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Endocrinology, doi:10.1210/en.2003-0569
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Endocrinology Vol. 144, No. 11 4763-4772
Copyright © 2003 by The Endocrine Society

Intratesticular Delivery of Tumor Necrosis Factor-{alpha} and Ceramide Directly Abrogates Steroidogenic Acute Regulatory Protein Expression and Leydig Cell Steroidogenesis in Adult Rats

Victoria Morales, Pino Santana, Raquel Díaz, Carlos Tabraue, Germán Gallardo, Félix López Blanco, Inmaculada Hernández, Luisa F. Fanjul and Carlos M. Ruiz de Galarreta

Departamento de Bioquímica, Biología Molecular y Fisiología. Facultad de Medicina, Universidad de Las Palmas de Gran Canaria, Las Palmas 35016, Spain

Address all correspondence and requests for reprints to: Carlos M. Ruiz de Galarreta, Department of Biochemistry, Molecular Biology, and Physiology, University of Las Palmas, School of Medicine, Las Palmas de GC 35061, Spain. E-mail: cruiz{at}dbbf.ulpgc.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Systemic or intratesticular release of TNF{alpha} and IL1ß have been implicated in the reduced testosterone biosynthesis and impaired production of competent spermatozoa found in human patients suffering from sepsis or chronic inflammation. Although in vitro and in vivo studies have demonstrated that TNF{alpha} and IL1ß intercept the hypothalamic-pituitary testis axis at different levels, the site(s) of action and relative contribution of each cytokine to the overall testicular failure associated to systemic inflammatory processes remains poorly defined. In this study we show that intratesticular delivery of TNF{alpha} induced a rapid (4 h) and sustained (up to 24 h) reduction in steroidogenic acute regulatory (StAR) protein expression and testosterone biosynthesis in nonstimulated or human chorionic gonadotropin-treated intact or hypophysectomized rats. Bilateral treatment with cell-permeant short-chain ceramides (C2-cer or C6-cer) reproduced the early (4 h) inhibitory action of TNF{alpha} on testosterone biosynthesis and testicular StAR expression. The inhibitory action of C2-cer or C6-cer was not observed in animals treated with inactive analogs (dihydroceramide), phosphorylcholine, sphingosine, or sphoingosine-1P. In sharp contrast to the previously described ability of IL1ß to prevent human chorionic gonadotropin-stimulated Leydig cell steroidogenesis in vitro, serum testosterone and testicular StAR protein expression remained unchanged in animals bilaterally injected with this cytokine. These data support the concept that TNF{alpha} triggers different effector mechanisms to directly inhibit Leydig cell StAR expression and steroidogenesis, which ultimately contribute to the global reproductive failure associated with chronic inflammation and sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMIC-PITUITARY-TESTIS axis ensures that the interstitial Leydig cells produce the appropriate amounts of testosterone required to maintain spermatogenesis and male secondary sex characteristics (reviewed in Refs.1, 2, 3). Biosynthesis of all types of steroid hormones is initiated in the inner mitochondrial membrane in which cholesterol is converted into the common precursor steroid pregnenolone by the P450 cholesterol side chain cleavage (P450scc) enzyme (reviewed in Refs.4, 5, 6, 7). Pregnenolone formation is restricted by the aqueous intermembrane space of the mitochondria, which prevents the access of the hydrophobic cholesterol substrate to the P450scc site of action (4, 5, 6, 7). To circumvent this limitation, steroid biosynthesis relies on a complex cAMP-dependent two-step process: mobilization of cholesterol from intracellular stores to the outer mitochondrial membrane and its subsequent translocation to the mitochondrial matrix (4, 5, 6, 7). This later event resulting in the accumulation of a metabolizable cholesterol pool at the P450scc site of action requires de novo synthesis of the steroidogenic acute regulatory (StAR) protein and represents the true rate-limiting step of the steroidogenic pathway (5, 6, 7). Pregnenolone is converted into progesterone by the 3ß-hydroxysteroid dehydrogenase enzyme and subsequently metabolized in testicular Leydig cells to androstenedione and testosterone by the sequential action the of P450c17–20 lyase (Cyp17) and 17ß-hydroxysteroid dehydrogenase enzymes (for a review see Ref.8).

Despite the well-established fact that testosterone biosynthesis is primarily regulated by the pulsatile release of pituitary LH, there are compelling evidences that Leydig cell steroidogenesis is additionally modulated by circulating and/or locally produced hormones, growth factors, and cytokines (1, 2, 3). In this setting, the severe hypogonadism and reduced plasma testosterone levels of males suffering from critical illness, burns, and sepsis (9, 10, 11) have been related to the elevated concentrations of macrophage-derived cytokines such as TNF{alpha} and IL1ß present in the serum of these patients (12, 13, 14). Such causal relationship is supported by the reduced steroidogenic activity observed after systemic treatment with TNF{alpha} (15, 16), when IL1ß is directly delivered into the cerebral ventricles (17, 18) or in lipopolysaccharide (LPS)-challenged animals (19, 20, 21, 22, 23).

Although cessation of steroidogenesis in LPS-treated animals (19, 20, 21, 22, 23) may be causally related to the high circulating levels of TNF{alpha} and IL1ß (12, 13, 14), the testis is a highly integrated cellular system (1, 2, 3) in which Leydig cells are in close contact with tubular Sertoli cells and resident testicular macrophages, which account for as much as 20% of the total cell population of the interstitial space (reviewed in Refs. 24 and 25). In this scenario, because TNF{alpha} (26, 27, 28) and IL1ß (28, 29) are released by LPS-activated testicular macrophages and both agents share the ability to abrogate LH-induced and cAMP-mediated steroidogenesis in cultured Leydig cells (30, 31, 32, 33, 34, 35, 36, 37, 38, 39), it has been proposed that locally produced cytokines may contribute to the overall steroidogenic failure observed in animal models of inflammation (24, 25).

In addition to stimulating a common set of signaling events involved in multiple redundant cellular responses, the TNF{alpha} receptors (p55TNFR and p75TNFR) and IL1ß receptors (p60IL1R and p80IL1R) are structurally unrelated transmembrane proteins that activate different effector mechanisms that ultimately mediate the wide array of specific cellular and systemic responses characteristic of each cytokine (reviewed in Refs.40, 41, 42).

Many overlapping cellular responses induced by TNF{alpha} and IL1ß are initiated by a receptor-mediated activation of sphingomyelinase (SMase) that in turn promotes the hydrolysis of sphingomyelin to generate the inactive by-product phosphocholine and a regulatory ceramide moiety (reviewed in Ref.43). We have described that the rapid activation of sphingomyelin (SM) hydrolysis triggers a relevant mechanism whereby TNF{alpha} and IL1ß prevent FSH- and cAMP-stimulated steroidogenesis in cultured granulosa cells (44, 45). In contrast, data on this ceramide-mediated effector mechanism have not been reported in normal Leydig cells and so far activation of the SM pathway appears to be restricted to the inhibitory action of TNF{alpha} on hCG-induced StAR expression in the Cyp17-defficient MA-10 Leydig tumor cell line (38).

Moreover, although the direct activation of the SM pathway with exogenous SMase or cell-permeant ceramides reproduces the inhibitory actions of both cytokines on human chorionic gonadotropin (hCG)-stimulated P450scc and Cyp17 gene expression (37, 38, 39), these treatments also abrogate gonadotropin-induced StAR expression, which is a TNF{alpha}-specific response to Leydig cells (36, 37, 38).

In addition to these direct inhibitory actions of TNF{alpha} and IL1ß on Leydig cell steroidogenesis in vitro (30, 31, 32, 33, 34, 35, 36, 37, 38, 39), the testicular failure observed in immune-challenged animals might also be causally related to alterations in serum gonadotropin levels (21, 46) and/or activation of the hypothalamic-pituitary-adrenal (HPA) axis (reviewed in Refs.47, 48, 49). Concerning this later situation, immune insults such as infection and chronic inflammation stimulate the pituitary release of ACTH and subsequently the biosynthesis and secretion of adrenal steroids (47, 48, 49). Although the release of adrenal steroids constitutes a defensive and ultimately protective mechanism against tissue damage during inflammation and sepsis, these antiinflammatory hormones also exert direct inhibitory actions on Leydig cell steroidogenesis (21, 22, 50, 51) and therefore may contribute to the reduced testicular function associated to these pathological situations (47, 48, 49).

Even though the above-mentioned in vivo and in vitro studies demonstrate that TNF{alpha} and IL1ß inhibit the hypothalamic-pituitary-testis axis at multiple levels, the precise mechanism(s), site(s) of action, and relative contribution of each cytokine to the overall testicular failure associated to local or systemic inflammation are poorly defined.

In this study we used intact and hypophysectomized rats to investigate whether direct intratesticular delivery of TNF{alpha} and IL1ß exerts similar inhibitory actions in vivo as those described in cultured hCG-stimulated Leydig cells (30, 31, 32, 33, 34, 35, 36, 37, 38, 39). In a more precise manner, we evaluated whether intratesticular treatment with TNF{alpha} and IL1ß abrogates StAR expression, which represents the true rate-limiting step in steroid biosynthesis (5, 6, 7). In addition, we also considered whether the direct activation of the SM pathway with short-chain ceramide analogs reproduce in intact animals the inhibitory responses on hCG-stimulated Leydig cell steroidogenesis reported in vitro (37, 38, 39).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and reagents
Human recombinant TNF{alpha} and IL1ß were from Calbiochem (Barcelona, Spain). Human chorionic gonadotropin was purchased from Farma-Lepori (Barcelona, Spain). Phosphorylcholine, phenylmethylsulfonyl fluoride, metal-free protease inhibitor cocktail (P8340), and LPS (from Escherichia coli, serotype 0127:B8) were purchased from Sigma Chemical Co. (St. Louis MO). Sphingosine, sphingosine-1P, and the membrane-permeable analogs of ceramide N-hexanoylsphingosine (C6-cer), N-acetylsphingosine (C2-cer), and N-acetylsphinganine [C2-dihydroceramide (DHC)] were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Ribonuclease inhibitor (n° N211), AMV reverse transcriptase (no. M 519), Thermus aquaticus (Taq) DNA polymerase (no. M166), and Sau3A I (R619) were obtained from Promega (Madison, WI). The polydeoxy-thymidine primers (pdT12–15), deoxynucleotide-triphosphates (dNTPs), the 1-kb molecular weight standard and {Phi}X174/HaeIII digestion product were from Pharmacia (Barcelona, Spain). Digoxigenin-11–2'-deoxy-uridine-5'-trisphosphate (DIG-dUTP) and the positively charged nylon membranes were obtained from Roche Molecular Biochemicals (Madrid, Spain).

Animals and treatments
Adult (300–350 g) Sprague Dawley rats were obtained from Pan Lab (Barcelona, Spain). The same source provided the age-mated (2–3 months old) hypophysectomized animals (270–290 g), which were delivered 2 wk after surgery. Animals were housed in groups of three rats per cage and maintained at least 1 wk before experiments under controlled temperature (22–24 C) and light/dark cycle (14 h light; lights switched on at 0700 h) with free access to tap water or 0.9% saline (hypophysectomized animals) and a standard pelleted diet (Purina, Barcelona, Spain). All experimental procedures were approved by the University of Las Palmas Committee on Animal Care and were conducted according to the European Union normative 86/609 concerning the handling and use of experimental animals. Intact and hypophysectomized animals were randomly assigned to experimental groups, lightly anesthetized with ether, and injected intratesticularly through the scrotal wall with cytokines [200 ng/testes·kg body weight (BW)] or identically treated with cell-permeant ceramides (C2-cer and C6-cer), DHC, phosphorylcholine, sphingosine, and sphingosine-1P (all used at the dose of 30 µg/testes·kg BW). Depending on the experiment, control animals received equivalent volumes (25 µl) of ethanol (EtOH) or pyrogen-free saline or were just subjected to the stress associated to the needle (27-gauge) penetration (52). In other experiments, intratesticular delivery of cytokines or other agents was followed (30 min later) by the ip administration of hCG (100 IU) or an equivalent volume (0.5 ml) of pyrogen-free saline.

Tissue collection and testosterone determination
Sequential blood samples from lightly anesthetized animals were obtained from the retroorbital plexus 2 h before treatment, immediately after treatment (time 0), and at the time points indicated in each experiment. This sequential blood sampling approach minimizes the number of animals per group because it circumvents the large variations in serum testosterone levels derived from the pulsatile release of LH and the circadian pattern of Leydig cell testosterone secretion (53, 54). In other experiments, animals were killed by CO2 asphyxiation, blood collected by heart puncture, and testes immediately removed for RT-PCR and Western blot analysis of StAR (see below). Blood samples were left for 1 h at room temperature and serum separated from the clots, transferred to new tubes, centrifuged (1000 x g at 4 C for 20 min) and the clean supernatants stored frozen (-20 C). Serum testosterone content was determined by RIA using commercially available reagents and following the instructions of the manufacturer (Amersham Corp., Arlington Heights, IL). The sensitivity of the assay was 10 pg/tube and the intraassay and interassay coefficients of variation of 4% and 9.5%, respectively.

RT-PCR of StAR
Total RNA was isolated using Ultraspec RNA (Biotecx, Houston, TX) and isopropyl alcohol precipitation. Equal amounts of RNA (1 µg) were incubated for 75 min at 42 C in 20 µl (final volume) of 1x PCR buffer containing 500 ng oligo (dT)12–15 primer, 1 mM dNTP, 5 U AMV reverse transcriptase, and 20 U RNAsin ribonuclease inhibitor, and reactions terminated by heating at 95 C for 5 min and cooling the tubes on ice. After an initial denaturation step (94 C for 3 min) each cDNA sample was amplified in a Perkin-Elmer/Cetus thermocycler in 25 µl PCR buffer containing (final concentration) 2.5 µM DIG-dUTP, 100 µM of dNTP, 1.5 mM Cl2Mg, 0.625 U Taq DNA polymerase, and 125 ng (10–15 pmol each) and the gene-specific oligonucleotides (forward: 5'-ACAACCAGGAAGGCTGGAAG-3'; reverse 5'-CCTCTGCGCTTGGTACAGC-3') synthesized and HPLC purified by DNA-International (Lake Oswego, OR). The primers complementary to nucleotides 341–360 and 632–650 of the known sequence of StAR (55, 56, 57) were designed to generate an amplification signal 309 bp of the region common to the three alternative spliced products of StAR cDNA containing an unique Sau3A I restriction site (GATC: 528–531) between bases 527 and 528 of the gene (35, 36). After an overnight digestion at 37 C in 20 µl buffer B (Promega) containing 3 U Sau3A, restriction products were resolved on 4.5% high-resolution MS-8 agarose (Hispanlab, Madrid, Spain) gels and produced identifiable bands of the predicted 186- and 123-bp size.

Oligonucleotides designated to generate a PCR product of 983 bp of glycerol-3-phosphate dehydrogenase (G3PDH) were obtained from Clontech (Palo Alto, CA) and used as amplification control. To analyze PCR products from each target gene, 10 µl of the amplified fragments were separated by electrophoresis on 1.8% agarose gels in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). The gels were incubated for 15 min in transference buffer (0.4 M NaOH, 1.5 M NaCl) and transferred to positively charged nylon membranes by positive pressure blotting (Posiblotter, Stratagene, La Jolla, CA). The membranes were cross-linked (1200 kJ/30 sec) on a Stratalinker UV cross-linker (Stratagene) and chemiluminescent detection performed with a commercially available DIG luminescent detection kit following the instructions of the manufacturer (Roche Molecular Biochemicals, no. 1363514). The quantitative data were obtained using a DIANA III densitometric scanning instrument (RAYTEST, Straubenhardt, Germany) with AIDA image analyzer software.

Immunoblot analysis of StAR
The tissues were mechanically homogenized (100 rpm, 25 passes) with a Potter Elvejelm homogenizer in TSE buffer (0.25 M sucrose, 10 mM Tris, 0.1 mM EDTA, pH 7.4) and mitochondria isolated by differential centrifugation as described (55, 56, 57). Mitochondrial proteins were extracted with 50 µl of 20 mM HEPES buffer (pH 7.4) containing (final concentration) 0.15 M NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and the recommended final concentration of protease inhibitor cocktail (100 µl/107 cells). Equal amounts of mitochondrial proteins (100 µg) were boiled 5 min in 2x sodium dodecyl sulfate (SDS) sample buffer [25 mM Tris-HCl (pH 6.8), 1% SDS, 5% ß-mercaptoethanol, 1 mM EDTA, 4% glycerol, and 0.001% bromophenol blue] separated on SDS-7.5% polyacrylamide gels and then electrophoretically transferred to polivinyldifluoride membranes (Bio-Rad Laboratories, Madrid, Spain). After incubation (1 h at room temperature) in PBS supplemented with 4% nonfat dry milk and 0.5% Tween 20 to block nonspecific binding sites, membranes were reincubated (12 h) in the same buffer containing anticytochrome C antibody used at the final dilution (1:1000) recommended by the manufacturer (Santa Cruz Biotechnology, Santa Cruz, CA) or a polyclonal anti-StAR antibody (final dilution 1:2000) raised against a peptide corresponding to residues 88–98 of the proposed N terminus of the published sequence of the mature (30 kDa) StAR protein (55, 56). The peptide was synthesized and conjugated to keyhole limpet hemocyanin by the water-soluble carbodiimide method and used to produce antibodies in rabbits on a fee-for-service basis by Genosys Biotechnologies Inc. (Cambridge, UK). Two rabbits developed antibodies with a high titer, and only one of them (designated AntiS-1) was used in all experiments described here. The membranes were washed (three times for 10 min each wash) and incubated (1 h at room temperature) in fresh blocking buffer containing an 1:4000 dilution of horseradish peroxidase-conjugated antirabbit IgG (Amersham Corp., Barcelona, Spain) and the specific StAR or cytochrome C signals detected using an ECL detection kit (American Life Science, Buckinghamshire, UK). Analysis of the integrated ODs was performed by scanning densitometry.

Statistical analysis
Results for serum testosterone levels are pooled data (mean ± SEM) derived from at least two experiments performed on different days using a minimum of five animals per experimental group. Comparisons were performed using ANOVA and as indicated the t test for comparison of the means. Differences with P > 0.05 were not considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-dependent inhibition of serum testosterone levels after intratesticular treatment with TNF{alpha} and C2-cer
As assessed in preliminary experiments, no significant differences in serum testosterone concentrations were observed in untreated rats as compared with animals subjected to needle penetration in both testes or bilaterally injected with equivalent volumes (25 µl) of saline or EtOH vehicles (results not shown). Hence, to reduce the number of rats, we used animals subjected to needle stress as single control group for the different vehicles.

Results from the first series of experiments illustrate that basal serum testosterone levels were similar 2 h before (6.5 ± 0.7 ng/ml) and immediately after (6.9 ± 0.9 ng/ml) intratesticular treatment with cytokines and C2-cer (Fig. 1AGo). Treatment with TNF{alpha} significantly reduced (30%) testosterone concentration 2 h later (P < 0.05), being a more pronounced inhibition (circa 70%) observed in animals bilaterally treated with the cell-permeant C2-cer analog. Testosterone concentrations were dramatically reduced by 4 h in animals treated with TNF{alpha} or C2-cer, whereas no significant differences were observed after intratesticular delivery of IL1ß or needle penetration (for clarity only IL1ß data are shown in Fig. 1AGo). Testosterone levels remained low throughout the entire experimental period in animals injected with TNF{alpha} but significantly recovered by 6 and 8 h in animals treated with cell-permeant ceramide. In additional experiments (Fig. 1BGo), we document that treatment with C6-cer reduced serum testosterone concentrations with a similar potency as C2-cer. In contrast, serum testosterone levels remained unchanged in rats bilaterally injected with the inactive ceramide analog DHC, the by-product of SMase action phosphorylcholine (43), sphingosine, or its phosphorylated metabolite sphingosine-1P.



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  		FIG. 1. Effect of intratesticular treatment with cytokines and short-chain ceramide (C2-cer) on serum testosterone levels in male rats. A, Blood samples from anesthetized rats were obtained 2 h before and immediately after intratesticular treatment (open circles). Animals (n = 8 per group) were injected in each testes with the same doses (200 ng/kg BW) of IL1ß (closed circles) and TNF{alpha} (open squares). Groups of animals were treated in a similar manner with 30 µg/kg BW of cell-permeant C2-cer (closed squares) or subjected to needle penetration in each testes (for clarity this latter group is not represented because the results overlapped those of animals treated with IL1ß). Sequential blood samples were obtained from the retroorbital plexus immediately after treatment (0 time) and at the time points indicated (2–8 h). In additional experiments (B), animals were subjected to needle stress or injected in each testis with equivalent concentrations (30 µg/kg BW) of another cell-permeant ceramide (C6-cer), the inactive analog DHC, phosphorylcholine, sphingosine (SP), or sphingosine-1P (SP1P). Results for serum testosterone concentrations are expressed as percent concentrations (mean ± SEM) of control values (upper panel) or in ng/ml (lower panel) and represent pooled data of two experiments performed on different days with the same number of animals per treatment. In several cases SEs are less than data point drawn, and groups with different letters are significantly different (P < 0.05).

 
Intratesticular treatment with TNF{alpha} and C2-cer abrogate StAR expression in unstimulated and hCG-treated rats
To determine the site(s) and mechanism(s) whereby TNF{alpha} and C2-cer abrogate testicular steroidogenesis in vivo, we used a sensitive RT-PCR procedure to detect StAR transcription (Fig. 2Go, A–C) and developed a specific anti-StAR polyclonal antibody (AS-1), which recognizes the mature (30 kDa) mitochondrial form of the protein (Fig. 2Go, D and E). An amplification product for StAR of the predicted 309-bp size was detected in steroidogenic glands (adrenals and testis), whereas the 983-bp signal of the constitutive G3PDH used as amplification control was also present in brain, liver, and kidney (Fig. 2AGo). Verification that the amplified product represents StAR transcripts was confirmed by the presence of the predicted 186- and 123-bp fragments after overnight digestion hydrolysis with Sau3A (Fig. 2BGo). Because of the high sensitivity of the chemiluminescence detection method, initial experimental conditions were established for optimal reverse transcription and amplification of these products in a range sensitive to the amount of RNA input and number of cycles (Fig. 2CGo). Using immunoblot analysis, we detected cytochrome c in mitochondrial proteins extracted from all tissues tested. In contrast, the 30-kDa band of StAR was apparent only in adrenals and testis (Fig. 2DGo) and specifically abrogated when membranes containing the mitochondrial proteins were probed with AS-1 antibody previously preadsorbed (5 h) with immunogen (Fig. 2EGo).



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  		FIG. 2. Characterization of StAR transcripts and specificity of the polyclonal anti-StAR antibody. A, Total RNA was extracted from rat tissues (T, testis, A, adrenals; B, brain; L, liver; K, kidney), reverse transcribed into cDNA, and amplified in the presence of DIG-dUTP for 20 or 18 cycles (for StAR or the internal amplification control G3PDH, respectively). Chemiluminescent detection of the specific 309 bp (StAR) and 983 bp (G3PDH) was performed as described in Materials and Methods. B, The 309-bp product was digested overnight with Sau3A I (3 U/20 µl) and fragments separated by electrophoresis on 2% agarose gels (B). Lanes 1 and 4 represent a 1-kb ladder and {Phi}X174/Hae III digestion products, respectively. After digestion hydrolysis with Sau3A I, the 306-bp signal of StAR (lane2) generated two fragments of the predicted size (186 and 123 bp) shown in lane 3. C, RT-PCR amplification of StAR and G3PDH using different template amounts (25–200 ng) obtained from LH-treated male rats amplified for 20 or 18 cycles (for StAR and G3PDH, respectively) or the detailed number of cycles for each gene product using 125 ng RNA input. Proteins were extracted from mitochondrial pellets isolated by differential centrifugation in TSE buffer from the same rat tissues (T, testis, Ad, adrenal; B, brain; L, liver; K, kidney) and separated by SDS-PAGE. Proteins were electrophoretically transferred to polivinyldifluoride membranes and probed with anti-StAR AS-1 or Cyt-c antibody (D). The specific 30-kDa signal of StAR was not apparent when the AS-1 antibody was preadsorbed (5 h) with 10 µg/ml of the immunogen (E).

 
With these approaches, we show that intratesticular delivery of TNF{alpha} significantly reduced 4 and 24 h later the specific mRNA signal of StAR and mitochondrial accumulation of the 30-kDa form of the protein, with no such actions observed in animals treated with IL1ß (Fig. 3Go). Likewise, bilateral treatment with equivalent volumes (25 µl) of saline or EtOh vehicle did not affect testosterone biosynthesis and StAR expression, compared with animals subjected to needle stress (results not shown). As expected from previous experiments (Fig. 1Go), intratesticular treatment with C2-cer-abrogated testosterone biosynthesis and StAR expression at the early time point studied (4 h) and no differences with control or IL1ß-treated animals were observed 24 h later.



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  		FIG. 3. Intratesticular treatment with TNF{alpha} and ceramide abrogates StAR expression and steroidogenesis in intact animals. Rats (n = 8/group) were subjected to needle stress (C) or injected in each testis with the same doses of cytokines or C2-cer used before. Animals were killed 4 h (open bars) or 24 h (dashed bars) later, blood samples obtained by heart puncture, and the testes rapidly removed and used for RT-PCR and Western blot analysis. Serum testosterone levels (mean ± SEM) represent pooled data of two experiments, using the same number of animals per group. Densitometric analysis of five different experiments and a representative RT-PCR (middle panel) and Western blot (lower panel) are depicted. Different letters denote significant differences between groups (P < 0.05).

 
Inhibitory action of TNF{alpha} and C2-cer on hCG-stimulated steroidogenesis and StAR expression in intact and hypophysectomized rats
To determine that the rapid and sustained inhibition of testicular steroidogenesis induced by TNF{alpha} is not a secondary response related to altered gonadotropin release (21, 46), in the next experiments we used hCG-stimulated animals (Fig. 4Go) pretreated (30 min) with cytokines or cell-permeant ceramides. As compared with unstimulated rats, testosterone concentrations dramatically augmented in hCG-treated animals 4 h later and remained elevated 24 h after gonadotropin stimulation (P < 0.05). The stimulatory action of hCG on testosterone biosynthesis paralleled the significant increase in mRNA levels of StAR and mitochondrial accumulation of the 30-kDa form of the protein. Although IL1ß had no apparent effect on gonadotropin-induced steroidogenesis, intratesticular delivery of TNF{alpha} and C2-cer prevented the large increase in testosterone concentrations and StAR expression observed 4 h after hCG administration. No differences in testosterone levels and StAR expression were observed 24 h later in animals injected with C2-cer as compared with hCG-stimulated controls. Of interest to note, although testicular steroidogenesis still remained low 24 h after TNF{alpha} treatment, testosterone concentration and StAR expression were higher in these animals than in unstimulated control rats.



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  		FIG. 4. Effect of TNF{alpha} and ceramide on gonadotropin-induced StAR expression and steroidogenesis. Equal number of rats (n = 8/group) were bilaterally injected with cytokines (200 ng/ kg BW per testis) or C2-cer (30 µg/kg BW per testis) and treated 30 min later with a single ip dose of hCG (100 IU) or 0.5 ml saline (controls). Blood samples were collected from the retroorbital plexus 4 h (open bars) and 24 h later (dashed bars) and serum obtained and stored frozen until assayed for testosterone concentrations. In additional experiments, animals were treated in a similar manner, blood samples obtained by heart puncture, and the testes rapidly removed and used for RT-PCR and Western analysis. Serum testosterone levels (mean ± SEM) represent pooled data of two experiments, using the same number of animals per group. Quantitation of five different RT-PCR (middle panel) and Western blot (lower panel) analysis, and a representative experiment showing mRNA and protein levels are depicted. Groups with different letters (a–d) are significantly different (P < 0.05).

 
Animals receiving the doses of cytokines or ceramide analogs used in these experiments exhibited no signs of local or general inflammation observed after systemic treatment with 5 mg/kg BW of bacterial LPS (results not shown). Nevertheless, these data may simply reflect that a single cytokine is unable to fully reproduce the general condition of illness (lethargy, piloerection, shivering, and increased rectal temperature) observed in LPS-treated rats (42) and do not exclude that, once delivered, these agents are cleared from the testis via the spermatic vein to the general circulation (52).

In this regard, although the release of ACTH and the subsequent secretion of antiinflammatory adrenal steroids (47, 48, 49) constitutes a defense mechanism against immune insults, it is also well established that glucocorticoids exert direct inhibitory actions on Leydig cell steroidogenesis (21, 22, 50, 51) and therefore may contribute to the reduced testicular function associated with chronic inflammation and sepsis (47, 48, 49). Compared with intact controls, preliminary experiments revealed that needle stress induced a slight increase in adrenal StAR expression in some animals (results not shown). Even though this response was unrelated to a specific agent delivered via the scrotal wall, in the next experiments we used hypophysectomized animals to confirm that the inhibitory actions of TNF{alpha} and C2-cer on Leydig cell StAR expression and steroidogenesis are unrelated to alterations in the HPA axis (Fig. 5Go). The extremely low serum testosterone levels (upper panel), Leydig cell mRNA (middle panel), and protein levels of StAR (lower panel) were significantly augmented 4 h after gonadotropin treatment and remained elevated even 24 h after hCG administration. As expected, gonadotropin-stimulated steroidogenesis remained unchanged in rats pretreated with IL1ß, but the direct delivery of TNF{alpha} and C2-cer dramatically blunted hCG-induced StAR expression 4 h later. By 24 h, this inhibitory effect was still apparent in animals bilaterally treated with TNF{alpha}, but similar to intact rats (Fig. 4Go), testosterone concentration, StAR message, and protein levels were significantly higher in these animals than in unstimulated controls.



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  		FIG. 5. Effect of TNF{alpha} and ceramides on hCG-induced StAR expression and testosterone biosynthesis in hypophysectomized rats. Hypophysectomized rats (n = 5 per group) were subjected to needle stress (C), injected in each testes with equivalent amounts of TNF{alpha} and IL-lß1 (200 ng/kg BW per testis) or with C2-cer (30 µg/kg BW per testis) and treated 30 min later with a single ip dose of hCG (100 IU). Blood was collected from the retroorbital plexus 4 h (open bars) and 24 h later (dashed bars) and serum obtained and stored frozen until assayed for testosterone concentrations (upper panel). In parallel experiments groups of animals (n = 5 per group) were killed 4 h or 24 h after treatment, and blood samples were collected and the testes were removed and used to analyze mRNA levels (middle panel) and mitochondrial protein levels (lower panel) of StAR. Serum testosterone levels (mean ± SEM) represent pooled data of both experiments, which were replicated two times with the same number of animals per group. Representative experiments and quantitation of StAR transcription and protein levels are shown in the middle and lower panels. Groups with different letters (a–d) are significantly different (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In contrast to the large amount of experimental evidence that demonstrates the ability of TNF{alpha} and IL1ß to directly abrogate hCG-stimulated Leydig cell steroidogenesis in vitro (30, 31, 32, 33, 34, 35, 36, 37, 38, 39), information concerning similar actions under in vivo conditions is less abundant. In this study we show that intratesticular delivery of TNF{alpha} and cell-permeant ceramides directly abrogate testosterone biosynthesis in intact and hypophysectomized and extend these findings to demonstrate for the first time that this response is causally related to an inhibitory action on basal and hCG-induced StAR expression in Leydig cells.

In contrast, and despite evidences demonstrating the capacity of IL1ß to prevent hCG-and cAMP-stimulated steroidogenesis in cultured Leydig cells (30, 32, 33, 36, 39), local delivery of this cytokine did not significantly reduce serum testosterone levels and StAR expression in unstimulated or gonadotropin-treated animals. These divergent results were somewhat unexpected because binding of TNF{alpha} and ILß to specific TNFR and IL1ßR expressed in the testis (37, 58, 59, 60) trigger a common set of inhibitory actions on hCG-stimulated testosterone biosynthesis in vitro (30, 31, 32, 33, 34, 35, 36, 37, 38, 39). Nevertheless, although testicular alterations in endotoxin-treated animals (20, 21, 22, 23) correlate with the systemic release of cytokines (9, 10, 11, 12, 13, 14), it must also be considered that the access of circulating IL1ß to the interstitial space is limited by the blood-testis barrier (60), and simultaneously endotoxin treatment dramatically reduces IL1ßR expression in the testis (61).

Otherwise intracerebroventricular delivery of IL1ß induced a rapid decrease in pituitary FSH and LH release and a drastic a reduction of testicular StAR expression and steroidogenesis (17, 18). Although the ability of circulating cytokines to traverse the blood-brain barrier remains controversial (49), recent evidence suggests that LPS induces a rapid increase in IL1ß content in the hypothalamic extracellular fluid that chronologically precedes the reduction in gonadotropin release and the elevated systemic concentrations of ACTH and corticosteroids (62). Nevertheless inhibition of StAR expression and steroidogenesis after intracerebroventricular delivery of IL1ß was unrelated to pituitary gonadotropin secretion or activation of the HPA axis (17, 18, 63). On the other hand, these studies revealed that basal steroidogenesis remained unchanged after systemic administration of IL1ß, but this treatment induced a small, albeit significant, reduction in testosterone concentrations in hCG-stimulated animals (18). Although the inhibitory action induced by iv treatment with the cytokine was accompanied by a dramatic increase in serum IL6 levels, administration of ILß into the cerebral ventricles abrogated steroidogenesis before increases in serum IL6 levels were noted (18). In considering that IL6 activates the HPA axis and induces Leydig cell resistance to LH action in vivo (64), it seems reasonable to conclude that the release of this cytokine may account for the reduced inhibitory action on hCG-stimulated steroidogenesis observed after systemic treatment with IL1ß.

These data, in conjunction with the limited ability of IL1ß to cross the blood-testis barrier (60) and the lack of inhibitory effect after intratesticular delivery of the cytokine presented in this study, strongly support the contention that the contribution of IL1ß to the general testicular failure associated with sepsis and inflammation may be exerted at the hypothalamic-pituitary level and/or mediated by the release of other factors that directly inhibit Leydig cell function.

Otherwise, the inhibitory effect induced after direct intratesticular administration of TNF{alpha} suggests that the local release of this cytokine by resident macrophages may significantly contribute to abrogate Leydig cell steroidogenesis during chronic inflammation (21, 22). In considering that administration of TNF{alpha} reduces testosterone biosynthesis in human volunteers and experimental animals (14, 15), the present data also support the notion that elevated systemic levels of this cytokine achieved during endotoxemia can transverse the blood-testis barrier and directly exert inhibitory actions on Leydig cell function.

Of interest to note, inhibition of testosterone biosynthesis and StAR expression after intratesticular delivery of TNF{alpha} occurred in a time frame (4–24 h) similar to that previously observed in LPS-challenged animals (20, 21, 22, 23). Nevertheless, inhibition of testicular steroidogenesis in LPS-treated mice was causally related to a rapid (2 h) inhibition of StAR mRNA translation into the mature form of the protein, this early action being accompanied by a reduction of the hormone-regulatable components P450scc and Cyp17 at the longer time periods (24 h) studied (20). The data presented herein however clearly demonstrate that TNF{alpha} prevents basal and hCG-stimulated StAR transcription and translation under in vivo conditions in rats (Figs. 3–5GoGoGo). These divergent results may reflect species differences and/or the systemic treatment with LPS used in other reports (20, 21, 22, 23), compared with intratesticular delivery of a single agent (TNF{alpha}) used in this study. It must be noted, however, that despite the fact that P450scc and Cyp17 enzymes are important components of the steroidogenic machinery in Leydig cells (8), StAR-mediated delivery of cholesterol to the inner mitochondrial membrane represents the first and true rate-limiting step for the biosynthesis of steroid hormones (5, 6, 7). Evidence for the paramount role of this initial step in steroid biosynthesis derived from experiments in which steroidogenic cells were cultured in the presence of the 22R-hydroxycholesterol or 25-hydroxycholesterol (25HC) analogs of the precursor (4, 5, 6, 7). These hydroxylated compounds freely diffuse across the mitochondrial intermembrane space, accumulate at the P450scc complex and are converted to pregnenolone (4, 5, 6, 7). Hence, under these experimental conditions steroidogenic cells produced high levels of steroids in the absence of hormone stimulation, indicating that full catalytic activity of P450scc is limited by the slow diffusion of cholesterol through the aqueous space between the outer and inner mitochondrial membrane (5).

But perhaps the most striking evidence of its paramount role in steroidogenesis concerns the demonstration that nonsense mutations of the human StAR gene leads to congenital adrenal hyperplasia (57), a fatal disease in which steroid biosynthesis is virtually eliminated. Hence, because inhibition of StAR expression is sufficient to explain the dramatic decrease in testosterone biosynthesis induced by TNF{alpha} and cell-permeant ceramides, no further attempts were made to elucidate whether these treatments intercept additional steps along the steroidogenic pathway. Because TNF{alpha} also abrogates P450scc and Cyp17 in cultured Leydig cells (31, 34, 35), these results do not exclude that treatment in vivo with TNF{alpha} intercepts additional components of the steroidogenic pathway located downstream of StAR activity (8).

This assumption deserves further consideration in view of recent reports that demonstrate that testicular macrophages express the 25-hydroxylase enzyme required to convert cholesterol into the polar secretory product 25HC (65, 66). Although the release of low levels of 25HC may facilitate testosterone biosynthesis in unstimulated Leydig cells (4, 5, 6, 7), these studies document that prolonged exposure to high concentrations of the oxysterol precursor abrogated LH responsiveness and induced nonspecific cytotoxic actions in testicular cells (66). Nevertheless, because concentrations of androgens similar to those found in the interstitial space abolished 25-hydroxylase transcription and oxysterol release by testicular macrophages (67), it has been proposed that 25HC and testosterone integrate a communication system between resident macrophages and Leydig cells, which could provide a physiologically relevant mechanism required to sustain basal and LH-stimulated steroidogenesis. If such a mechanism operates in vivo, the early inhibition of testosterone biosynthesis induced by TNF{alpha} and C2-cer may simultaneously up-regulate 25-hydroxylase expression and subsequently the release of 25HC (67), which is then converted to testosterone in a StAR-independent manner in surrounding Leydig cells that retain functional P450scc and Cyp17 enzymes (4, 5, 6, 7). Hence, the recovery of serum testosterone levels after ceramide treatment suggests a functional steroidogenic machinery downstream of StAR protein. On the contrary, the prolonged inhibition of steroidogenesis observed in TNF{alpha}-challenged rats sustains the notion that this cytokine induces additional inhibitory actions at the P450scc and Cyp17 enzyme steps.

Ceramide generation in response to Fas/Apo1 ligation or p55TNFR activation has been associated with the cytotoxic and/or apoptotic actions mediated by the TNF{alpha}/Fas system (41, 43). In contrast to the well-established role of a ligand-regulated SM pathway in mediating cellular function and life-and-death decisions in the ovary, there is little information concerning a similar role for ceramide in the testis (reviewed in Ref.68). This study shows that testosterone biosynthesis and StAR expression gradually recovered (6–8 h) and reached control levels 24 h after treatment with C2-cer (Figs. 1Go and 3Go) a time point at which testicular steroidogenesis still remained low in animals treated with TNF{alpha}. Nevertheless in TNF{alpha}-treated and hCG-stimulated animals, testicular testosterone biosynthesis and StAR expression remained higher than in saline-injected controls (Figs. 3Go and 4Go). Based on these data, it seems reasonable to conclude that the direct inhibition of testicular steroidogenesis reported in this study is not related to nonspecific toxic actions of TNF{alpha} or its putative second-messenger ceramide moiety.

The ability of C2-cer and C6-cer to reproduce the short-term inhibitory actions of TNF{alpha} is a ceramide-specific event because no such effect was observed in animals treated with DHC, the by-product of SMase action phosphorylcholine (43) or the two ceramide metabolites sphingosine and sphingosine-1P (reviewed in Ref.69). Nevertheless, these data do not demonstrate that inhibition of testosterone biosynthesis and StAR expression in vivo is causally related to a ligand-induced activation of the SM pathway. Activation of the SM pathway is specifically coupled to the p55TNFR and has been demonstrated in a variety of cellular models, but such a relationship is difficult to assess in organs and tissues of TNF{alpha}-challenged animals (41, 43).

In addition to the cellular complexity of the testis, analysis of the SM pathway in vivo is further complicated because ligand-induced ceramide generation may accumulate in cells for variable periods of time, be recycled back to SM, or be degraded to sphingosine by ceramidase and further transformed by sphingosine kinase into the sphingosine-1P moiety (43, 69). Although the role of sphingolipid metabolites in the testis remains poorly defined (68), it has been recently shown that the total testicular concentration of sphingosine-1P is considerable higher (up to 100 nmol/g) than in other tissues tested (70). In agreement with these findings, metabolic labeling experiments performed with MA-10 Leydig tumor cells (38) and primary cultures of pig Sertoli cells (71) demonstrated that ceramide generated in response to TNF{alpha} was rapidly converted to sphingosine-1P in these cellular models. Furthermore, although cell-permeant ceramides reproduced the inhibitory action of TNF{alpha} on StAR expression in MA-10 Leydig cells (38), the conversion of ceramide to sphingosine was required for TNF{alpha}-induced glucose metabolism and LDH expression in Sertoli cells (71).

In this study we show that intratesticular delivery of cell-permeant ceramides induced a rapid and transient inhibition on testicular steroidogenesis (Fig. 1AGo), but no such effect was observed after bilateral treatment with sphingosine or sphingosine-1P (Fig. 1BGo). If it is assumed that the sequential action of testicular ceramidase and sphingosine kinase can convert exogenous short-chain ceramides to sphingosine-1P (43, 70), such a scenario provides a plausible explanation for the transient inhibition of steroidogenesis observed after intratesticular treatment with the C2-cer analog. Conversely, in considering that C2-cer reproduced only the early (up to 4 h) actions of TNF{alpha} on testicular steroidogenesis, it must be assumed that additional effector mechanisms unrelated to the SM pathway are ultimately responsible of the long-term abrogation of testosterone biosynthesis and StAR expression. Finally, because sphingosine-1P exerts stimulatory actions on cell growth and survival (43, 70), the release of sphingosine and its conversion to sphingosine-1P may constitute a physiological and ultimately protective mechanism to counteract the deleterious actions of ceramide and TNF{alpha} on germ cell survival and Leydig cell steroidogenesis.

In conclusion, the present study highlights that the intratesticular delivery of agents provides a useful model to evaluate the physiological relevance of putative intragonadal regulatory agents. This study also revealed several novel elements concerning the testicular response to inflammation. In the first place, we demonstrate that the direct intratesticular delivery of TNF{alpha} prevents basal or hCG-stimulated steroidogenesis and identified StAR as the target for the inhibitory action of this cytokine under in vivo conditions. In addition, we document the ability of cell-permeant ceramides to reproduce the early inhibitory actions of TNF{alpha} on StAR gene transcription and protein expression in unstimulated or hCG-treated animals. Although this study does not establish a causal relationship between TNF{alpha}-induced SM hydrolysis and testicular function in vivo, the present data demonstrate for the first time a direct and specific inhibitory action of ceramide on testicular StAR expression and steroidogenesis in intact animals.


   Footnotes
 
This work was supported by the Dirección General de Enseñanza Superior e Investigación Científica y Técnica (DGESICT) Grants 98/0233 (to C.M.R.D.G.) and 98/0234 (to L.F.F.). The Ministry of Science and Education of Spain supported V.M. and R.D.

Abbreviations: AS-1, Anti-StAR polyclonal antibody; BW, body weight; C2-cer, N-acetyl sphingosine; c6-cer, N-hexanoyl ceramide; DHC, N-acetyl sphinganine; DIG, digoxigenin; dNTP, deoxynucleotide-triphosphate; dUTP, 11–2'-deoxy-uridine-5'-trisphosphate; EtOH, ethanol; G3PDH, glycerol-3-phosphate dehydrogenase; 25HC, 25-hydroxycholesterol; hCG, human chorionic gonadotropin; HPA, hypothalamic-pituitary-adrenal; LPS, lipopolysaccharide; P450scc, P450 cholesterol side chain cleavage; SDS, sodium dodecyl sulfate; SM, sphingomyelin; SMase, sphingomyelinase; StAR, steroidogenic acute regulatory protein.

Received May 7, 2003.

Accepted for publication July 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pescovitz OH, Srivastava CH, Breyer PR, Monts BA 1994 Paracrine control of spermatogenesis. Trends Endocrinol Metab 5:126–132[CrossRef][Medline]
  2. Saez JM 1994 Leydig cells: endocrine, paracrine and autocrine regulation. Endocr Rev 15:574–626[Abstract/Free Full Text]
  3. Gnessi L, Fabri A, Spera G 1997 Gonadal peptides as mediators of development and functional control of the testis: an integrated system with hormones and local environment. Endocr Rev 18:541–609[Abstract/Free Full Text]
  4. Miller WL 1995 Mitochondrial specificity of the early steps in steroidogenesis. J Steroid Biochem Mol Biol 55:607–616[CrossRef][Medline]
  5. Watermann MR 1995 A rising StAR: an essential role in cholesterol transport. Science 267:1780–1781[Free Full Text]
  6. Stocco DM, Clark BJ 1996 Role of the steroidogenic acute regulatory protein (StAR) in steroidogenesis. Biochem Pharmacol 51:197–205[CrossRef][Medline]
  7. Stocco DM 2000 The role of the StAR protein in steroidogenesis: challenges for the future. J Endocrinol 164:247–253[Abstract]
  8. Miller WL 2002 Androgen biosynthesis from cholesterol to DHEA. Mol Cell Endocrinol 198:7–14[CrossRef][Medline]
  9. Buch JP, Havlovec SK 1991 Variation in sperm penetration assay related to viral illness. Fertil Steril 55:844–846[Medline]
  10. Handelsmann DJ 1994 Testicular disfunction in systemic disease. Endocrinol Metab Clin North Am 23:839–856[Medline]
  11. Cutolo M, Balleari E, Giusti M, Monachesi M, Accardo S 1996 Sex hormone status of patients with rheumatoid arthritis: evidence of low serum concentrations at baseline and after human chorionic gonadotropin stimulation. Arthritis Rheum 31:1314–1317
  12. Damas T, Reuter A, Gysen P, Demonty J, Lamy M, Franchimont P 1989 Tumor necrosis factor and interleukin-1 serum levels during severe sepsis in humans. Crit Care Med 17:975–978[Medline]
  13. Cannon JG, Tompkins RG, Gelfand JA, Michie HR, Standford GG, Van der Meer JWM, Endres S, Lonnemann G, Corsetti J, Chernow B, Wilmore DW, Wolff SM, Burke JF, Dinarello C 1990 Circulating interleukin-1 and tumor necrosis factor in septic shock and experimental endotoxin fever. J Infect Dis 161:79–84[Medline]
  14. Mantovani G, Maccio A, Mura L, Massa A, Mudu MC, Mulas C, Lusso MR, Madeddu C, Dessi A 2000 Serum levels of leptin and proinflammatory cytokines in patients with advanced-stage cancer at different sites. J Mol Med 78:554–561[CrossRef][Medline]
  15. Van der Pool T, Rominj JA, Endert E, Sauerwein HP 1993 Effects of tumor necrosis factor on the hypothalamic-pituitary-testicular axis in healthy men. Metabolism 42:303–307[CrossRef][Medline]
  16. Mearly K, Robinson B, Millette CF, Majzoub J, Wilmore DW 1990 The testicular effects of tumor necrosis factor. Ann Surg 211:470–475[Medline]
  17. Ogilvie KM, Hales KH, Roberts ME, Hales DB, Rivier C 1999 The inhibitory effect of intracerebroventricular injection of interleukin-1ß on testosterone secretion in the rat: role of steroidogenic acute regulatory protein (StAR). Biol Reprod 60:527–533[Abstract/Free Full Text]
  18. Turnbull UV, Rivier C 1997 Inhibition of gonadotropin-induced testosterone secretion by the intracerebroventricular injection of interleukin-1ß in the male rat. Endocrinology 138:1008–1013[Abstract/Free Full Text]
  19. Tulassay Z, Vicziánn M, Böjthe L, Czeizel A 1970 Quantitative histological studies on the injury of spermatogenesis induced by endotoxin in rats. J Reprod Fertil 22:161–164[Abstract/Free Full Text]
  20. Bossmann HB, Hales HK, Li Z, Stocco DM, Hales DB 1996 Acute in vivo inhibition of testosterone by endotoxin parallels loss of steroidogenic acute regulatory (StAR) protein in Leydig cells. Endocrinology 137:4522–4525[Abstract]
  21. O’Bryan MK, Schlatt S, Philips DJ, de Krestner DM, Hedger PM 2000 Bacterial lipopolysaccharide-induced inflammation compromises testicular function at multiples levels in vivo. Endocrinology 141:238–246[Abstract/Free Full Text]
  22. Hales KH, Thorsten D, Ginde S, Shankar BK, Roberts M, Bossman HB, Hales DB 2000 Diametric effects of bacterial endotoxin lipopolysaccharide on adrenal and Leydig cell steroidogenic acute regulatory protein. Endocrinology 141:4000–4012[Abstract/Free Full Text]
  23. Sharma AC, Bossman HB, Motew SJ, Hales KH, Hales DB, Ferguson JL 1996 Steroid hormone alterations following induction of chronic intraperitoneal sepsis in male rats. Shock 6:150–154[Medline]
  24. Hutson JC 1994 Testicular macrophages. Int Rev Cytol 149:99–143[Medline]
  25. Hedger MP 1996 Testicular leukocytes: what are they doing? Rev Reprod 2:38–47
  26. Hutson JC 1993 Secretion of tumor necrosis factor-{alpha} by testicular macrophages. J Reprod Immunol 23:63–72[CrossRef][Medline]
  27. Moore C, Hutson JC 1994 Physiological relevance of tumor necrosis factor in mediating macrophage-Leydig cell interactions. Endocrinology 134:63–69[Abstract/Free Full Text]
  28. Kern S, Robertson SA, Mau VJ, Maddocks S 1995 Cytokine secretion by macrophages in the testis. Biol Reprod 53:1407–1416[Abstract]
  29. Hayes R, Chalmers SA, Nicolic-Paterson DJ, Atkins RC, Hedger MP 1996 Secretion of bioactive interleukin-1 by rat testicular macrophages in vitro. J Androl 17:41–49[Abstract/Free Full Text]
  30. Lin T, Wang D, Nagpal ML, Calkins JH, Chang W, Chi R 1991 Interleukin-1 inhibits steroidogenesis and P450 cholesterol side chain cleavage enzyme (P450scc) in primary cultures of Leydig cells. Endocrinology 129:1305–1311[Abstract/Free Full Text]
  31. Mauduit C, Hartmann DJ, Chauvin MA, Revol A, Morera AM, Benahmed M 1991 Tumor necrosis factor-{alpha} inhibits gonadotropin actions in cultured porcine Leydig cells: site(s) of action. Endocrinology 129:2933–2940[Abstract/Free Full Text]
  32. Hales DB 1992 Interleukin-1 inhibits Leydig cell steroidogenesis primarily by decreasing 17{alpha}-hydroxylase/C17–20 lyase cytochrome P450 expression. Endocrinology 131:2165–2172[Abstract/Free Full Text]
  33. Hales DB, Xiong Y, Tur-Kaspa I 1992 The role of cytokines in regulating Leydig cell P450c17 gene expression. J Steroid Biochem 43:907–914[CrossRef]
  34. Xiong Y, Hales DB 1993 The role of tumor necrosis factor-{alpha} in the regulation of mouse Leydig cell steroidogenesis. Endocrinology 132:2438–2444[Abstract/Free Full Text]
  35. Li X, Youngblood GL, Payne AH, Hales DB 1995 Tumor necrosis factor-{alpha} inhibition of 17{alpha}-hydroxylase C17–20 Lyase gene (Cyp17) expression. Endocrinology 136:3519–3526[Abstract]
  36. Lin T, Wang D, Stocco DM 1998 Interleukin-1 inhibits Leydig cell steroidogenesis without affecting steroidogenic acute regulatory protein (StAR) messenger RNA or protein levels. J Endocrinol 156:461–467[Abstract]
  37. Mauduit C, Gasnier F, Rey C, Chauvin MA, Stocco DM, Louisot P, Benahmed M 1998 Tumor necrosis factor-{alpha} inhibits Leydig cell steroidogenesis through decreases in steroidogenic acute regulatory protein expression. Endocrinology 139:2863–2868[Abstract/Free Full Text]
  38. Budnik LT, Jahner D, Mukhopadhyay AK 1999 Inhibitory effect of TNF{alpha} on mouse tumor Leydig cells: possible role of ceramide in the mechanism of action. Mol Cell Endocrinol 150:39–46[CrossRef][Medline]
  39. Meroni SB, Pellizzari EH, Canepa DF, Cigorraga SB 2000 Possible role of ceramide in the regulation of rat Leydig cell function. J Steroid Biochem 75:307–313[CrossRef][Medline]
  40. Sims JE, Giri JG, Dower SK 1994 The two interleukin-1 receptors play different roles in L-1 activities. Clin Immunol Immunopathol 72:9–14[CrossRef][Medline]
  41. Wallach D, Varvolomeev EE, Malinin NL, Goltsev YV, Kovalenko AV, Boldin MP 1999 Tumor necrosis factor receptor and Fas signaling mechanisms. Annu Rev Immunol 17:361–367
  42. Dinarello CA 2000 Proinflammatory cytokines. Chest 118:503–508[Abstract/Free Full Text]
  43. Kolesnick R 2002 The therapeutical potential of modulating the ceramide-sphingomyelin pathway. J Clin Invest 110:3–8[CrossRef][Medline]
  44. Santana P, Llanes L, Gallardo G, Quintana J, Gonzalez J, Estevez F, Ruiz de Galarreta CM, Fanjul LF 1996 Interleukin-1ß stimulates sphingomyelin hydrolysis in cultured granulosa cells: evidence for a regulatory role of ceramide on progesterone and prostaglandin biosynthesis. Endocrinology 137:2480–2489[Abstract]
  45. Santana P, Llanes L, Gallardo G, Quintana J, Gonzalez J, Estevez F, Ruiz de Galarreta CM, Fanjul LF 1995 Ceramide mediates tumor necrosis factor effects on P450-aromatase activity in cultured granulosa cells. Endocrinology 136:2345–2348[Abstract]
  46. Gow RM, O’Bryan MK, Canny BJ, Ooi GT, Hedger MP 2001 Differential effects of dexamethasone treatment on lipopolysaccharide-induced testicular inflammation and reproductive hormone inhibition in adult rats. J Endocrinol 168:193–201[Abstract]
  47. Rivier C, Rivier S 1991 Effect of stress on the activity of the hypothalamic-pituitary-adrenal axis: peripheral and central mechanisms. Biol Reprod 45:523–532[Abstract]
  48. Kapcala LP, Chautard T, Eskay RL 1995 The protective role of the hypothalamic-pituitary-adrenal axis against lethality produced by immune, infectious and inflammatory stress. Ann NY Acad Sci 771:419–437[Medline]
  49. John CD, Buckingham JC 2003 Cytokines: regulation of the hypothalamo-pituitary-adrenocortical axis. Curr Opin Pharmacol 3:78–84[CrossRef][Medline]
  50. Hales DB, Payne AH 1989 Glucocorticoid-mediated repression of P450scc mRNA expression and de novo synthesis in cultured Leydig cells. Endocrinology 124:2099–2114[Abstract/Free Full Text]
  51. Gao H, Shan L, Monder C, Hardy MP 1996 Suppression of endogenous corticosterone levels in vivo increases the steroidogenic capacity of purified Leydig cells in vitro. Endocrinology 137:1714–1718[Abstract]
  52. Dalterio E, Bartke A, Brodie A, Mayfield D 1983 Effects of testosterone, estradiol, aromatase inhibitor, gonadotropin and prolactin on the response of mouse testes to acute gonadotropin stimulation. J Steroid Biochem 18:391–396[CrossRef][Medline]
  53. Ellis GB, Desjardins C 1982 Male rats secrete luteinizing hormone and testosterone episodically. Endocrinology 110:1618–1627[Abstract/Free Full Text]
  54. Mock EJ, Norton HW, Frankel AI 1978 Daily rhythmicity of serum testosterone concentration in the male laboratory rat. Endocrinology 103:1111–1121[Abstract/Free Full Text]
  55. Clark BJ, Wells J, King SR, Stocco DM 1994 The purification, cloning and expression of a novel luteinizing hormone-induced mitochondrial protein in MA-10 mouse Leydig tumor cells: characterization of the steroidogenic acute regulatory protein (StAR). J Biol Chem 269:28314–28322[Abstract/Free Full Text]
  56. Clark BJ, Soo SC, Caron KM, Ikeda Y, Parker KY, Stocco DM 1995 Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Mol Endocrinol 9:1346–1355[Abstract/Free Full Text]
  57. Lin D, Sugawara T, Strauss III JF, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL 1995 Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267:1828–1831[Abstract/Free Full Text]
  58. Cunningham ET, Wada E, Carter DB, Tracey DE, Battey JF, De Souza EB 1992 Distribution of type I interleukin-1 receptor messenger RNA in testis: an in situ histochemical study in the mouse. Neuroendocrinology 56:94–99[Medline]
  59. Takao T, Mitchell WM, Tracey DE, De Souza EB 1990 Identification of interleukin-1 receptors in mice testis. Endocrinology 127:251–258[Abstract/Free Full Text]
  60. Banks WA, Kastin AJ 1992 Human interleukin-1{alpha} crosses the blood-testis barriers of the mouse. J Androl 13:254–259[Abstract/Free Full Text]
  61. Takao T, Culp SG, De Souza EB 1993 Reciprocal regulation of interleukin-1ß (IL1ß) and IL1 receptors by lipopolysaccharide (endotoxin) treatment in the mouse brain-endocrine-immune axis. Endocrinology 132:1497–1504[Abstract/Free Full Text]
  62. Ma XC, Chen LT, Oliver J, Horvath E, Phelps CP 2000 Cytokine and adrenal axis responses to endotoxin. Brain Res 861:135–142[CrossRef][Medline]
  63. Rivest S, Rivier C 1993 Centrally injected interleukin-1ß inhibits hypothalamic LHRH secretion and circulating LH levels via prostaglandins in rats. J Neuroendocrinol 5:445–450[CrossRef][Medline]
  64. Spath-Schwalbe E, Born J, Schrezenmeier H, Bornstein SR, Stromeyer P, Dreshler S, Fehm HL, Porzsolt F 1994 Interleukin-6 stimulates the hypothalamus-pituitary-adrenocortical axis in man. J Clin Endocrinol Metab 79:1212–1214[Abstract]
  65. Lukyanenko YO, Chen JJ, Hutson JC 2001 Production of 25-hydroxycholesterol by testicular macrophages and its effects on Leydig cells. Biol Reprod 64:790–796[Abstract/Free Full Text]
  66. Chen JJ, Lukyanenko YO, Hutson JC 2002 25-Hydroxycholesterol hydroxycholesterol is produced by testicular macrophages during the early postnatal period and influences differentiation of Leydig cells in vitro. Biol Reprod 66:1336–1341[Abstract/Free Full Text]
  67. Lukyanenko YO, Chen JJ, Hutson JC 2002 Testosterone regulates 25-hydroxycholesterol production in testicular macrophages. Biol Reprod 67:1435–1438[Abstract/Free Full Text]
  68. Tilly JL, Kolesnick RN 1999 Sphingolipid signalling in gonadal development and function. Chem Phys Lipids 102:149–155[CrossRef][Medline]
  69. Spiegel S, English D, Milstein S 2002 Sphingosine-1P signaling: providing cells with a sense of direction. Trends Cell Biol 12:236–242[CrossRef][Medline]
  70. Yatomi Y, Welch RJ, Igarashi Y 1997 Distribution of sphingosine-1P, a bioactive sphingolipid in rat tissues. FEBS Lett 404:173–174[CrossRef][Medline]
  71. Grataroli R, Boussuoar F, Benahmed M 2000 Role of sphingosine in tumor necrosis factor-{alpha} stimulatory effect on lactate dehydrogenase A expression and activity in porcine Sertoli cells. Biol Reprod 63:1473–1481[Abstract/Free Full Text]



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