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Endocrinology Vol. 138, No. 3 994-998
Copyright © 1997 by The Endocrine Society

Articles

Nitric Oxide Production by Cultured Rat Leydig Cells

Noboru Tatsumi, Masato Fujisawa, Masanori Kanzaki, Yoshihiro Okuda, Hiroshi Okada, Soichi Arakawa and Sadao Kamidono

Department of Urology, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) has emerged as an intracellular and intercellular messenger in a number of biological systems. In the present study, we demonstrated that NO was produced by cultured rat Leydig cells, and that inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) was expressed in Leydig cells. NO was measured as nitrite with the method of Griess. Although unstimulated Leydig cells produce little NO, interleukin-1ß (IL-1ß) markedly increased NO production. NO production was inhibited by the NOS inhibitor, NG-monomethyl-L-arginine. Northern blot analysis showed that iNOS mRNA was little expressed in freshly isolated immature Leydig cells, but that iNOS mRNA levels were increased by the addition of IL-1ß in a dose-dependent manner at the concentration up to 10 ng/ml. The levels of iNOS mRNA were increased as early as 3 h after the addition of IL-1ß and persisted for up to 24 h. In adult Leydig cells, IL-1ß stimulated iNOS mRNA expression. Immunocytochemical analysis demonstrated iNOS-like immunoreactivity in the cytoplasm of Leydig cells. These results indicate that NO is produced in Leydig cells and suggest that NO might be involved in the physiological function of Leydig cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NITRIC OXIDE (NO) is a free radical generated from L-arginine by nitric oxide synthase (NOS), and it is recognized that NO acts as an intracellular and intercellular messenger in a number of biological systems (1, 2). NOS has been classified into three isoforms, such as neuronal NOS, inducible NOS (iNOS), and endothelial NOS (3). iNOS is Ca2+/calmodulin independent and induced by cytokines and endotoxin. It is well known that iNOS exists in macrophages and that NO participates in their cytotoxic function by inhibiting of a number of key enzymes in phagocitized cells that contain iron in their catalytic centers, including ribonucleotide reductase, iron sulfur cluster-dependent enzymes (complexes I and II) involved in mitochondrial electron transport, and cis-aconitase involved in the citric acid cycle (4). Moreover, it has become clear that iNOS acts in many cell types, including smooth muscle cells, hepatocytes, and chondrocytes (4). It has been reported that rat serum testosterone levels were increased by the administration of a NOS inhibitor and were suppressed by agents that promote NO production (5, 6, 7). These data suggest that NO may participate in some physiological modulation of testicular cell function. Recently, it has been reported that iNOS messenger RNA (mRNA) was expressed in cultured rat Sertoli cells (8). In the present study, we examined whether iNOS exists in cultured rat Leydig cells and whether Leydig cells produce NO.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Immature (20-day-old) and adult (55- to 65-day-old) male Crj:S-D rats were purchased from Charles River Japan (Hino, Japan). Collagenase/dispase was obtained from Boehringer Mannheim (Mannheim, Germany), and human recombinant Interleukin-1ß (IL-1ß) was purchased from R&D Systems (Minneapolis, MN). Reagents for nitrite measurement by the Griess method were obtained from Wako (Osaka, Japan). Rabbit anti-iNOS polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rat iNOS complementary DNA (cDNA) probe was a gift from Dr. Y. Kawahara (Kobe University School of Medicine, Kobe, Japan). GeneScreen Plus membrane was obtained from DuPont (New England Nuclear, Boston, MA). Mouse antirat macrophage monoclonal antibody was obtained from Serotec (Oxford, UK). Other culture reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Preparation of Leydig cells
All rats were killed by asphyxiation using CO2. All studies were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Immature Leydig cell-enriched cultures (>85% staining positive for 3ß-hydroxysteroid dehydrogenase) were prepared as described previously (9).

Adult rat Leydig cells (>97% staining positive for 3ß-hydroxysteroid dehydrogenase) were isolated by the method of Klinefelter et al. (10) with minor modifications. In brief, the testes were perfused with medium 199, followed by collagenase digestion (0.25 mg/ml) of the decapsulated testis at 34 C in a shaking water bath for 15 min. The crude interstitial cells were then fractionated by size using a Beckman Elutriator rotor (model J2-21M, Beckman, Palo Alto, CA) with a 5-ml chamber at a speed of 2,000 rpm and a flow rate of 16 ml/min. The fractions enriched for Leydig cells were then centrifuged through a 55% Percoll gradient at 20,000 x g at 4 C for 60 min. After centrifugation, purified Leydig cells were divided into a heavier than 1.068 g/ml fraction. The cells were plated onto 100-mm polystyrene dishes for RNA isolation or into 24-multiwell tissue culture plates for the assay of nitrite and cultured at 34 C in a humidified atmosphere of 5% CO2 and 95% air with DMEM nutrient with F-12 mixture (DMEM/F-12) containing 2.5 µg/ml insulin, 5 µg/ml transferrin, and 10 µg/ml penicillin-streptomycin (immature Leydig cells) or DMEM/F-12 containing 10 µg/ml penicillin-streptomycin (adult Leydig cells). The concentrations of Leydig cells were 5 x 106/100-mm dish or 2.5 x 105/well. At 48 h, the medium was removed, the cells were washed, serum-free medium containing IL-1ß (0.01–100 ng/ml) was added to monolayers, and the cells were maintained for various periods up to 24 h. A rat macrophage antibody (ED2) was used to check for contamination by macrophages in the Leydig cell preparations, as described previously (11). Less than 1% of the cells stained positive for macrophages.

Measurement of nitrite production
NO was measured as nitrite according to the method of Griess (12). In brief, the Leydig cell culture medium was added to an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid). The absorbance at 550 nm was measured, and the nitrite concentration was determined from a calibration curve of sodium nitrite standards.

Northern blot analysis
Total RNA was isolated as described previously (13). Total RNA (20 µg) was electrophoresed through a 1.0% agarose gel containing formaldehyde and transferred directly to a GeneScreen Plus membrane. The membranes were incubated with a random primed, [32P]deoxy-CTP-labeled rat iNOS cDNA probe containing 1033 bp of iNOS-specific sequence. Nonspecific binding of the radioactive probe was removed by washing in 2 x SSPE (1 x SSPE = 150 mM NaCl, 10 mM NaH2PO4, and 1 mM EDTA) for 30 min at room temperature followed by 2 x SSPE-1% SDS for 90 min at 65 C and 0.1 x SSPE for 30 min at room temperature. The filters were then analyzed using the BAS 2000 Fujix Bio-imaging Analyzer. After determining the level of iNOS mRNA, the filters were subsequently stripped and rehybridized with a random primed [32P]deoxy-CTP-labeled ß-actin cDNA probe as a internal control (Clontech, Palo Alto, CA).

Immunocytochemistry
Freshly isolated Leydig cells were placed in chamber slides (Lab-Tek, Nunc, Naperville, IL) and cultured for 48 h at 34 C. The cells were fixed with 4% paraformaldehyde-PBS for 30 min at 4 C and permeabilized with Triton X-100. The cells were then processed for the avidin-biotin complex (ABC) method (14) and incubated for 2 h at 37 C with a rabbit anti-iNOS polyclonal antibody diluted to 1:1000. Nonimmune rabbit serum was employed in place of primary antiserum to determine the level of nonspecific immunoreactivity.

Data analysis
The significance of results was determined using Student’s t test, ANOVA, and Dunnett’s multiple comparison test for comparison of means where applicable. P <= 0.05 was considered to indicate significance. Values are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of cytokines and hormone on immature Leydig cell nitrite production
IL-1ß induced nitrite production of Leydig cells for 24 h in a dose-dependent manner at concentrations ranging from 0.01–100 ng/ml (Fig. 1Go). On the other hand, other factors (TNF{alpha}, forskolin, and hCG) did not stimulate nitrite production. Figure 2Go shows the time course of nitrite production by Leydig cells cultured in control medium, medium containing 1 mM NG-monomethyl-L-arginine (l-NMMA), medium containing IL-1ß (1 ng/ml), or medium containing IL-1ß (1 ng/ml) and l-NMMA (1 mM). IL-1ß increased nitrite production of the cells in proportion to the culture period, whereas l-NMMA consistently inhibited nitrite production.



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  		Figure 1. Nitrite production by immature rat Leydig cells cultured in control medium and medium containing cytokines and hormone. Nitrite is produced by unstimulated Leydig cells and IL-1ß ({blacksquare}) increases nitrite production in a dose-dependent manner, but other factors [TNF{alpha} (), forskolin ({square}), and hCG ()] did not stimulate nitrite production.

 


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  		Figure 2. Time course of nitrite production by immature rat Leydig cells cultured in control medium ({diamond}), medium with IL-1ß (1 ng/ml; {square}), medium with l-NMMA (1 nM; {circ}), and medium with the both of them ({triangleup}). NO production increases in proportion to culture period, but l-NMMA consistently inhibits nitrite production in Leydig cells.

 
Effect of cytokines and hormone on adult Leydig cell nitrite production
IL-1ß stimulates nitrite production by adult Leydig cells in a dose-dependent manner at concentrations ranging from 0.01–100 ng/ml (Fig. 3Go).



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  		Figure 3. Effect of IL-1ß on nitrite production by adult rat Leydig cells. Although nitrite production is suppressed somewhat at a concentration of 0.01 ng/ml, it is increased in proportion to the concentration up to 100 ng/ml.

 
Effect of IL-1ß on iNOS mRNA expression in immature Leydig cells
To determine the expression of iNOS mRNA in Leydig cells, total RNA extracted from Leydig cells treated with or without IL-1ß (1 ng/ml) was analyzed by Northern blot hybridization. Figure 4Go shows the dose-dependent iNOS mRNA expression of the cells cultured for 12 h in control medium and in medium containing IL-1ß (0.01–100 ng/ml).



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  		Figure 4. Dose-dependent relationship of IL-1ß-stimulated iNOS mRNA expression. Total RNA (20 µg) isolated from cultured immature rat Leydig cells stimulated by IL-1ß for 12 h at a concentration of 0.01–100 ng/ml was analyzed by Northern blot hybridization using labeled rat iNOS and ß-actin cDNA. A, iNOS mRNA is induced by IL-1ß in a dose-dependent manner at concentrations up to 10 ng/ml. The positions of the 18S and 28S ribosomal RNA submits are indicated at the right. B, The relative level of iNOS mRNA was calculated by dividing by the total amount of RNA present (using actin images). The ratio of iNOS/ß-actin in samples treated with IL-1ß (1 ng/ml) is arbitrarily defined as 1.

 
Prominent transcripts of iNOS mRNA are recognized at about 5.0 kilobases in length. The basal level of iNOS mRNA was barely detectable, but IL-1ß stimulated iNOS mRNA expression in Leydig cells in a dose-dependent manner, and the expression reached its peak at a concentration of 10 ng/ml.

Figure 5Go depicts the time course of IL-1ß stimulation at a concentration of 1 ng/ml for 0, 3, 6, 12, and 24 h. The levels of iNOS mRNA were low in unstimulated Leydig cells in each culture period, but they began to increase as early as 3 h after the addition of IL-1ß and continued for up to 24 h.



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  		Figure 5. Time course of iNOS mRNA expression in immature rat Leydig cells stimulated by IL-1ß. A, Total RNA (20 µg) isolated from cultured immature rat Leydig cells stimulated by IL-1ß (1 ng/ml) was analyzed by Northern blot hybridization using labeled rat iNOS and ß-actin cDNA. The positions of the 18S and 28S ribosomal RNA submits are indicated at the right. B, The relative level of iNOS mRNA was calculated by dividing by the total amount of RNA present (using actin images). The ratio of iNOS/ß-actin in samples treated with IL-1ß (1 ng/ml) at 6 h is arbitrarily defined as 1. Although only a small amount of iNOS mRNA was expressed by the Leydig cells cultured in control medium ({circ}) in each culture period, iNOS mRNA was markedly induced in the cells treated with IL-1ß ({square}) as early as 6 h after the addition of IL-1ß and persisted for up to 24 h.

 
Effect of IL-1ß on iNOS mRNA expression in adult Leydig cells
Figure 6Go shows the time course of IL-1ß stimulation at a concentration of 1 ng/ml for 0, 6, and 12 h. The levels of iNOS mRNA were low in unstimulated Leydig cells in each culture period, but increased 6 and 12 h after the addition of IL-1ß to the same degree.



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  		Figure 6. Time course of iNOS mRNA expression in adult rat Leydig cells stimulated by IL-1ß (1 ng/ml). A, Total RNA (20 µg) isolated from cultured adult rat Leydig cells stimulated by IL-1ß (1 ng/ml) was analyzed by Northern blot hybridization using labeled rat iNOS and ß-actin cDNA. The positions of the 18S and 28S ribosomal RNA submits are indicated at the right. Although only a small amount of iNOS mRNA was expressed by unstimulated rat Leydig cells in each culture period, iNOS mRNA was markedly induced 6 and 12 h after the addition of IL-1ß. B, The relative level of iNOS mRNA was calculated by dividing by the total amount of RNA present (using actin images). The ratio of iNOS/ß-actin in samples treated with IL-1ß (1 ng/ml) at 6 h is arbitrarily defined as 1. Although only a small amount of iNOS mRNA was expressed by unstimulated rat Leydig cells in each culture period ({circ}), iNOS mRNA was markedly induced 6 and 12 h after the addition of IL-1ß ({square}).

 
Localization and detection of iNOS-like immunoreactivity by immunocytochemistry
iNOS-like immunoreactivity was examined in cultured immature and adult Leydig cells stimulated by IL-1ß. Using the ABC method (13), small granules were distributed in the cytoplasm of Leydig cells (Fig. 7Go).



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  		Figure 7. Immunocytochemical localization of iNOS protein in cultured rat Leydig cells. A, The specificity of staining with the ABC method in immature Leydig cells was confirmed using nonimmune rabbit serum (x400 ). B, Most of the immature Leydig cells exhibited iNOS-like immunoreactivity with use of the ABC method (x400). C, The specificity of staining with the ABC method in adult Leydig cells was confirmed using nonimmune rabbit serum (x400). D, Most of the adult Leydig cells exhibited iNOS-like immunoreactivity with use of the ABC method (x400).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In addition to the relaxing of vascular smooth muscle, the cytotoxic action of macrophages, and synaptic plasticity in the nervous system, it has been clearly shown that NO plays a number of roles in many biological systems (1, 2). Several studies have shown that the inhibitor of NOS increased steroidogenesis (5, 15), and another study reported that a NO-related agent suppressed testosterone secretion (6). These results suggest that NO acts as an inhibitor of steroidogenesis in the reproductive system. In the present study, we demonstrate for the first time that NO is produced by cultured immature and adult rat Leydig cells. NO was produced in low levels by unstimulated rat Leydig cells, but was markedly increased by IL-1ß. Northern blot analysis supports this result by detection of an increased level of iNOS mRNA expression, and immunological staining shows the existence of iNOS in the cytoplasm of Leydig cells.

It was formerly thought that the function of Leydig cells was modulated only by LH from the pituitary, but now it is clear that there are other regulating system(s) within the testis (16). Previous studies indicate that various cytokines are produced by testicular cells and are involved in the modulation of these functions of testicular cells (17, 18, 19, 20). Rat testis produces large amounts of IL-1-like factor (21), and potential sources of IL-1-like factor may be macrophages, Sertoli cells, peritubular cells, and Leydig cells (21). Especially, it is known that testicular macrophages take part in the modulation of the development and function of Leydig cells with cytokines such as IL-1 and TNF{alpha} (18, 19, 22, 23). As we demonstrate in this study, NO production by Leydig cells is stimulated by IL-1ß in dose-dependent manner. Many studies have reported the effect of IL-1 on Leydig cell steroidogenesis. IL-1ß or IL-1{alpha} inhibits LH/hCG-stimulated testosterone production in immature and adult rat Leydig cells (18, 24). On the other hand, basal steroidogenesis of immature Leydig cells is stimulated by IL-1ß (17, 25), but adult Leydig cells are not stimulated (17, 24). At present, the published data concerning the effect of IL-1 on Leydig cell steroidogenesis are conflicting (22). Provided that IL-1ß inhibits steroidogenesis in Leydig cells, our findings indicate that NO, which is produced by iNOS induced in the presence of IL-1ß, may be involved in some part of the regulation of Leydig cell steroidogenesis. Further studies will be required to explain the correlation between NO and the other cytokines in the modulation of Leydig cell steroidogenesis.


   Footnotes
 
Address requests for reprints to: Dr. Masato Fujisawa, M.D., Department of Urology, Kobe University School of Medicine, 7–5-1, Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

Received July 26, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  5. Förstermann U, Closs EI, Pollock JS, Nakane M, Schwarz P, Gath I, Kleinert H 1994 Nitric oxide control of steroidogenesis:endocrine effect of NG-nitro-L-arginine and comparisons to alcohol. Life Sci 50:PL35–PL40
  6. Adams ML, Meyer ER, Sewing BN, Ciero TJ 1994 Effect of nitric oxide-related agents on rat testicular function. J Pharmacol Exp Ther 269:230–237[Abstract/Free Full Text]
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  8. Stephan JP, Guillemois C, Jegou B, Bauche F 1995 Nitric oxide production by Sertoli cells in response to cytokines and lipopolysaccharide. Biochem Biophys Res Commun 213:218–224[CrossRef][Medline]
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  10. Klinefelter GR, Hall PF, Ewing LL 1987 Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36:769–783[Abstract]
  11. Lin T, Wang D, Nagpal M 1993 Human chorionic gonadotropin induces interleukin-1 gene expression in rat Leydig cells in vivo. Mol Cell Endocrinol 95:139–145[CrossRef][Medline]
  12. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR 1982 Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126:131–138[CrossRef][Medline]
  13. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by scid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  14. Hsu SM, Raine L, Fanger H 1981 Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 29:577–580[Abstract]
  15. van Voorhis BJ, Dunn MS, Snyder GD, Weiner CP 1994 Nitric oxide: an autocrine regulator of human granulosa-luteal cell steroidogenesis. Endocrinology 135:1799–1806[Abstract]
  16. Sharpe RM 1990 Intratesticular control of steroidogenesis. Clin Endocrinol (Oxf) 33:787–807[Medline]
  17. Verhoeven G, Cailleau J, van Damme J, Billiau A 1988 Interleukin-1 stimulate steroidogenesis in cultured rat Leydig cells. Mol Cell Endocrinol 57:51–60[CrossRef][Medline]
  18. Calkins JH, Sigel MM, Nankin HR, Lin T 1988 Interleukin-1 inhibits Leydig cell steroidogenesis in primary culture. Endocrinology 123:1605–1610[Abstract/Free Full Text]
  19. Calkins JH, Guo H, Sigel MM, Lin T 1990 Tumor necrosis factor-{alpha} enhances inhibitory effects of interleukin-1ß on Leydig cell steroidogenesis. Biochem Biophys Res Commun 166:1313–1318[CrossRef][Medline]
  20. Okuda Y, Sun X-R, Morris PL 1994 Interleukin-6 (IL-6) mRNAs expressed in Leydig and Sertoli cells are regulated by cytokines, gonadotropins and neuropeptides. Endocr J 2:617–624
  21. Khan SA, Söder O, Syed V, Gustafsson K, Lindh M, Ritzen EM 1987 The rat testis produces large amount of an interleukin-1-like factor. Int J Androl 10:495–503[Medline]
  22. Sun X, Hedger MP, Risbridger GP 1993 The effect of testicular macrophages and interleukin-1 on testosterone production by purified adult rat Leydig cells cultured under in vitro maintenance conditions. Endocrinology 132:186–192[Abstract/Free Full Text]
  23. Yee JB, Hutson JC 1985 Effect of testicular macrophage-conditioned medium on Leydig cells in culture. Endocrinology 116:2682–2684[Abstract/Free Full Text]
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