 2. Effect of sodium nitroprusside (SNP) on capacitation of bull spermatozoa. C: control; H: heparin; SNP: sodium nitroprusside (␮M). Samples were incubated for 45 min at 38 ◦ C in complete TALP medium. Different letters indicate significant differences (P < 0.05), n = 5.

Capacitation (%)

P.C. Rodriguez et al. / Animal Reproduction Science 85 (2005) 231–242 40 35 30 25 20 15 10 5 0

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Fig. 3. Effect of nitric oxide synthase inhibitors on capacitation with heparin of bull spermatozoa. C: control; H: heparin; l-NA: N␻-nitro-l-arginine (␮M); l-NAME: N␻-nitro-l-arginine methyl ester (␮M). Samples were incubated for 45 min at 38 ◦ C in complete TALP medium. Different letters indicate significant differences (P < 0.05), n = 5.

Capacitation (%)

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Fig. 4. Effect of nitric oxide scavengers on sperm capacitation induced with heparin. C: control; H: heparin; MB: H + methylene blue (␮M); Hg: H + hemoglobin (␮g/ml). Samples were incubated for 45 min at 38 ◦ C in complete TALP medium. Different letters indicate significant differences (P < 0.05), n = 5.

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Fig. 5. Effect of nitric oxide scavengers on sperm capacitation induced with SNP. C: control; SNP: sodium nitroprusside (0.05 ␮M); MB: SNP + methylene blue (␮M); Hg: SNP + hemoglobin (␮g/ml). Samples were incubated for 45 min at 38 ◦ C in complete TALP medium. Different letters indicate significant differences (P < 0.05), n = 5.

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Capacitation (%)

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H-89

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GEN

Fig. 6. Effect of specific inhibitors of protein kinase A (H-89), protein kinase C (BM) and protein tyrosine kinase (GEN) on capacitation induced by SNP in bovine spermatozoa. C: control; SNP: sodium nitroprusside 0.05 ␮M; H-89: SNP + H-89; BM: SNP + bisindolylmaleimide I; GEN: SNP + genistein. Samples were incubated for 45 min at 38 ◦ C in complete TALP medium. Different letters indicate significant differences (P < 0.05), n = 5.

A significant decrease was observed in amount of capacitation in response to the specific inhibitors of protein kinases: H-89 (PKA), BM (PKC) and genistein (PTK; Fig. 6). The relevance of other reactive oxygen species (superoxide anion and hydrogen peroxide) was studied with the experimental model utilized. When spermatozoa were incubated with SNP (0.05 ␮M), in the presence of different catalase or superoxide dismutase concentrations, significant changes were not observed in capacitation percentage (Fig. 7). Because NO• binds to the heme group of the catalase, reversibly inhibiting H2 O2 breaking down with a Ki approximately 0.2 ␮M of NO• (Brown, 1995), the degree of catalase inhibition at the concentrations added in the present experiments was studied with SNP 0.05 ␮M as capacitation inducer. A 50% inhibition of catalase activity was obtained, because of the presence of SNP 0.05 ␮M when 20 ␮g/ml of the enzyme were used and, 10% of inhibition when 40 ␮g/ml of the enzyme were used, meaning that NO• fails to totally inhibit the capacity to break down H2 O2 of the catalase added during capacitation with SNP.

Capacitation (%)

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Fig. 7. Effect of catalase and superoxide dismutase (SOD) on capacitation induced by SNP in bull spermatozoa. C: control; SNP: sodium nitroprusside 0.05 ␮M; SOD: SNP + SOD ( mg/ml); CAT: SNP + catalase (␮g/ml). Samples were incubated for 45 min at 38 ◦ C in complete TALP medium. Same letters indicate that there are no significant differences (P > 0.05), n = 5.

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4. Discussion A variety of biological and pharmacological agents are capable of inducing capacitation in mammalian spermatozoa (Thundathil et al., 2002). The wide distribution of NOS in diverse cells of the female and male reproductive organs (Rosselli, 1997) suggests a major role for NO• in the physiological regulation of reproduction. The influence of exogenous NO• on the motility, viability, metabolism and mechanisms leading to fertilization has been studied in human and rodent sperm (Tomlinson et al., 1992; Hellstrom et al., 1994; Weinberg et al., 1995; Zini et al., 1995; Herrero et al., 1999). Sperm motility is regarded as an essential requirement to achieve oocyte fertilization. Sperm require ATP generated by the glycolytic pathway and mitochondrial respiration for the maintenance of motility. There are inconsistent data in the literature regarding the effect of nitric oxide on motility; Weinberg et al. (1995) demonstrated that 1 mM of SNP inhibited the motility of human sperm, these results agree with Tomlinson et al. (1992) who showed that SNP diminished motility and that hemoglobin inhibited this effect. Hellstrom et al. (1994) suggested, however, a beneficial effect of SNP on the maintenance of motility and viability in human sperm after thawing. Results from the present study with bull sperm show that lesser concentrations of SNP fail to affect either motility or sperm viability, but concentrations greater than 400 ␮M reduced motility in a dose-dependent manner (Fig. 1). These data imply that greater NO• concentrations can cause sperm cytotoxicity, possibly due to the inhibitory effect of nitric oxide when binding to the heme group contained in enzymes of cellular respiration. The nitration of those proteins, for example cytochrome c oxidase, results in a decrease in the production of ATP required as an energy source by spermatozoa. Additionally, NO• could interact with the O2 •− to form the peroxynitrite anion. This molecule is very reactive and could act on cytosolic or membrane compounds such as lipids or proteins with thiol groups, affecting the integrity of the sperm membrane. Moreover, at the mitochondria, peroxynitrite anion inhibits complexes I and III of the respiratory chain (Torres et al., 1995), thus altering mitochondrial function and ATP production. Recent publications suggest that the peroxynitrite at lesser concentrations would also have a physiological function, producing the nitration of sperm protein tyrosine and leading to enhanced capacitation of human spermatozoa (Herrero and Gagnon, 2001). In the present study, NO• generated from low concentrations of SNP acts as a capacitation inducer in cryopreserved bovine spermatozoa, reaching concentrations similar to those of heparin treated samples (Fig. 2). The capacity of the NO• scavengers, hemoglobin and methylene blue to inhibit capacitation occurring in the presence of SNP, indicates the participation of NO• as an inducer of this process. The inhibition of motility but not viability by methylene blue indicates that the activity of guanylyl cyclase and cGMP is essential for the maintenance of the physiological sperm conditions in our experimental model. NOS has been detected in the acrosome and the tail of mouse and human spermatozoa (Lewis et al., 1996) and has been shown to have a role in the acrosome reaction induced by progesterone in mouse spermatozoa in vitro (Herrero et al., 1997). Results from the present study indicate that NOS and NO• participate in bull sperm capacitation; however, the mechanism by which NOS is activated during this process is not yet clear. It is speculated

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that the presence of serum albumin causes alterations in sperm membrane fluidity (Go and Wolf, 1985) helping to accelerate ionic changes involved in capacitation, such as the entry of calcium, for which the intracellular increase could modulate the sperm calcium-dependent NOS isoform. The capacity of superoxide dismutase to inhibit capacitation induced by heparin in bull spermatozoa, indicates the participation of the superoxide anion in this process (O’Flaherty et al., 1999). Several possible pathways have been described by which NO• interacts with O2 •− and H2 O2 . Zini et al. (1995) proposed that the three reactive oxygen species are involved in sperm capacitation. According to data from the present study, the addition of superoxide dismutase and catalase fails to modify capacitation, indicating that with the employed values of SNP, neither the presence of O2 •− nor of H2 O2 is required for the action of NO• , or alternatively that NO• stimulates capacitation independently of O2 •− generation. The mechanism by which NO• modulates capacitation remains to be established. It is known that NO• acts as an intracellular signal activating guanylyl cyclase and thus cGMP, leading to physiological responses mediated by the activation of cGMP-dependent protein kinase (PKG). It has also been shown that NO• acts via cAMP/PKA (Visconti et al., 1995), and cAMP increase is one of the well-established events that occur during capacitation (White and Aitken, 1989; Leclerc et al., 1996). The blockade of SNP-induced capacitation in the presence of a specific PKA inhibitor demonstrates that the activation of this enzyme is involved in mechanisms triggered by NO• , regulating concentrations of cAMP and, therefore, capacitation. In tissues such as the heart, muscle and kidney, NO• causes an intracellular cAMP increase due to the inhibition of its hydrolysis by a phosphodiesterase inhibited by cGMP (Mery et al., 1993). PTK is an enzyme that phosphorylates tyrosine amino acids that are part of the proteins associated with capacitation (Leclerc et al., 1996). The reduction of capacitation in the presence of a specific PTK inhibitor suggests the participation of this enzyme in capacitation induced by NO• . The activation of PTK could occur due to PKA-mediated phosphorylation, in agreement with data obtained in human spermatozoa (Leclerc et al., 1997). It has been demonstrated that PKC activation takes place in human (Lowenstein et al., 1994) and bovine spermatozoa (Cordoba et al., 1997) during capacitation, which is consistent with present findings using a specific PKC inhibitor and NO• as inducer of capacitation in bovine spermatozoa. In conclusion, the present study demonstrates that nitric oxide induces capacitation by means of intracellular mechanisms that seem to involve the activation of protein kinases in cryopreserved bull spermatozoa and that the concentrations of SNP used to induce capacitation fail to affect either progressive motility or sperm viability.

Acknowledgements We are deeply grateful to Genética Los Nogales for semen sample supplies. This research was supported by the Secretar´ıa de Ciencia y Técnica de la Universidad de Buenos Aires (V016, UBACYT).

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