Mol. Hum. Reprod. Advance Access originally published online on April 30, 2004
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Molecular Human Reproduction, Vol. 10, No. 7, pp. 543-552, 2004
Molecular Human Reproduction vol. 10 no. 7 © European Society of Human Reproduction and Embryology, 2004; all rights reserved
Comparative analysis between cyclic GMP and cyclic AMP signalling in human sperm
Institute for Hormone and Fertility Research at the University of Hamburg, Grandweg 64, D-22529 Hamburg, Germany
1 To whom Correspondence should be addressed.; Email: mueller{at}ihf.de
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Abstract |
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The principal involvement of cyclic nucleotides in regulating sperm functions is well established, but the factors controlling their generation and actions have not yet been entirely resolved. In particular, specific roles for cyclic (c)GMP in mammalian sperm are poorly understood. In this study, we have characterized comparatively the cAMP and cGMP signalling systems in ejaculated human sperm. Mean concentrations of cGMP (0.1 µmol/l) were found to be 100-fold lower than those of cAMP in non-stimulated cells, and adenylyl cyclase (AC) activities predominate by far guanylyl cyclase (GC) activities in both particulate and soluble protein fractions. By different experimental approaches (photoaffinity labelling, cyclase assays, immunoblotting), we provide evidence for the presence (guanylyl cyclase-A, soluble guanylyl cyclase, regulatory and catalytic subunits of cAMP-dependent protein kinase) or absence (guanylyl cyclase-B, natriuretic peptide clearance receptor, neuronal nitric oxide synthase, cGMP-dependent protein kinsae I) of different factors involved in either cAMP or cGMP pathways. Functional studies showed that cGMP, at high concentrations, can enhance sperm protein tyrosine phosphorylation but not serine phosphorylation of glycogen synthase kinase. This study reveals that human sperm are characterized by an exceptional predominance of cAMP signalling and indicates potential roles for cGMP.
Key words: cGMP/GC-A/GSK-3/NPR-C/NOS
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Introduction |
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After ejaculation into the female genital tract, mammalian sperm must undergo complex maturational processes as a prerequisite to be capable of fertilizing an oocyte. These processes, referred to as capacitation, affect the sperm motility and include the acquisition of the ability to induce the acrosome reaction (de Lamirande et al., 1997
).
There is well-established evidence that cyclic nucleotides and alterations in protein phosphorylation play pivotal roles in molecular events leading to capacitation (Visconti and Kopf, 1998). In particular, cyclic AMP (cAMP), generated by adenylyl cyclases, has been abundantly shown to act as a signalling molecule in these processes. Moreover, there is compelling evidence (reviewed recently by Urner and Sakkas, 2003) for a crucial involvement of cAMP-dependent protein kinase A (PKA) and so-called PKA-anchoring proteins (AKAP, A kinase anchoring proteins).
In contrast, potential roles for cyclic GMP (cGMP) in mammalian sperm are less clearly defined. At least in marine invertebrates, cGMP acts as the primary intracellular messenger in sperm chemotaxis which is based on interaction of oocyte-released peptides with sperm receptor guanylyl cyclases (Kaupp et al., 2003; Matsumoto et al., 2003).
The aim of the present study was to characterize at a molecular and functional level the cGMP signalling system compared with that of cAMP in ejaculated human sperm. Comparative analyses served to examine the basal content of cGMP versus that of cAMP in sperm samples from a variety of healthy individuals. To assess comparatively the presence of cyclic nucleotide-generating enzymes, we determined guanylyl cyclase (GC) and adenylyl cyclase (AC) activities associated with particulate and soluble fractions of sperm homogenates.
Considering previous reports that atrial natriuretic peptide (ANP) is a component of ovarian follicular fluid (Anderson et al., 1994), that it can act as a chemoattractant of human sperm (Zamir et al., 1993; Anderson et al., 1995) and induce the acrosome reaction (Anderson et al., 1994; Zamir et al., 1995; Rotem et al., 1998), we re-evaluated the expression of the ANP receptor, guanylyl cyclase-A (GC-A), on sperm membranes (Silvestroni et al., 1992). Functional studies were used to elucidate the capability of ANP and of the related peptides, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), to elevate cellular cGMP levels. Investigations of the two latter peptides were based on recent evidence for the expression in testis of a BNP-specific receptor (Goy et al., 2001) and on the identification of CNP in seminal plasma (Chrisman et al., 1993).
In addition to hypothesized functions of peptide receptor guanylyl cyclases, localized to the sperm membrane, there is considerable evidence (Revelli et al., 2002) for roles in sperm capacitation of the soluble GC (sGC) which is activated by the free radical nitric oxide (NO). Reported studies refer to functions of NO on sperm motility (Lewis et al., 1996; Donnelly et al., 1997; O'Bryan et al., 1998), in acrosomal reaction (Revelli et al., 1999, 2001) and identified effects on tyrosine phosphorylation of sperm proteins (Herrero et al., 1999; Thundathil et al., 2003). However, it is not clear whether all of these effects are mediated via sGC/cGMP and which type(s) of the three known NO-generating enzymes, endothelial NO synthase (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS), are involved. To address this issue in part, we examined (i) the presence and functional activity in human sperm of sGC, (ii) the expression of eNOS and nNOS, and (iii) whether cGMP, as compared to cAMP, can alter the amount of protein tyrosine phosphorylation in sperm cells.
To assess the ability of cGMP to induce serine phosphorylation of significant sperm proteins, we selected glycogen synthase kinase-3 (GSK-3) as an experimental target molecule. This kinase was shown to represent a key element in sperm physiology (Vijayaraghavan et al., 2000), and its activity is regulated by both tyrosine and serine/threonine phosphorylation (Pearl and Barford, 2002). In this study, we specifically investigated the effects of cGMP/cAMP on phosphorylation at serine9 of the GSK-3ß isoform.
Since cGMP signalling is thought to depend on the presence of specific target proteins, we also characterized the expression of cyclic nucleotide-binding proteins in human sperm. In these studies, particular attention was concentrated on cGMP-dependent protein kinase (PKG I), representing a key mediator of cGMP effects (Schlossmann et al., 2003) and on PKA, the major cellular target for cAMP.
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Materials
Sodium nitroprusside (SNP), bovine serum albumin (BSA, fraction V), creatine phosphate, creatine phosphokinase, and EDTA were purchased from Sigma (Germany), 3-isobutyl-1-methylxanthine (IBMX) from BIOMOL (Germany), 8-bromo-cAMP and 8-bromo-cGMP from BIOLOG (Germany), and GTP and ATP from Amersham (Germany). The synthetic peptides ANP (‘1–28’), BNP (‘BNP-32’), CNP (‘CNP-22’) and C-ANF were purchased from Bachem (Weil am Rhein, Germany), 125I-ANP (IM 186; 2 kCi/mmol) from Amersham (Braunschweig, Germany). 125I-[Tyr0]CNP32–53 (rat, human), 2.2 kCi/mmol, was purchased from Peptide Radioiodination Service Center (USA) and purified from bovine serum albumin by ultrafiltration through a Centricon-10 device (Amicon, Germany) prior to use.
Semen collection and sperm preparations
Human semen samples were obtained by masturbation from healthy adult volunteers after 
2 days of abstinence. The samples were examined immediately after liquefaction (30–60 min at room temperature), and only samples exhibiting normal semen parameters, according to World Health Organization (World Health Organization, 1999) guidelines, were used. Sperm samples were washed twice with minimal essential medium (MEM; Life Technologies, UK)/25 mmol/l HEPES, pH 7.4, by centrifugation at 800 g for 10 min. In most cases, the final sperm pellet was processed by the swim-up procedure (World Health Organization, 1999) using MEM/25 mmol/l HEPES, supplemented with 4% (w/v) BSA as medium. The upper (‘swim-up’) layer, containing the motile sperm, was removed, centrifuged at 400 g for 15 min, washed twice, and then used for further experiments. The ‘bottom’ layers after swim-up procedures were collected and used in further experiments when microscopic evaluations revealed a cellular content of >90% sperm. To separate sperm proteins into particulate and soluble fractions, pooled sperm samples were homogenized in an ice-cold solution (1 ml per 1–5 x 107 cells) of 50 mmol/l Tris–HCl, pH 7.5, 1 mmol/l EDTA, 1 mmol/l dithiothreitol and 0.1 mmol/l PMSF, by 20 strokes, in a Potter–Elvehjem homogenizer. After sedimentation for 2 min at 3000 g, the supernatants were centrifuged at 4°C for 40 min at 100 000 g. The final supernatant (‘soluble/cytosolic’) fractions were collected and the pellets resuspended in 50 mmol/l Tris, pH 7.5 (‘particulate/membrane’ fractions). Protein concentrations were determined by using a kit from Bio-Rad Laboratories (Germany).
Rat and human tissues
We used certain rat (derived from 3 month old male Wistar rats; Müller et al., 2004) and human (provided by the Institute of Anatomy, University of Hamburg; Middendorff et al., 2002) tissues for comparative analyses. Tissues were stored at –80°C until the preparation of soluble and particulate protein fractions as described before (Middendorff et al., 2002; Müller et al., 2002). In brief, frozen tissues were first pulverized in a mortar and then homogenized in a Potter–Elvehjem homogenizer. After centrifugation at 3000 g for 8 min at 4°C to remove cell debris and nuclei, the samples were centrifuged for 30 min at 100 000 g. The resulting supernatant fractions (‘soluble/cytosolic proteins‘) and the crude membrane pellets after resuspension in 50 mmol/l Tris–HCl, pH 7.5 (‘particulate/membrane proteins’) were stored at –80°C until experimental usage. Protein concentrations were determined by using a kit from Biorad (Germany) with BSA (Sigma, fraction V) as standard.
Measurement of cGMP and cAMP content in individual sperm samples
Individual swim-up sperm samples, consisting of 107 cells in 600 µl MEM, were agitated with 2 ml ice-cold ethanol and kept for 10 min at 4°C. After centrifugation at 1000 g for 30 min at 4°C, the supernatant fractions were collected, vacuum-evaporated and resolved in 200 µl of 50 mmol/l HEPES, pH 7.5. After acetylation (by addition of 10 µl acetic anhydride/triethylamide, 1:2, vol/vol), the cGMP and cAMP concentrations in the same solutions were determined in aliquots of 25 or 50 µl by using commercially available (IHF, Germany) specific immunoassays. The cross-reactivities of the cGMP-specific enzyme-linked immunosorbent assay (ELISA) were: 0.00004 (cAMP), 0.00003 (guanosine), <0.00001 (other nucleotides/nucleosides). Cross-reactivities of the cAMP-specific ELISA: 0.00028 (cGMP), 0.00020 (adenosine), <0.00001 (other nucleotides/nucleosides). The measurement ranges were 0.5–80 fmol/50 µl (cGMP-ELISA) and 1–250 fmol/50 µl (cAMP-ELISA).
Guanylyl and adenylyl cyclase assays
Incubations with membrane or cytosolic proteins (10 µg each) were carried out at 32°C for 30 min in total volumes of 200 µl in the presence (5 mmol/l) of either MgCl2 or MnCl2. The GC assay buffer contained 50 mmol/l Tris–HCl (pH 7.5), 1 mmol/l dithiothreitol (DTT), 1 mg/ml BSA, 0.5 mmol/l IBMX, 10 mmol/l creatine phosphate, 13.2 IU/ml creatine phosphokinase, 0.2 mmol/l EGTA and 1 mmol/l GTP. The AC assay buffer contained 40 mmol/l Tris–HCl (pH 7.5), 1 mmol/l EDTA, 1 mmol/l ATP, 0.5 mmol/l IBMX, and 1 mmol/l DTT. Reactions were terminated by addition of 1.5 ml of ice-cold 100% ethanol, and cyclic nucleotide levels in the ethanol extracts were determined by immunoassays as described above.
Affinity labelling of natriuretic peptide receptors by 125I-labelled ANP and CNP
Protocols for photoaffinity labelling of natriuretic peptide receptors in crude membranes by 125I-ANP (Müller et al., 2002) or by 125I-[Tyr0]CNP (Müller et al., 2000) have been described before. In brief, membranes were incubated with 125I-labelled peptides, ligand/receptor cross-links were induced by UV light irradiation, and reaction products were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions in 7.0% acrylamide separation gels followed by autoradiography at –70°C using Kodak XAR-5 films and intensifying screens. The results shown in Figure 3 are representative of five experiments, performed with three different pooled sperm samples.
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Treatment of sperm with GC-activating agents
Sperm samples, consisting of 107 swim-up sperm in 450 µl of MEM/HEPES, pH 7.4, 0.01% BSA and 0.5 mmol/l IBMX, were incubated for 90 min at 36°C in either the absence or presence of natriuretic peptides (0.5 µmol/l) or sodium nitroprusside (0.5 mmol/l). Reactions were stopped by addition of 2 ml of ice-cold ethanol. After centrifugation at 2000 g for 10 mins at 4°C, supernatant fractions were vacuum-dried, resolved in 200 µl of 50 mmol/l HEPES, pH 7.5, and aliquots were used to determine cGMP levels by ELISA.
Assessment of natriuretic peptide effects on sperm membranes
To evaluate ligand-induced stimulations of membrane receptor GC, particulate fractions of sperm homogenates (2.5 or 5 µg of protein) were incubated at 37°C for 12 min in total volumes of 75 µl containing 25 mmol/l HEPES, pH 7.4, 5 mmol/l MgCl2, 1 mmol/l ATP, 50 mmol/l NaCl, 0.5 mmol/l 3-isobutyl-1-methylxanthine, 5 mmol/l creatine phosphate, 5 IU creatine phosphokinase, and 1 mmol/l GTP in either the absence or presence (1 µmol/l) of ANP, BNP or CNP. Reactions were terminated and analysed (by cGMP-ELISA) as described (Müller et al., 2004).
Immunoblotting
After separation of proteins by SDS–PAGE in 6% (nNOS, eNOS) or 9% (all other antigens) acrylamide gels, immunoblotting was performed using either PVDF (Millipore, Germany: for pTyr, GSK-3) or nitrocellulose (Amersham, Germany: for all other) membranes. Blocking was carried out with Boehringer blocking reagent (Roche Diagnostics, Germany) except for GSK-3 blots (‘I-block’; Tropix, USA). Anti-nNOS (‘610308’; www.bdbiosciences.com), anti-eNOS (‘N30020’; Dianova, Germany), anti-sGC (‘210-724-1’; recognizing predominantly the ß1 subunit; Alexis, USA), anti-pTyr (‘4G10’; Upstate, USA), anti-GSK-3
/ß (‘KAM-ST002E’; StressGen, Canada), anti-serine9-phosphorylated GSK-3ß (Chemicon, USA), anti-PKG I (‘539729’; Calbiochem, USA), and anti-PKAc (‘sc-905’; raised against the catalytic subunit 
of PKA, but partially cross-reactive with the and ß subunits; Santa Cruz, www.scbt.com) were used as primary, anti-mouse or anti-rabbit IgG, linked to peroxidase (Pierce, USA), as secondary antibodies. Signals were detected using enhanced chemiluminescence (Amersham RPN 2105) on Fuji (13862 C) X-ray films.
Affinity labelling of cyclic nucleotide-binding proteins by [32P]cGMP
Soluble or particulate fractions of sperm homogenates were incubated for 15 min at 0°C with [32P]cGMP (3 nmol/l) in a total volume of 50 µl of 25 mmol/l Tris–HCl buffer, pH 7.5, containing 100 mmol/l KCl, 2.5 mmol/l EDTA, 1 mmol/l dithiothreitol, and 10 µmol/l IBMX in either the absence or presence (1 µmol/l) of unlabelled cAMP. Samples were then irradiated in the dark for 12 min at room temperature on a UV table (UV transilluminator model TM-36; UVP Inc., USA). Reactions were terminated by chilling and the addition of 300 µl of ice-cold 10% (v/v) trichloroacetic acid. After 20 min at 0°C, samples were centrifuged at 4°C for 5 min at 15 000 g, and the protein pellets were resolved in 50 µl of SDS–PAGE sample buffer consisting of 0.125 mol/l Tris–HCl, pH 6.8, 66.6 mmol/l DTT, 10% (v/v) glycerin, 5% (w/v) SDS and 0.02% (w/v) Bromophenol Blue. Samples were boiled for 3 min prior to analysis by SDS–PAGE under reducing conditions in 9% polyacrylamide separation gels. Gels were dried after staining and then exposed to Kodak XAR-5 films between intensifying screens at –70°C for 6–12 days. Generally, the findings presented are based on thorough investigations, including detailed assessments of competing effects of the two unlabelled cyclic nucleotides and the usage of clear-cut control proteins (PKA, PKG preparations). The data shown in Figure 7 is representative of four analyses performed with different pools of human sperm.
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Determination of cGMP/cAMP effects on sperm protein phosphorylation
Sperm samples (5 x 106 cells) were incubated for different time periods (30–90 min) at 36°C in 450 µl of MEM/25 mmol/l HEPES (pH 7.4), 0.5 mmol/l IBMX, 0.01% BSA in the absence or presence (0.5 to 2.0 mmol/l) of 8-bromo-cGMP or 8-bromo-cAMP. Reactions were stopped by addition of 150 µl of 4-fold strength electophoresis sample buffer (‘Roti-Load’; Roth, Germany). Each sample was boiled for 5 min prior to SDS–PAGE, and the effects on protein phosphorylation were visualized by Western blots, using antibodies against tyrosine-phosphorylated proteins (pTyr) or serine9-phosphorylated GSK-3ß (see immunoblotting).
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Results |
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To clarify the cGMP content in human sperm, we examined ejaculated semen samples from healthy donors. These studies (see Figure 1A
for a representative analysis) revealed values of 1–90 fmol cGMP per 106 sperm cells, indicating very low cellular concentrations. There was considerable variability between different semen samples (provided every 3–4 weeks) from the same donor. In addition, we recognized remarkable individual differences (note the elevated cGMP content in case of no. 2). Comparative analyses of cAMP generally revealed much higher values, ranging from 200 fmol up to 2.3 pmol (Figure 1B). There appears to be no correlation between cellular cGMP and cAMP levels in individual sperm samples. Estimating that human sperm have on average a volume of 50 fl, the mean cyclic nucleotide concentrations are 0.1 (cGMP) and 10 (cAMP) µmol/l.
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We next used pools of semen samples to characterize guanylyl cyclase (GC) activities present in human sperm. For comparison, analogous determinations were performed using two human tissues, testis and cerebral cortex. The cell/tissue homogenates were separated into membrane (M) and cytosolic (C) fractions to assess particulate and soluble enzyme activities. Moreover, assays were performed in the presence of either Mg2 + or Mn2 + to reveal potential differences in cation dependency. These studies revealed a significant production of cGMP by both membrane and cytosolic fractions and showed that Mn2 + is more effective than Mg2 + in stimulating GC activity (Figure 2
). However, as compared to testis and cerebral cortex, the basal values of sperm GC activity are very low. In contrast, adenylyl cyclase (AC) activities associated with sperm protein fractions are higher and comparable to those determined in the two tissue extracts. Regarding the specific amounts of AC/GC activities measured in the human reference tissues, we note that post-mortem decreases in enzyme levels/activities may have occurred and that such phenomena can affect differentially the types (AC versus GC) and cation dependencies (Mg2 + versus Mn2 + ) of these enzymes (Nicol et al., 1981). In conclusion, cGMP content and basal GC activities in human sperm appear very low as compared to the two human tissues examined and as compared to cAMP levels and basal AC activities in the same sperm samples.
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Based on previously published studies showing that ANP, which binds to and stimulates the membrane GC, GC-A (Kuhn, 2003
), has physiologically relevant functions in human sperm (indicated specifically in the Introduction section), we specifically examined the presence of GC-A in human sperm. To identify this protein at a molecular level, a photoaffinity labelling approach capable of visualizing the ANP receptor in rat (Müller et al., 2002) and human (Middendorff et al., 2002) tissues was used. These experiments revealed a low, but clearly detectable expression of GC-A in membrane preparations from ejaculated human sperm (Figure 3A
). In contrast, we failed to find by analogous approaches (Müller et al., 2000) any expression of the CNP receptor, GC-B, in these membranes (Figure 3B). Both analyses in addition showed that sperm membranes do not contain verifiable levels of a third receptor subtype, designated as NPR-C, which lacks GC activity, binds ANP, BNP and CNP with similar affinities and serves as a natriuretic peptide clearance receptor (Kuhn, 2003).
To assess the functional activity of GC-A, we incubated purified sperm samples in either the absence or presence of ANP prior to determinations of cGMP. Analogous incubations were carried out in the presence of the ANP-related peptides, BNP (binds to the same receptor, but with a 10-fold lower affinity) and CNP (binds to GC-B), as well as in the presence of the NO donor, SNP, representing an exogenous stimulator of sGC. These experiments (Figure 4) did not show any effects of ANP or of the two other natriuretic peptides (BNP, CNP) on sperm cGMP levels. In contrast, significant (2.8-fold) increases were found in response to SNP. These findings revealed the presence and functional activity of sGC but failed to demonstrate an increase in sperm cGMP in response to natriuretic peptides. Immunoblot analyses proved the expression of sGC (Figure 5A) and verified previously published results (Donnelly et al., 1997; Revelli et al., 1999) indicating the expression of the NO-generating enzyme eNOS (data not shown). However, we were unable to find, by analogous approaches, any detectable expression of nNOS in human sperm (Figure 5B). Note that two tissues characterized by relatively low expression levels of nNOS (rat penis, human testis) served as positive controls in addition to cerebellum (high abundance of nNOS) in this experiment.
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With regard to the apparent functional inactivity of the peptide receptor guanylyl cyclases, as assessed using intact sperm, we also performed GC assays with isolated sperm membranes. Neither ANP, nor BNP and CNP (at 1 µmol/l each) were capable of enhancing cGMP production as compared to basal values (data not shown).
We next examined, by Western blot analyses, the expression in human sperm of the cGMP-dependent protein kinase I (PKG I), representing the key mediator of cellular cGMP effects (Schlossmann et al., 2003). Using rat tissues and human testis as positive controls, expression of this enzyme remained undetectable in human sperm (Figure 6A). Thus, this kinase apparently is not implicated in mediating effects of cGMP in human sperm. In contrast to PKG I, catalytic subunits of PKA were found to be abundantly expressed in both particulate and soluble fractions of sperm homogenates (Figure 6B).
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To assess further the presence in human sperm of potential cGMP target proteins, we used an assay which is based on UV light-induced affinity cross-linking of [32P]cGMP to cyclic nucleotide-binding proteins. Since this approach also reveals highly expressed cAMP-binding proteins such as the regulatory subunits of PKA (Tang et al., 1993
), reactions were performed in either the absence or presence of unlabelled cAMP. Under these conditions (Middendorff et al., 2002), known physiological target proteins for cGMP are detectable in the presence of excess cAMP and can be discriminated from preferentially cAMP-binding proteins (detectable only in the absence of cAMP). Figure 7 demonstrates major amounts of [32P]cGMP-labelled proteins in both soluble and particulate fractions of sperm homogenates, but only in the absence of competing cAMP. In contrast to previously examined human testicular tissues (Middendorff et al., 2002) and other cell types (our unpublished results), we were unable to find any expression of preferentially cGMP-binding proteins (lanes ‘+ cAMP’). Since PKG I is well detectable by this approach (Middendorff et al., 2002), the experiment confirmed the absence in human sperm of this kinase. On the other hand, PKA regulatory subunits (RI: 49; RII: 55 kDa) and proteolytic fragments of these (38 kDa) are thought to represent the bulk of the [32P]cGMP-binding proteins found (Tang et al., 1993), with RI apparently representing the predominantly labelled polypeptide within the soluble sperm protein fractions. Consistently, similar apparent molecular masses were reported previously for these proteins/fragments (Schoff et al., 1982; Eppenberger and Fabbro, 1984) after photolabelling of human sperm proteins by 8-N3-[32P]cAMP.
Considering that protein tyrosine phosphorylation plays a key role in sperm physiology (Aitkin et al., 1995), we next investigated whether cGMP can affect this reaction. While 0.5 mmol/l cAMP elicits significant increases in sperm protein phosphorylation, cGMP is ineffective at this concentration and shows weak effects only at relatively high (2 mmol/l) levels (Figure 8).
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As a control, we in addition analysed cyclic nucleotide-induced alterations in serine phosphorylation of GSK-3. This kinase is thought to play a pivotal role in sperm motility regulation (Vijayaraghavan et al., 2000
). Our studies revealed increased serine phosphorylation of sperm GSK-3ß in response to cAMP in a time- and concentration-dependent manner (Figure 9A), whereas cGMP, even at high (2 mmol/l) levels, was incapable of inducing any effects (Figure 9B). The expression of GSK-3 was confirmed by using phosphorylation-independent antibodies, demonstrating the presence of the two isoforms, GSK-3 and GSK-3ß, in both swim-up (SU) and bottom (BO) fractions of human sperm (Figure 9C).
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Discussion |
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This study served to estimate the relative roles of cGMP- and cAMP-dependent signalling in human sperm. The assessment of basal cyclic nucleotide levels in ejaculated sperm revealed high cellular concentrations (mean value: 10 µmol/l) of cAMP and relatively low (0.1 µmol/l) average values for cGMP. The absence of any correlation between cGMP and cAMP content in individual sperm samples is indicative of an independent regulation of synthesis and/or degradation of the two cyclic nucleotides. We noted a pronounced variability between different ejaculates of the same donor as well as between semen samples of different individuals. Large inter-sample variations have frequently been reported, and there are many possible explanations, some of which are discussed comprehensively in a recent publication (Harrison, 2003
).
Interestingly, the mean cGMP concentration in sperm is several-fold (0.1 as against 0.4 µmol/l) lower and cAMP levels are markedly higher (10 as against 4.4 µmol/l) than in human platelets (Eigenthaler et al., 1992). This comparison is significant since influences on cyclic nucleotide levels by processes related to DNA, mRNA and protein synthesis are thought to play no role or at least minor roles in both cellular compartments. Based on recent evidence that the [cAMP]:[cGMP] ratio can be crucial in controlling cellular functions (Nishiyama et al., 2003), our data, indicating a 100-fold excess of cAMP on cGMP (as compared to an 11-fold excess in platelets), refers in general to a predominant role for cAMP-dependent signalling pathways in human sperm. It remains to be emphasized that sperm cyclic nucleotide levels may rapidly change during movement within the female genital tract and that local (sub)cellular effects, based on compartmentalized activities of membrane or soluble adenylyl/guanylyl cyclases, can hardly be evaluated by whole cell content measurements.
Determinations of basal (i.e. ligand-independent) cyclase activities revealed markedly higher amounts of cAMP- as compared to cGMP-generating enzymes in sperm cells. Most significantly, GC activities in sperm are very poor when compared with those in two human reference tissues (testis, cerebral cortex), whereas sperm AC activities are similar to those in the two tissues examined. Thus, human sperm appear to be distinguished by particularly low concentrations of cGMP-synthesizing enzymes. Note that a major portion of both AC and GC activity was associated with membrane (particulate) sperm protein fractions. Assuming that Mn2 + acts as an equally effective stimulator of all cyclase activities present in sperm, our findings refer to a predominantly membrane rather than cytosolic localization of these enzymes. The identities, subcellular localizations and physiological roles of various AC isoforms represent a current topic of research activities (Harrison, 2003; Wade et al., 2003).
Photoaffinity labelling experiments demonstrated an expression, although at low levels, of the ANP receptor, GC-A, in sperm membranes. These findings seemed to support previous studies (see Introduction), showing that ANP can modulate certain sperm functions. However, an ANP-induced accumulation of cGMP in ejaculated sperm was not detectable in our study. Two possible explanations are (i) that cGMP elevations were very rapid but transient (Kaupp et al., 2003) and not measurable under the specific conditions of our experiments or (ii) that GC-A was unresponsive to hormonal stimulation. Although incubations were performed in the presence of the phosphodiesterase (PDE) inhibitor, IBMX, we cannot exclude the presence of IBMX-insensitive cGMP-degrading activities, localized to subcellular sites of GC-A expression. Our findings that ANP cannot stimulate cGMP generation on isolated sperm membranes, however, do not support this possibility but rather suggest a hormonal unresponsiveness of GC-A. In fact, desensitization of the ANP receptor, which is elicited by dephosphorylation, represents a well-established regulatory mechanism (reviewed in Kuhn, 2003). The kinase(s) and phosphatase(s) implicated in GC-A sensitization/desensitization are not yet defined, and hence, data on the presence or absence of these enzymes in human sperm are not available. However, our findings that PKG I is absent in sperm cells at least rules out a potential involvement (Airhart et al., 2003) of this kinase. Provided that desensitization (by dephosphorylation at the intracellular kinase homology domain) of GC-A is responsible for the apparent functional inactivity of the receptor in ejaculated sperm, it remains, however, conceivable that cellular signalling events, elicited by oocyte-released factors and/or associated with the acrosome reaction, can lead to a receptor re-activation within the female genital tract. The hitherto reported sperm functions of ANP would be consistent with this possibility.
In addition we show that the so-called natriuretic peptide clearance receptor (NPR-C) is not expressed, at least at substantial levels, in human sperm. This receptor, 60 kDa in size, lacks a GC domain and is thought to control the local availability (and hence bioactivity) of natriuretic peptides, although direct modulatory effects on cellular cAMP levels have also been reported (Kuhn, 2003). Since NPR-C is well detectable by the photoaffinity labelling approach in reference tissues (Figure 3) and previously examined membranes (Müller et al., 2004), our present findings refer to the absence of a meaningful NPR-C expression in human sperm cells. Like ANP, the two related peptides, BNP and CNP, were found to be ineffective in elevating sperm cGMP levels. Thus, our study does not provide any evidence for the expression of either a recently proposed BNP-specific receptor (Goy et al., 2001) or of the CNP receptor, GC-B, in sperm. Whether GC-A accounts for all of the basal GC activity found in sperm membranes or whether other enzymes contribute to this activity, remains to be resolved. In this context, it has to be noted that at least seven mammalian membrane GC have been identified, of which four have presently unknown ligands (Kuhn, 2003).
This study confirms the presence and functional activity of sGC in human sperm (Revelli et al., 2001) and provides further evidence that eNOS as the local NO-generating enzyme (Donnelly et al., 1997; O'Bryan et al., 1998). The failure to detect nNOS expression is in alignment with a previous report, demonstrating the expression of eNOS but not that of nNOS or iNOS by western blot analyses (Revelli et al., 1999). Our findings, on the other hand, are not consistent with immunohistochemical data, indicating a localization of nNOS to human sperm (Herrero et al., 1996; Lewis et al., 1996).
Comparative analyses of the major cellular target proteins for cGMP (PKG) and cAMP (PKA) revealed an abundant expression in human sperm of catalytic subunits of PKA, whereas PKG (contains catalytic and regulatory subunits in the same polypeptide chain) expression remained undetectable. The failure to find any immunoreactivity against PKG I (we used an antibody directed against this kinase isoform) is consistent with normal fertilizing properties of sperm in PKG I-deficient mice (Hedlund et al., 2000). Data obtained by [32P]cGMP cross-linking confirmed the absence of PKG I and showed high expression levels of cAMP-binding proteins. The most prominently labelled species are thought to represent regulatory subunits of PKA as well as proteolytic fragments of RI and/or RII (Schoff et al., 1982; Eppenberger and Fabbro, 1984; Tang et al., 1993). Thus, both catalytic and regulatory subunits of PKA are abundantly present in human sperm, and their cellular levels appear higher (see Figure 6B for PKA catalytic subunit) than in three tissues examined by comparison. The failure to detect cGMP-binding proteins by [32P]cGMP at least refers to a lower abundance of these species in sperm than in human testis (Middendorff et al., 2002). Based on the limited sensitivity of the assay, our findings, however, do not exclude the presence in sperm of target proteins (such as cGMP-gated ion channels) which are expressed at relatively low cellular concentrations.
Functional studies showed that cAMP elicits a concentration-dependent increase in tyrosine phosphorylation of the two major phosphotyrosine-containing proteins of human sperm (Leclerc et al., 1996; Ficarro et al., 2003). To a certain degree, this reaction could be induced also by high concentrations (2 mmol/l) of cGMP. The latter is of potential interest with regard to recently reported (Thundathil et al., 2003) NO-mediated increases in tyrosine phosphorylation of the two proteins. Our observations that cGMP is effective only at high concentrations is most reasonably explained by a cross-activation of PKA. However, since an involvement of this kinase was questioned in a previous study (Thundathil et al., 2003), the distinct signalling pathways responsible for NO- and/or cGMP-mediated increases in sperm protein tyrosine phosphorylation still remain to be elucidated.
In contrast to influences on tyrosine phosphorylation, we did not find any effects of cGMP on serine phosphorylation, as assessed by using antibodies specific to serine9-phosphorylated GSK-3ß. Comparative analyses revealed a dose- and time-dependent phosphorylation in response to cAMP, indicating the existence of pathways for this reaction in human sperm. The data suggest that, in addition to tyrosine phosphorylation of GSK-3 (Vijayaraghavan et al., 2000), serine phosphorylation is involved in controlling GSK-3 activities in mature sperm. Whether this type of phosphorylation is also implicated in regulating sperm motility, as reported for GSK-3 tyrosine phosphorylation in bovine sperm (Vijayaraghavan et al., 2000), has yet to be investigated. Our findings that cGMP, even at high concentrations, does not have the capability to mediate GSK3ß serine phosphorylation at least indicates that proposed functions of NO on sperm motility (Lewis et al., 1996; Donnelly et al., 1997; O'Bryan et al., 1998) are not linked to this reaction. However, physiological implications of cGMP in eliciting serine/threonine phosphorylation of other sperm proteins are certainly conceivable. We note in addition the demonstration of both GSK-3 isoforms in human sperm, and that their apparent relative expression levels (GSK-3 immunoreactivity is more abundant than GSK-3ß immunoreactivity) were found to be similar to those in bovine sperm (Vijayaraghavan et al., 1996).
In conclusion, the present study indicates that cAMP signalling to an exceptional extent predominates over cGMP signalling in human sperm. This concerns the cellular content of the two cyclic nucleotides in non-stimulated cells, the cyclic nucleotide-generating enzymes and the main cyclic nucleotide target proteins, PKA and PKG. Findings that the (cGMP-specific) phosphodiesterase (PDE) type 5 represents only a small fraction of the whole PDE activity in sperm and that inhibition by sildenafil triggers physiological effects via elevated cAMP (Lefievre et al., 2000) are consistent with a predominant usage of cAMP-dependent pathways. Recent observations that cAMP but not cGMP analogues can induce human sperm capacitation (de Vries et al., 2003) further support this. The apparently complete absence of PKG I is remarkable and rules out an implication of this kinase in mediating physiological effects of NO or ANP (and other potential activators of guanylyl cyclases) in human sperm. Reports on mammalian sperm cyclic nucleotide-gated ion channels that respond to cGMP (Weyand et al., 1994; Wiesner et al., 1998; Ren et al., 2001) and the identification of novel ion channels that are expressed specifically in sperm (Quill et al., 2001) raise the possibility that such proteins represent important target molecules for cGMP in human sperm.
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We thank members of our institute for providing semen samples and Dr Ralf Middendorff (Institute of Anatomy, University of Hamburg) for human tissues. This work was supported by the Innovationsstiftung Hamburg and by grants from the Deutsche Forschungsgemeinschaft (Iv 7/4-3-8 to D.M. and A.K.M.).
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Submitted on February 25, 2004; resubmitted on March 31, 2004; accepted on April 6, 2004.
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