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REPRODUCTION-DEVELOPMENT |
Division of Reproductive Biology (H.C., B.R.Z.), Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205; and Population Council (M.P.H.), New York, New York 10021
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
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Abstract |
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Introduction |
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In young rats, experimental suppression of serum LH levels results in decreases in Leydig cell volume and T production (5). Reduced Leydig cell volume and T production also characterize aged Leydig cells (4), suggesting the possibility that age-related changes in LH might be responsible for the reduced steroidogenesis that characterizes old Leydig cells. Indeed, although serum LH concentrations in Brown Norway rats do not decline with age (4, 6), changes in LH pulse interval and amplitude do occur (7), and this could have deleterious effects on Leydig cell steroidogenesis.
In a previous study (8), we hypothesized that if reduced T production resulted from age-related changes in LH, the administration of LH to aged rats should restore T production to the higher levels seen in young rats. Exogenously administered LH failed to increase T production by old Leydig cells (8), suggesting that deficient LH stimulation may not be the underlying cause of age-related declines in Leydig cell steroidogenesis. However, other considerations point to a need for continued testing of the role of LH action. First, the administration of comparable doses of LH to young and old rats will not necessarily result in identical intratesticular LH concentrations. For example, testicular regression occurs in a significant proportion of the testes in rats at age 20 months and older (1, 9, 10), which could influence blood flow within the testes and thus affect the delivery of LH to Leydig cells. Second, an extensive extracellular matrix surrounds old but not young Leydig cells (our unpublished data), and this might prevent LH from access to the old cells.
Herein, our objective was to determine whether the exposure of old Leydig cells to LH in vitro would increase the ability of these cells to produce T at rates equivalent to young cells. To this end, Leydig cells from the testes of young and old rats were isolated and cultured long-term with LH. We hypothesized that if the aged-related reduction of T production resulted from LH deficits and not from deficits of the Leydig cells themselves, it should be possible to restore Leydig cell steroidogenic function with LH. We further hypothesized that if the old cells failed to respond to LH, the relative insensitivity of these cells might be due to changes in LH receptor number and affinity, and/or the ability of the cells to produce 3',5'-cAMP in response to LH.
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Materials and Methods |
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Leydig cell purification
Leydig cells were isolated as previously described (12). 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, 15 min). The dissociated cells were then subjected to centrifugal elutriation and Percoll gradient centrifugation purification, as previously described (12). The final purity of the Leydig cells obtained this way, determined by staining the cells for 3ß-hydroxysteroid dehydrogenase activity, consistently was about 95%. Cell viability, assessed by trypan blue exclusion, was over 95%.
Culture of isolated Leydig cells with LH in vitro
Leydig cells were cultured according to the procedure described by Klinefelter and Ewing (13). Briefly, purified Leydig cells were resuspended (106/ml) in M-199 medium (Life Technologies, Inc., Grand Island, NY) supplemented with 2.2 g/liter NaHCO3, 2.4 g/liter HEPES, 0.1% BSA, and 12.5 mg/liter gentamycin sulfate, pH 7.4. One-microliter of cell suspension (1.0 x 106) then was added to a Corning, Inc. (Corning, NY) 24-well culture plate 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, LH (USDA-bLH-B-6, USDA Animal Hormone Program, Beltsville, MD) was added to a final concentration of 0.5 ng/ml. To determine the maximal 24 h T production, cells were stimulated with 100 ng/ml LH for the final 24 h of culture. This concentration of LH was selected based on the results of previous studies of Leydig cells isolated from Sprague Dawley rats (13), and from preliminary studies of Brown Norway rats. The final culture volume was adjusted to 2.0 ml with M-199 culture medium, and the cultures were maintained at 34C in 5% CO2:5% O2:90% N2.
Media were changed every 24 h. To this end, 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 T assay. The beads, with Leydig cells attached, were then resuspended in fresh culture medium and placed in the reduced-oxygen environment. At the end of culture, Leydig cells attached to the beads were lysed with TES buffer (10 mM Tris, pH 8.0; 1 mM EDTA; 1% SDS; 100 mM KCl) at 50 C for 30 min. DNA was assayed fluorometrically with 4',6-diamidino-2-phenylindole (14).
LH receptor analysis
The human CG (hCG) binding assay was performed as previously described (15). In brief, aliquots of 0.5 x 106 Leydig cells were incubated in 0.5 ml culture medium (DMEM-Ham’s F-12, Sigma, supplemented with 0.1% BSA) containing 40,000 cpm of [125I]hCG (NEN Life Science Products, Boston, MA), using 12 x 75 mm polypropylene test tubes. A displacement curve was formed by the addition of cold hCG (0.1–20 ng) to triplicate assay tubes. The tubes were then incubated at 4 C overnight in a multipurpose rotator (Scientific Industries Inc., Bohemia, NY). The tubes were centrifuged at 250 x g for 10 min at 4 C. The pellet was washed twice with 2 ml of cold culture medium and centrifuged. The final pellet was solubilized with 0.5 ml of 0.5 N NaOH. The tubes were then capped and radioactivity was measured in a 
-counter.
Bound ligand per mole of total hCG was plotted against moles of total hCG to free ligand, and the results were subjected to linear regression analysis. The fitted lines were then used to estimate the dissociation constant (Kd), according to the equation: Kd = (-1/slope)/(liter); and the number of binding sites per cell (B), according to the equation: B = (Bmax) (Avogadro’s number)/(no. of cells per tube), where Bmax= X intercept (16, 17).
cAMP and T 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, 0.1% BSA, pH 7.4. The cells (1 x 105/200 µl) were preincubated in 96-well Falcon culture plates (Becton Dickinson and Co., Franklin Lakes, NJ) under 5% CO2:95% air at 34 C for 2 h. The medium was then carefully removed and 50 µl fresh phenol-red-free M-199 medium, containing LH or forskolin (Sigma), was added to the plates. For the time-course study, the cells were incubated with 100 ng/ml LH for 5–20 min. For the zero time point, cells were not incubated with LH, but instead lysed with 50 µl TET buffer (0.05 M Tris; 4 mM EDTA; 2 mg/ml theophylline, pH 7.5) after a 2-h preincubation and then frozen in liquid N2 and stored at -80 C. For the dose-response studies, cells were incubated with 50 µl medium containing LH (0–100ng/ml) or forskolin (1–500 µM, diluted from 0.25 M in dimethylsulfoxide) for 20 min after the 2-h preincuabtion. Media were then removed and 50 µl TET buffer was added immediately to the plate. Preparations were frozen in liquid N2 and kept at -80 C until cAMP and T assay. For some cells, isobutyl-methylxanthine (IBMX, Sigma) was included in the medium to inhibit phosphodiesterase activity. cAMP was assayed with a cAMP [3H] assay system (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s directions. The sensitivity of the assay was 0.05 pmol per assay tube. T was assayed by RIA (T antibody from ICN Biomedicals, Costa Mesa, CA; 3H-T from NEN Life Science Products) in the same medium being assayed for cAMP. The sensitivity, intraassay and interassay coefficients of variation of the RIAs for T were 13 pg/tube, 8.9% and 13.6%, respectively.
Statistical analyses
Data are expressed as the mean ± SEM. One-way ANOVA followed by the Scheffé multiple-range test was used to identify differences between groups. Values were considered significant at P < 0.05.
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Results |
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shows the capacity of isolated Leydig cells to produce T in response to LH over the course of a 24 h (1 d) culture period, and in the last 24 h of a 3-d culture period. Leydig cells were isolated from young (YC) and old (OC) rats, and from young rats that had received LH-suppressive T implants for 7 d before the isolation (YT). On d 1, young Leydig cells from untreated rats produced significantly more T than old cells or cells from LH suppressed young rats, and cells from old rats produced more T than cells from the LH suppressed rats. With 3 d of culture, cells from young rats were found to have maintained their ability to produce T at high levels, whereas those from old rats did not increase in their ability to produce T. In contrast, the cells from young LH-suppressed rats, which produced only low levels of T on d 1, had a significantly increased ability to produce T by d 3 of culture.
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compares hCG binding sites (2A) and Kd (2B) in young and old Leydig cells, and in cells from LH-suppressed young rats. The number of binding sites per cell and Kd in young control cells are 2.24 ± 0.90 x 104 and 1.42 ± 0.56 nM, respectively. In comparison to the young control cells, decreases in binding sites and in Kd of 50–70% were seen in old cells and in cells from LH-suppressed young rats.
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) and T (Fig. 4) production, cells isolated from the three groups of rats were incubated with maximally stimulating LH (100 ng/ml) for 0–20 min (Figs. 3A and 4A), or for 20 min with increasing LH concentrations (Figs. 3B and 4B). With maximally stimulating LH (100 ng/ml), cAMP concentrations reached a plateau within 15 min in all three cell types (Fig. 3A). However, the cells from old rats produced significantly less cAMP than cells from both young control and young LH suppressed animals. Interestingly, the cells from the young control and young LH-suppressed rats produced same, relatively high amounts of cAMP despite the significant reduction in LH receptor numbers in cells from LH-suppressed rats (Fig. 2A
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, incubation of the Leydig cells with maximally stimulating LH resulted in linear increases in T production in all three groups after an initial 5-min delay (Fig. 4A). Young control Leydig cells produced significantly more T than old control cells; young LH-suppressed cells produced even less T than the old cells. Under stimulation by different concentrations of LH, T production reached plateaus in the three cell types at about 1 ng/ml LH (Fig. 4B). Leydig cells from young LH-suppressed rats had an EC50 that was lower than control young and old cells, but the differences were not significant.
To determine whether impeded function of adenylate cyclase in old cells was responsible for the reduced cAMP production, we incubated cells with forskolin (20 min), an agent known to increase cAMP production by activating adenylate cyclase in Leydig cells (18). Figure 5 shows the effects of forskolin on cAMP production. At each forskolin concentration from 0–500 µM, cAMP production was equivalent among the three groups of cells, suggesting that adenylate cyclase is maintained in old Leydig cells.
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shows cAMP production by cells incubated with LH plus IBMX (Fig. 6A), or with forskolin plus IBMX (Fig. 6B). In the presence of IBMX, the total cellular cAMP produced by the three types of cells increased somewhat, by 5–30%, whether the cells were stimulated with LH (Fig. 6A) or with forskolin (Fig. 6B). Upon stimulation with LH, old Leydig cells, whether incubated with IBMX or not, produced less cAMP than young control cells or young cells from LH-suppressed rats. In contrast, the three types of cells produced equivalent amounts of cAMP when they were incubated with forskolin, whether in the presence of absence of IBMX. These results suggest that lower cAMP production by old Leydig cells results from lower rates of production and not from increased rates of degradation.
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Discussion |
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Young Leydig cells sustained their ability to produce T over a 3-d culture period, and, as expected, cells from the LH-suppressed rats increased their T production over the 3-d period almost to control levels. In contrast, culture of the steroidogenically hypofunctional old Leydig cells with LH failed to increase their T production. These results indicate that, whatever the cause of the reduced steroidogenesis that characterizes old Leydig cells, LH stimulation, by itself, apparently is unable to reverse the deficit(s) within 3 d. Moreover, the results support the contention that LH understimulation is unlikely to be responsible for age-related reductions in steroidogenesis by these cells.
Why, then, do old Leydig cells fail to produce high levels of T when cultured with LH, while cells from young LH-deficient cells respond to LH treatment by producing high T levels? We have shown herein that Leydig cells from both old control rats and from young LH-suppressed rats have reduced numbers of LH binding sites. However, whereas the cells from young LH-suppressed rats produced cAMP at the high levels of young control cells, the old cells produced far less cAMP. These results indicate that old Leydig cells may have defects in the LH-cAMP signaling cascade that reduces their responsiveness to LH stimulation. In turn, the reduced ability of these cells to produce cAMP is likely to result in downstream effects, including reductions in StAR (20) and in the steroidogenic enzymes involved in T production (21).
As indicated above, the number of binding sites on Leydig cells, and the affinity of these sites for LH (hCG), change with age. It seems unlikely, however, that the reduced ability of old Leydig cells to produce cAMP results from a deficiency in LH binding. The number of binding sites and binding affinity both changed to an even greater extent in cells from young, LH-suppressed rats than in old cells, but the ability of the former cells to produce cAMP in response to LH did not change. The fact that these cells maintained their ability to produce cAMP despite reduced LH receptor number is not particularly surprising, given a substantial literature indicating that maximal cAMP production can be achieved by LH or hCG binding to only a small fraction of the LH receptors present in Leydig cells (22, 23).
This leads to the question: What changes in the signal transduction pathway of old Leydig cells might cause reductions in cAMP production? LH receptors are coupled to adenylate cyclase through G proteins (24). Forskolin can activate adenylate cyclase by directly binding to the enzyme, thus by-passing the hormone receptor-G protein signal transduction pathway (25). We show here that, under forskolin stimulation, old cells are able to produce the same amount of cAMP as young control and young LH suppressed cells, suggesting that adenylate cyclase is maintained in old cells. Thus the most likely problem(s) is in the quality of the receptors, in the G proteins, and/or in their coupling.
How might aging result in changes in the membrane of old Leydig cells that would result in ineffecient signal transduction? During normal metabolism, cells produce reactive oxygen species that can damage DNA, protein and lipids (26). There is extensive evidence that free radical damage may contribute to cell aging (27, 28). If there were free radical damage to LH receptors or G proteins, the result might be the reduced ability of LH to stimulate cAMP production. Free radical damage to lipids in the cell membrane might influence membrane fluidity (29, 30), and this also might render the LH-cAMP cascade less efficient (31). Indeed, it has been shown that rat aortic endothelial cell membrane fluidity decreases with aging (32). Perhaps more relevant, reduction in rat corpus luteum cell membrane fluidity induced by oxygen radicals has been shown to disrupt LH-stimulated cAMP production (33). Whether or not such a mechanism is involved in Leydig cell aging is under study in our laboratory.
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Acknowledgments |
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Footnotes |
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Abbreviations: hCG, Human CG; IBMX, isobutyl-methylxanthine; Kd, dissociation constant.
Received October 29, 2001.
Accepted for publication January 24, 2001.
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