Journal of Histochemistry and Cytochemistry, Vol. 48, 1163-1172, September 2000, Copyright © 2000, The Histochemical Society, Inc.


ARTICLE

Immunolocalization of A1 Adenosine Receptors in Mammalian Spermatozoa

A. Minellia, C. Allegruccia, P. Piombonib, R. Mannuccic, C. Lluisd, and R. Francod
a Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione Biochimica Cellulare, Università di Perugia, Perugia, Italia
b Istituto di Biologia Generale, Università e Centro per lo Studio delle Cellule Germinali, CNR, Siena, Italia
c Laboratorio Analisi di Immagine, Dipartimento di Medicina Clinica e Sperimentale, Sezione Medicina Interna e Scienze Oncologiche, Università di Perugia, Perugia, Italia
d Departament de Bioquimica i Biologia Molecular, Institut Pi i Sunyer d'Investigacion Biomediques, Universitat de Barcelona, Barcelona, España

Correspondence to: A. Minelli, Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sez. Biochimica Cellulare, Università di Perugia, Via del Giochetto, 06123 Perugia, Italia. E-mail: albami@tin.it


  Summary
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The presence of A1 adenosine receptors (A1AR) in mammalian spermatozoa was previously demonstrated by radiochemical and immunochemical detection. This study was performed to investigate the cellular location of the A1AR to determine whether these receptors were somehow connected with ecto-adenosine deaminase and to evaluate their function in calcium uptake. By immunofluorescence staining we showed that in mammalian spermatozoa A1AR were constantly localized in the acrosomal region. This finding was confirmed by immunogold detection. Confocal analyses with anti-A1 and anti-ADA antibodies showed a high degree of co-localization. Calcium loading assay showed that this association was functional and affected calcium accumulation in mammalian spermatozoa. Therefore, we concluded that the acrosomal localization of A1AR was a constant feature in mammalian sperm. Moreover, these A1 receptors were functionally coupled to ecto-ADA and were able to modulate calcium uptake into an IP3-gated store.

(J Histochem Cytochem 48:1163–1171, 2000)

Key Words: A1 adenosine receptors, ecto-ADA, mammalian spermatozoa, localization, calcium uptake


  Introduction
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The action of adenosine in discriminating input from the extracellular environment is effected through a series of cell membrane-heptaspanning proteins, i.e., A1, A2A, A2B, and A3 receptors (AR). Many physiological processes are modulated by the interaction of the endogenous nucleotide with its specific receptors, G-proteins, Ca2+ mobilization, inositol phosphate hydrolysis, and/or cAMP production. Via A1 adenosine receptor (A1AR), adenosine reduces heart rate (Belardinelli et al. 1989 ), bronchoconstriction (Nyce and Metzger 1997 ; Abebe and Mustafa 1998 ), respiratory rythm (Mironov et al. 1999 ), glomerular filtration rate and renin release (Spielman and Arend 1991 ), inhibits lipolysis (Pippig et al. 1995 ), regulates the physiological state of pituitary tumor cells (Navarro et al. 1999 ), and increases the chemotaxis of tumor cells (Woodhouse et al. 1998 ). On the other hand, in several instances, the biological role of A1AR is yet to be defined. In rat testis (Bhat et al. 1998 ), the expression of A1AR is clearly age-dependent so that the age-dependent deficit in the A1AR signal transduction pathway predicts a concomitant reduction in the action of adenosine. The physiological role of A1AR in the testes is still not clear. New perspectives and more questions concerning the complex physiological role of A1AR are provided by the finding that ecto-adenosine deaminase (ecto-ADA), the enzyme that degrades adenosine to inosine on the cell surface (Franco et al. 1997 ), is capable of modulating ligand binding and signaling through A1AR. Irrespective of its catalytic activity, ecto-ADA appears to be necessary for high-affinity binding of agonists to A1AR (Saura et al. 1996 ). In addition, it has also been shown that A1AR are regulated in a heterologous way in response to other hormones, i.e., epidermal growth factor (EGF) in a pituitary-derived cell line (Navarro et al. 1999 ). To date, the state of adenosine receptors in mammalian spermatozoa has not been thoroughly defined. The existence of A2AAR has been reported on the basis of the rank potency of pharmacological effectors and of their positive effects on motility and capacitation (Fraser and Duncan 1993 ; Shen et al. 1993 ; Fenichel et al. 1996 ). The existence of A1AR has been reported after radiochemical and immunochemical detection (Minelli et al. 1995 , Minelli et al. 1997 , Minelli et al. 1999 ).

Bearing this in mind and because of the existence of ecto-ADA in mammalian spermatozoa membrane (Minelli et al. 1999 ), in this study we analyzed the localization of A1AR in mammalian sperm cells, investigated their distribution in relation to ecto-ADA molecules, and studied their functional state in an attempt to add new details to the understanding of their physiological role in the fertilization process.


  Materials and Methods
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Materials and Methods
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Materials
PBS, BSA, Hepes, DMSO, 5-carboxyfluorescein diacetate (CFDA), propidium iodide (PI), sodium piruvate, sodium lactate, calcium ionophore A23187 (calcimycin), rabbit IgG, rabbit IgG conjugated to fluorescein isothiocyanate (FITC), rabbit IgG conjugated to tetramethylrhodamine isothicyanate (TRITC), goat anti-rabbit IgG–FITC, goat anti-rabbit IgG conjugated to 10-nm gold, normal goat serum (NGS), paraformaldehyde, glutaraldehyde, digitonin, DTT, oligomycin, antimycin A, creatine phosphate, creatine kinase, polyethylenimine, adenosine deaminase (ADA), D-myoinositol-1,4,5-trisphosphate (IP3), N6-R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), 2-chloro-N6-cyclopentyladenosine (CCPA), N6-cyclopentyl-9-methyladenine (N-0840), 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamido-adenosine (CGS21680), 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxilate (MRS1191), and N6- (4-aminobenzyl)-9-[5-(methylcarbonyl)-ß-D-ribofuranosyl]adenine (AB-MECA) were from Sigma (St Louis, MO). 4-(2-[7-amino-2-(2-furyl)- [1,2,4]triazolo[2,3-a][1,3,5]triazin, 5-ylamino]ethyl)phenol (ZM 241385) was from Tocris Cookson (Bristol, UK). Penicillin G and streptomycin were from GIBCO BRL (Gaithersburg, MD). 45CaCl2 (25 mCi/mg) was from NEN Life Science Products (Boston, MA). Immuno-fluore mounting medium was from ICN Pharmaceuticals (Costa Mesa, CA). COMPLETE protease inhibitor cocktail was from Boehringer–Mannheim (Mannheim, Germany). Bio-Rad protein assay was from Bio-Rad Laboratories (Hercules, CA). All other reagents were of the highest available quality.

Sperm Isolation and Preparation
Adult bovine epididymes were removed and spermatozoa collected by flushing the cauda with 100 mM NaCl, 3.1 mM KCl, 0.3 mM K2PO4, 25 mM NaHCO3, 1.5 mM MgCl2, 1 mM sodium pyruvate, 21.6 mM sodium lactate, 5000 U/ml penicillin G, 5 mg/ml streptomycin, 10 mM Hepes, pH 7.4 (Tyrode's modified medium, TALP). Spermatozoa number was determined by a Thoma chamber (Brand; Wertheim, Main, Germany) and viability was evaluated by fluorescent microscopy (Olympus CH-2) with 5-carboxyfluorescein diecetate and PI.

Rat epididymes were removed and spermatozoa collected by microflushing the cauda, then treated as described.

Rabbit ejaculates (provided by rabbittry of Department of Zootechnical Sciences, University of Perugia), horse ejaculates (provided by Associazione Regionale Allevatori Marche, Macerata), and human ejaculates from fertile donors (provided by Echevarne Laboratorios, Barcelona) were centrifuged at 800 x g for 10 min at RT and sperm resuspended as described.

For the 45Ca2+ loading assay, bovine sperm were dispersed and washed in PBS containing 2 mM EGTA, 1 mM ß-mercaptoethanol and COMPLETE protease inhibitor cocktail. After pelleting at 800 x g for 10 min, sperm were resuspended in 20 mM Hepes–KOH, pH 7.4, containing 1 mM ß-mercaptoethanol, followed by the addition of digitonin to a final concentration of 10 µM. Samples were incubated on ice for 10 min before addition to the 45Ca2+ loading buffer.

Antibodies
Fluorescent and non-fluorescent affinity-purified polyclonal antipeptide antibody against A1AR (PC21-FITC and PC21) and affinity-purified polyclonal antibody against adenosine deaminase (anti-ADA–TRITC and anti-ADA) were a gift from Prof. Franco and have been thoroughly characterized elsewhere (Aran et al. 1991 ; Ciruela et al. 1995 ).

Confocal Microscopy
Bovine sperm cells (1 x 106 ) were adhered to glass coverslips, rinsed in PBS, and fixed with methanol at -20C for 3 min or with 4% paraformaldehyde at RT for 15 min. These preparations were rinsed in PBS, 20 mM glycine, and were incubated for 15 min with PBS, 20 mM glycine, 1% BSA, and 0.05% NaN3 (blocking buffer).

The coverslips were incubated for 1 hr at 37C with polyclonal rabbit PC-21–FITC antibody (70 µg/ml) in blocking buffer. Double immunofluorescence staining was performed by treating with a mixture of 70 µg/ml PC21–FITC and 70 µg/ml anti-ADA–TRITC for 1 hr at 37C. The coverslips were then rinsed for 40 min in blocking buffer and mounted with immuno-fluore mounting medium. Negative controls were obtained by treating the preparations with 70 µg/ml nonimmune rabbit IgG–FITC and nonimmune rabbit IgG–TRITC. Ten fields of about 20 cells were observed with a Leica TCS 4D (Leica Laser TechniK; Heidelberg, Germany) confocal scanning laser microscope adapted to an inverted Leitz DMIRBE microscope.

The extent of co-localization of the two labelings was assessed by computerized image analysis (KS 300; Kontron, Muchen, Germany). A couple of images of the same field stained with the two labelings was analyzed at each time. In each image, the specific staining was discriminated from the nonspecific background by the threshold function. The discriminated images of the two labelings were superposed and subtracted by the AND Boolean operator function. Using this function, a new image, containing only pixels positive in both original discriminated images, is created. The percentage of coexistence is obtained by expressing the number of positive pixels in the new image as the percentage of the number of positive pixels in each original discriminated image.

Immunogold Detection
Bovine spermatozoa (50 x 106), fixed in methanol at -20C for 10 min, were washed three times for 10 min with PBS and incubated in PBS/1% BSA/5% normal goat serum. This blocking step was followed by an overnight incubation at 4C of the spermatozoa with PC21 diluted in PBS/0.1% BSA/1% normal goat serum (120 µg/ml). Samples were washed three times with PBS and fixed in 2% paraformaldehyde–PBS for 20 min at RT. After treatment with PBS–0.5 M glycine for 30 min at RT, samples were incubated for 2 hr at RT with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (1:10 in PBS/0.1% BSA/1% normal goat serum). After washing with PBS (three times for 10 min), the preparation was fixed with 2.5% glutaraldehyde–PBS for 1 hr at 4C and postfixed in 1% buffered osmium tetroxide. After dehydration, samples were embedded in Epon–Araldite (Fluka Chemie; Buchs, Switzerland). The ultrathin sections were observed in a transmission electron microscope (CM10; Philips Electronic Instruments, Mahwah, NJ). Negative controls were obtained either by omitting the primary antibody in the procedure or by using a nonimmune rabbit IgG at the same concentration of PC21 antibody.

Flow Cytometry
Bovine sperm cells (3 x 106) were fixed with 4% paraformaldehyde in PBS for 15 min at RT, washed with PBS–20 mM glycine, and then incubated for 15 min in blocking buffer. Immunofluorescence staining was performed by treating the cells with PC21 antibody (70 µg/ml) at 37C for 1 hr. Washed cells were then incubated for 45 min at RT with goat anti-rabbit IgG–FITC (1:50). Cells, washed three times with the blocking buffer, were suspended in PBS and analyzed using an Epics profile flow cytometer (Coulter; Hialeah, FL). Histograms corresponding to data from 12,000 cells were processed using Immuno-4-Software (Coulter). Negative controls were performed by treating the cells with a nonimmune rabbit IgG and a goat anti-rabbit IgG–FITC.

45Ca2+ Loading Assay
Bovine spermatozoa (70 x 106), incubated with 2 U/ml ADA for 30 min at RT and permeabilized by 10 µM digitonin, were loaded with 45Ca2+ by incubation for 45 min at 37C in the uptake buffer [20 mM Hepes–KOH, 75 mM potassium oxalate, 3% polyethylene glycol (average molecular weight 8000), 1 mM MgCl2, 2 mM ATP, 10 mM DTT, and COMPLETE protease inhibitors, pH 7.4] containing 45CaCl2 2 µCi/ml. The incubation mixture was supplemented with the mitochondrial inhibitors oligomycin (1 µg/ml), antimycin A (2 µg/ml), and 5 mM sodium azide plus an ATP-regenerating system (10 mM creatine phosphate and 10 U/ml creatine kinase). Experiments were performed in the presence of 10 µM A23187, 10 µM IP3, 5 nM CPA, 5 nM CCPA, 5 nM R-PIA, 50 nM CGS21680, 5 nM AB-MECA, 100 nM N0840, 500 nM ZM241385, 300 nM MRS1191, and a non-ATP control.

The 300-µl reactions were terminated by rapid filtration through 0.5% polyethylenimine-coated glass fiber filters (Whatman GF-C). Filters were washed twice with 1.5 ml of the stop solution (100 mM KCl, 10 mM Hepes–KOH, 5 mM MgCl2, 1 mM EGTA, pH 7.4) at RT. Filter disks were allowed to elute overnight in 5 ml of scintillant (Ultima Gold Packard; Canberra, CT) and counted with a Packard Tri-Carb scintillation counter at 60% efficiency.

Statistical Analysis
Statistical comparison between mean values of the treated groups was always performed against the control without the addition of the tested compound, using a two-tailed Student's t-test.


  Results
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Localization of A1AR
Immunocytochemical techniques using a specific anti-A1 antibody resulted in the localization of A1AR in intact and permeabilized mammalian spermatozoa (Fig 1). In bovine spermatozoa (Fig 1a), fluorescence localization showed that A1ARs were present in the acrosomal membranes, in the postacrosomal region, and in the connecting and middle piece, although the fluorescence of this area was of weak intensity. Rabbit spermatozoa (Fig 1b) showed fluorescence in the acrosomal and postacrosomal regions and in the middle piece. Equine spermatozoa (Fig 1c) showed a diffuse fluorescence at the acrosome and a strong immunostaining at the postacrosomal region. No fluorescence was detected either at the middle or at the connecting piece. Human spermatozoa (Fig 1d) showed diffuse fluorescence of the acrosomal domain as well as a sharp and strong fluorescence at the equatorial segment and at the middle piece. In rat spermatozoa (Fig 1e), the fluorescence was mainly localized at the postacrosomal region, whereas a feeble and diffuse fluorescence appeared at the acrosome. Intact bovine spermatozoa fixed with paraformaldehyde (Fig 1f) showed fluorescence in the plasma membranes overlying the acrosome and in the postacrosomal region, but did not show any fluorescence at the connecting and middle piece. In the observed fields, the percentage of stained cells was 100%. Treatment of the sperm cells, either permeabilized or non-permeabilized, with nonimmune rabbit IgG resulted in totally nonfluorescent images (data not shown).



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Figure 1. Localization of A1AR in mammalian spermatozoa. Spermatozoa were adhered to glass coverslips, rinsed in PBS, and fixed with methanol at -20C for 3 min or with 4% paraformaldehyde. The immunofluorescence staining was performed as described in Materials and Methods, using PC21–FITC (70 µg/ml) antibody. Fluorescence at the cell surface was observed by confocal microscopy. Representative images corresponding to a horizontal section at the middle of the cells are shown. (a) Bovine (bar = 12 µm); (b) rabbit (bar = 10 µm); (c) equine (bar = 20 µm); (d) human (bar = 15 µm); (e) rat (bar = 15 µm) spermatozoa. (f) Bovine spermatozoa fixed with 4% paraformaldehyde and treated as described. Bar = 12 µm.

Ultrastructural localization of A1AR in methanol-treated bovine spermatozoa is shown in Fig 2. The figure shows the localization in one section and is representative of all investigated sections. Gold particles were distributed at the outer acrosomal membrane (Fig 2a) and at the inner postacrosomal domain (Fig 2b). No labeling of sperm exposed to control rabbit serum was detected (data not shown). Ultrastructural localization of A1AR in paraformaldehyde-treated bovine spermatozoa was not possible due to the absence of labeling with either pre-embedding or postembedding procedures. The sperm cell population was homogeneous, as indicated by the plot of side vs forward light scatter (Fig 3a). Non-permeabilized cells, labeled with PC21 antibody or with irrelevant rabbit antibody, were analyzed by flow cytometry. The majority of cells (>95%; Fig 3b) expressed A1AR on their plasma membranes. Spermatozoa were not permeabilized in these observations because treatment with methanol resulted in a non-homogeneous cell population (Fig 3c).



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Figure 2. Ultrastructural localization of A1AR in bovine spermatozoa. Spermatozoa treated with PC21 (120 µg/ml) and goat anti-rabbit IgG–gold conjugate (10 nm) as described in Materials and Methods were embedded in Epon–Araldite and observed in a transmission electron microscope. Gold particles labeled the outer acrosomal membrane (a) and the inner postacrosomal domain (b). Bars = 0.2 µm.



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Figure 3. Flow cytometric characterization of bovine spermatozoa and immunodetection of A1AR. Spermatozoa (3 x 106) fixed with paraformaldehyde (a,b) or with methanol (c) as described in Materials and Methods were immunostained with PC21 antibody (70 µg/ml) and goat anti-rabbit IgG–FITC. (a) Side vs forward light scatter of bovine spermatozoa. Number of analyzed cells was 12,000. (b) Flow cytometric analysis of A1AR expression. Spermatozoa labeled with PC21 antibody (gray) or with rabbit nonimmune antibody (white). (c) Side vs forward light scatter of methanol-treated bovine spermatozoa.

Immunostaining observations with anti-ADA demonstrated this enzyme at the surface of mammalian spermatozoa (Fig 4a and Fig 4b). Co-localization of A1AR and ADA was seen in all cells observed. Confocal microscopy of double immunofluorescence staining showed a very high degree of co-localization (88%) between ADA and A1AR, as indicated by the intensity of yellow and its position far from the axis origin of the cytofluorogram.



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Figure 4. Distribution of A1AR and ADA at the surface of mammalian spermatozoa. Spermatozoa were adhered to glass coverslips, rinsed in PBS, fixed, and permeabilized with methanol at -20C for 3 min. Double immunofluorescence staining as described in Materials and Methods was performed by treatment with a mixture of PC21–FITC (70 µg/ml) and anti-ADA–TRITC (70 µg/ml). The coverslips were used for confocal microscopy observations. (a) Bovine spermatozoa (bar = 10 µm). (b) Human spermatozoa (bar = 50 µm). In a and b, the images at right correspond to the superposition of the two fluorescences (yellow).

45Ca2+ Loading of Digitonin-permeabilized Bovine Spermatozoa
Digitonin-permeabilized bovine spermatozoa were loaded with 45Ca2+ to examine the effects of agonists and antagonists of adenosine receptors on calcium release from intracellular stores (Fig 5). Oligomycin, antimycin A, and sodium azide were included in the reaction buffer at concentrations known to inhibit mitochondrial 45Ca2+ accumulation (Bourguignon et al. 1994 ). 45Ca2+ accumulated in the presence of ATP, which activates Ca-ATPase loading of intracellular stores (Verma et al. 1990 ). No calcium accumulation occurred in the absence of ATP, and calcium ionophore A23187 caused a marked reduction in calcium uptake. IP3 reduced the amount of calcium loaded into the sperm cells to a value close to that found in the presence of specific A1AR agonists. Specific A1 antagonist N0840 was almost ineffective. Concomitant treatment with R-PIA and N0840 resulted in a moderate effect on calcium accumulation. The specific A2AR agonist CGS21680, the antagonist ZM241385, and a mixture of the two were ineffective. The specific A3 agonist AB-MECA, the antagonist MRS1191, and a mixture of the two did not modify sperm calcium uptake at all.



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Figure 5. Effects of adenosine receptor agonists and antagonists on 45Ca2+ loading in permeabilized bovine sperm cells. Spermatozoa (75 x 106 cells) permeabilized with 10 µM digitonin were incubated at 37C for 45 min in the uptake buffer. A1 agonists: 5 nM R-PIA (Ki 1.17 nM); 5 nM CPA (Ki 1 nM); 5 nM CCPA (Ki 0.4 nM). A1 antagonist: 100 nM N0840 (Ki 10 nM). A2A agonist: 50 nM CGS21680 (Ki 15 nM). A2A antagonist: 500 nM ZM241385 (Ki 83 nM). A3 agonist: 5 nM AB-MECA (Ki 1.5 nM). A3 antagonist: 300 nM MRS1191 (Ki 31 nM); 10 µM IP3; 10 µM A23187. The amount of 45Ca2+ loaded by ATP-treated permeabilized spermatozoa was regarded as the maximal level of 45Ca2+ loading signal and other experimental results were compared to this maximal loading. Data are the means ± SD of (n = 4) independent experiments run in quadruplicate. * p<0.01.


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This is the first report on the localization of A1AR in mammalian spermatozoa. These heptaspanning receptors, known to be located at the surface of the cells, have been previously characterized in bovine spermatozoa both in the membrane-bound form and in the solubilized form (Minelli et al. 1995 , Minelli et al. 1997 ). The A1 receptors have been shown to exist in a single high-affinity state and to be tightly coupled to G-proteins. Immunofluorescence staining and flow cytometry of intact cells confirmed that A1AR are located on the plasma membranes overlying the acrosomal and postacrosomal regions. The immunocytochemistry was also performed in permeabilized cells because of the particular morphology of the spermatozoa (Allen and Green 1997 ). In all species studied, the A1 receptors were always found in the acrosomal membranes and in the postacrosomal region. The localization at the middle piece appeared to be a more variable finding. We focused our attention on head localization because, despite some differences among the species, the head always showed a strong fluorescence indicating the presence of A1AR. The consistent location of these receptors was believed to be linked to an essential role played by A1 in the function of mammalian spermatozoa. These A1 sperm receptors are functionally active and affect calcium uptake by spermatozoa. We have shown that A1 agonists modulate the amount of calcium that can be accumulated in the sperm cells. Our experiments have been carried out with epididymal bovine spermatozoa which, in contrast to ejaculated sperm, are capable of capturing calcium ions readily (Cordoba et al. 1997 ). The involvement of A2A receptors, whose existence has already been reported (Fraser and Duncan 1993 ; Shen et al. 1993 ; Fenichel et al. 1996 ), was ruled out by the fact that the highly specific A2A agonist was totally ineffective in modulating calcium influx of spermatozoa. The possible involvment of A3 receptors was also excluded by investigating the effects of the specific A3 agonist on calcium loading. To date, A3 receptors in mammalian spermatozoa have not been reported but their existence has been hypothesized on the basis of the high A3 expression in rat testis (Meyerhof et al. 1991 ). Moreover, in the heart, the activation of both A1 and A3 receptors was recently found to be essential for mediating the cardioprotective effect of adenosine released during cardiac ischemia (Liang and Jacobson 1998 ). In view of these findings, the concomitant presence of A1 and A3 receptors in mammalian spermatozoa is possible. However, only A1-selective agonists were effective in modulating calcium uptake. Sperm intracellular calcium plays a major role in motility, capacitation control, and acrosome reaction. Although multiple signal transduction components have been identified as important for acrosomal exocytosis, the specific nature of the ligand–receptor interaction, the relevant signaling pathway, and the sequence of transduction events are still poorly understood (Cheng et al. 1994 ; Roldan et al. 1994 ; Walensky and Snyder 1995 ). The physiological acrosome reaction is calcium-dependent and has long been known to require extracellular calcium (Yanagimachi and Usui 1974 ). Ligand-mediated generation of IP3 followed by calcium release from IP3-gated internal stores promotes extracellular calcium influx across the plasma membrane (Berridge and Irvine 1989 ; Berridge 1995 ), but the mechanism of these events is still not completely clear (Publicover and Barratt 1999 ). However, the requirement of extracellular calcium to induce sperm acrosomal exocytosis strongly suggests that IP3-gated calcium release may be an important mechanism for activating the extracellular calcium influx required for the acrosome reaction. Our data on calcium accumulation indicate that spermatozoa can load calcium into an IP3-gated store and that A1 agonists mimic the IP3 effect. Therefore, it can be suggested that the initial A1 agonist-induced IP3 generation is followed by acrosomal calcium release which, in turn, triggers the extracellular calcium influx required for the acrosome reaction. The finding that in mammalian spermatozoa A1 receptors are highly co-localized with ecto-ADA adds a new intriguing element to the already complex physiological role of the A1 receptors. Ecto-ADA was shown to have an extraenzymatic functional role by interacting with ADA-binding proteins, i.e., CD26 and A1AR (Kameoka et al. 1993 ; Ciruela et al. 1996 ; Franco et al. 1997 ; Minelli et al. 1999 ) The functional association of A1AR and ADA was shown to regulate the physiological state of pituitary tumor cells by inhibiting L-type voltage-dependent calcium channels (Navarro et al. 1997 ; Zapata et al. 1997 ). Voltage-sensitive L-like type channels, now known as T-type channels (Florman 1994 ; Florman et al. 1998 ), are present in mammalian spermatozoa. They are activated and opened by sperm membrane depolarization produced by a small and transient influx of calcium into the sperm head (Storey et al. 1992 ; Florman 1994 ). Because IP3 receptors are present in sperm acrosomes (Walensky and Snyder 1995 ), a mechanism of action might involve T-channel activation that produces a transient calcium influx which, in turn, initiates a downstream process of Ca2+-induced Ca2+ release leading to the acrosome reaction (Arnoult et al. 1996 ; Florman et al. 1998 ). It is known that spontaneous exocytosis must be strictly controlled in sperm because secretion must be coordinated with egg contact. Specialized regulatory mechanisms are required so that spontaneous secretory events are suppressed. It is tempting to speculate that the functional association of A1AR with ADA may represent the specialized regulatory mechanism whereby adenosine, present in the female reproductive tract (Samuelson et al. 1985 ), would regulate the physiological responsiveness of the spermatozoa to the spontaneous acrosome reaction. This suggestion implies that adenosine may constitute an automatic pathway for short-term desensitization when the autacoid accumulates in the extracellular space. Further studies are required to test this suggested mechanism.


  Acknowledgments

Supported by Italian CNR 97/9803839 and by the Spanish Commission of Science and Technology CICYT PB97/0984-SAF 97/0066.

We thank Prof B.T. Storey (University of Pennsylvania, Philadelphia) for helpful discussion and Dr M. Zoli (Dipartimento di Scienze Biomediche, Università di Modena, Italia) for the computerized analyses of co-localization images. We are grateful to Dr Mary Kerrigan for valuable linguistic suggestions.

Received for publication February 10, 2000; accepted March 15, 2000.


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Introduction
Materials and Methods
Results
Discussion
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