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a The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037
b Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-1ß, or interleukin 6 (IL-6) delivered to the brain ventricles significantly decreases secretion of GnRH and LH [5–8] and, in addition, dramatically blunts testicular responsiveness to exogenous hCG [9–11]. Previous data from our laboratory show that this effect, though time-dependent, occurs before significant elevation of IL-6 in the peripheral bloodstream and does not depend on adrenal activation or changes in LH concentrations [10]. These data led us to hypothesize a direct connection between the brain and testis (perhaps neuronal, as suggested by others [12–14]) by which IL-1ß inhibits the testicular response to hCG [11]. Unfortunately, a direct test of this hypothesis in vivo is precluded by the decreases in basal and hCG-stimulated testosterone secretion that follow complete testicular denervation [15, 16] or the local application of lidocaine (unpublished observation). In view of the impossibility of using this approach, we reasoned that if icv IL-1ß blunted the testosterone response to hCG via a neural brain-testicular pathway, changes in testicular physiology should be evident shortly after IL-1ß administration (i.e., before measurable increases of cytokines in the blood) and should be demonstrable in the absence of circulating cytokines, which can inhibit testosterone secretion directly [17–23]. In the experiments described here, we collected testes from rats that had received injections of vehicle or IL-1ß icv 30 or 90 min earlier, and determined whether blunted testosterone secretion depended on decreased binding of hCG/LH or on inhibited biosynthetic capacity of the Leydig cell.
Testosterone biosynthesis depends on the action of StAR protein [24]. StAR facilitates the transfer of cholesterol from the outer to the inner mitochondrial membrane to cholesterol side-chain cleavage P450, which catalyzes its conversion to pregnenolone. Pregnenolone then diffuses to the smooth endoplasmic reticulum, where it is converted to testosterone via the actions of 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17-hydroxylase/C17–20 lyase P450 (P450c17), and 17ß-hydroxysteroid dehydrogenase. Thus, central administration of IL-1ß could restrain synthesis of testosterone by inhibiting LH/hCG binding or by decreasing any of the proteins involved in the availability and conversion of cholesterol to testosterone. While our studies do not provide a definitive answer to the question of the mechanisms through which icv IL-1ß induces these changes, they indicate that the StAR protein plays a role in the phenomenon we had observed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Harlan Sprague Dawley rats weighing 180–200 g were obtained from a commercial supplier (Harlan, San Diego, CA) and maintained under controlled lighting (12L:12D, lights-on at 0600 h) with food and water available continuously. Under anesthesia (0.6–0.7 ml/animal of a mixture of acepromazine, 1 mg/ml [Fermenta Animal Health Co., Kansas City, MO]; xylazine 5 mg/ml [Phoenix Scientific, Inc., St. Joseph, MO]; and ketamine 25 mg/ml [Fort Dodge Laboratories, Fort Dodge, IA]), a guide cannula (Plastics One, Roanoke, VA) was implanted in the right lateral cerebral ventricle using a stereotaxic frame and coordinates derived from Paxinos and Watson [25] (0.4 mm caudal, 1.4 mm lateral, and 3.8 mm ventral to the skull at the bregma). The cannula was held in place using cranioplastic cement (Plastics One) and dental screws. A dummy cannula was placed in the lumen of the guide cannula, and an antibacterial powder (KenVet, Ashland, OH) was applied. The animals were caged individually in plastic boxes with pine shavings and allowed one week to recover from surgery before experimentation.
On the day of an experiment, animals were removed to a sound-proof room and housed individually in opaque buckets with their cannulae connected to injectors and extended with polyethylene tubing so that animals could be injected without being handled. Injections were given at least 3 h after rehousing to allow hormone levels to return to basal. All protocols were approved by the Salk Institute IACUC.
In Vivo Treatment
Recombinant human IL-1ß (a gift from Dr. S. Gillis, Immunex, Seattle, WA) was dissolved in 0.01% BSA/apyrogenic water for injection (The Butler Co., Columbus, OH) at a dose of 80 ng/5 µl. Controls received the BSA vehicle. The icv treatment, delivered at the rate of 1 µl/10 sec, was given 90 or 30 min before decapitation. Testes were removed, placed into 50-ml conical tubes containing ice-cold M199 complete (see below), and processed for ex vivo treatment with hCG as described below. Intracerebroventricular injections were staggered so that no more that 20 min elapsed between the decapitation of the first and the last animal.
Ex Vivo Treatment
Cell culture medium was M199 complete (M199 [31100–035; Gibco BRL, Grand Island, NY], supplemented with 10 mM HEPES [H7523; Sigma, St. Louis, MO], 26 mM NaHCO3 [S8875; Sigma], 100 IU/ml penicillin, and 100 µg/ml streptomycin [95070–030; Gibco BRL], 1% BSA) adjusted to pH 7.4 and sterile filtered (0.2-µm Nalgene filter apparatus, Nalge Company, Rochester, NY).
Testicular tissue was either decapsulated or subjected to enzymatic dissociation followed by a Percoll density gradient to semi-purify Leydig cells. To decapsulate the testes, the capsule opposite the testicular vein was cut with a razor blade, and the whole testis was placed into a well containing 5 ml M199 complete [26]. Graded doses of hCG (C1063; Sigma) were added to testes and incubated in a humidified chamber at 35°C for 3 h. Media were collected into polypropylene tubes and stored frozen until assayed for testosterone.
To obtain an enriched preparation of Leydig cells, testes were first decapsulated as described above. Both testes from 2 animals (given the same in vivo treatment) were pooled in 1 dissociation tube containing 18 ml M199 complete, 2 ml 10% BSA, and 100 µl collagenase (200 mg/ml, CLS4; Worthington, Lakewood, NJ). Dissociation tubes were incubated at 37°C for 10 min with shaking to separate interstitial cells from the seminiferous tubules. After incubation, 30 ml M199 complete was added to each tube, and tubes were inverted several times and then chilled on ice for 2 min. The resultant supernatant was collected, filtered through organza, and centrifuged to pellet cells. Cells were resuspended in M199 complete (2.5 ml/tube) and subjected to a density gradient (on 9 parts Percoll [17–0891–01; Pharmacia Biotech], 1 part 8-strength Dulbecco's PBS [D6650; Sigma], and 0.1% BSA) at 12 000 rpm for 15 min. After centrifugation, 1-ml fractions (#1–8) were collected from the bottom of each gradient tube. Preliminary experiments had shown these fractions to contain the majority of cells that secreted testosterone as well as the greatest number of cells that stained for 3ß-HSD activity. Percoll was removed by dilution in M199 complete (40 ml), and the fractions were centrifuged at low speed and resuspended in 5 ml of M199 complete. Cells were counted with a hemacytometer, and dilutions were made so that 1 ml of media contained 106 cells. Triplicate aliquots of cells were incubated with hCG for 3 h at 35°C to determine their capacity to synthesize testosterone. Separate aliquots of cells were pelleted, frozen on dry ice, and shipped to University of Illinois at Chicago for determination of LH/hCG binding and levels of StAR protein. Each experiment was performed over a period of 3 days (2 animals/in vivo treatment group per gradient tube per day), and the entire experiment was performed twice. Because results from each experiment were similar, the data are pooled and presented in Results.
Binding of hCG
Highly purified hCG (batch CR127) was obtained from the National Hormone and Pituitary Program, NIDDK (Rockville, MD) and radiolabeled with carrier-free Na125I using the chloramine T method as described by Dufau et al. [27]. Briefly, 25 µg hCG and 12.5 µg chloramine T were added to 1 mCi 125I, vortexed, and incubated on ice for 30 sec. After termination of the reaction by adding 0.125 mg sodium metabisulfite containing 1.0% KI, the purified iodinated hCG was obtained by gel filtration on a Sephadex G-50 column that had been equilibrated with 50 mM Tris buffer containing 1 mM EDTA and 0.1% BSA. The specific activity, as determined by self-displacement analysis, was 3 µCi/µg protein from mouse testis homogenate.
After cells were thawed on ice, they were dispersed in 0.25 ml PBS containing 0.1% BSA and filtered through 3 layers of organza. The resulting suspension was diluted to a final volume of 6.5 ml and used for binding studies. Incubation of 300-µl cell suspensions with [125I]hCG (100 000 cpm in 750 µl) in the presence of increasing concentrations of unlabeled hCG was performed in polystyrene tubes at 27°C. After 18 h, the suspensions were washed twice with cold PBS-BSA and centrifuged at 20 000 x g for 20 min. After aspiration of the second supernatant, radioactivity was determined by counting for 1 min in a gamma counter. Specific binding was determined by subtracting the total amount of radioactivity bound from the radioactivity present when excess unlabeled hCG was added. Binding data were analyzed by the method of Scatchard [28]. Samples from each interval after treatment (i.e., 30 min or 90 min post-injection) were measured in the same assay.
Measurement of StAR and P450c17
Western blots for measurement of StAR and P450c17 protein were performed as previously described [1, 18] using polyclonal rabbit anti-mouse sera prepared as described [18, 29, 30]. Data were corrected for total protein loaded in each lane (quantitated after staining with cypro-red).
Treatment with R22-Hydroxycholesterol
To test whether in vivo treatment with IL-1ß suppressed testosterone biosynthesis via inhibition of StAR function, decapsulated testes from animals treated with vehicle or IL-1ß (see above) were incubated ex vivo in the presence or absence of R22 hydroxycholesterol (20 µM; Sigma). This form of cholesterol is water-permeable and does not require functional StAR protein for transport to the inner mitochondrial membrane. Its use therefore allowed us to test the hypothesis that icv IL-1ß-inhibition of testosterone secretion is at the level of cholesterol transport and is mediated by decreased StAR protein.
Testosterone Assay
Testosterone content in cell culture media was measured using a commercially available kit (TKTT; DPC, Los Angeles, CA). The interassay coefficients of variation, determined at 20%, 50%, and 80% binding, were 11.9, 17.8, and 25.6%, respectively.
Statistical Analysis
Data on testosterone secretion were analyzed by ANOVA with in vivo (vehicle vs. IL-1ß) and ex vivo (dose hCG) treatments as the variables. Since hormone measurements were collected from 3 aliquots of each gradient tube at each dose of hCG, this level of replication was treated as a repeated measure. When indicated by the results of the ANOVA procedure, Fisher's probable least-squares difference or least-squares means testing were employed in post-hoc analyses. Levels of StAR and P450c17 proteins were measured in duplicate, and the means of the two estimates were subjected to a two-way ANOVA with in vivo treatment and interval after injection (30 or 90 min) as the variables.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although decapsulated testes from both vehicle and IL-1ß-treated rats responded robustly to hCG stimulation ex vivo (p < 0.0001, ANOVA), prior exposure to the cytokine in vivo resulted in significant suppression of testosterone secretion at either 30 or 90 min before the animals were killed (p < 0.0001, ANOVA; Fig. 1).
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Effect of In Vivo icv Treatment with Vehicle or IL-1ß on Ex Vivo Secretion of Testosterone from Semi-Purified Leydig Cells
All isolated Leydig cells also responded to hCG stimulation in a dose-dependent manner (p < 0.0001, main effect, rm ANOVA). Furthermore, the results obtained qualitatively paralleled those from decapsulated testes in that in vivo exposure to IL-1ß suppressed the ex vivo response to hCG (p < 0.0001, main effect, rm ANOVA). The time intervals chosen (30 and 90 min before the animals were killed) did not impact the degree of inhibition observed (Fig. 2).
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Effect of In Vivo icv Treatment with Vehicle or IL-1ß on LH/hCG Binding
In the limited number of samples analyzed for hormone binding (n = 2), icv exposure to IL-1ß for 30 min in vivo resulted in a slight reduction in LH/hCG binding affinity (Ka, 3.28 ± 0.16 x 109 M-1 for vehicle versus 5.4 ± 1.7 x 109 M-1 for IL-1ß-treated animals) and a 29% reduction in maximal binding [Bmax, 7.21 ± 0.57 x 1018 (M/µg protein)-1 versus 5.12 ± 1.6 x 1018 (M/µg protein)-1]. Leydig cells isolated 90 min after IL-1ß also had reduced affinity (1.28 ± 0.85 x 109 M-1 for vehicle versus 2.20 ± 1.2 x 109 M-1 for IL-1ß-treated animals) and a 39% decrease in maximal binding [2.47 ± 1.3 x 1017 (M/µg protein)-1 versus 1.50 + 0.82 x 1017 (M/µg protein)-1]. Comparison of maximal binding between time points cannot be made since samples were analyzed in separate binding assays.
Effect of In Vivo icv Treatment with Vehicle or IL-1ß on Expression of StAR
Compared to vehicle treatment, in vivo exposure to IL-1ß decreased levels of StAR protein detected in isolated Leydig cells within 30 min of injection (p = 0.0094, main effect, ANOVA). A longer interval between injection and killing of the animals did not result in a greater inhibition of StAR expression (Fig. 3).
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Effect of In Vivo icv Treatment with Vehicle or IL-1ß on Expression of P450c17
In contrast to its ability to down-regulate StAR protein, icv treatment with IL-1ß did not significantly alter expression of P450c17 (vehicle versus IL-1ß: p = 0.9862 at -30, p = 0.1042 at -90, least-squares means post-hoc test; Fig. 4).
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Effect of In Vivo icv Treatment with Vehicle or IL-1ß on the Ability of R22-Hydroxycholesterol to Induce Secretion of Testosterone
As shown above, decapsulated testes from animals treated in vivo with IL-1ß and stimulated with hCG ex vivo had suppressed testosterone secretion in comparison to testes from animals treated with vehicle (in vivo vehicle/hCG/no R22 vs. in vivo IL-1ß/hCG/no R22, p = 0.0055, least-squares means). However, the presence of R22-hydroxycholesterol enhanced testosterone secretion from these tissues and narrowed the impact of the in vivo treatment to nonsignificant levels (in vivo vehicle/hCG/R22 vs. in vivo IL-1ß/hCG/R22, p = 0.0793; Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since one of the physiological changes evoked by hCG administration is increased blood flow through the testis [33], and since testicular blood flow is well correlated with the levels of testosterone in systemic circulation [34, 35], it might have been hypothesized that the effect of icv-injected IL-1ß on testicular function was mediated by alterations in testicular blood flow. However, the persistence of a diminished response to hCG ex vivo in these experiments suggests that the effect of centrally administered IL-1ß does not rely exclusively on this mechanism. Furthermore, in this and other models of immune-mediated inhibition of Leydig cell function, there was no evidence of impaired blood flow to the testes. In fact, we have shown recently that during chronic intraperitoneal sepsis in rats, there is an increase in testicular blood flow concomitant with increased production of nitric oxide [36]. However, as the effect of icv IL-1ß administration on testosterone secretion is not as pronounced ex vivo as that observed in intact animals [10, 11], we cannot rule out the possibility that changes in testicular perfusion might play a role. Taken together, the preceding data indicate that central administration of IL-1ß can evoke prolonged (on the order of hours) inhibition of Leydig cell function that persists ex vivo in the absence of high circulating levels of cytokines and corticosteroids.
On the basis of the foregoing observations, we had hypothesized that IL-1ß administered into the cerebral ventricles might evoke changes in the capacity of Leydig cells to bind hCG/LH. Although we did observe a decrease in the binding of radiolabeled LH to cell membranes from rats treated in vivo with IL-1ß (approximately 35%), there remained substantial capacity to bind this hormone. Since full stimulation has been documented with as little as 5% of the normal complement of receptors [37], it seems unlikely that reduced LH/hCG binding is the major mechanism by which icv IL-1ß inhibits testicular function.
The first step in the biosynthesis of testosterone is the movement of cholesterol from the outside of the cell to the inner mitochondrial membrane; this step depends on functional StAR protein. Because systemic administration of LPS impairs testosterone secretion in parallel with decreased expression of StAR [1], we had hypothesized that icv administration of IL-1ß might also restrain testosterone synthesis by impairing expression of StAR. In support of this hypothesis, we found that levels of StAR were reduced to 60% of control levels 30 or 90 min after icv injection of IL-1ß. In addition, administration of R22-hydroxycholesterol narrowed the difference in hCG-stimulated testosterone secretion between testes of controls and those of IL-1ß-treated animals from a reduction of 44% to a reduction of 25%. However, the impact of icv IL-1ß on levels of StAR is more modest than that observed after in vivo treatment with LPS [1, 38], and the amount of testosterone released by testes harvested from IL-1ß-treated animals tended to be smaller, regardless of ex vivo treatment. Furthermore, administration of R-22-hydroxycholesterol failed to completely reverse the ability of icv exposure to IL-1ß to suppress ex vivo secretion of testosterone. Thus, though our data support a role for StAR, they suggest that icv injection of IL-1ß also alters the expression or activity of one or more of the downstream steroidogenic enzymes that synthesize testosterone from cholesterol. Since P450c17 is the most sensitive of the biosynthetic enzymes to IL-1ß in vitro [17], we also examined the effects of icv administration of the cytokine on levels of P450c17 protein. Our data show that administration of IL-1ß directly into the cerebral ventricles does not significantly decrease the amount of P450c17 expressed, suggesting that some other component of the testosterone biosynthetic pathway may be inhibited. This finding also provides support for the contention that the effects of icv administered IL-1ß are not mediated by circulating levels of this cytokine.
Collectively, our data show that exposure in vivo to icv administration of IL-1ß results in long-term suppression of testicular testosterone secretion. Two points need to be emphasized. First, this inhibition persists ex vivo, indicating that decreased testicular blood flow and/or circulating cytokines are not primarily responsible for the physiological response. Furthermore, our data suggest that the mechanism does not rely on the down regulation of LH binding. Our data do support the hypothesis that icv IL-1ß impairs StAR function, but that decreases in testicular synthesis of testosterone via inhibition of an enzyme (or enzymes) within the biosynthetic cascade that occurs within the mitochondria or smooth endoplasmic reticulum may also be involved. The second point is that, though present, the difference in the responsiveness of the isolated testes from vehicle- or IL-1ß-treated rats is significantly less than we have reported in the intact rat [10]. If we accept the hypothesis that icv IL-1ß influences testosterone secretion through a neural pathway that connects the brain to the male gonads, severing this connection by isolating the testes will remove the inhibitory influence of the cytokine. We would then expect that, indeed, testes tested ex vivo will exhibit a less pronounced blunting in the steroidogenic capacity.
There is no doubt that during antigenic stimulation, cytokines present in the brain alter the testicular function through mechanisms involving both decreases in LH output and LH-independent blunting of androgen production. On the other hand, we have presented recent evidence that the CNS-testicular connection we hypothesize involves inhibitory catecholamines [9]. Because many stresses increase CNS catecholamine levels [39–42], our data may provide an explanation for hitherto unexplained cases of stress-induced decreases in testosterone release in the absence of significant changes in LH levels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Catherine Rivier, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037. FAX: 619 552 1546; crivier{at}salk.edu
3 Current address: Department of Pharmacology, Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, CA 92121.
Accepted: September 29, 1998.
Received: April 17, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase/C17–20 lyase cytochrome P450 expression. Endocrinology 1992; 131:2165–2172.[Abstract] in the regulation of mouse Leydig cell steroidogenesis. Endocrinology 1993; 132: 2438–2444. inhibits gonadotropin action in cultured porcine Leydig cells: site(s) of action. Endocrinology 1991; 129:2933–2940.[Abstract] inhibition of 17-hydroxylase/C17–20 lyase gene (CYP17) expression. Endocrinology 1995; 136:3519–3526.[Abstract]This article has been cited by other articles:
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  M. Herman and C. Rivier Activation of a Neural Brain-Testicular Pathway Rapidly Lowers Leydig Cell Levels of the Steroidogenic Acute Regulatory Protein and the Peripheral-Type Benzodiazepine Receptor while Increasing Levels of Neuronal Nitric Oxide Synthase Endocrinology, January 1, 2006; 147(1): 624 - 633. [Abstract] [Full Text] [PDF]
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D. J. Selvage, S. Y. Lee, L. H. Parsons, D. O. Seo, and C. L. Rivier A Hypothalamic-Testicular Neural Pathway Is Influenced by Brain Catecholamines, But Not Testicular Blood Flow Endocrinology, April 1, 2004; 145(4): 1750 - 1759. [Abstract] [Full Text] [PDF]
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V. Morales, P. Santana, R. Diaz, C. Tabraue, G. Gallardo, F. L. Blanco, I. Hernandez, L. F. Fanjul, and C. M. Ruiz de Galarreta Intratesticular Delivery of Tumor Necrosis Factor-{alpha} and Ceramide Directly Abrogates Steroidogenic Acute Regulatory Protein Expression and Leydig Cell Steroidogenesis in Adult Rats Endocrinology, November 1, 2003; 144(11): 4763 - 4772. [Abstract] [Full Text] [PDF]
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 H. Schwarzenbach, P. R. Manna, D. M. Stocco, G. Chakrabarti, and A. K. Mukhopadhyay Stimulatory Effect of Progesterone on the Expression of Steroidogenic Acute Regulatory Protein in MA-10 Leydig Cells Biol Reprod, March 1, 2003; 68(3): 1054 - 1063. [Abstract] [Full Text] [PDF]
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D. J. Selvage and C. Rivier Importance of the Paraventricular Nucleus of the Hypothalamus as a Component of a Neural Pathway between the Brain and the Testes that Modulates Testosterone Secretion Independently of the Pituitary Endocrinology, February 1, 2003; 144(2): 594 - 598. [Abstract] [Full Text] [PDF]
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S. Lee, R. Miselis, and C. Rivier Anatomical and Functional Evidence for a Neural Hypothalamic-Testicular Pathway that Is Independent of the Pituitary Endocrinology, November 1, 2002; 143(11): 4447 - 4454. [Abstract] [Full Text] [PDF]
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C. Mauduit, I. Goddard, V. Besset, E. Tabone, C. Rey, F. Gasnier, F. Dacheux, and M. Benahmed Leukemia Inhibitory Factor Antagonizes Gonadotropin Induced-Testosterone Synthesis in Cultured Porcine Leydig Cells: Sites of Action Endocrinology, June 1, 2001; 142(6): 2509 - 2520. [Abstract] [Full Text] [PDF]
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K. Held Hales, T. Diemer, S. Ginde, B. K. Shankar, M. Roberts, H. B. Bosmann, and D. B. Hales Diametric Effects of Bacterial Endotoxin Lipopolysaccharide on Adrenal and Leydig Cell Steroidogenic Acute Regulatory Protein Endocrinology, November 1, 2000; 141(11): 4000 - 4012. [Abstract] [Full Text] [PDF]
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M. K. O'Bryan, S. Schlatt, D. J. Phillips, D. M. de Kretser, and M. P. Hedger Bacterial Lipopolysaccharide-Induced Inflammation Compromises Testicular Function at Multiple Levels in Vivo Endocrinology, January 1, 2000; 141(1): 238 - 246. [Abstract] [Full Text] [PDF]
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