Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, Illinois 62794-1222
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ABSTRACT |
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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
i-1,2-,
i-3- and
-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
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INTRODUCTION |
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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.
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METHODS |
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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.
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 · min1 · 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).
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
-tubulin cDNA. The relative band intensities of
A1 AR and
-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
-tubulin mRNA levels.
Data analysis. Statistical differences among means were determined using ANOVA followed by Tukey's post hoc analyses.
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RESULTS |
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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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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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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
i- and
-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
i-1- and
i-2-subunits in preparations
that express these subunits (14, 27). Figure 4 provides evidence of
labeling of a single protein band, presumably
i-2. This conclusion is based
on the limited distribution of the
i-1 protein outside of the
central nervous system. An age-dependent decrease in the expression of
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
-subunits were significantly reduced by 18 mo but
increased toward control levels by 25 mo.
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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 -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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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