Age-dependent reductions in A1 adenosine receptor expression in rat testes

Satyanarayan G. Bhat, Marty Wilson, and Vickram Ramkumar

Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-1222

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The rat testis expresses high levels of A1 adenosine receptors (A1 AR) that couple to the inhibition of adenylyl cyclase activity. However, the physiological role of these receptors in the testis is not clear. Previous studies have documented a number of changes in the testis associated with the aging process. The goal of this study was to assess whether alteration in the expression and function of the testicular A1 AR occurs in aging, using the Fischer 344 rats as an aging model. Quantitation of A1 AR expression by radioligand binding of [3H]1,3-dipropyl-8-cyclopentylxanthine, an antagonist radioligand, indicates reductions in receptor number by 35 ± 13.3 and 53 ± 18.2% in 18- and 25-mo-old rats, respectively, compared with 3-mo-old rats. Similar reductions in A1 AR expression were determined using Western blotting and receptor autoradiography. Quantitation of the Gi proteins using selective antibodies indicate age-dependent reductions in the levels of alpha i-1,2-, alpha i-3- and beta -subunits. Furthermore, the modulatory influences of guanosine 5'-O-(3-thiotriphosphate) on the binding of agonist and antagonist radioligands to the A1 AR were substantially reduced. Northern blotting analysis of rat testicular poly(A)+ RNA indicates both a 3.4-kb transcript and a 5.6-kb transcript that hybridized to the canine A1 AR cDNA probe. The levels of the 5.6-kb transcript were decreased by 24 ± 18 and 52 ± 3% in the 18- and 25-mo-old rats, respectively, compared with the 3-mo-old rats. These results indicate age-dependent deficits in the A1 AR signal transduction pathway in the testes and predict concomitant reductions in the action of adenosine.

purinergic receptor; G proteins; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ADENOSINE MEDIATES ITS physiological effects in a variety of tissue, in part, through activation of G protein-coupled receptors termed adenosine receptors (AR). To date, four subtypes of AR have been identified, these being the A1, A2A, A2B, and A3 receptors (34). These receptors are differentially distributed in the central nervous system, heart, testes, adipose tissue, liver, kidney, smooth and skeletal muscles, and blood cells and platelets (25). Both the A1 and A3 AR couple to Gi proteins and thereby promote inhibition of adenylyl cyclase (32). In contrast, the A2A and A2B AR positively couple to adenylyl cyclase through the Gs proteins. Other effectors shown to be regulated following activation of AR include K+ channels, Ca2+ channels (3), and phospholipase C (15, 20).

Studies by Murphy et al. (22) were the first to demonstrate the presence of AR binding sites in rat testes by autoradiography. In that study, [3H]cyclohexyladenosine binding was localized to spermatocytes in the seminiferous tubule epithelium. Subsequent work by Stiles and co-workers (33) provided evidence of A1 AR in the rat testes negatively coupled to adenylyl cyclase. Interestingly, the testicular A1 AR appeared to be larger on SDS-PAGE than the rat brain receptor (33), apparently as a result of increased glycosylation (24). Recently, Rivkees (29) provided evidence for differential distribution of A1 and A3 AR in the rat testes. The A1 AR were localized to the Sertoli's cells, whereas the more abundant A3 AR were localized primarily to the germ cells. However, no A2 AR was detected, despite a previous report that this receptor was present on mouse sperms, where it regulates sperm motility (9).

Several observations provide indirect evidence supporting a physiological role of AR in rat testes. Blockade of these receptors by caffeine led to changes in sperm motility, respiration (12), metabolism (13), and their ability to penetrate the ovum (30). High doses of methylxanthine antagonists resulted in testicular atrophy (11). In cultures of Sertoli's cells, activation of A1 AR leads to inhibition of follicle-stimulating hormone (FSH)-mediated cAMP production and the aromatization of androgen to estrogen (4).

The male reproductive system undergoes significant changes during aging. These changes include decrease in sexual activity, testosterone levels, ejaculate volume, and total sperm production (23, 35). To obtain a better insight into the role of this receptor subtype in the testes, we assessed the levels and G protein interactions of the A1 AR in the rat testes in aging. Our results indicate substantial reductions in A1 AR in the testes during aging, which might reflect decreases in the steady-state levels of mRNA encoding this receptor subtype.

    METHODS
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Introduction
Methods
Results
Discussion
References

Animals. Animals used in this study were Fischer 344 rats of three different age groups: 3, 18, and 25 mo. These rats were obtained from Harlan (Indianapolis, IN) and were maintained on regular food and water. The protocol for the use of animals in this study was approved by the Laboratory Care and Use Committee of the Southern Illinois University School of Medicine. Animals were allowed to recover for at least 1 wk before being killed.

Sample collection. Rats were killed using a guillotine, and the testes were rapidly dissected free of epididymis and frozen in liquid nitrogen for radioligand binding, Northern blotting, and Western blotting assays. For autoradiography and histological studies, testes were fixed to chucks and then frozen on dry ice. Twenty-micrometer sections were obtained using a sliding microtome (International Equipment, Needham Heights, MA), fixed to microscope slides, and then stored at -20°C for 1-2 days before performance of the relevant studies.

During dissections, testes were grossly examined for the presence of tumors and those that indicated evidence of tumors were discarded. About 60% of the 25-mo-old rats had tumors in at least one testis. Furthermore, testes were sectioned before preparation of membranes for studies (described below) and if tumors were observed in these "normal" testes, these were also discarded.

Membrane preparation. The rat testicular membranes were prepared exactly as previously described (33). In brief, frozen testes were thawed and placed in ice-cold 50 mM Tris · HCl (pH 7.4) containing 10 mM MgCl2, 1 mM EDTA, 10 µg/ml soybean inhibitor, 10 µg/ml benzamidine, and 2 µg/ml pepstatin (buffer A). The tissue was then homogenized by a Polytron (Brinkmann; setting 7) for 40 s at 4°C. After centrifugation at 1,000 g for 10 min, the supernatant was centrifuged at 40,000 g for 15 min. The resulting pellet was suspended in buffer A to a final protein concentration of 1 mg/ml. Before performance of radioligand binding assays, crude plasma membrane preparations were incubated with adenosine deaminase (5 U/ml) at 37°C for 10 min to eliminate endogenous adenosine.

Radioligand binding assay. The levels of A1 AR in rat testicular membranes were determined using a selective antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX). The assays were performed by incubating membranes (75 µg protein) at 37°C for 1 h with various concentrations of [3H]DPCPX in absence (total binding) or presence (nonspecific binding) of theophylline (0.5 mM), in a total volume of 250 µl of buffer A. After incubations, samples were filtered through GF/B glass fiber filters using a cell harvester (Brandel, Gaithersburg, MD) and quickly washed with 9 ml of ice-cold buffer A containing 0.01% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Bound radioactivity was determined using a liquid scintillation counter. Experiments utilizing 125I-labeled aminophenylethyladenosine (APNEA) were performed in a similar fashion, and radioactive counts were determined using a gamma counter. Nonspecific binding (non-A1 AR sites) was defined by using 100 nM DPCPX to saturate the A1 AR sites. Saturation curves were analyzed by a computer-based curve-fitting program, described previously (7, 16), equipped with a statistical package.

Receptor autoradiography. Slides were thawed, preincubated with adenosine deaminase for 15 min at room temperature in buffer A, and then incubated with 5 nM [3H]DPCPX (21.8 Ci/mmol). Nonspecific binding was defined using adjacent sections that were incubated with the radioligand together with 0.5 mM theophylline. Incubations were terminated with four washes of ice-cold buffer containing 0.1% CHAPS, and slides were allowed to air dry. Care was taken to limit the time of exposure of the sections with the wash solution to ~5 s/wash. Autoradiograms were generated by apposing the slides to 3H-sensitive Hyperfilm (Amersham Life Sciences, Evanston, IL). After exposure for 2 wk at 4°C, the films were developed manually using Kodak D19 and were fixed and air dried. The autoradiographic films were quantified by computer-assisted densitometry, using the MCID imaging system (Imaging Research, Saint Catharines, ON, Canada). The average optical density was determined by taking multiple-density readings (~10) from different areas of the section. Background readings were obtained in a similar fashion, using sections incubated with the radioligand plus 0.5 mM theophylline.

Histological examinations of the sections were performed by staining the slides with cresyl violet. Data obtained by counting the number of seminiferous tubules in one microscope field of each histological section indicate reductions of 38 and 34% for the 18- and 25-mo-old rats, respectively, compared with the 3-mo-old animals.

SDS-PAGE and Western blotting. Testicular membranes were solubilized in SDS-PAGE buffer at a concentration of 2 µg protein/µl. Samples (75 µg) were electrophoresed by SDS-PAGE according to the method of Laemmli (18). The proteins were then transferred to nitrocellulose filters using a Nova Blot apparatus (Pharmacia Biotech, Piscataway, NJ), blocked in Blotto solution (130 mM NaCl, 2.7 mM KCl, 1.8 mM Na2HPO4, 1.5 mM KH2PO4, 0.1% NaN3, and 5% low-fat skim milk) containing 0.1% Triton X-100 and incubated at 4°C overnight with G protein antisera (14, 27). These antibodies were obtained from Dr. Tom Gettys (Medical University of South Carolina, Charleston, SC). After incubations, the blots were washed 5 times (10 min each) with Blotto solution and incubated with 125I-labeled goat anti-rabbit IgG [300,000 counts · min-1 · ml-1 (cpm/ml)] for 1 h at room temperature. Blots were then washed five times (10 min each) with Blotto containing 1% Triton X-100 before exposure to autoradiographic films or analysis using a GS-250 molecular imager (Bio-Rad, Hercules, CA).

The levels of A1 AR were also determined by Western blotting using a polyclonal antiserum (Alfa Diagnostic International, San Antonio, TX). Testicular membranes, prepared as described above, were gently homogenized on ice in buffer A containing CHAPS, using a CHAPS-to-protein ratio of 2.5:1. Samples were then stirred on ice for 1 h and centrifuged at 40,000 g for 15 min. The supernatants were desalted on a Sephadex G-25 column and equilibrated with buffer A containing 0.1% CHAPS. One hundred micrograms of protein from each desalted fraction were solubilized in SDS-PAGE buffer, resolved by SDS-PAGE, and used in Western blotting studies (as above). The levels of actin were determined by Western blotting studies using a monoclonal anti-actin antibody (Sigma Chemical, St. Louis, MO) and an enhanced chemiluminescence horseradish peroxidase detection kit (Amersham Life Sciences) according to the manufacturer's protocol. The relative expression of A1 AR and G proteins in each lane was obtained after normalization to actin. Appropriate quantitations of Western blots (both iodinated and luminescent proteins) were performed using a phosphorimager.

Preparation of RNA and Northern blotting. Experiments dealing with isolation of total RNA and selection of poly(A)+ messenger RNA were performed as described (6). Poly(A)+ RNA samples (10 µg) were electrophoresed on a 1% agarose gel containing 0.5× MOPS buffer (5× MOPS buffer contains 200 mM MOPS, 50 mM sodium acetate, and 5 mM EDTA) and 3% formaldehyde. After electrophoresis, RNA was transferred to nylon membranes using a Stratagene ultraviolet cross-linker. Prehybridization mixture contained 5× saline sodium citrate (SSC), 2× Denhardt's, 1% SDS, 0.2 mg/ml salmon sperm DNA, and 50% formamide. The hybridization solution (10 ml) was essentially the same as the prehybridization solution, except that it contained 2.5× Denhardt's and random primer 32P-labeled canine A1 AR cDNA probes at a concentration of 1-2 × 106 cpm/ml. Blots were incubated with prehybridization solutions in a 42°C shaking water bath for 4 h. This was followed by hybridization for 16-24 h at 42°C. After incubation, blots were washed twice (15 min each) at room temperature in wash buffer containing 2× SSC and 0.1% SDS and twice at 62°C (20 min each) in buffer containing 0.1× SSC and 0.1% SDS. Blots were then exposed to autoradiographic films (Kodak X-OMAT LS, Sigma) and stored at -80°C for 1-4 days. For normalization, blots were stripped with and reprobed with chick alpha -tubulin cDNA. The relative band intensities of A1 AR and alpha -tubulin mRNA on the blots were subsequently quantitated by exposure of blots to phosphorimager screens for 6-12 h and densitometric scanning using a phosphorimager. The levels of A1 AR mRNA were normalized with alpha -tubulin mRNA levels.

Data analysis. Statistical differences among means were determined using ANOVA followed by Tukey's post hoc analyses.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Histological examination of testes from rats of different ages. Figure 1 demonstrates cresyl violet staining of testicular cross sections obtained from the rat. A well-defined interstitial cell layer separating the seminiferous tubules is evident in sections obtained from 3-mo-old animals (Fig. 1a). The seminiferous tubules are filled with spermatids and spermatocytes, which occupy the entire lumen of these tubules. Sections from an 18-mo-old animal (Fig. 1b) provide evidence for some disruption in the interstitial cell layer and also a decrease in the number of sperms present in the seminiferous tubules. In addition, the seminiferous tubules are larger in diameter than those obtained from 3-mo-old rats. Sections obtained from 25-mo-old rats indicate dramatic disruption in the integrity of the seminiferous tubules and interstitial cell layers and provide evidence of tubular sclerosis. In addition, a substantial reduction in spermatocytes in seminiferous tubules is readily apparent.


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Fig. 1.   Histological examination of cross sections of rat testes with aging. Testes were isolated and frozen onto metal tissue holders using dry ice. Tissues were stored at -80°C before sectioning. Twenty-micrometer sections were obtained using a sliding microtome, and these sections were fixed to microscope slides and stored at -20°C. Sections were stained with cresyl violet within 1-2 days of preparation. a, b, and c: Light microscopic representations of sections from 3-, 18-, and 25-mo-old animals, respectively. Note integrity of interstitial cell layer (icl) and sizes of seminiferous tubules (st). Arrowheads indicate areas with disruption of interstitial cell layer (b) and sclerosis of seminiferous tubules (c). Scale bar (bottom right), 100 µm.

Decrease in the expression of A1 AR in rat testes with age. Saturation plots, performed using the antagonist radioligand [3H]DPCPX, indicate age-dependent reductions in A1 AR in the testes. The specific binding obtained averaged ~70% of total binding at concentrations of [3H]DPCPX around the equilibrium dissociation constant (Kd). A Scatchard representation of the data is shown in Fig. 2B and indicates age-dependent reductions in total receptor number (Bmax) without changes in Kd. Values for Bmax were 353 ± 37, 233 ± 47, and 168 ± 64 for the 3-, 18-, and 25-mo-old rats, respectively (Fig. 2, A and B). The values obtained for the 18- and 25-mo-old rats were statistically significant from those observed for the 3-mo-old rats (P < 0.05). No significant alteration in the Kd was observed. The respective Kd values were 1.0 ± 0.2, 1.0 ± 0.2, and 1.7 ± 0.9 nM for the 3-, 18-, and 25-mo-old animals.


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Fig. 2.   Age-dependent change in A1 adenosine receptor (AR) expression in rat testes. A: crude plasma membranes (75 µg protein) prepared from 3-, 18-, and 25-mo-old rat testes were incubated with increasing concentrations of [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0-5 nM) for 1 h at 37°C. Specific binding was calculated as difference between total binding and nonspecific binding (in presence of 0.5 mM theophylline). Saturation curves were best fitted to a 1-state model, using a computer-based curve-fitting program (7, 16). This saturation is representative of 4 experiments, each performed in triplicate. B: transformation of data in A by Scatchard analysis. C: quantitation of A1 AR in rat testes by Western blotting. Each lane was loaded with 100 µg of membrane protein and was resolved by SDS-PAGE using 12% acrylamide gels. A1 AR was visualized by autoradiography following incubation of filters with 125I-labeled goat anti-rabbit IgG [300,000 counts · min-1 · ml-1 (cpm/ml)]. Normalization of blots was performed by incubating A1 AR blots with a monoclonal antibody against actin (lower band). Testicular A1 AR was purified by affinity chromatography (24) and was used as a positive control.

Western blotting studies for the A1 AR indicate labeling of a 42-kDa protein in both testicular membranes and affinity-purified preparations from testes, comparable to the molecular size of the A1 AR reported previously (24). Compared with testicular preparations from the 3-mo-old animals, the levels of A1 AR in the 18- and 25-mo-old rats were reduced by 19 ± 6 and 78 ± 7%, respectively (Fig. 2C). Both of these values were statistically significant from that obtained from the 3-mo-old animals (P < 0.05).

Autoradiographic studies localized the [3H]DPCPX binding sites to the tubular epithelium of the seminiferous tubules but not to spermatocytes contained in the lumen (Fig. 3). This is clearly depicted in Fig. 3b, where labeling surrounds a central hollow (the lumen of the seminiferous tubules). Radioligand binding in the presence of 0.5 mM theophylline (nonspecific binding) was negligible (Fig. 3, d-f ), indicating that specific binding of the radioligand was to the A1 AR. Specific labeling of the A1 AR was determined by densitometric scanning of different (at least 10 readings) regions of autoradiograph with similar tubule density using an MCID camera. The average relative levels of A1 AR estimated (3-mo value equals 100%) for sections obtained from 18- and 25-mo-old rats were 100 ± 12 and 61 ± 6%, respectively. The reason for the lack of change in A1 AR in the 18-mo-old rat testes in the autoradiographic sections is not clear. However, it might point to a lack of sensitivity of this technique to detect differences in receptor expression. Although the 25-mo-old sections indicate fewer sites for labeling, the labeling intensity per tubule appeared to be greater. This probably implies a greater number of A1 AR per tubule in the testes from old rats.


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Fig. 3.   Age-dependent changes in A1 AR expression determined by autoradiography. Twenty-micrometer sections of testes from rats aged 3 mo (a and d), 18 mo (b and e), and 25 mo (c and f) were prepared using a sliding microtome, and these were fixed to microscope slides and stored at -20°C until use. Slides were incubated with [3H]DPCPX (5 nM) for 2 h at room temperature in absence (a-c) and presence (d-f) of 0.5 mM theophylline. Autoradiograms were obtained by exposing labeled sections to 3H-sensitive film for 2 wk at 4°C. Sections are representatives of at least 5 individual sections per treatment condition. Scale bar, 500 µm.

Decrease in Gi protein subunit expression during aging. Because the functions of the A1 AR are mediated through coupling to G proteins, studies were performed to determine whether age-dependent changes exist in expression of these G proteins in testes. The levels of these proteins were determined by Western blotting, using polyclonal antibodies for the alpha i- and beta -subunits. Each lane was loaded with the identical amount of plasma membrane proteins. The levels of G protein subunits were normalized to actin and expressed as percentages of 3-mo values (Fig. 4). Antibody 453 recognizes both alpha i-1- and alpha i-2-subunits in preparations that express these subunits (14, 27). Figure 4 provides evidence of labeling of a single protein band, presumably alpha i-2. This conclusion is based on the limited distribution of the alpha i-1 protein outside of the central nervous system. An age-dependent decrease in the expression of alpha i subtypes in testicular membrane preparations was observed. Significant decreases in Gi subunits were identified by 18 mo (Table 1), with only slight changes from these levels obtained in the 25-mo-old group (Fig. 4). The levels of beta -subunits were significantly reduced by 18 mo but increased toward control levels by 25 mo.


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Fig. 4.   Age-dependent alterations in G protein subunit expression in rat testes. Testicular membrane proteins (75 µg) were solubilized and electrophoresed on SDS-PAGE, as described in METHODS. Western blotting was performed essentially as described in METHODS, using 125I-labeled goat anti-rabbit IgG as secondary antibody. Relative intensities of bands (normalized to actin) were determined using a phosphorimager. Blot is representative of 3-4 independent experiments that showed similar results.

                              
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Table 1.   Expression of G protein subunits in rat testes in aging

Both agonist and antagonist binding to the A1 AR appear to be sensitive to GTP analogs (10, 26). To assess A1 AR-G protein coupling with aging, the ability of guanine nucleotide to regulate receptor binding in testicular membrane preparations from different age groups was assessed. In these experiments the hydrolysis-resistant GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) was used. Incubation of membranes with GTPgamma S increased the binding of [3H]DPCPX in testicular membrane preparations obtained from a 3-mo-old rat (Fig. 5A). Increase in antagonist binding obtained with 0.1 µM GTPgamma S in the 3-mo-old rat testes was 21 ± 5%. A similar concentration of GTPgamma S was ineffective in regulating [3H]DPCPX binding in membranes obtained from 18- and 25-mo-old rats. Unlike the antagonist, guanine nucleotides promote uncoupling of the A1 AR from their associated G proteins, resulting in substantially lower affinity of the radioligand for the receptor. In testicular preparations obtained from 3-mo-old animals, GTPgamma S (0.1 µM) reduced the binding of 125I-APNEA to the A1 AR to 41 ± 2% of control, indicative of uncoupling of the receptor from its G protein(s) (Fig. 5B). In the presence of GTPgamma S, 125I-APNEA binding was reduced to 40 ± 1 and 78 ± 10%, respectively, in the 18- and 25-mo-old groups.


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Fig. 5.   Age-dependent changes in interactions between A1 AR and Gi proteins in rat testes. Membranes (75 µg protein) were incubated for 1 h with guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S; 0.1 µM) and either 125I-aminophenylethyladenosine (APNEA) (B) or [3H]DPCPX (A) at 37°C. Specific binding of radioligands was determined as difference in binding in samples treated without and with 100 nM DPCPX (for 125I-APNEA binding) or 0.5 mM theophylline (for [3H]DPCPX). For each age group, 100% level of binding was defined in absence of GTPgamma S. Levels of specific 125I-APNEA detected in absence of GTPgamma S (100%) in testes of 3-, 18-, and 25-mo-old rats were 206 ± 72, 81 ± 23, and 57 ± 29 fmol/mg protein. Respective levels of [3H]DPCPX binding were 138 ± 66, 102 ± 13, and 89 ± 33 fmol/mg protein in 3-, 18-, and 25-mo age groups. Data are means ± SE of 4 independent experiments. * Statistically significant difference vs. 3-mo-old testes.

Decrease in mRNA encoding the A1 AR in aging. RNA samples were prepared from rat testes and used in Northern blot analyses to determine whether decreases in A1 AR-specific mRNA encoding this receptor could account for decreases in the expression of the protein in aging. Due to the low levels of A1 AR-specific RNA in the testes, poly(A)+ preparations were first prepared and used for Northern blotting. Blots were hybridized with a random primer cDNA probe for the canine A1 AR, and two transcripts were detected, these being 3.4 and 5.6 kb (Fig. 6). The lower 3.4-kb band was barely detectable in the 3-mo-old group and was not used for quantitation. For normalization, blots were first stripped and reprobed with a cDNA encoding the chick alpha -tubulin. After normalization, the steady-state levels of the 5.6-kb RNA encoding the A1 AR in the 18- and 25-mo-old animals were 76 ± 18 and 48 ± 2%, respectively, of the 3-mo-old animals.


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Fig. 6.   Age-dependent change in rat testicular A1 AR mRNA. Poly(A)+ RNA samples prepared from testes from 3-, 18-, and 25-mo-old rats were resolved on 1% MOPS-formaldehyde-agarose gel and blotted on nylon membranes. Blots were probed with a random primer labeled cDNA for canine A1 AR at a concentration of 1-2 × 106 cpm/ml. Prehybridization, hybridization, and wash conditions are as described in METHODS. Blots were then subjected to autoradiography for 1-2 days. Relative band intensities were determined with a phosphorimager. Normalization of these blots was performed using a random primer labeled chick alpha -tubulin cDNA. Similar findings were obtained in at least 4 different animals for each age group.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

This study demonstrates deficits in the A1 AR signal transduction pathway in the testes in aging. Decreases in the expression of both the A1 AR and Gi proteins were observed, as well as diminished receptor-G protein coupling. Changes in the levels of the A1 AR were associated with, and may be explained by, decreases in the steady-state levels of mRNA encoding this receptor subtype.

AR are localized to discrete regions of the testes. Previous studies by Murphy et al. (22) indicated that the binding of [3H]cyclohexyladenosine, an agonist that interacts with both A1 and A3 AR, to rat testicular sections was localized within the seminiferous tubules, where it appeared to be associated with sperm and/or other supporting cells. Manipulations that resulted in a decrease in Leydig cells did not change the extent of radioligand binding. However, manipulation that led to reductions in sperm production decreased the level of radioligand binding. These data suggest that the binding of [3H]cyclohexyladenosine was restricted to Sertoli's cells and spermatocytes. In a recent study, Rivkees (29) localized the A1 AR to Sertoli's cells and the A3 AR to germ cells.

Age-related changes in testicular functions have previously been documented. A decrease in total sperm production and an increase in nonviable and abnormal spermatocytes have been detected with aging. Microscopic examinations of testicular sections obtained from different age groups indicate age-dependent reductions in spermatocytes present in the seminiferous tubules. Data obtained in the rat (see Fig. 1) in this study confirm this latter finding. In addition, reductions in the number of Sertoli's cells have also been detected in aging, which correlated with the reduction in sperm production (17, 31). On the basis of these data and the observation that the A1 AR is localized to Sertoli's cells, it is possible that the decrease in A1 AR in the rat testes in aging could reflect, in part, a decrease in the number of Sertoli's cells and/or in the intensity of labeling of these cells.

Reductions in the expression of the A1 AR in Sertoli's cells might compromise the physiological role(s) of this receptor subtype in the testes. Studies by Davenport and Heindel (8) indicate that A1 AR in Sertoli's cells inhibit the actions of FSH, presumably by inhibiting adenylyl cyclase. As such, adenosine acts as a tonic inhibitor of FSH, such that desensitization of the A1 AR in Sertoli's cells augments the action of FSH. Similarly, downregulation and uncoupling of the A1 AR during aging (as observed in this study) would be expected to reduce the inhibitory action of adenosine, resulting in enhanced FSH activity.

A reciprocal interaction between Sertoli's cells and germ cells has been proposed (4). In this model, adenosine produced by germ cells activates inhibitory AR on Sertoli's cells, leading to inhibition of FSH-stimulated responses. This leads to regulation (either increase or decrease) in secretion of factors to influence the germ cells. In this respect, the action of adenosine might closely regulate germ cell function. Alteration in the function of A1 AR on Sertoli's cells, therefore, might lead to dysregulation of Sertoli's cell-germ cell function, and this might account for deficiency in spermatogenesis accompanying the aging process.

In addition to changes in receptor expression, decreases in Gi protein subunits were also observed. Decreases in G protein subunits were quantitated by Western blotting and were functionally assessed by guanine nucleotide regulation of radioligand binding to the A1 AR. Like other G protein-coupled receptors, the binding of agonist radioligands to the A1 AR is reduced by guanine nucleotides, due to uncoupling of the receptor from its G proteins. Interestingly, the reverse appears to be true for antagonist binding. Guanine nucleotides increase the binding of antagonist radioligands to the A1 AR (10, 26). It has been concluded that, whereas the agonist interacts preferentially with the G protein-coupled A1 AR, the binding of the antagonist is facilitated by receptor uncoupling (10, 26).

The mechanism(s) underlying the decrease in A1 AR-specific RNA in testes during aging is unclear at present. A decrease in the steady-state level of mRNA encoding this receptor likely results from a decrease in transcription of the receptor gene and/or a decrease in the stability of the mRNA. At present, however, we cannot distinguish between these two possibilities. In the case of transcriptional regulation, the promoter region of the human A1 AR gene possesses consensus sequences for activating protein 1 transcription factors (28). However, the identity of an endogenous factor that promotes gene activation via AP-1 transcription factors is not yet known. Several pieces of evidence indicate decreases in the DNA binding activity of AP-1 transcription factors with aging (2, 19, 36), and as such this could contribute to decreased transcription of the A1 AR gene.

One complication in studying changes in A1 AR expression in aging is that the production of sex steroids, such as testosterone (35), also decreases in aging. At present it is unclear whether the expression of the A1 AR is under control of such sex steroids. If this were the case, a decrease in the levels of testosterone would directly lead to a decrease in expression of the A1 AR.

Regulation of the A1 AR during aging has been demonstrated in several different tissues. In the rat brain, for example, age-dependent reductions in levels of A1 AR were observed in the hippocampus and cortex but not in the striatum (5). Similar age-dependent changes were observed in the mouse cortical, hippocampal, and cerebellar membranes. In the gerbil, decreases in agonist binding were observed in the hippocampus and cerebellum, whereas increases in binding were obtained in the neocortex and striatum (1). Recent studies in the heart indicate increases in A1 AR density in the rabbit aging models (21). This correlated well with increased sensitivity of the senescent heart to the negative inotropic action of adenosine in this tissue.

In summary, the present study provides evidence for age-dependent decrease in A1 AR expression, presumably linked to decreases in the steady-state levels of mRNA encoding this receptor. The significance of this finding to testicular function awaits future studies.

    ACKNOWLEDGEMENTS

We thank Valerie Free for assistance in preparing the manuscript for submission. We also acknowledge the technical assistance of Dr. Robert Helfert, Dr. Lenny Maroun, Wendy Terry, Tina Holder, and Terry Sommers.

    FOOTNOTES

Address for reprint requests: V. Ramkumar, SIU School of Medicine, Box 19230, Springfield, IL 62974-1222.

Received 30 September 1997; accepted in final form 5 January 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 274(4):C1057-C1064
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society




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