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Dibutyryl Cyclic Adenosine Monophosphate Restores the Ability of Aged Leydig Cells to Produce Testosterone at the High Levels Characteristic of Young Cells

Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. Haolin Chen, Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205. E-mail: hchen{at}jhsph.edu.

	Abstract

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The wealth of knowledge about the function and regulation of adult Leydig cells, the cells within the mammalian testis that produce testosterone, make these cells ideal for studying principles and mechanisms of aging. A hallmark of mammalian aging is decreased serum testosterone concentration. In the Brown Norway rat, this has been shown to be associated with the reduced ability of aged Leydig cells to produce testosterone in response to LH. Herein, we demonstrate that culturing the aged cells with dibutyryl cAMP, a membrane-permeable cAMP agonist that bypasses the LH receptor-adenlyly cyclase cascade, restores testosterone production to levels comparable to those of young cells and also restores steroidogenic acute regulatory protein and P450scc, the proteins involved in the rate-limiting steps of steroidogenesis. These results strongly suggest that signal transduction deficits are responsible for reduced steroidogenesis by aged Leydig cells and that bypassing signal transduction reverses the steroidogenic decline by the aged cells.

	Introduction

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IN RATS, AS IN MEN, reduced serum testosterone concentration is a hallmark of aging (1, 2). The decreases in serum testosterone may be accompanied by a constellation of symptoms, sometimes termed andropause, that include sexual dysfunction, lack of energy, loss of muscle and bone mass, increased frailty, loss of balance, cognitive impairment, and decreased general well-being (3). We have shown in the Brown Norway rat, which is now a well-established model for human male reproductive aging, that age-related reduction in serum testosterone results from the reduced production of testosterone by individual Leydig cells (4). The aged cells are characterized by a number of deficits in the steroidogenic pathway, including reductions in LH receptor number, cAMP production (5), the cholesterol transport proteins steroidogenic acute regulatory protein (StAR) (6) and peripheral benzodiazepine receptor (7), and the steroidogenic enzymes of the mitochondria and smooth endoplasmic reticulum that convert cholesterol to testosterone (8). As yet, the roles played by particular deficits in the reduced steroidogenic capacity of aging Leydig cells and the mechanism by which these deficits arise are uncertain.

In young rats, experimental suppression of serum LH levels results in decreases in Leydig cell volume and testosterone production (9), qualities that also characterize aged Leydig cells (4). However, serum LH concentrations in Brown Norway rats do not decline significantly with age (4, 6), and the administration of exogenous LH to old rats in vivo or the incubation of purified, aged Leydig cells with LH in vitro failed to increase testosterone production by old Leydig cells (5, 10), suggesting that deficits in LH are unlikely to represent the underlying cause of age-related decline in Leydig cell steroidogenesis.

The observation that LH-stimulated old Leydig cells produce far less cAMP than young cells (5) suggests that the old cells have defects in the LH-cAMP signaling cascade. Recognizing the critical role played by LH-stimulated cAMP in steroidogenesis (11), we hypothesized that reduced cAMP production might cause age-related reductions in steroidogenesis. To test this hypothesis, we reasoned that if the steroidogenically hypofunctional aged cells could be induced to produce testosterone at the high levels of young adult cells by incubating the cells with dibutyryl cAMP (dbcAMP), then we would conclude that aged-related reductions in the steroidogenesis most likely result from defects in the LH signaling pathway leading to reduced cAMP.

	Materials and Methods

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Animals
Male Brown Norway rats at ages 4–6 months (young) and 21–24 months (old) were obtained through the National Institute on Aging and supplied by Harlan Sprague Dawley Inc. (Indianapolis, IN). Rats were housed in controlled light (14 h light and 10 h dark) and temperature (22 C), with free access to rat chow and water. All procedures were in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with protocols approved by the Johns Hopkins Animal Care and Use Committee.

Suppression of LH
To experimentally suppress LH in young rats, the rats were administered testosterone via sc polydimethylsiloxane (SILASTIC brand; Dow Corning, Midland, MI) implants. Details of the fabrication of the implants have been described previously (12). Briefly, rats received implants of 3 cm placed sc into the interscapular region for 5 d to suppress Leydig cell steroidogenesis through feedback suppression of serum LH.

Leydig cell purification
Leydig cells were isolated as previously described (13). In brief, the testicular artery was cannulated and perfused with collagenase (1 mg/ml, Type 3; Worthington, Freehold, NJ) in dissociation buffer (M-199 medium with 2.2 g/liter HEPES, 1.0 g/liter BSA, 25 mg/liter trypsin inhibitor, 0.7 g/liter sodium bicarbonate, pH 7.4) to clear blood from the testes. Testes were decapsulated and digested in collagenase (0.25 mg/ml, 34 C) with shaking (90 cycles/min, 30 min). The dissociated cells were then subjected to centrifugal elutriation and Percoll gradient centrifugation purification, as previously described (13). The final purity of the Leydig cells obtained this way, determined by staining the cells for 3ß-hydroxysteroid dehydrogenase activity, was consistently about 95%. Cell viability, assessed by trypan blue exclusion, was over 95%.

Culture of isolated Leydig cells with LH and dbcAMP in vitro
Leydig cells were cultured according to the procedure described by Klinefelter and Ewing (14). Briefly, purified Leydig cells were resuspended (10⁶/ml) in M-199 medium (GIBCO, Grand Island, NY) supplemented with 2.2 g/liter NaHCO₃, 2.4 g/liter HEPES, 0.1% BSA, and 12.5 mg/liter gentamicin sulfate (pH 7.4). One milliliter of cell suspension (1.0 x 10⁶) was then added to a low attachment 24-well culture plate (catalog no. 3473; Corning Inc., Acton, MA) containing 0.2 ml of Cytodex 3 beads (Sigma, St. Louis, MO). Bovine lipoprotein (Sigma) was added to provide a final concentration of 0.5 mg/ml. For cells that were cultured for more than 24 h, either LH (final concentration, 0.5 ng/ml; USDA-bLH-B-6, United States Department of Agriculture Animal Hormone Program, Beltsville, MD) or dbcAMP (final concentration, 1 mM) was added. In preliminary studies, we demonstrated that these LH and dbcAMP concentrations maintained testosterone production at about 50% of the steroidogenic capacity of the cells. To determine the maximal 24-h testosterone production, cells were stimulated with either 100 ng/ml LH or 5 mM dbcAMP for the final 24 h of culture. These concentrations of LH and dbcAMP were selected based on the results of previous studies (14) and from preliminary experiments. The final culture volume was adjusted to 2.0 ml with M-199 culture medium, and the cultures were maintained at 34 C in 5% CO₂-5% O₂-90% N₂.

Media were changed every 24 h. For this purpose, the Cytodex 3 beads with Leydig cells attached were allowed to settle to the bottom of the culture wells, and the supernatants were collected and frozen for testosterone assay. The beads, with Leydig cells attached, were washed once with fresh culture medium and then resuspended in fresh culture medium and placed in the reduced-oxygen environment. At the end of culture, media were collected and stored at –80 C. Testosterone in the medium was assayed by RIA (testosterone antibody from ICN, Costa Mesa, CA; and ³H-testosterone from NEN Life Science Products, Boston, MA). The sensitivity and intraassay and interassay coefficients of variation of the RIA were 13 pg/tube and 8.9 and 13.6%, respectively. Leydig cells attached to the beads were lysed with TES buffer (10 mM Tris, pH 8.0; 1 mM EDTA, 1% sodium dodecyl sulfate, and 100 mM KCl) at 50 C for 30 min. DNA was assayed fluorometrically with 4',6-diamidino-2-phenylindole (15). Testosterone production was normalized by the amounts of DNA recovered from the beads.

cAMP production
Purified Leydig cells were resuspended (5 x 10⁵/ml) in M-199 medium supplemented with 2.2 g/liter NaHCO₃, 2.4 g/liter HEPES, and 0.1% BSA (pH 7.4). The cells (1 x 10⁵/200 µl) were preincubated in 96-well Falcon culture plates (Becton Dickinson, Franklin Lakes, NJ) under 5% CO₂-95% air at 34 C for 4 h. The medium was then carefully removed, and 50 µl fresh phenol red-free M-199 medium, containing LH (100 ng/ml) or forskolin (500 µM; Sigma), was added to the plates. These concentrations of LH and forskolin stimulate cAMP production maximally (5). The medium also contained 100 µM isobutyl-methylxanthine (IBMX; Sigma) to inhibit phosphodiesterase activity. After 15 min of incubation, 50 µl Tris buffer (0.05 M, pH 7.5; containing 4 mM EDTA and 2 mg/ml theophylline) was added to the culture medium, and the plates were frozen by dry ice and stored at –80 C until cAMP assay. In some experiments, 500 ng/ml cholera toxin (Calbiochem, La Jolla, CA) or 100 ng/ml pertussis toxin (Sigma) were included in the medium during the preincubation period. We found that these concentrations of toxins maximally inhibited G protein function or stimulated G protein function without affecting overall cell viability. cAMP was assayed with a cAMP [³H] assay system (Amersham, Piscataway, NJ) according to the manufacturer’s directions. The sensitivity of the assay was 0.05 pmoles per assay tube.

Adenylyl cyclase activity
Adenylyl cyclase activity was assayed by incubating Leydig cell membrane fractions at 34 C for 15 min and monitoring the conversion of ATP to cAMP (16). In brief, about 5 x 10⁶ cells were homogenized in 200 µl of HEPES buffer (50 mM, pH 8) containing 1 mM EDTA and protease inhibitor cocktail (Sigma) on ice. After centrifugation at 400 x g for 2 min, the supernatant was centrifuged at 100,000 x g for 15 min at 4 C. To assay the adenylyl cyclase activity, 10 µg of membrane protein were incubated in the 100 µl reaction solution (50 mM HEPES, pH 8; 1 mM EDTA, 10 mM MgCl₂, 0.5 mM ATP, 1.5 mM K⁺phosphoenolpyruvate, 10 µg/ml pyruvate kinase, 0.1 mg/ml BSA, 100 µM IBMX, and 100 µM forskolin) at 34 C for 15 min. cAMP in the final reaction solution was assayed with a cAMP [³H] assay system (Amersham) according to the manufacturer’s directions. Enzyme activity was proportional to the concentration of membranes and the time of incubation.

Western blot analysis
To analyze the stimulatory G (Gs) protein, the cell membrane fractions were lysed in solubilization buffer (100 mM Tris-HCl, pH 6.8; 1% Triton X-100, 100 mM dithiothreitol, and 10% protease inhibitor cocktail; Sigma) at 4 C for 30 min. To analyze the StAR and P450scc proteins, the purified cells were lysed in the same solubilization buffer at 4 C for 60 min. After centrifugation (15,000 x g for 15 min at 4 C), the supernatant was mixed with 3x SDS sample buffer (New England BioLabs Inc., Beverly, MA). Twenty micrograms of protein from each sample were separated on a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane. After incubation with primary antibodies (1:1000) and horseradish peroxidase-conjugated secondary antibody (1:5000; Amersham), antibodies bound to the proteins were detected with the SuperSignal West Pico Luminescence kit (Pierce, Rockford, IL). For the Gs -subunit, membranes were analyzed with antibody (catalog no. 371731) from Calbiochem (La Jolla, CA), and the result was confirmed with antibody (SA-131) from Biomol (Plymouth Meeting, PA). StAR antibody was a gift from Dr. Douglas M. Stocco (Texas Tech University Health Sciences Center, Lubbock, TX). Cytochrome P450 cholesterol side-chain cleavage (P450scc) antibody (AB1294) was purchased from Chemicon International (Temecula, CA).

Statistical analyses
Data are expressed as the mean ± SEM. Statistical differences were determined by one-way ANOVA. If group differences were revealed by ANOVA (P < 0.05), then differences between individual groups were determined using the Student-Newman-Keuls test (P < 0.05) by using SigmaStat software (Systat Software Inc., Richmond, CA). Values were considered significant at P < 0.05.

	Results

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Figure 1 shows the capacity of isolated Leydig cells to produce testosterone when stimulated maximally with LH or dbcAMP for 24 h in vitro (d 1) or in the last 24 h of a 3-d culture period (d 3). Leydig cells were isolated from young control and old control rats and from young rats that had received LH-suppressive testosterone implants for 5 d. The latter cells served as a positive control because high levels of testosterone production would be expected to be restored in these cells with LH stimulation (17). Young Leydig cells from untreated rats produced significantly more testosterone than old cells or cells from young LH-suppressed rats, whether the cells were incubated with LH or dbcAMP for 24 h (d 1). With 3 d of culture with either LH or dbcAMP, testosterone production by cells from young rats maintained their ability to produce testosterone at high levels. With LH for 3 d, the cells from young LH-suppressed rats, which produced very low levels of testosterone on d 1, had a significantly increased ability to produce testosterone; and with dbcAMP for 3 d, the ability of these cells to produce testosterone increased even more. With 3 d of culture with LH, the old cells did not increase in their ability to produce testosterone. In striking contrast, the ability of old Leydig cells to produce testosterone increased to close to young levels when the cells were incubated with dbcAMP for 3 d. Note that the old cells cultured with dbcAMP for 3 d produced twice the testosterone of cells cultured with LH.

View larger version (31K): [in this window] [in a new window]   FIG. 1. Testosterone-producing capacity of Leydig cells in vitro in response to LH or dbcAMP for 1 or 3 d. Leydig cells were isolated from young rats (YC), young rats that received LH-suppressive testosterone for 5 d before isolation (YT), and old rats (OC). The cells were cultured for 1 (Day 1) or 3 (Day 3) d with either LH (100 ng/ml) or dbcAMP (5 mM). For cells cultured for 3 d, lower concentrations of LH (0.5 ng/ml) or dbcAMP (1 mM) were used for the first 2 d. Each bar represents the mean ± SEM of four experiments. a, Significant differences from the same cells treated with LH at d 1. b, Significant differences from the same cells treated with dbcAMP at d 1.

View larger version (23K): [in this window] [in a new window]   FIG. 2. A, Representative Western blots of P450scc and StAR protein. Leydig cells isolated from young rats (YC), young rats that received LH-suppressive testosterone for 5 d (YT), and old rats (OC) were incubated with LH for 1 d or with LH or dbcAMP (DC) for 3 d. B, Activities of 17-hydroxylase and C17-20 lyase, which convert progesterone to 17-hydroxyprogesterone and androstenedione, respectively. Each bar represents the mean ± SEM of four experiments.

These results suggest that reduced cAMP plays a central role in the reduced steroidogenic ability of old Leydig cells. Consequently, we wished to determine the mechanism by which aging results in reduced cAMP production. Figure 3A confirms that, with maximally stimulating LH, Leydig cells isolated from aged rats produced about half the cAMP of cells isolated from young rats. To determine whether impeded function of adenylyl cyclase in old cells was responsible for the reduced cAMP production, cells were incubated with forskolin, an agent known to increase cAMP production by directly activating adenylyl cyclase in Leydig cells. When the young and aged cells were stimulated with forskolin, cAMP production was equivalent between the two groups of cells (Fig. 3A), suggesting that adenylyl cyclase is maintained in old Leydig cells. To determine whether the inhibitory G (Gi) proteins were responsible for the reduced cAMP production by aged cells, cells were cultured with pertussis toxin, which inhibits Gi proteins in Leydig cells. The difference between cAMP production of young and aged cells persisted, suggesting that changes in Gi protein are unlikely to be responsible for the reduced cAMP production in aged cells. To determine whether the Gs proteins were responsible for the reduced cAMP production, cells were cultured with cholera toxin, which activates Gs proteins. Under this condition, the ability of the aged cells to produce cAMP significantly increased. Indeed, cAMP production was equivalent between young and aged cells after cholera toxin treatment. To further examine whether Gs protein content changes with age, the -subunits of the Gs protein of young and aged cells were analyzed by Western blots (Fig. 3B). No differences were seen between the two ages, confirming that Gs proteins do not themselves change with age. These results suggest that Gs protein is maintained in old cells but that, in contrast to young cells, it may not be activated efficiently by LH in the membranes of aged cells.

View larger version (18K): [in this window] [in a new window]   FIG. 3. A, cAMP production by Leydig cells isolated from young and old rats in response to LH, forskolin (F), pertussis toxin (P+LH), or cholera toxin (C+LH). Purified Leydig cells were incubated for 15 min with LH (100 ng/ml) or forskolin (500 µM) in the presence of IBMX (100 µM) after a 4-h preincubation period with or without pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml). Values represent the mean ± SEM of four experiments. B, Western blot of Gs protein -subunit from young (Y1, Y2, and Y3) and old (O1, O2, and O3) cells. C, cAMP production by cell membranes isolated from young and old Leydig cells and incubated without (basal) or with forskolin and with IBMX (100 µM). *, Significant differences from young and old cells treated with LH alone.

	Discussion

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Although aging male Brown Norway rats exhibit defects on both testicular and hypothalamic/pituitary levels, as is the case in humans (18, 19), the primary cause of reduced testosterone production by aged Leydig cells resides in deficits at the testicular level (5, 10, 18). Previous studies have shown that under LH stimulation, old Leydig cells produce far less cAMP than young cells (5). These differences in cAMP production persist when cells are cultured with the phosphodiesterase inhibitor, IBMX, suggesting that old Leydig cells have deficits in cAMP production and not in its metabolism. The first question we addressed was whether the defect in LH signaling pathway is responsible for the age-related reduction in Leydig cell steroidogenesis. The ability of young Leydig cells to produce testosterone was sustained over a 3-d culture period with either LH or dbcAMP stimulation. Testosterone production by cells isolated from the LH-suppressed young rats increased with both LH and dbcAMP, indicating that the in vitro system was capable of restoring steroidogenic function. Culturing the old Leydig cells with LH failed to increase testosterone production and thus was unable to reverse the steroidogenic deficits of old Leydig cells. In striking contrast, culturing these cells with dbcAMP, which bypasses the defect in the LH signaling pathway, resulted in the restoration of testosterone production to young levels thus, in effect, reversing aging effects. These results provide strong evidence that inefficient LH signal transduction, leading to reduced cAMP production, is responsible for the reduced steroidogenesis that characterizes aged Leydig cells.

Accompanying the restoration of testosterone production by old cells, two of the key proteins in the steroidogenic pathway, StAR and P450scc, were restored to the levels of young cells. Interestingly, however, the individual activities of P450c17, namely 17-hydroxylase and C17-20 lyase activities, which convert progesterone to 17-hydroxyprogesterone and androstenedione, respectively, and which were reduced by LH suppression of the young cells and by age, were not restored to young levels by LH or dbcAMP, despite the fact that dbcAMP restored testosterone production to these cells. These results indicate that testosterone production can be maximal even in the face of reductions in some of the steroidogenic enzymes or, alternatively, that under some circumstances, testosterone production can be regulated by P450c17-independent pathways (20).

The results with dbcAMP clearly suggest that changes in the signal transduction pathway of old Leydig cells cause reductions in cAMP production and, in turn, in testosterone production. Therefore, we addressed the issue of which changes in signal transduction might be responsible for reduced cAMP. LH receptors are coupled to adenylyl cyclase through G proteins (11). Forskolin can activate adenylyl cyclase by directly binding to the enzyme, thus bypassing the hormone receptor-G protein signal transduction pathway (21). When stimulated with forskolin, old cells produced the same amount of cAMP as young cells, suggesting that adenylyl cyclase is maintained in the old cells. This was confirmed by direct assay of adenylyl cyclase activity in the cell membrane fractions. As in other cells, LH receptors are coupled to adenylyl cyclase mainly through the Gs protein in Leydig cells (11). In addition to Gs protein, a group of Gi proteins may also be involved in the negative modulation of adenylyl cyclase activity in rat Leydig cells (22). We asked whether the reduced cAMP production in old cells results from changes in Gs and/or Gi proteins. Pretreatment of old cells with pertussis toxin, which inhibits Gi proteins, did not restore the LH-stimulated cAMP production to the level of young cells, suggesting that changes in Gi protein are unlikely to be the cause of old cells producing less cAMP than young cells. Pretreatment with cholera toxin, which bypasses the LH receptor and activates Gs proteins directly, increased the ability of old cells to produce cAMP almost to the levels of young cells, suggesting that Gs protein itself is well maintained in old cells. This was confirmed by Western blot analysis of Gs protein -subunit, which indicated that Gs protein does not change quantitatively with age.

We have reported previously (5) that, although LH receptor number decreases in old Leydig cells, such changes in LH receptor number are unlikely to account for decrease in cAMP production. This and the observations herein that the Gs and Gi proteins and adenylyl cyclase are well maintained in old cells suggest that deficits in these proteins are not responsible for reduced cAMP production in old cells. Rather, we suggest that reduced cAMP production results from defects in the LH receptor itself or in coupling of the LH receptor to adenylyl cyclase through Gs proteins. What would cause such defects? During normal metabolism, cells produce reactive oxygen species that can damage DNA, protein, and lipids (23). There is extensive evidence that free radical damage may contribute to cell aging (24, 25). We have reported that aged Leydig cells produce more reactive oxygen than young cells (26), and unpublished studies from our lab indicate that the major scavengers of reactive oxygen (superoxide dismutase 1 and 2, glutathione peroxidase, catalase, and glutathione) are reduced in aged cells. If free radical damage affected membrane fluidity (27, 28), the LH receptor-G protein-adenylyl cyclase coupling cascades probably would become less efficient (29). Indeed, it has been shown that rat aortic endothelial cell membrane fluidity decreases with aging (30) and that changes in rat corpus luteum cell membrane fluidity in response to oxygen radicals disrupt LH-stimulated cAMP production (31). Whether or not this is the mechanism by which signal transduction is made inefficient in old cells and, if so, whether age-related deficits result from extrinsic changes that impinge upon the Leydig cells or from changes intrinsic to the cells themselves remain uncertain.

	Footnotes

 
This work was supported by National Institutes of Health Grant AG21092 from the National Institute on Aging.

Abbreviations: dbcAMP, Dibutyryl cAMP; Gi, inhibitory G; Gs, stimulatory G; IBMX, isobutyl-methylxanthine; StAR, steroidogenic acute regulatory protein.

Received May 19, 2004.

Accepted for publication June 22, 2004.

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