ARTICLE |
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
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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 48:11631171, 2000)
Key Words: A1 adenosine receptors, ecto-ADA, mammalian spermatozoa, localization, calcium uptake
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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 (
Bearing this in mind and because of the existence of ecto-ADA in mammalian spermatozoa membrane (
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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 IgGFITC, 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 BoehringerMannheim (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 HepesKOH, 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-ADATRITC and anti-ADA) were a gift from Prof. Franco and have been thoroughly characterized elsewhere (
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-21FITC antibody (70 µg/ml) in blocking buffer. Double immunofluorescence staining was performed by treating with a mixture of 70 µg/ml PC21FITC and 70 µg/ml anti-ADATRITC 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 IgGFITC and nonimmune rabbit IgGTRITC. 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% paraformaldehydePBS for 20 min at RT. After treatment with PBS0.5 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% glutaraldehydePBS for 1 hr at 4C and postfixed in 1% buffered osmium tetroxide. After dehydration, samples were embedded in EponAraldite (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 PBS20 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 IgGFITC (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 IgGFITC.
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 HepesKOH, 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 HepesKOH, 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.
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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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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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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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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 (
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Discussion |
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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 (
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Acknowledgments |
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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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