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Endocrinology Vol. 139, No. 5 2217-2222
Copyright © 1998 by The Endocrine Society

ARTICLES

Interferon-{gamma} Inhibits the Steroidogenic Acute Regulatory Protein Messenger Ribonucleic Acid Expression and Protein Levels in Primary Cultures of Rat Leydig Cells1

Tu Lin, Jianxin Hu, Deli Wang and Douglas M. Stocco

Medical and Research Services, W. J. B. Dorn Veterans Affairs Medical Center, and the Department of Medicine, University of South Carolina School of Medicine (T.L., J.H., D.W.), Columbia, South Carolina 29208; and Department of Cell Biology and Biochemistry, Texas Tech University School of Medicine Health Sciences Center (D.M.S.), Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Tu Lin, M.D., Department of Medicine, University of South Carolina School of Medicine, Medical Library Building, Suite 316, Columbia, South Carolina 29208.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} (IFN{gamma}) is an immunomodulating cytokine that has profound effects on reproductive function. IFN{gamma} inhibits steroidogenesis both in vivo and in vitro. The mechanism by which IFN{gamma} inhibits Leydig cell steroidogenesis remains unclear. In the present study, we evaluated the effect of IFN{gamma} on the expression and regulation of the steroidogenic acute regulatory protein (StAR) gene in primary cultures of rat Leydig cells. StAR facilitates the efficient production of steroid hormone by regulating the translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of the cytochrome P450 side-chain cleavage (P450scc) enzyme system that converts cholesterol to pregnenolone. IFN{gamma} inhibited hCG-induced StAR messenger RNA (mRNA) levels in a dose-dependent manner. The addition of IFN{gamma} in a concentration of 500 U/ml decreased hCG-induced 3.8- and 1.7-kilobase StAR mRNA by 78% and 70%, respectively. IFN{gamma} also reduced hCG-stimulated P450scc mRNA levels by 69%. The inhibitory effects of IFN{gamma} on StAR mRNA levels were confirmed by ribonuclease protection assay. As early as 12 h after the addition of IFN{gamma}, hCG-induced StAR mRNA levels decreased by more than 44%. To evaluate the effects of IFN{gamma} on StAR protein levels, Western blot analyses were performed. hCG in a concentration of 10 ng/ml increased StAR protein by 5.6-fold. Treatment of Leydig cells with IFN{gamma} (500 U/ml) decreased hCG-induced StAR protein by 44%. In contrast, interleukin-1 and murine tumor necrosis factor-{alpha} reduced hCG-induced P450scc mRNA expression without inhibiting StAR mRNA or protein levels. In conclusion, IFN{gamma} inhibits Leydig cell steroidogenesis by down-regulating StAR gene expression and protein production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTERFERONS (IFNs) are potent biological response modifiers. In addition to their antiviral activity, IFNs exert various biological effects, including inhibition of cell growth, activation of the immune system and induction of messenger RNA (mRNA) synthesis of various genes (1). Four major classes of IFNs have been identified. The leukocyte IFNs are designated IFN{alpha} and IFN{omega}, fibroblast IFN is designated IFNß, and immune IFN is designated IFN{gamma} (1). IFNs have also been found to have profound effects on reproductive function. Treatment of mice with IFN{gamma} results in altered germinal epithelium and decreases spermatogenesis (2, 3). Male mice that are transgenic for IFNs display alteration of the spermatogenic process and are sterile (4, 5). IFN{gamma} also inhibits gonadal steroidogenesis both in vivo and in vitro (6, 7, 8, 9). However, the mechanisms by which IFN{gamma} inhibits Leydig cell steroidogenesis remain unclear. Inhibitory effects of IFN{gamma} can either be partially reversed (in porcine Leydig cells) or completely reversed (in murine Leydig cells) by the addition of hydroxylated analogs of cholesterol, which can readily diffuse across cell and mitochondrial membranes and can be used to replace cholesterol as substrates for cytochrome P450 side-chain cleavage (P450scc) (8, 9). These findings suggest that IFN{gamma} probably inhibits the substrate availability for P450scc.

The acute response of steroidogenic cells to tropic hormone stimulation is characterized by an increase in the rate of transfer of cholesterol from the outer to the inner mitochondrial membrane, where resides the first enzyme in the biosynthetic pathway, P450scc (10). This process is mediated by a short-lived cycloheximide-sensitive protein (11, 12, 13). A number of candidate proteins have been proposed to facilitate the transport of cholesterol, including sterol carrier protein 2 (14), steroidogenesis-activator polypeptide (15), and the peripheral benzodiazepine receptor and its ligand, the diazepam-binding inhibitor (16). However, considerable evidence now suggests that the steroidogenic acute regulatory protein (StAR), a specific 30-kDa mitochondrial protein, mediates this critical function of cholesterol transport for steroidogenesis (17, 18). Transient transfection of both steroidogenic and nonsteroidogenic cells with StAR complementary DNA (cDNA) directly stimulated steroid production in the absence of trophic hormone stimulation (17, 19, 20). Furthermore, in patients with lipoid congenital adrenal hyperplasia, adrenal and gonadal steroidogenesis is impaired because of inefficient transport of cholesterol into the mitochondria. The defects responsible for this disease are mutations in the StAR gene that generate truncated and nonfunctional protein (21, 22, 23). To date, mutations of the StAR gene are the only known causes of this autosomal recessive disorder (21, 22, 23). As the inhibitory effects of IFN{gamma} on Leydig cell steroidogenesis could be reversed by the addition of hydroxylated cholesterol, suggesting that IFN{gamma} might affect the transport of cholesterol into the mitochondria, our present study evaluated the effects of IFN{gamma} on StAR mRNA levels and protein production by rat Leydig cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The StAR cDNA probes were prepared as reported previously (17). P450scc cDNA probe was provided by Dr. JoAnne Richards (Houston, TX). [{alpha}-32P]Deoxy-CTP (3,000 Ci/mmol) and [{alpha}-32P]CTP (3,000 Ci/mmol) were obtained from ICN Biochemical (Costa Mesa, CA). Murine IFN{gamma} and tumor necrosis factor-{alpha} (TNF{alpha}) were purchased from Genzyme Corp. (Cambridge, MA). Human recombinant interleukin-1ß (IL-1ß) was provided by Lilly Research Laboratories (Indianapolis, IN). Highly purified hCG (13,000 U/mg) was provided by Dr. Patricia Morris (The Population Council, Rockefeller University, New York, NY).

Isolation and culture of Leydig cells
Male Sprague-Dawley rats (55–60 days old) were obtained from Charles River (Raleigh, NC). Highly purified Leydig cells were isolated from rat testes using the combination of arterial perfusion, collagenase digestion, centrifugal elutriation, and Percoll gradient centrifugation as described previously with minor modifications (24, 25). By 3ß-hydroxysteroid dehydrogenase staining, over 97% of the cells were stained positive for Leydig cells (26). The protocol was approved by the local animal study subcommittee.

Purified Leydig cells were resuspended in DMEM-Ham’s F-12 with 0.5% BSA, 15 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin. Leydig cells (8–10 x 106 cells) were plated in 50-mm culture wells (Costar, Cambridge, MA) and incubated at 37 C in a humidified atmosphere of 95% air-5% CO2, and IFN{gamma} (10–500 U/ml), IL-1ß (10 ng/ml), or TNF{alpha} (10 ng/ml) was added. After 24 h in culture, the medium was removed and replaced with fresh medium. IFN{gamma}, IL-1ß, TNF{alpha}, and/or hCG (10 ng/ml) were added, and cultures were continued for an additional 4 h. Total cellular RNA was then extracted. To evaluate the effects of IFN{gamma} on the conversions of steroid precursors to testosterone, purified Leydig cells (1.5 x 105 cells/ml) were cultured with or without IFN{gamma} (500 U/ml) for 24 h. After medium change, cells were cultured with or without IFN{gamma} (500 U/ml), hCG (10 ng/ml), and 22R-hydroxycholesterol, pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone (DHEA), or androstenedione in concentrations of 1 µM. Cultures were continued for an additional 24 h. Culture media were then centrifuged, and the supernatants were saved at -20 C for testosterone assay. More than 95% of the cells remained viable, as determined by trypan blue exclusion.

RNA extraction and Northern blot analysis
Total cellular RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (27). Northern blot analyses were performed as previously reported (28). Hybridization was carried out with 1–5 x 107 cpm of the StAR, P450scc, or ß-actin cDNA probes labeled with [{alpha}-32P]deoxy-CTP (3000 Ci/mmol) using a Random Primers DNA Labeling System (Life Technologies, Grand Island, NY). The membranes were then exposed to Fuji RX x-ray film (Fuji, Tokyo, Japan) with an intensifying screen at -70 C. The autoradiograms were quantified by densitometric scanning using a Bio-Rad video densitometer (model 620, Bio-Rad, Richmond, CA). The expression of ß-actin mRNA, which was not affected by the treatment, was used as the internal control for each specimen (29). Results are expressed as arbitrary units of StAR or P450scc/actin messenger RNA (mRNA) ratios.

Ribonuclease (RNase) protection assay
A 180-bp mouse StAR cDNA fragment was PCR amplified using primers corresponding to the mouse cDNA sequence bases 542–562 and 702–722 (sense primer, 5'-AGAGGATTGGAAAAGACACGG-3'; antisense primer, 5'-GCTCTGATGACACCACTCTGC-3') (9) and cloned into a pCRII vector using the TA cloning kit (Invitrogen Corp., San Diego, CA). The insert was sequenced manually by the dideoxynucleotide chain termination method (Sequenase version 2.0 kit, U.S. Biochemical Corp., Cleveland, OH). The StAR antisense riboprobe (complementary RNA), approximately 288 bases in length, was transcribed using the Maxiscript in vitro transcription kit (Ambion, Austin, TX). Before transcription, the DNA template was linearized with BamHI. Transcription was carried out for 1 h at 37 C using 0.5 µg DNA template with [{alpha}- 32P]CTP (ICN) and T7 polymerase. Rat ß-actin RNA antisense riboprobe was synthesized using Ambion’s pTRI-ß-actin-125-rat template, T7 polymerase, and the Maxiscript in vitro transcription kit (Ambion).

RNase protection assays were performed using the HybSpeed RPA kit (Ambion). In brief, preliminary experiments were performed to determine saturating quantities of each riboprobe used in subsequent experiments. Total RNA (10 µg) from rat Leydig cells was resuspended in hybridization buffer containing saturating concentrations of StAR riboprobes and rat ß-actin riboprobes. The mixture was heated to 95 C for 5 min, and samples were hybridized for 1 h at 68 h. Samples were then treated with RNase A/T1 mixture for 1 h at 37 C, precipitated, resuspended in formamide-containing gel loading buffer, and run on 5% polyacrylamide-8 M urea gels. In each gel, five [{alpha}- 32P]CTP-labeled RNA transcripts synthesized from Century Marker Templates (Ambion) with lengths of 100, 200, 300, 400, and 500 bases were run simultaneously with samples in a separate lane as size standards. Gel were exposed overnight and for up to 3 days to Fuji RX film at -70 C with intensifying screens.

Isolation of mitochondria and Western blot analysis
Leydig cells were homogenized in TSE buffer [0.25 M sucrose, 10 mM Tris (pH 7.4), and 0.1 mM EDTA] on ice using 20 strokes in a Dounce homogenizer (Kontes Co., Vineland, NJ). Lysates were centrifuged at 600 x g for 15 min at 4 C. The supernatant was centrifuged at 10,000 x g for 15 min at 4 C. The resulting pellets were then resuspended in 1 ml ice-cold buffer. A 100-µl aliquot of this was used for protein quantitation by the Bio-Rad protein dye assay. The remaining mitochondrial suspension was pelleted by centrifugation at 10,000 x g for 15 min at 4 C and then lyophilized. Western blot analyses were performed as previously described, using a mouse polyclonal antisera to a 10-amino acid segment (amino acids 88–98) of the StAR protein (17). The specific signal was detected by chemiluminescence using the Renaissance kit from DuPont-New England Nuclear (Boston, MA). The integrated ODs of the bands were quantitated using the BioImage Visage 2000 computer-assisted image analysis system (BioImage, Ann Arbor, MI).

All experiments were repeated at least three times. One-way ANOVA followed by Newman-Keuls multiple comparison tests were used for statistical analyses (GraphPad Prism, version 2.01). P <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We evaluated the effects of IFN{gamma} on the hCG-induced StAR mRNA level (Fig. 1Go). Leydig cells were cultured with or without IFN{gamma} (10, 100, or 500 U/ml) for 24 h. After a medium change, cells were cultured with or without IFN{gamma} or hCG (10 ng/ml) for an additional 4 h. IFN{gamma} inhibited hCG-induced testosterone formation (hCG only, 175 ± 12 ng/ml; 500 U/ml IFN{gamma} plus hCG, 68 ± 5 ng/ml; P < 0.01). StAR mRNA is expressed in rat Leydig cells as two major transcripts, 3.8 and 1.7 kb. IFN{gamma} inhibited hCG-induced StAR mRNA expression in a dose-dependent manner (Fig. 1Go, A and B). IFN{gamma} at a concentration of 500 U/ml inhibited hCG-induced 3.8-kb StAR by 78% and 1.7-kb mRNA by 70%. hCG-induced P450scc mRNA levels were also reduced by IFN{gamma} by 69% (Fig. 1Go, A and B). In contrast, even though both IL-1 and TNF{alpha} are potent inhibitors of Leydig cell steroidogenesis (30, 31, 32, 33, 34, 35), we found that IL-1ß and TNF{alpha} did not decrease hCG-stimulated StAR mRNA levels (Fig. 2Go, A–C). In some of the experiments, StAR mRNA levels actually increased in the presence of TNF{alpha} or IL-1ß. Table 1Go shows the effects of IFN{gamma} on the conversions of steroid precursors to testosterone. IFN{gamma} (500 U/ml) inhibited hCG-stimulated testosterone formation and the conversion of 22R-hydroxycholesterol to testosterone, whereas the conversions of pregnenolone, 17-hydroxypregnenolone, DHEA, and androstenedione to testosterone were not affected.



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  		Figure 1. Effects of IFN{gamma} on hCG-induced StAR and P450scc mRNA levels. Purified Leydig cells were cultured with or without IFN{gamma} (10–500 U/ml) for 24 h. After a medium change, cells were treated with IFN{gamma} and hCG (10 ng/ml) for an additional 4 h. Total RNAs were then extracted for Northern blot analyses. A total of 20 µg RNA were loaded in each lane. A, A representative Northern blot. B, Results are the mean ± SE for StAR or P450scc mRNA/actin ratios of three separate experiments. *, P < 0.05; **, P < 0.01 (compared with respective controls).

 


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  		Figure 2. Effects of IL-1ß and TNF{alpha} on hCG-induced StAR and P450scc mRNA levels. Purified Leydig cells were cultured with or without IL-1ß (10 ng/ml) or TNF{alpha} (10 ng/ml) for 24 h. After a medium change, cells were treated with or without IL-1ß, TNF{alpha}, and hCG (10 ng/ml) for an additional 4 h. Total RNAs were then extracted for Northern blot analyses. This is a representative Northern blot (A and B). Results are the mean ± SE mRNA/actin ratios of three separate experiments (C). *, P < 0.05 compared with cells treated with hCG only.

 

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  		Table 1. Effects of IFN{gamma} on the conversions of steroid precursors to testosterone

 
The effects of IFN{gamma} on StAR mRNA expression were confirmed by RNase protection assay assay. Hybridization with 32P-labeled StAR complementary RNA followed by RNase digestion and PAGE resulted in protected StAR and ß-actin RNA bands corresponding to approximately 180 and 125 bp, respectively. As early as 12 h after the addition of IFN{gamma}, hCG-induced StAR mRNA levels decreased by more than 44% (Fig. 3Go, A and B). Twenty-four-hour treatment with IFN{gamma} reduced hCG-induced StAR mRNA by 56% (Fig. 3Go, A and B).



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  		Figure 3. RNase protection assay. A, A representative autoradiogram showing the effects IFN{gamma} on StAR mRNA expression. Rat Leydig cells were treated with or without IFN{gamma} (500 U/ml) for 12 or 24 h. hCG (10 ng/ml) was then added, and cultures were continued for an additional 4 h before RNA extraction. Ten-microgram aliquots of total RNA were subjected to RNase protection assay as described in Materials and Methods. Lanes 1 and 2 show the mouse StAR riboprobe and the rat ß-actin riboprobe, which were carried through the assay in the presence of only nontarget yeast RNA and were subsequently incubated without and with RNase, respectively. Probes in lane 1 were diluted before loading. B, StAR/actin mRNA ratios in arbitrary units. Comparable results were obtained in two other separate experiments.

 
To evaluate whether the IFN{gamma}-induced decrease in StAR mRNA is associated with altered StAR protein levels, we carried out Western blot analyses. Leydig cells were treated with or without IFN{gamma} (500 U/ml) for 24 h. After a medium change, cells were treated with IFN{gamma} and hCG (10 ng/ml) for an additional 4 h. StAR protein levels were measured in isolated mitochondria. The addition of hCG (10 ng/ml) increased StAR protein levels by 5.6-fold. IFN{gamma} decreased hCG-induced StAR protein by 44% (Fig. 4Go). Neither IL-1ß nor TNF{alpha} had any effect on hCG-induced StAR protein levels (data not shown).



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  		Figure 4. Western immunoblot of StAR protein. Mitochondrial protein (90 µg) was analyzed in each sample. Mitochondria were isolated from Leydig cells as described in Materials and Methods. Lane 1, Control; lane 2, hCG (10 ng/ml); lane 3, hCG (10 ng/ml) plus IFN{gamma} (500 U/ml). Similar results were observed in three other separate experiments. This is a representative blot. However, because of the nature of the Western immunoblot analysis using chemiluminescence, it is not possible to perform statistical analysis on samples analyzed at different times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that IFN{gamma} decreases hCG-induced StAR mRNA in a dose-dependent manner. Decreased StAR mRNA levels are associated with a decrease in StAR protein levels. These results indicate that IFN{gamma} inhibits Leydig cell steroidogenesis by down-regulating StAR gene expression.

IFNs have profound effects on male reproductive function. IFNs can be produced in the testis and have autocrine and paracrine effects. IFN{alpha} protein and corresponding mRNA are expressed by peritubular, Sertoli, and germ cells (36). Both IFN{gamma} mRNA and protein are found in early spermatids (36). Transgenic mice carrying extra mouse IFN genes under the control of a mouse metallothionein I promoter offer an excellent model to investigate the biological functions of IFNs (4, 5). High levels of the metallothionein-IFN fusion gene were expressed constitutively in the testis without heavy metal induction. In some of the animals, biologically active IFNs could also be detected in testicular homogenates. These mice were sterile. Histological examination of the testicular tissue showed degeneration of spermatogenic cells, leading to calcium deposits and complete atrophy of the seminiferous tubules (4, 5).

Intratesticular injection of mice with human recombinant IFN{gamma} in a dose of 20 µg/testis caused desquamation of the germinal epithelium and reduction of germinal cell height and tubular diameter (2). Quantitative studies showed a significant decrease in the number of Sertoli cells, stage 7 spermatids, and stage 16 spermatozoa. Body weight and the weights of the testis and epididymis were not affected (2). Male mice receiving 2 mg/kg·day recombinant murine IFN{gamma} on postnatal days 8–60 showed delayed sexual maturation, reduced epididymal and testicular weights, reduced sperm count and concentration, and sperm abnormalities (3). Mating performance and fertility were also reduced (3). Intraperitoneal injection of IFN{alpha}-2b reduced oligosaccharide, ß-galactoside {alpha}-2,6-sialyltransferase, gene expression in rat testis (37). The oligosaccharide moieties of sperm surface glycoproteins are important for the production of functionally mature spermatozoa (38, 39).

IFN{gamma} inhibits gonadal steroidogenesis both in vivo and in vitro (6, 7, 8, 9). Administration of IFN{gamma} decreases the serum testosterone concentration (6). Pretreatment of porcine Leydig cells with IFN{gamma} for 24 h decreased hCG-stimulated testosterone formation by 77% (8). Hydroxylated cholesterol derivatives, 22R-hydroxycholesterol and 20-hydroxycholesterol, were able to partially reverse the inhibitory effect of IFN{gamma}. Incubation with IFN{gamma} also decreases basal P450scc and P450c17 mRNA levels by 45% and 35%, respectively (8). In isolated murine Leydig cells, IFN{gamma} inhibited hCG-stimulated testosterone formation (9). However, the inhibitory effect of IFN{gamma} could be completely reversed by the addition of 22R-hydroxycholesterol, 17{alpha}-hydroxyprogesterone, or DHEA (9). This suggests that in murine Leydig cells, IFN{gamma} affects events in cholesteryl ester hydrolysis or cholesterol transport before the side-chain cleavage of cholesterol (9). Our present study demonstrates that in rat Leydig cells, IFN{gamma} inhibits hCG-induced testosterone formation and that these inhibitory effects persist in the presence of 22R-hydroxycholesterol. The conversions of pregnenolone, 17-hydroxypregnenolone, DHEA, and androstenedione to testosterone were not affected by IFN{gamma}. Furthermore, IFN{gamma} decreased hCG-induced StAR protein levels. This suggests that in rat Leydig cells, the major inhibitory effects of IFN{gamma} are at the steps of StAR and P450scc.

Lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, is a potent activator of the immune system that induces local inflammation, antibody production, and, in severe infection, septic shock (40). LPS has been used extensively to study the effects of experimental endotoxinemia. When adult male mice were treated with LPS ip, there was a greater than 90% decrease in serum testosterone level within 2 h. However, there was no inhibition of StAR mRNA expression 2 or 24 h after LPS injection (41). P450scc and P450c17 mRNA levels decreased at 24 h, but not at 2 h. An acute reduction of serum testosterone levels was associated with decreased StAR protein, and this inhibition appeared to occur at the level of translation (41). As endotoxins can induce the production and release of various cytokines, including ILs, TNF{alpha}, and IFN{gamma} (40), inhibitory effects of LPS on testosterone production are most likely mediated by the combination of these cytokines. Indeed, TNF{alpha} and IL-1 have been shown to inhibit testosterone formation by repression of P450scc and P450c17 mRNA and protein levels in cultured murine Leydig cells (32, 34). The major inhibitory effects of TNF{alpha} and IL-1 were on the reduction of P450c17 mRNA and protein levels (32, 34). However, there appears to be species differences in the responses to various cytokines. In immature porcine Leydig cells, TNF{alpha} inhibited hCG-induced testosterone formation, and the inhibitory effect could be reversed by 22R-hydroxycholesterol (33). This suggests that the major inhibitory effect of TNF{alpha} in porcine Leydig cells is the decrease in the availability of cholesterol substrate in the mitochondria (33). We reported previously that TNF{alpha} inhibits rat Leydig cell steroidogenesis by inhibiting P450scc mRNA levels (35). The inhibitory effect could not be reversed by the addition of 20-hydroxycholesterol, whereas conversions of pregnenolone, 17-hydroxypregnenolone, DHEA, and androstenedione to testosterone were not affected (35). These data suggest that the major inhibitory effect of TNF{alpha} in rat Leydig cells is at the step of P450scc. IL-1ß also inhibited hCG-stimulated testosterone production and P450scc mRNA levels in a dose-dependent manner in purified rat Leydig cells in culture (28). In the present study, we found that even though IFN{gamma}, IL-1ß, and TNF{alpha} inhibited Leydig cell function, only IFN{gamma} decreased hCG-induced StAR gene expression and protein production, whereas IL-1ß and TNF{alpha} had no effect.

IFN{gamma} exerts its biological function by binding to its cell surface receptors (1). The IFN{gamma}-receptor complex has two subunits: a ligand-binding {alpha}-chain (IFN{gamma}R{alpha}), which cannot generate signal transduction, and a ß-chain (IFN{gamma}Rß), which is required for signaling (1). Binding of IFN{gamma} leads to activation of JAK1 (Janus kinase 1) and JAK2, which are associated with the intracellular domain of the IFN{gamma}R{alpha} and IFN{gamma}Rß chains, respectively. Activation of JAK1 and JAK2 causes activation of STAT-1 by phosphorylation of tyrosine. STAT-1 dimerizes through reciprocal interactions of phosphotyrosine and an Src homology 2 domain and enters the nucleus to regulate transcription of many different genes containing {gamma}-activated sequence elements (1, 42). As both IFN{gamma}R{alpha} and IFN{gamma}Rß mRNAs have been identified in the rat testis (43), and IFN{gamma} has been shown to regulate immediate early genes in Sertoli cells by phosphorylation of STAT-1 protein (44, 45), it is possible that inhibitory effects of IFN{gamma} on StAR gene expression may be mediated by similar JAK/STAT pathways.

Male hypogonadism and decreased androgen levels are frequently associated with acute and chronic inflammation (46). High local concentrations of androgen are required in the development of functional spermatozoa and accessory duct function (47, 48). It is likely that impaired spermatogenesis and fertility in these inflammatory conditions are the results of an activated immune system with subsequent impaired reproductive function (49). In the present study we have provided evidence that IFN{gamma}, an immune IFN, inhibits Leydig cell steroidogenesis by down-regulating StAR gene expression and protein production. This may be one mechanism by which testosterone production is impaired in inflammatory disorders. Furthermore, IFN{gamma} has been localized in the testis; thus, IFNs may also play an important physiological role in the regulation of testicular steroidogenesis and spermatogenesis.


   Footnotes
 
1 This work was supported by the Department of Veterans Affairs Medical Research Fund (to T.L.) and NIH Grant HD-17481 (to D.M.S.). Back

Received October 24, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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