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Testis |
in the Pathogenesis of Autoimmune Orchitis in Rats1
Instituto Multidisciplinario de Biología Celular,3 CONICET, La Plata, Argentina
Centro de Investigaciones en Reproducción,4 Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
Instituto de Biología y Medicina Experimental,5 CONICET, Buenos Aires, Argentina
Facultad de Ciencias Exactas,6 Universidad Nacional de La Plata, La Plata, Argentina
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF) is involved in germ cell apoptosis and in Leydig cell steroidogenesis. The EAO was induced in adult male Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. In the experimental group, a severe orchitis was observed 80 days after the first immunization. ED1- and ED2-positive macrophages were quantified by immunohistochemistry. The TNF concentration of conditioned media from testicular macrophages (TMCM) was determined by ELISA. The number of apoptotic TNF receptor 1 (TNFR1)-positive germ cells was identified by combining in situ end labeling of apoptotic DNA and immunohistochemical techniques. The effect of TNF on Leydig cell testosterone production was determined by RIA. In rats with EAO, we observed a significant increase in the number of TNF-positive testicular macrophages, the TNF concentration in TMCM, and the number of TNFR1-positive germ cells. Sixty percent of TNFR1-positive germ cells were apoptotic. These results suggest that TNF could be involved in the pathogenesis of EAO. Acting together with other local factors such as Fas-FasL, TNF could trigger germ cell apoptosis. We also demonstrated that TNF inhibited in vitro testosterone production in basal and hCG-stimulated Leydig cells from rats with orchitis.
apoptosis, cytokines, immunology, testis, testosterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular macrophages interact with a variety of cell types, including lymphocytes and Leydig cells. The latter have been shown to regulate the number of testicular macrophages [3]. On the other hand, macrophages and their products have the ability to modulate Leydig cell steroidogenesis [4].
Although the role of testicular macrophages in the immunologically privileged status of the normal testis is not clear, it has been demonstrated that when macrophages are immunoactivated, they acquire an inflammatory pattern expressed by their ability to secrete several proinflammatory cytokines, such as tumor necrosis factor- (TNF), interleukin (IL)-1, and IL-6 [5].
The TNF is a pleiotropic cytokine that modulates inflammatory and immunoendocrine responses and is involved in apoptotic cell death. It exists in two biologically active forms: as a propeptide, associated to the cellular membrane; and as a soluble peptide, generated by the action of specific metalloproteases. It exerts its biological activity by binding to specific receptors localized on the cell surface. Two different receptors are known: TNFR1 (55 kDa), which is mainly involved in the death pathway; and TNFR2 (75 kDa). In mouse testis, pachytene spermatocytes and round spermatids were found to express TNF mRNA, and the bioactive cytokine was mainly produced by the round spermatids [6]. In the normal testis, TNF has been found to modulate spermatogenesis [4, 6–8] and Leydig cell steroidogenesis in vitro [9–12] and to stimulate the expression of cell adhesion molecules [13].
Controversial results have been reported in vivo concerning the role of this cytokine in the development of experimental autoimmune orchitis (EAO). Teuscher et al. [14], injecting neutralizing antibodies to TNF in mice with autoimmune orchitis, suggested that TNF is not a major factor in the pathogenesis of the disease. In contrast, Yule and Tung [15] were able to reduce testis damage by using the same TNF antibody therapy in another EAO model, thereby highlighting the importance of this cytokine in the pathogenesis of EAO.
We previously studied different aspects of EAO induced in rats by active immunization with spermatic antigens and adjuvants. In this experimental model, we described an increase in the number of major histocompatibility complex (MHC) II-positive interstitial cells, mainly macrophages [16], and we observed numerous interdigitations and specialized junctions between these cells and Leydig cells [17]. We also reported that testicular macrophage-conditioned media from rats with EAO modulated Leydig cell steroidogenesis [18].
The main target of the immunological attack in this model is the germ cells that undergo sloughing and death. Recently, we demonstrated that germ cell death occurs through apoptosis and that the Fas-FasL system is involved [19]. Because not all apoptotic germ cells expressed Fas, another pathway could be involved in this process. The aim of the present study was to characterize testicular macrophage subpopulations, to study the involvement of TNF in germ cell apoptosis, and to examine the effect of this cytokine on Leydig cell steroidogenesis in EAO.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (age, 50–60 days) were kept at 22°C with a 14L:10D photoperiod and were fed standard food pellets and water ad libitum. The animals were killed according to protocols for animal use, in agreement with NIH guidelines for care and use of experimental animals and approved by the local ethical committee (IBYME-CONICET).
Immunization Schedule
Rats in the experimental (E) group were immunized with testicular homogenate (TH) prepared as previously described [20]. Briefly, rat testes were decapsulated, diluted in an equal volume of saline, and disrupted in an Omni mixer (I. Sorvall, Inc., Norwalk, CT) for 30 sec. The final concentration was 500 mg/ml wet weight. Rats in the E group were injected three times with 200 mg wet weight of TH/dose at 14-day intervals. Antigen (0.4 ml) emulsified with complete Freund adjuvant (CFA; 0.4 ml) was injected intradermally in footpads and at multiple sites near ganglion regions. The first two immunizations were followed by an i.v. injection of 0.5 ml of Bordetella pertussis (strain 10536; Instituto Malbrán, Buenos Aires, Argentina) containing 1010 microorganisms, and the third immunization was followed by an i.p. injection of 109 microorganisms. Rats in the control (C) group were injected with an emulsion of saline, CFA, and B. pertussis in the same conditions as the E group. The E, C, and normal (N) untreated rats were killed at different time periods (0–35, 50–60, 70–110, and 120–150 days) after the first immunization. Blood was collected and sera stored at -70°C until use. Testes were removed, weighed, fixed in Bouin solution, and embedded in paraffin or quickly frozen for cryostat sections to be used in histopathology or immunohistochemistry, respectively. The procedure to isolate testicular and peritoneal macrophages and Leydig cells is described below.
Histopathology
The histopathology of the testis was studied in sections obtained from three different levels and stained with hematoxylin-eosin.
Immunohistochemistry
A mouse monoclonal antibody, ED1, that recognizes a cytoplasmic antigen in rat monocytes, macrophages, and dendritic cells and a mouse monoclonal antibody, ED2, that recognizes a membrane antigen of tissue macrophages were used to identify circulating and resident macrophages, respectively. Serial cryostat testis sections fixed for 10 min in cold acetone were washed in PBS and incubated with 0.3% H2O2 in methanol for 30 min to block endogenous peroxidase. After incubation with 3% normal horse serum for 30 min at room temperature, sections were treated with Avidin/Biotin Blocking Solution (Vector Laboratories, Burlingame, CA) and then incubated with ED1 (10 µg/ml; 554954; BD Pharmingen, San Diego, CA), with ED2 (5 µg/ml; 550573; BD Pharmingen) in 0.1 M PBS with 0.03% Triton-X 100 and 5% normal rat serum, or with ED1 and ED2 for 1 h at room temperature in a humidified chamber. A biotinylated horse anti-mouse rat-adsorbed immunoglobulin (Ig) G (2 µg/ml; Vector Laboratories) was used as secondary antibody. The reaction was amplified with a Vectastain Elite ABC Kit (Vector Laboratories), and the reaction product was visualized by adding diaminobenzidine substrate (Vector Laboratories). For negative control, the first antibody was omitted. Sections were counterstained with hematoxylin. For each antibody, positively immunostained cells were counted using a 25x objective, and the ocular (12.5x) was fitted with a quadratic grid with a total area of 96 100 µm2. Numerical density (number of macrophages per unit volume) was determined by counting positive macrophages (Na) occurring within the total grid boundary. The total number of grid fields counted for each section was 30, and four animals per group were studied. Average nuclear diameter (D) was measured with a 100x Zeiss (Oberkochen, Germany) objective and an ocular micrometer calibrated with a stage micrometer [21]. The values of D (mean ± SEM) obtained from the N, C, and E groups were as follows for ED1: N group, 5.09 ± 0.20 µm; C group, 4.76 ± 0.31 µm; and E group: 5.25 ± 0.20 µm. The values of D (mean ± SEM) obtained from the N, C, and E groups were as follows for ED2: N group, 5.37 ± 0.20 µm; C group, 5.61 ± 0.07 µm; and E group, 5.89 ± 0.12 µm. The final numerical density or number of macrophages per unit volume testis (Nv) was then calculated by the Floderus equation [22]: Nv = Na/D + T - 2h, where T is the section thickness (6 µm) and h is a correction factor to calculate nuclei diameters. The number of macrophages per testis was calculated by multiplying the final numerical density by the testis volume. The latter was calculated as previously described [23]. Some macrophages express ED1 and ED2 antigen. Incubating tissue sections with ED1 antibodies, we identified ED1-positive/ED2-negative and ED1-positive/ED2-positive subsets, and incubating tissue sections with ED2 antibodies, we detected ED2-positive/ED1-negative and ED1-positive/ED2-positive subsets. By combined ED1 and ED2 immunohistochemistry, we obtained the total number of macrophages.
To detect TNF and TNFR1, the immunoperoxidase technique described for ED1 and ED2 was applied to paraformaldehyde-fixed, isolated testicular macrophages and to paraffin testis sections. A rabbit polyclonal antibody anti-mouse TNF that cross-reacts with rat TNF (1:200; P-350, Endogen, Woburn, MA) and a goat polyclonal antibody anti-mouse that cross-reacts with rat TNFR1 (1:80; sc-1069; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used as primary antibodies. A rabbit anti-goat IgG (4 µg/ml; Vector Laboratories) and a goat anti-rabbit IgG (5 µg/ml; Vector Laboratories) were used as secondary antibodies, and normal goat or rabbit sera were used to avoid nonspecific staining for TNF and TNFR1, respectively. As negative controls, the first antibodies were replaced by PBS or by respective rabbit or goat IgG isotypes. The TNFR1-positive germ cells were counted in 100 seminiferous tubules of three nonconsecutive sections from four to five animals per group using a 25x objective.
Localization of TNFR1 in Apoptotic Cells
The TNFR1 expression in apoptotic germ cells was studied by combining immunohistochemistry of TNFR1 and in situ end labeling of apoptotic DNA (ISEL) techniques. For the ISEL technique, DNA fragmentation in individual cells was visualized by indirect immunoenzyme detection of digoxigenin-labeled genomic DNA. To facilitate antigen retrieval, deparaffinized and hydrated tissue sections (thickness, <5 µm) were heated in a microwave (5 min at 370 W) in sodium citrate buffer (10 mM, pH 6.0) and quickly cooled in PBS. The procedure for the ISEL technique was performed as previously described [19].
To detect TNFR1 in apoptotic cells, we combined immunoperoxidase and ISEL techniques in testis serial sections as described above, and we also used ISEL/immunofluorescence (IF) techniques in the following procedure: testis sections were processed as indicated above for ISEL, with minor modifications. After incubation with the enzyme and digoxigenin-11-2',3'-dideoxy-uridine-5'-triphosphate (dig-ddUTP), sections were washed and incubated with 1.5% normal sheep serum (NSS) in 2% BSA for 30 min at room temperature. Then, sections were incubated with anti-TNFR1 (1:30; Santa Cruz Biotechnology) for 1 h at room temperature. A fluorescein isothiocyanate-conjugated antidigoxigenin (1:8; Roche Molecular Biochemicals, Mannheim, Germany) and a rhodamine-conjugated anti-IgG (1:70; Sigma Chemical Co., St. Louis, MO) were used to detect ISEL-positive and TNFR1-positive cells, respectively. The antibodies were diluted in PBS containing 1% blocking reagent, 0.5% BSA and 1.5% NSS. The TNFR1-positive germ cells, ISEL-positive (apoptotic) germ cells, and double-positive germ cells were counted in 100 seminiferous tubules of two nonconsecutive testis sections from four rats with orchitis. The percentage of TNFR1 apoptotic germ cells was calculated as follows: the number of double-positive germ cells multiplied by 100 and then divided by the number of TNFR1-positive germ cells. The percentage of apoptotic cells expressing TNFR1 was calculated as the number of double-positive cells multiplied by 100 and then divided by the number of ISEL-positive cells.
Isolation and Culture of Testicular Macrophages
The isolation procedure was similar to the one described by Yee and Hutson [24], with minor modifications. Rats from the N, C, and E groups (killed 70–150 days after the first immunization) were perfused with cold, sterile saline solution until tissues were pale. Interstitial cells were obtained by collagenase digestion or mechanical dispersion in PBS under sterile and low-endotoxin conditions as previously described [18]. Briefly, decapsulated testes were incubated with collagenase (0.25 mg/ml; Worthington Biochemical Corporation, Freehold, NJ) plus 0.1% BSA (fraction V; Sigma) in a Dubnoff shaking water bath at 34°C for 15 min. Tubes were filled with cool PBS and placed on ice for 3–4 min to allow the tubules to settle, then the supernatant was centrifuged at 300 x g for 5 min at 4°C to allow cell sedimentation. The pellet was resuspended in PBS, plated on polystyrene Petri dishes (2 ml/plate; plate diameter, 35 mm; Nunc, Inc., Naperville, IL) and on round coverslips and then incubated at 34°C for 6–10 min. The unattached cells were aspirated and the dishes vigorously rinsed three times with PBS. To obtain conditioned media, the adherent cells were cultured in Medium 199 (M199; Sigma) plus penicillin-streptomycin solution (1 ml/100 ml; Gibco-BRL, Life Technologies, Rockville, MD) for 24 h at 34°C in a humidified atmosphere of 95% air and 5% CO2 in the absence or presence of lipopolysaccharide (LPS; 50 µg/ml; Sigma). Purity of macrophage preparations was evaluated in cell smears by counting the number of adherent cells that incorporated trypan blue or latex beads (diameter, 0.81 µm; Difco Laboratories, Detroit, MI) after 1-h incubation at 34°C. From 90% to 95% of adherent cells incorporated trypan blue or latex beads.
Peritoneal Macrophage Isolation
Macrophages were obtained by lavage of the peritoneal cavity with 15 ml of sterile PBS. The fluid collected in conical tubes was incubated with trypsin (2.5 mg/ml; Worthington) in a Dubnoff shaking bath at 34°C for 10–15 min. Then, 10 ml of PBS were added, and the rest of the procedure was the same as that described for testicular macrophages.
Macrophage-Conditioned Media
Macrophage-conditioned media (MCM) was collected from cultures of testicular or peritoneal macrophages with a Pasteur pipette in sterile conditions, centrifuged at 3000 rpm for 10 min, and stored at -70°C before TNF determination by ELISA. The total number of adherent cells per plate was calculated by a cell adhesion colorimetric microassay using 0.5% crystal violet [18].
Immunoassay for Rat TNF (ELISA)
A solid-phase sandwich ELISA for rat TNF (KRC 3011; Biosource International, Camarillo, CA) was used to quantify TNF in rat serum and in testicular MCM (TMCM) and peritoneal MCM (PMCM) from the N, C, and E groups. All samples were centrifuged before immunoassay. Briefly, samples including standards of known TNF content were incubated in 96-well plates coated with an antiserum to TNF (90 min). After removal of excess second antibody, streptavidin-peroxidase was added. After incubation (45 min) and washing to remove all unbound enzyme, a substrate solution (tetramethyl benzidine) was added for 30 min in the dark. After the addition of stop solution, plates were read within 2 h in an ELISA plate reader at 450 nm. The minimum detectable dose of rat TNF with this cytoscreen kit is less than 4 pg/ml. Samples from the N, C, and E groups obtained in several immunization protocols were run in the same assay, and each sample was measured three times.
In Vitro Testosterone Production by Leydig Cells
Purified Leydig cell preparations were obtained from rats of the N, C, and E groups as previously described [25, 26]. Briefly, testes were removed, decapsulated, and digested with collagenase (0.25 mg/ml; Worthington). The digestion procedure was stopped by dilution with fresh medium. Two successive washes and sedimentations were then performed. The supernatants were pooled and interstitial cells collected by centrifugation at 220 x g for 10 min. Crude cell preparations were purified on a discontinuous five-layer Percoll (Pharmacia, Uppsala, Sweden) density gradient (21%, 26%, 34%, 40%, and 60%). The gradient was centrifuged at 800 x g for 30 min, and the interphase between 40% and 60% was collected and washed with medium to remove Percoll [26]. In all experiments, cell viability, as assessed by trypan blue exclusion method, was approximately 95%. Purity of cell preparation, as determined by a positive cytochemical reaction for 3ß-hydroxysteroid dehydrogenase was 85%. Leydig cells (105 cells/tube) were incubated in M199 with or without hCG (10 ng/ml; CR-127 recombinant hCG; NIH, Bethesda, MD) in a Dubnoff shaking water bath for 3 h at 34°C in an atmosphere of 95% O2 and 5% CO2. The effect of TNF on Leydig cell steroidogenesis was evaluated by adding recombinant rat TNF (150 pg/ml; Biosource International) to Leydig cells. Cell viability was approximately 95% before and after TNF incubation. Media were collected and centrifuged, and the supernatants were stored at -20°C. Testosterone was measured by RIA in the incubation media using testosterone (1,2-3H(N); 60 Ci/mmol; New England Nuclear, Boston, MA) and a specific antibody from Immunotech Diagnostic (Montreal, Canada) as previously described and validated [27]. The sensitivity of the assay was 12.5 pg/ml. The within-assay and interassay coefficients of variation were 7% and 11%, respectively.
Statistical Data Analysis
Statistical evaluations were done using one-way or two-way ANOVA and the Tukey test for testosterone results, TNF analysis of conditioned media, and morphometric studies. The nonparametric Mann-Whitney rank test was used to analyze quantitative data of TNFR1-positive cells. A value of P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A dark-brown reaction product was observed in the cytoplasm of ED1-positive and ED2-positive macrophages present in the interstitium of the testis of all groups of rats studied (Fig. 1). Some ED2-positive macrophages exhibited a more intense reactivity in the periphery of cell cytoplasm.
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In normal rats, most testicular macrophages expressed ED2 antigen, whereas a minor proportion expressed ED1 antigen only (Table 1). An increase in the total number of macrophages was observed in rats from the E and C groups compared to rats from the N group. Compared to rats in the N group, the increase in the total number of macrophages of rats in the C group mainly corresponds to the arrival of monocytes-macrophages from the circulation (ED1-positive cells). In contrast, rats with orchitis showed a significant increase in both populations of macrophages (ED1-positive/ED2-negative and ED2-positive/ED1-negative). It has been suggested [2] that ED1-positive/ED2-positive cells represent a transient subset of macrophages.
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The procedure to isolate testicular macrophages from rats injected with trypan blue 48 h before death showed that 90–95% of adherent cells incorporated trypan blue (Fig. 2A). The TNF was observed in the cytoplasm of testicular macrophages (Fig. 2B). The percentages of TNF-positive testicular macrophages were 18% and 77.5% in the C and E groups, respectively.
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As shown in Figure 3A, a significant increase (P < 0.05) in TNF content of TMCM from rats with EAO was observed compared to the N or C groups in the absence and in the presence of LPS. In addition, a significant difference was observed between TNF content in TMCM of the C and N groups. Similar results were obtained in TMCM of macrophages isolated by mechanical dispersion or by collagenase digestion. No significant differences in TNF content of PMCM was observed among the N, C, and E groups (Fig. 3B). As shown in Figure 3, only peritoneal macrophages of the N, C, and E groups responded to LPS stimulation by an increase in TNF secretion. We were unable to detect TNF by ELISA in the sera of rats from the N, C, and E groups.
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By immunohistochemistry, we demonstrated that TNFR1 is expressed in macrophages, lymphocytes, and Leydig cells in all groups of rats. We also observed TNFR1 immunoreactivity in a scarce number of spermatocytes in testis sections from the C group and in numerous degenerating spermatocytes and spermatids in rats with EAO (Fig. 4). In these rats, the number of TNFR1-positive cells significantly increased from Day 70 onward compared to rats in the C group (Fig. 5). By combining ISEL and immunohistochemistry in serial testis sections, we detected TNFR1 expression in apoptotic ISEL-positive cells (Fig. 6). Moreover, by IF, we observed apoptotic germ cells expressing TNFR1 (Fig. 7). We observed that 40.1% ± 11.2% of apoptotic germ cells expressed TNFR1, whereas 64.0% ± 15.0% of TNFR1-positive germ cells were also ISEL-positive.
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Leydig cells from rats with EAO showed an enhanced in vitro testosterone production in basal and hCG-stimulated conditions (Fig. 8) compared to those from rats in the N and C groups (P < 0.01), confirming previous results [25]. Figure 8 shows that TNF inhibited testosterone production in basal and hCG-stimulated Leydig cells from the N, C, and E groups. However, the percentage of inhibition of TNF on testosterone production by hCG-stimulated Leydig cells from the E group was significantly higher (70%) compared to the N and C groups (50%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the cytoplasm of testicular macrophages and observed an increase in the percentage of TNF-positive macrophages in rats with orchitis compared to controls. Recent data obtained by reverse transcription-polymerase chain reaction in human testicular biopsy specimens of pathologies involving inflammation also showed an increase in the number of interstitial macrophages and TNF expression in testicular macrophages [29]. We also observed that testicular macrophages are able to secrete TNF in vitro, confirming previous results [30]. In rats with EAO, we detected a significant increase of TNF content in the TMCM of the E group compared to the C group. Also, rats injected with adjuvants exhibited a higher TNF content in the TMCM compared to normal rats, probably because of the inflammatory effect induced by adjuvants.
Kern et al. [5] reported a poor immunoaccessory function of testicular macrophages compared to peritoneal macrophages. Concordantly, we observed a response to LPS stimulation by peritoneal, but not by testicular, macrophages. However, we speculate that a change in the subpopulations and in the immune function of testicular macrophages occurs in EAO when the immune system encounters testis-specific autoantigens and macrophages have been primed by lymphokines. In fact, in rats with EAO, we observed an activation of testicular macrophages and an increase of the TNF released by these cells and not by peritoneal macrophages. We also detected an increase of IL-6 production by testicular macrophages [31] and an increase of Fas-FasL and cell adhesion molecule expression [19, 32] that could be explained largely by the effects of TNF and other proinflammatory cytokines. In accordance with these data and those reported using another experimental model [33], we postulate that in rats with EAO, TNF is probably involved in the progression of the disease because of its ability to increase endothelial cell permeability, to facilitate lymphomonocyte extravasation in the testis, and to activate T cells and macrophages through paracrine and autocrine mechanisms.
In autoimmune orchitis, a severe damage of seminiferous tubules characterized by degeneration, death, and sloughing of germ cells, mainly spermatids and spermatocytes, occurs. We recently demonstrated that in EAO, germ cell death occurs by apoptosis and the Fas-FasL system is involved in this process [19]. We reported that approximately 44% of apoptotic germ cells express Fas, suggesting that other death pathways could be involved, as has been demonstrated by Richburg et al. [34] in gld mice (which lack functional Fas). In the present study, we observed that with the increase of TNF secretion by testicular macrophages, a simultaneous increase of TNFR1-positive germ cells occurs in rats with EAO. The observation that approximately 40%–50% of apoptotic germ cells expressed TNFR1 suggests that the TNF-TNFR1 system is involved together with the Fas-FasL system in germ cell apoptosis. It is possible that TNF released by interstitial macrophages could enter the seminiferous tubules, reaching the adluminal compartment, as has been demonstrated for IL-1 [35, 36]. Moreover, Hellani et al [37] reported that TNF may favor the opening of Sertoli cell tight junctions, inhibiting oligodendrocyte-specific protein (OSP)/claudin 11 cell expression. It has been demonstrated that OSP has the ability to form tight-junction strands [38], and Gow et al [39], studying OSP/claudin 11-null mice, suggested a key role for OSP in formation of the blood-testis barrier (BTB). We can speculate that the high production of TNF by testicular macrophages in EAO could eventually alter BTB permeability by inhibiting OSP expression. In addition, alterations of BTB have been reported in guinea pigs treated with CFA [40]. In EAO, sloughing of germ cells could also alter this barrier.
Previous work [23] showed low or normal serum testosterone levels and signs of hypogonadism in rats with EAO. However, we observed [25; present study] increased in vitro testosterone production by purified Leydig cells obtained from rats with EAO. This apparent paradox has been reported in several experimental models, with increased in vitro testosterone production being associated with severely damaged seminiferous tubules [41]. Alterations in blood flow or decrease in capillary permeability in damaged testes or in the metabolic clearance of testosterone could explain this fact.
Expression of TNFR1 by Leydig cells observed in the present report suggests, as has been reported in the literature [4, 42], that TNF can modulate Leydig cell steroidogenesis. Numerous studies described an inhibitory effect of TNF on testosterone production by Leydig cells [9, 11, 12]. Most authors suggested that TNF appears to act as a transcriptional repressor of steroidogenic enzyme gene expression [4, 42] and through a decrease in steroidogenic acute regulatory gene expression, probably mediated via TNFR1 [12]. Most of these studies were performed in normal Leydig cells. In a previous study [25] and in the present one, we observed that in contrast to normal rats, Leydig cells from rats with orchitis show in vitro hyperresponsiveness to hCG and the highest sensitivity to TNF under stimulated conditions. We can speculate that in contrast to controls, the inflammatory microenvironment caused by a complex network of cytokines and other factors in the testis of rats with EAO could induce an increase in the in vitro steroidogenic response and in the number or the avidity of TNF receptors in Leydig cells. These data extend our previous work, in which we reported that TMCM of rats with EAO induced a bimodal effect on normal Leydig cell steroidogenesis, depending on the TMCM concentration [18]. The present results suggest that TNF could be one of the molecules responsible for the inhibitory effect.
In conclusion, we postulate that in an in vivo model of autoimmune orchitis, TNF is involved in the perpetuation of the inflammatory state, in the induction of germ cell apoptosis, and in the regulation of Leydig steroidogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Livia Lustig, Centro de Investigaciones en Reproducción, Facultad de Medicina, Paraguay 2155, Piso 10, C1121 ABG Buenos Aires, Argentina. FAX: 54 11 59509612; llustig{at}fmed.uba.ar
Received: 19 September 2002.
First decision: 15 October 2002.
Accepted: 18 December 2002.
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in mouse spermatogenic cells. Endocrinology 1993 133:389-396 down-regulates the Fas ligand and inhibits germ cell apoptosis in the human testis. J Clin Endocrinol Metab 2001 86:4480-4488 enhances inhibitory effects of interleukin-1ß on Leydig cell steroidogenesis. Biochem Biophys Res Commun 1990 166:1313-1318[CrossRef][Medline] in the regulation of mouse Leydig cell steroidogenesis. Endocrinology 1993 132:2438-2444 inhibits Leydig cells steroidogenesis through a decrease in steroidogenic acute regulatory protein expression. Endocrinology 1998 139:2863-2868 by testicular macrophages. J Reprod Immunol 1993 23:63-72[CrossRef][Medline] crosses the blood testis barriers of the mouse. J Androl 1992 13:254-259 across the blood-brain barrier and blood-testis barrier in rats. Brain Res 2000 881:57-61[CrossRef][Medline]This article has been cited by other articles:
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  M.S. Theas, C. Rival, S. Jarazo-Dietrich, P. Jacobo, V.A. Guazzone, and L. Lustig Tumour necrosis factor-{alpha} released by testicular macrophages induces apoptosis of germ cells in autoimmune orchitis Hum. Reprod., August 1, 2008; 23(8): 1865 - 1872. [Abstract] [Full Text] [PDF]
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 C. Rival, V. A. Guazzone, W. von Wulffen, H. Hackstein, E. Schneider, L. Lustig, A. Meinhardt, and M. Fijak Expression of co-stimulatory molecules, chemokine receptors and proinflammatory cytokines in dendritic cells from normal and chronically inflamed rat testis Mol. Hum. Reprod., December 1, 2007; 13(12): 853 - 861. [Abstract] [Full Text] [PDF]
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 M. W M Li, W. Xia, D. D Mruk, C. Q F Wang, H. H N Yan, M. K Y Siu, W.-y. Lui, W. M Lee, and C Y. Cheng Tumor necrosis factor {alpha} reversibly disrupts the blood-testis barrier and impairs Sertoli-germ cell adhesion in the seminiferous epithelium of adult rat testes. J. Endocrinol., August 1, 2006; 190(2): 313 - 329. [Abstract] [Full Text] [PDF]
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M.S. Theas, C. Rival, S.J. Dietrich, V.A. Guazzone, and L. Lustig Death receptor and mitochondrial pathways are involved in germ cell apoptosis in an experimental model of autoimmune orchitis Hum. Reprod., July 1, 2006; 21(7): 1734 - 1742. [Abstract] [Full Text] [PDF]
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 X. Li, L. Strauss, A. Kaatrasalo, A. Mayerhofer, I. Huhtaniemi, R. Santti, S. Makela, and M. Poutanen Transgenic Mice Expressing P450 Aromatase as a Model for Male Infertility Associated with Chronic Inflammation in the Testis Endocrinology, March 1, 2006; 147(3): 1271 - 1277. [Abstract] [Full Text] [PDF]
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 V A Guazzone, B Denduchis, and L Lustig Involvement of CD44 in leukocyte recruitment to the rat testis in experimental autoimmune orchitis Reproduction, May 1, 2005; 129(5): 603 - 609. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. respective group without LPS
