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Biology of Reproduction 64, 1708-1712 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Signaling Pathway of Nitric Oxide-Induced Acrosome Reaction in Human Spermatozoa1

Alberto Revelli2,a,c, Costanzo Costamagnab, Federica Moffaa, Elisabetta Aldierib, Simona Ochettia, Amalia Bosiab, Marco Massobrioa, Bo Lindblomc, and Dario Ghigob

a Department of Obstetrical and Gynecological Sciences, S. Anna Hospital, University of Torino, 10126 Torino, Italy b Department of Genetics, Biology, and Biochemistry, University of Torino, 10126 Torino, Italy c Department of Obstetrics and Gynecology, Akademiska Sjukhuset, University of Uppsala, 75185 Uppsala, Sweden

ABSTRACT

Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+-free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.

acrosome reaction, cGMP, kinases, nitric oxide, sperm, sperm capacitation

INTRODUCTION

Nitric oxide (NO), a highly reactive gas with a short half-life, is synthesized from the enzymatic conversion of L-arginine to L-citrulline by NADPH-dependent NO synthases (NOSs) [1]. Two major classes of NOS have been identified: Ca2+/calmodulin-dependent constitutive isoforms (eNOS and bNOS), and a Ca2+/calmodulin-independent-inducible isoform (iNOS) [1].

The NO-generating system has been demonstrated in the human reproductive tract, where NO plays a role in a variety of reproductive functions (reviewed in [2]). In vitro studies have shown that low concentrations of NO enhance the motility of mouse [3], hamster [4], and human [5, 6] spermatozoa, the acrosome reaction (AR) of mouse [7] and bull [8] spermatozoa, and the zona pellucida-binding ability of human spermatozoa [9]. Accordingly, sperm motility is inhibited by the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) [10] and by the NO scavenger, methylene blue [11]; furthermore, NO-releasing compounds prime spermatozoa to respond earlier to human follicular fluid, whereas NOS inhibitors decrease the percentage of AR-positive cells [12]. On the other hand, higher NO concentrations seem to exert opposite effects on the motility, viability, and metabolism of human spermatozoa in vitro [6, 13–15]. Some studies have provided evidence for the presence of eNOS [10, 16, 17] and bNOS [10, 18] in human spermatozoa. NO synthesis and eNOS expression are higher in normozoospermic subjects than in asthenozoospermic subjects [10]. Abnormally shaped human spermatozoa exhibit aberrant eNOS immunostaining, which is associated with decreased sperm motility [16]. In a previous paper, we demonstrated that the activation of eNOS in human spermatozoa is involved in the follicular fluid-induced AR, and that several NO donors are able to stimulate the AR of human spermatozoa in a specific and dose-dependent way [17].

Although it is well established that several NO actions on smooth muscle cells, platelets, and endothelial cells are mediated via activation of soluble guanylate cyclase (sGC) and synthesis of cGMP [19], the mechanisms through which NO influences sperm function have not been clarified to date.

MATERIALS AND METHODS

Reagents

Fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), sodium nitroprusside (SNP), EDTA, Hepes (sodium salt), 6-anilino-5,8-quinolinedione (LY83583), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), 8-bromo-cGMP, Earle balanced salt solution (EBSS) medium, bovine serum albumin (BSA, fraction V), Eosin Y, calphostin C, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma Chemical Company (St. Louis, MO). The inhibitors of cGMP-dependent protein kinase (PKG), 8-bromoguanosine-3',5'-monophosphorothioate Rp-isomer (Rp-8-Br-cGMPS) and 8-(4-chlorophenylthio)guanosine-3',5'-monophosphorothioate Rp-isomer (Rp-8-pCPT-cGMPS) were from Biolog Life Science Institute (Gremen, Germany). Fluoromont was from Serva (Heidelberg, Germany). The [3H]cGMP radioimmunoassay kit was from Amersham International (Bucks, U.K.).

Collection and Preparation of Sperm Samples

Semen samples were obtained by masturbation after 3–5 days of sexual abstinence from donors of proven recent fertility (n = 48), whose partners had conceived within the last 2 yr. Each donor gave informed consent about the use of his semen for our experiments. All samples were allowed to liquefy for at least 30 min at 37°C, and were then evaluated for sperm concentration, motility, and morphology according to World Health Organization guidelines [20]. Only specimens with normal parameters [20] were used in the experiments. Motile spermatozoa were harvested by the swim-up technique (37°C for 3 h in air atmosphere) [20] using EBSS medium supplemented with 0.5% BSA. The presence of round cells (spermatogonia, spermatocytes, spermatids, and leucocytes) was initially less than 1 x 106/ml in all sperm samples, and it was minimal if not absent after the swim-up technique in the final suspension. After swim-up, the motile sperm-rich fraction was centrifuged at 600 x g for 10 min at room temperature, and sperm concentration was adjusted to approximately 80 x 106 cells/ml by removing excess medium.

Measurement of Intracellular cGMP

Aliquots of sperm suspensions in EBSS + 0.5% BSA, 250 µl each, containing 15 x 106 cells, were incubated for 4 h with the phosphodiesterase inhibitor, IBMX (100 µM), in the absence or presence of 100 µM SNP (final concentration) or an equal volume of EBSS (controls). After incubation, tubes were centrifuged in an Eppendorf microcentrifuge at 12 000 x g for 2 min. Supernatants were discarded and 50 µl of absolute ethanol was added to the pellets. Ethanol was evaporated by vacuum centrifugation, and 300 µl of Tris/EDTA buffer (50 mM Tris-HCl, 4 mM EDTA pH 7.5) was added. After 10 min, 100 µl of supernatant was tested for the cGMP level with a [3H]cGMP immunoassay system. Cyclic GMP content was expressed as pmol/106 cells. Cross-reactivity of the [3H]cGMP immunoassay system with cAMP was less than 0.001%.

Measurement of Acrosome Reaction

Individual post-swim up samples were divided into aliquots to form replicates, and were incubated for 4 h with different agonists and inhibitors, at the concentrations indicated below. Aliquots incubated with EBSS medium were used as controls. To test the effects of SNP during a complete Ca2+ deprivation, some normospermic samples were processed with a standard swim-up technique in Hepes-buffered solution without Ca2+ (145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM Hepes, and 10 mM glucose pH 7.4) containing 0.5% BSA, and post-swim up samples were further supplemented with 0.1 mM EDTA. As controls we used normospermic samples from the same donors, processed with swim-up in Hepes-buffered solution containing 0.5% BSA and 1 mM Ca2+; in this case, post-swim up samples were not supplemented with EDTA. In all experiments, the assessment of AR was performed immediately after the 4-h incubation. FITC-PSA, a fluoresceinated lectin showing specific affinity for terminal {alpha}-D-mannosyl and {alpha}-D-glucosyl residues of glycoproteins, was used to detect acrosomal status. Briefly, 50-µl aliquots of the sperm suspensions were used to prepare smears. After air-drying and dipping into absolute methanol for 30 min, smears were again air-dried at room temperature. Afterward, they were incubated in a dark, humidified chamber with PBS (pH 7.4) containing FITC-PSA (50 µg/ml) for 30 min at 37°C. Slides were then washed in tap water and mounted with Fluoromont. They were observed with an epifluorescent microscope (Zeiss, Germany) at 400x magnification immediately or within 5 days, during which they were kept in dark boxes. The percentage of acrosome-reacted spermatozoa in each slide was calculated on 200 cells, and independently assessed in blind by two different observers. Because the percentage of motile sperm was similar in the test suspensions (74% ± 5%, n = 38) and in controls (78% ± 7%, n = 38), and contemporaneous tests using the supravital stain, Eosin Y, showed a <20% loss of sperm viability in our experimental conditions, the observed ARs were assumed to be independent from cell death. The labeling patterns were classified according to the method of Cross et al. [21]: spermatozoa with fluorescence limited to the equatorial segment or with no detectable fluorescence in the head were considered as reacted, and cells with fluorescent acrosomes were considered as nonreacted. Spermatozoa with patchy fluorescent acrosomes were not assigned to this bimodal distribution, although it has been claimed that they represent early stages of AR [22], but were classified in a third group, which, however, did not exceed 10% of cells and was equally distributed in the experimental and control groups. Positive controls for AR were obtained by freezing and thawing semen three times.

Statistical Analysis

Each experimental point has been performed in duplicate or triplicate per experiment; all data in the text and figures are given as means ± SEM. Statistical analysis was carried out using the Student t-test for unpaired data, after having normalized the data by square root transformation.

RESULTS

Role of cGMP in SNP-Induced AR

After a 4-h incubation, SNP induced a significant increase in intracellular cGMP levels (0.085 ± 0.014 pmol/106 cells; n = 10) in comparison with control (0.044 ± 0.006 pmol/106 cells; n = 10; P < 0.05). Also, the percentage of AR induced by SNP after the same incubation time was significantly higher than it was for controls (Fig. 1); the AR-inducing effect of SNP was significantly reduced by the addition of the guanylate cyclase inhibitor, LY83583, whereas LY83583 had no effect per se. Because LY83583 has been found to inhibit NOS, to elicit NO auto-oxidation and to generate superoxide anion, which could scavenge NO [23], we performed a second set of experiments using the more specific inhibitor of guanylate cyclase, ODQ (Fig. 1); the AR-inducing effect of SNP was significantly reduced by the addition of ODQ, whereas ODQ per se exhibited no significant effect. Addition of the cGMP analogue, 8-bromo-cGMP, to the incubate containing SNP and ODQ restored the AR. Indeed, 8-bromo-cGMP increased the AR of human spermatozoa per se (Fig. 1). In order to ascertain the possible role of PKG in the cGMP-mediated AR, we incubated human spermatozoa with the PKG inhibitors, Rp-8-Br-cGMPS or Rp-8-pCPT-cGMPS; both of them blocked the SNP-induced AR without exerting a significant modification of control AR per se (Fig. 2).



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  		FIG. 1. Effect of LY83583, ODQ, and 8-bromo-cGMP on SNP-induced acrosomal reactivity (AR). SNP (100 µM) effect was tested on 14 post-swim up human sperm samples after a 4-h incubation, in the presence or absence of LY83583 (LY, 10 µM), ODQ (10 µM), and/or 8-bromo-cGMP (8-Br-cGMP, 1 mM). Significance versus control (ctrl): *P < 0.05, **P < 0.01 versus SNP: •, P < 0.05; ••, P < 0.01



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  		FIG. 2. Effect of the PKG inhibitors, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, on SNP-induced acrosomal reactivity. SNP (100 µM) and/or Rp-8-Br-cGMPS (PKGI-1, 10 µM) or Rp-8-pCPT-cGMPS (PKGI-2, 10 µM) effect was tested on five post-swim up human sperm samples after a 4-h incubation. Significance versus control (ctrl): *P < 0.05 versus SNP: •, P < 0.05; ••, P < 0.02

Role of Protein Kinase C and Ca2+ in SNP-Induced AR

The AR-inducing effect of SNP was significantly reduced by the addition of the PKC inhibitor, calphostin C (P < 0.05; Fig. 3), which had no effect on the AR per se. Furthermore, SNP failed to induce the AR of human spermatozoa when it was added to spermatozoa in a Ca2+-free medium (Fig. 4).



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  		FIG. 3. Effect of calphostin C on SNP-induced acrosomal reactivity. The effect of SNP (100 µM), calphostin C (calphC, 50 nM), or both was tested on 13 post-swim up human sperm samples after a 4-h incubation. Significance versus control (ctrl): *P < 0.001 versus SNP: •, P < 0.005



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  		FIG. 4. Effect of extracellular Ca2+ deprivation on SNP-induced acrosomal reactivity. SNP (100 µM) effect was tested on 6 post-swim up human sperm samples after a 4-h incubation in a Ca2+-free medium containing 100 µM EDTA (no Ca) or in Ca2+-containing medium (Ca). Significance versus control (ctrl): *P < 0.005 versus SNP: •, P < 0.001

DISCUSSION

An increasing amount of data concerning the effects of NO on human sperm function have been accumulating in the recent past; nevertheless, the intracellular mechanisms by which NO exerts these effects have not been elucidated to date. In several somatic cell systems, the effects of NO are mediated via activation of sGC and induction of cGMP synthesis. This intracellular transduction pathway is known to mediate the effects of NO, for instance, in vascular smooth muscle cell relaxation and growth, platelet aggregation, neurotransmission, and adherence of neutrophils to endothelial cells [19]. However, a growing amount of experimental data indicate that NO can induce its biological effects even via non-cGMP-dependent pathways (e.g., binding to heme-containing proteins other than sGC or S-nitrosylating the thiol group of a variety of proteins, such as glyceraldehyde-3-phosphate dehydrogenase) [1]. It has also been shown that NO elicits a wide spectrum of intracellular effects depending on its concentration, the cellular redox state, and the amount of metal ions, protein thiols, glutathione, and other nucleophilic targets present [1].

In the present study, the NO donor SNP was observed to increase both the intracellular cGMP levels of human spermatozoa and the percentage of AR-positive sperm cells. Zhang and Zheng [6] have observed that capacitated human spermatozoa synthesize cGMP after a 3-h SNP treatment; this result was obtained with an SNP concentration lower (100 nM) than that used in our experiments (100 µM). Zhang and Zheng found no SNP effect at higher concentrations. This discrepancy can be accounted for by the use, in our cGMP measurements, of the phosphodiesterase inhibitor, IBMX; indeed NO released from SNP has been shown to desensitize sGC [24] leading to a progressive decrease of cGMP synthesis, and the use of IBMX could have counterbalanced this phenomenon by decreasing the cGMP hydrolysis, thus allowing us to appreciate an increased cGMP production at a higher SNP concentration. SNP was used at a 100 µM concentration because this drug amount had previously exerted the maximal AR effect [17]. Moreover, in our experiments, the SNP-induced cGMP synthesis was linked to AR occurrence because the sGC inhibitors, LY83583 and ODQ, abolished the AR-inducing effect of SNP, which was recovered by adding 8-bromo-cGMP. The evidence of cGMP synthesis and involvement during NO-induced AR in human spermatozoa agrees with the observation of an intracellular cGMP rise during NO-dependent stimulation of AR in capacitated bull spermatozoa [8]. Cyclic GMP has been also implicated in the AR induced in vitro in capacitated human spermatozoa by atrial natriuretic peptide (ANP), which increases intracellular cGMP by activating the membrane isoform of guanylate cyclase; indeed, ANP effect is blocked by LY83583 [25]. Our experiments suggest that exogenous NO elicits AR in human sperm by increasing the intracellular cGMP level. One of the molecular targets of cGMP is PKG; two different cGMP-dependent protein kinases (PKGI and PKGII) have been identified in mammals [26, 27]. In our experiments, the PKG inhibitors, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, blocked the SNP-induced AR, suggesting that the NO/cGMP pathway, which is activated by SNP stimulation, needs the activation of a PKG in order to trigger AR in human sperm.

Furthermore, we demonstrate herein that the NO-induced AR does not occur in a Ca2+-free medium or in the presence of a PKC inhibitor. The need for extracellular Ca2+ in mammalian sperm AR has been repeatedly observed (reviewed in [28]). Both physiological stimuli of the human sperm AR; the zona pellucida protein, ZP3; and follicular fluid-derived progesterone elicit the influx of extracellular Ca2+. The subsequent, abrupt rise in intracellular Ca2+ concentration triggers a variety of signaling pathways, which ultimately leads to AR (reviewed in [28, 29]). A similar Ca2+ dependence has been reported during induction of human sperm AR by ANP [30], although it must be remarked that at high concentrations, ANP elicits the AR even in noncapacitated human spermatozoa, in the absence of Ca2+, or both [25]. In several cell types (e.g., gut interstitial cells, macrophages, pancreatic ß-cells) NO increases the intracellular Ca2+ level via the activation of a ryanodine receptor, which allows the ion efflux from the inositol-1,4,5-trisphosphate-insensitive Ca2+ pools [31]; in sea urchin eggs NO and cGMP seem to mobilize Ca2+ from intracellular stores by inducing synthesis of cyclic ADP-ribose, a putative agonist of the ryanodine receptor [31]. Differently from these cell types, several forms of physiological and pharmacological evidence indicate that the influx of external Ca2+ via opening of voltage-dependent calcium channels plays a crucial role in the induction of the AR in mammalian sperm [32]. Our experiments demonstrate that NO-elicited AR stimulation depends on the presence of extracellular Ca2+. It could be hypothesized that NO promotes the activation of plasma membrane Ca2+ channels, or inhibits a Ca2+ pump, or both. Cyclic nucleotide-gated channels have been found to be expressed in mammalian sperm [33] where they can regulate a Ca2+ entry pathway, which responds more sensitively to cGMP than to cAMP [34]. Moreover, NO inhibits the skeletal muscle sarcoplasmic reticulum Ca2+-ATPase [35] and the ATP-dependent Ca2+ uptake into platelet membrane vesicles [36]. NO could act in the same way in human spermatozoa. Indeed, it has been demonstrated that plasma membrane Ca2+-ATPase antagonists promote AR in guinea pig sperm, leading to cytosolic accumulation of Ca2+ [37].

The involvement of PKC in the AR of human spermatozoa has been previously demonstrated [28, 38], as has the effectiveness of calphostin C in blocking the PKC-mediated AR of these cells [39]. Both the PKC activators, 12-O-tetradecanoylphorbol-13-acetate and 1-oleoyl-2-acetylglycerol, are powerful inducers of the AR in capacitated human spermatozoa [40]. A recent study also showed that the ANP-induced AR of human spermatozoa involves PKC, because it may be inhibited by the PKC inhibitors, staurosporine and GF-109203X [30]. To this regard, extracellular Ca2+ could be necessary for NO and ANP to activate PKC and/or other protein kinases, such as Ca2+/calmodulin-dependent kinase II.

In conclusion, the present study demonstrates that the NO-induced AR of human spermatozoa, similarly to the ANP-elicited AR, requires the presence of extracellular Ca2+ and is mediated via the synthesis of cGMP and the activation of PKG and PKC. In our laboratory further experiments are in progress to detail the cross-talk between these signaling molecules, to identify the PKG isoform involved, and to clarify the role played by Ca2+ in the transduction of the AR-inducing signal triggered by NO in human sperm.

ACKNOWLEDGMENTS

The authors acknowledge Dr. Elena Delpiano and Miss Maria Luisa Rullo for their precious help in organizing the work and preparing this manuscript.

FOOTNOTES

First decision: 14 August 2000.

1 This study was supported financially by Gruppo di Studio in Cronoendocrinologia Ginecologica, Turin, Italy. Back

2 Correspondence: Alberto Revelli, Department of Obstetrical and Gynecological Sciences, S. Anna Hospital, University of Torino, Via Ventimiglia 3, 10126 Torino, Italy. FAX: 39 011 6964022; fertisave{at}yahoo.com Back

Accepted: January 25, 2001.

Received: July 5, 2000.

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