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Endocrinology Vol. 143, No. 2 621-626
Copyright © 2002 by The Endocrine Society

REPRODUCTION-DEVELOPMENT

Expression of 11ß-Hydroxylase in Rat Leydig Cells

Gui-Min Wang, Ren-Shan Ge, Syed A. Latif, David J. Morris and Matthew P. Hardy

Center for Biomedical Research, The Population Council, and Rockefeller University (G.M.W., R.S.G., M.P.H.), New York, New York 10021; and Department of Pathology and Laboratory Medicine, The Miriam Hospital, Brown University School of Medicine (S.A.L., D.J.M.), Providence, Rhode Island 02906

Address all correspondence and requests for reprints to: Dr. Matthew P. Hardy, The Population Council, 1230 York Avenue, New York, New York 10021. E-mail: hardy{at}popcbr.rockefeller.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
11ß-Hydroxy (11ß-OH) derivatives of certain steroids function as inhibitors of 11ß-hydroxysteroid dehydrogenase isoform 1 (11ßHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11ß-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11ß-hydroxylation of steroids and thus producing 11ß-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11ß-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11ß-hydroxylated steroids inhibit Leydig cell 11ßHSD1 activities. 11ß-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11ß-hydroxylase protein in Leydig cells. Moreover, 11ß-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the Km and maximum velocity values were 7.28 ± 0. 92 µM and 1.13 ± 0.04 µmol/106 cell·h, respectively. When assayed in Leydig cells, several 11ß-hydroxylated steroids were efficient inhibitors of 11ßHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11ß-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11ßHSD1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS INHIBIT testosterone (T) biosynthesis in Leydig cells through a GR-mediated mechanism (1). Ligand-activated GR represses basal and cAMP-induced gene expression of T biosynthetic enzymes, leading to reduced T production (2, 3). In Cushing’s disease or during stress, exposure to excess glucocorticoid is associated with reduced circulating T levels and reproductive dysfunction (4, 5, 6, 7). The glucocorticoid-metabolizing enzyme 11ß-hydroxysteroid dehydrogenase isoform 1 (11ßHSD1) catalyzes a reversible interconversion of active 11ß-OH-containing glucocorticoids [cortisol in humans and corticosterone (CORT) in rodents] and inert 11-keto forms (cortisone and 11-dehydro-CORT), and thus alters the Leydig cell’s sensitivity to glucocorticoid inhibition of T biosynthesis (8, 9, 10, 11).

Two isoforms of 11ßHSD (no. 1 and 2) have been characterized. 11ßHSD1 is a bidirectional enzyme, has a wide tissue distribution, and functions as either a dehydrogenase (CORT->11-dehydro-CORT) or an oxidoreductase (11-dehydro-CORT->CORT) in several tissues. In contrast, 11ßHSD2 functions as a unidirectional dehydrogenase that inactivates glucocorticoids (12). Adult rat Leydig cells express high levels of 11ßHSD1 oxidative activity (8, 13). However, in other glucocorticoid target tissues (e.g. liver and lung), 11ßHSD1 acts predominantly as a reductase and functions principally to locally regenerate active glucocorticoids from inactive 11-dehydro glucocorticoids (14). Whether 11ßHSD1 is the only isoform present in Leydig cells is not yet certain, and it may be that 11ßHSD1 behaves differently in different glucocorticoid target tissues (15, 16, 17). The physiological role played by 11ßHSD1 in Leydig cells is not fully understood. However, the bidirectional nature of 11ßHSD1 may well serve to control the magnitude of glucocorticoid action in this cell type.

The licorice derivative, glycyrrhetinic acid (GA), has been the most widely studied of the exogenous 11ßHSD inhibitors. Early studies by Monder and co-workers (8, 9) showed that inhibition of 11ßHSD1 oxidative activity in Leydig cells by GA increases the potency of CORT in suppression of T output. Recently, the 11ß-hydroxylated steroid derivative, 11ß-OH-progesterone (11ß-OH-Prog), has been shown to be as potent as GA in the inhibition of 11ßHSD oxidative activity (18, 19). This discovery raises the possibility that locally produced 11ßHSD inhibitors, if they are synthesized in the testis, may affect the resultant Leydig cell 11ßHSD1 activity.

The early steps of steroidogenesis in adrenal and testis are common and consist of the sequential conversion of cholesterol to pregnenolone by cytochrome P450 side-chain cleavage and of prenenolone to progesterone by 3ßHSD. Subsequent to these steps, 21-hydroxylase catalyzes the conversion of Prog to DOC in the rat adrenal cortex, where mitochondrial 11ß-hydroxylase, specifically localized in the zona fasciculata, converts DOC to CORT.

In the present study we investigated whether 11ß-hydoxylase is present in Leydig cells and whether 11ßHSD1 dehydrogenase (or possibly even oxidoreductase) inhibitors can be synthesized in the testis. We report the presence of 11ß-hydroxylase mRNA, protein, and enzyme activity in rat Leydig cells. To further assess the physiological role of 11ß-hydroxylase, the inhibitory potencies of a variety of 11ß-OH- and 11-keto-steroids on 11ßHSD1 activities (both dehydrogenase and oxidoreductase) in Leydig cells were measured, and several were found to be significant at nanomolar concentrations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats, weighing 200–250 g, were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All animal procedures were approved by the institutional animal care and use committee of Rockefeller University (Protocol 91200-R2).

RT-PCR analysis of 11ß-hydroxylase (CYP11B1) mRNA
Purified Leydig cells were isolated from adult rat testes as described previously (20). Cell preparations were judged to be more than 95% pure by histochemical staining for the Leydig cell-specific marker 3ßHSD (21). The adrenal gland and uterus were used as positive and negative controls, respectively. Total RNA was extracted by a single step procedure using phenol/guanidinium isothiocyanate (Molecular Research Center, Inc., Cincinnati, OH). RNA samples were retained for analysis if the OD260/280 ratio was above 1.8. First strand cDNA was transcribed using AMV reverse transcriptase (Promega Corp., Madison, WI).

PCR using the specific primer pair, 5'-GCTGGAGAATGTTCATGG-3' and 5'-CTCTGCCAGTTCGCGATA-3', was prepared with sequences as previously published (22) and resulted in a 312-bp fragment corresponding to nucleotides 528–840 of rat adrenal CYP11B1 cDNA. Amplification of the PCR products was performed under identical conditions, with a total of 30 cycles by denaturation at 94 C for 30 sec, primer annealing at 60 C for 1 min, and extension at 72 C for 1 min. PCR products were analyzed by electrophoresis on 1.5% agarose gels and submitted to Rockefeller University Protein DNA Technology Center for automated sequence analysis.

Localization of 11ß-hydroxylase by immunohistochemistry
After being perfused with Bouin’s fixative solution, the testes and adrenal glands were postfixed overnight in the same fixative solution, then embedded in paraffin. Antigen retrieval was carried out by microwave treatment for 10 min in 10 mM (pH 6.0) citrate buffer before immunostaining. Endogenous peroxidase activity was quenched by incubation with 0.3% H2O2 in absolute methanol for 20 min. Specific mouse antirat 11ß-hydroxylase monoclonal antibody (Chemicon International, Inc., Temecula, CA) was used at a 1:300 dilution overnight at 4 C. Antibody-antigen complexes were detected using a Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). The specificity of the antibody was assessed by positive staining in the zona fasciculata of the adrenal cortex (23) and lack of staining when the primary antibody was substituted by nonimmune mouse IgG. Localization in Leydig cells was also confirmed after their elimination from the testis using ethane dimethane sulfonate (EDS) (24).

Western blot assay with 11ß-hydroxylase antibody
Purified Leydig cells and adrenal cortex were homogenized in 3 vol ice-cold lysis buffer (1 x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonylfluoride, and 30 µl/ml aprotinin) and then centrifuged at 15,000 x g for 15 min at 4 C. Supernatants were collected and centrifuged twice. Protein concentrations were measured by the Bradford assay using a kit (Bio-Rad Laboratories, Inc., Hercules, CA). Aliquots of the detergent extracts (30 µg adrenal cortex and 100 µg Leydig cells) were subjected to 10% SDS-PAGE. Proteins were transferred electrophoretically onto nitrocellulose membranes (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). The blots were probed with the same antibody that was used for immunohistochemistry. The resulting bands were visualized by chemiluminescence (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Assay of 11ß-hydroxylase activity in Leydig cells
11ß-Hydroxylase activity was evaluated by measuring the rate of conversion of DOC to CORT. The reaction mixture was prepared in phenol red-free Leydig cell medium that contained [3H]DOC at final concentrations ranging from 1–150 µM. Triplicate samples were preincubated for 10 min at 34 C, 0.2 x 106 cells were added, and incubation was continued for 2 h. Reactions were terminated by adding ice-cold ether to the mixture. Steroids were then extracted twice with ether, the organic layer was dried under nitrogen, and the residues were analyzed by HPLC. Elution was monitored simultaneously at 240 nm with a variable wavelength detector and a flow scintillation analyzer (Packard 500 TR series, Downers Grove, IL). The stationary phase was an ODS column (Chromegabond MC18, 5 µm, 50 A, 25 cm, 4.6 cm). The mobile phase consisted of tetrahydrofuran/methanol/water (120:100:180), and the flow rate was 1 ml/min. Under these conditions, DOC and its possible metabolites, CORT, 11-dehydro-CORT, and aldosterone, eluted at 6.5, 5.9, 5, and 4 min, respectively.

Formation of CORT by purified intact Leydig cells was evaluated further using gas chromatography/mass spectrometry (GC/MS) by Dr. Cedric H. Shackleton (Children’s Hospital-Oakland Research Institute, Oakland, CA). The incubation was conducted as described above, but with 5 µM DOC in 50 ml culture medium containing 25 x 106 cells for 4 h. Methyloxime-trimethylsilyl ethers were made of the extracts before GC/MS analysis, which was carried out on a Hewlett-Packard Co. 5971 MSD instrument housing a nonpolar capillary column. The extracts were introduced by splitless injection, and the oven temperature was programmed to rise from 220 to 320 C during the run. Scanning was conducted over a 100–650 amu mass range.

Determination of kinetic constants
Kinetic analysis was performed by fitting initial velocity data as a function of substrate concentration to the Michaelis-Menten equation with EnzFitter, a nonlinear curve-fitting program (Biosoft, Ferguson, MO).

Assay of 11ßHSD enzyme kinetics and directionality in Leydig cells
Experiments were conducted with either intact cells or homogenates prepared from purified rat Leydig cells. In intact Leydig cells, the inhibitory activities of 11ß-OH-Prog and 11ß-OH-T as well as their 11-keto derivatives were assessed by incubating intact Leydig cells (containing 56,000 or 100,000 cells) in cell culture medium with 25 nM [3H]CORT (80 Ci/mmol; NEN Life Science Products, Boston, MS) or 31 nM [3H]11-dehydro-CORT (80 Ci/mmol; synthesized from [3H]CORT according to previously reported methods) (25, 26) as substrate at 34 C for 10 min.

Purified rat Leydig cells were homogenized in 25 mM HEPES buffer (pH 7.4) and rehomogenized after addition glycerol. 11ßHSD1 activities were assayed by incubating homogenates (13,000 cells equivalent) with 100 nM [3H]CORT in the presence of 3 mM NADP or homogenates (52,000 cells equivalent) with 600 nM [3H]11-dehydro-CORT in the presence of 3 mM NADPH at 37 C for 30 min. The reactions were stopped by the addition of methanol and centrifuged, and the steroid present in the supernatant was separated by HPLC using a DuPont Zorbax C8 column. The separated radioactive products (CORT and 11-dehydro-CORT) were detected and quantitated by flow scintillation analysis. The assay was conducted in the presence of varying concentrations (0.1–100 µM) of 11ß-OH-Prog, 11keto-Prog, 11ß-OH-T, 11-keto-T, 11ß-OH-AD, 11-keto-AD, 11ß-OH-dehydroepiandrosterone (11ß-OH-DHEA), T, or 3{alpha},5{alpha}-metabolite of T (3{alpha},5{alpha}-OH-THT; Steraloids, Newport, RI) for assessing IC50 values against 11ßHSD1 activity (both dehydrogenase and oxidoreductase).

Data analysis
Each experiment was repeated at least three times using three independently extracted RNA, protein, and tissue samples from different animals. The IC50 value was used for analysis of inhibitory activity of steroids on enzyme activity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of CYP11B1 mRNA in purified rat Leydig cells
Gel electrophoresis (Fig. 1AGo) revealed a 312-bp PCR fragment for CYP11B1 in Leydig cells and the adrenal gland, but not in uterus. The bands corresponding to CYP11B1 in Leydig cells were fainter than those in the adrenal gland. The identities of the PCR products were confirmed by sequencing. There was 100% concordance between the observed and predicted bases at the diagnostic CYP11B1 nucleotides (27). These results confirmed that CYP11B1 gene transcripts are present in Leydig cells.



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  		Figure 1. A, RT-PCR analysis of CYP11B1 gene transcripts in rat Leydig cells. Ethidium bromide-stained PCR products were detected after gel electrophoresis. A 312-bp segment for the CYP11B1 gene in Leydig cells and adrenal gland (arrow). There is no band corresponding to CYP11B1 in uterus, a negative control. B, Immunoblot of 11ß-hydroxylase protein in purified Leydig cells. Immunoblot analysis of Leydig cell protein homogenates revealed a 51.5-kDa band corresponding in size to rat 11ß-hydroxylase (arrow) in lysates of adrenal cortex. The band intensity for Leydig cells was weaker than that for the positive control, adrenal cortex.

 
Localization of 11ß-hydroxylase in rat testis
11ß-Hydroxylase protein localization in the testis was studied by immunohistochemistry, which provided evidence that 11ß-hydroxylase immunoreactivity was selectively localized in Leydig cells (Fig. 2CGo), because the testicular 11ß-hydroxylase immunoreactivity disappeared in animals treated with EDS, a drug that selectively destroys Leydig cells (Fig. 2DGo). The method was validated by testing the specificity of the antibody in the adrenal cortex, which showed the expected distribution of 11ß-hydroxylase immunoreactivity restricted to the zona fasciculata (Fig. 2AGo). Immunostaining was not seen in negative control sections in which the primary antibody was replaced by nonimmune mouse IgG (Fig. 2BGo). Furthermore, after SDS-PAGE and immunoblotting, the anti-11ß-hydroxylase antibody identified a protein band with a relative molecular mass of 51.5 kDa in both Leydig cells and adrenal cortex (Fig. 1BGo), which was consistent with the published molecular mass of rat adrenal 11ß-hydroxylase (28). The intensity of 11ß-hydroxylase immunoreactivity in Leydig cells was lower than that in adrenal cortex.



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  		Figure 2. Localization of 11ß-hydroxylase in rat testis and adrenal cortex. A, Positive staining for 11ß-hydroxylase in the zona fasciculata (ZF) of the adrenal cortex and absence of staining in zona glomerulosa (ZG). B, Absence of staining in a control adrenal section incubated with nonimmune mouse IgG (magnification, x200). C, Positive staining for 11ß-hydroxylase (arrow) in Leydig cells. D, Absence of staining for 11ß-hydroxylase (arrow) in EDS-treated testes (magnification, x200). Scale bar (bottom right panel), 50 µm.

 
Biochemical characteristics of 11ß-hydroxylase in Leydig cells
Purified Leydig cells were incubated with tritium-labeled DOC for timed intervals in the presence of increasing concentrations of substrate. A Michaelis-Menten relationship between the concentration of substrate and the rate of product formation was observed, with the Km for CORT synthesis being 7.28 ± 0.92 µM, and the maximum velocity equal to 1.13 ± 0.04 µmol/106 cells·h (Fig. 3Go).



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  		Figure 3. 11ß-Hydroxylase activity in intact Leydig cells. Rat Leydig cells converted DOC into CORT during incubations in vitro. A, Km and maximum velocity values of 11ß-hydroxylase in Leydig cells. B, Time curve of 11ß-hydroxylase in Leydig cells.

 
Synthesis of CORT from DOC was also confirmed by GC/MS. The molecular ion of CORT is at m/z 548, and loss of the oxime is seen at m/z 517. A peak in the analyzed incubate possessed a mass spectrum and retention time identical to that of the CORT standard. These results showed that there was a sufficient amount of 11ß-hydroxylase in Leydig cells to biosynthesize 11ß-OH compounds.

Effects of 11ß-hydroxylated steroids on 11ßHSD1 activity in Leydig cells
In cell homogenates, the 11ß-OH compounds inhibited 11ßHSD1 dehydrogenase reaction with a hierarchy of inhibitory activities in order of decreasing potency: 11ß-OH-Prog > 11ß-OH-T > 11ß-OH-AD, with corresponding IC50 values of 0.4, 1.7, and 70.0 µM, respectively. In contrast, their corresponding 11-keto derivatives were inactive as inhibitors of dehydrogenase activity with IC50 values of greater than 100 µM and suppressed the oxidoreductase reaction of 11ßHSD1 with IC50 values of 9.5, 18, and 21 µM, respectively (Table 1Go). In contrast, the 11ß-OH compounds were relatively inactive with respect to 11ßHSD1 oxidoreductase activities, with IC50 values exceeding 100 µM.


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  		Table 1. IC50 (micromolar concentration) values for androgen and progesterone metabolites assayed against 11ß-HSD1 present in rat Leydig cell homogenates

 
Of the other hydroxylated compounds tested, 11ß-OH-DHEA (a {Delta}5–6 compound) inhibited 11ßHSD1 dehydrogenase activity with an IC50 value of 3.3 µM and oxidoreductase activity with an IC50 value greater than 100 µM. T, which possesses no 11-OH function, inhibited both forward and backward enzymatic reactions of 11ßHSD, with IC50 values of 20 and 33 µM, respectively. 3{alpha},5{alpha}-THT was more potent than the parent compound, T, with IC50 values of 6.1 and 20.0 µM for the dehydrogenase and oxidoreductase activities, respectively (Table 1Go). This implies that 5{alpha} ring reduction of T increased its inhibitory potency relative to that of the parent steroid hormone, T.

When incubated with the whole cell preparations, in which much lower concentrations of substrates were used, 11ß-OH-T was a more potent inhibitor of 11ßHSD1 for the oxidative reaction compared with 11ß-OH-Prog, with IC50 values of 30 vs. 65 nM. However, the 11-keto derivatives were equally potent inhibitors of oxidoreductase activity in whole cell preparations, with IC50 values of 1.4 and 1.5 µM, respectively (Table 2Go). The inhibitory activity in whole cells was more potent compared with results obtained in the homogenate preparations.


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  		Table 2. IC50 (micromolar concentration) values for androgen and progesterone metabolites assayed against 11ßHSD1 present in whole cell preparations of rat Leydig cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The predominant catalytic activity of 11ßHSD1 locally controls the magnitude of glucocorticoid action in testis. By acting as a dehydrogenase it may serve to protect Leydig cells from the effects of excessive glucocorticoids. In contrast, by acting as an oxidoreductase, 11ßHSD1 could enhance glucocorticoid action by reactivation of 11-dehydroglucocorticoid back to active glucocorticoid. The biochemical determinants of the direction of the 11ßHSD1 reaction are not fully understood. The overall 11ßHSD1 activity in Leydig cells is most likely modulated by a variety of biochemical mechanisms, including different levels of enzyme expression, cofactor concentration, and redox state. The licorice derivative, GA, is a potent inhibitor of both 11ßHSD1 and 11ßHSD2 dehydrogenase as well as a moderate inhibitor of 11ßHSD1 oxidoreductase (29, 30). An increasing body of evidence indicates that some endogenously synthesized 11ß-hydroxylated derivatives of steroids, such as 11ß-OH-Prog, originally described as an inhibitor of placental 11ßHSD are potent inhibitors of both 11ßHSD1 and 11ßHSD2 dehydrogenase (19, 31).

Earlier experiments have shown that 11ß-OH-Prog inhibited 11ßHSD1 dehydrogenase, and 11-keto-Prog inhibited 11ßHSD1 oxidoreductase in vascular smooth muscle cells (32, 33). Rat testicular Leydig cell preparations display both dehydrogenase and oxidoreductase enzymatic activities of this enzyme, and to date, only 11ßHSD1 mRNA and protein have been shown to be present (15, 16, 17). In this study we considered the possibility that endogenous 11ßHSD inhibitors are synthesized in the testis and investigated whether 11ß-oxygenated products derived from progesterone, T, DHEA, or androstenedione inhibit either rat Leydig cell 11ßHSD1 dehydrogenase or oxidoreductase activity.

We have now demonstrated that 11ß-hydroxylase gene transcripts and immunoreactivity are selectively localized in rat Leydig cells by RT-PCR and immunohistochemistry, which was further confirmed by sequencing and the Western blot results. Extraadrenal expression of 11ß-hydroxylase in benign Leydig cell tumors and in eel testes has been reported previously (34, 35). This enzyme is also found in blood vessels, heart, and brain (36, 37, 38, 39). Taken together, the selective localization of 11ß-hydroxylase in Leydig cells suggests that the enzymatic machinery necessary for local production of 11ß-oxygenated inhibitory substances from steroids is present in the testis.

11ß-Hydroxylase gene transcription and protein expression do not provide unequivocal evidence that a functional protein is present. However, the results of the HPLC analysis demonstrate 11ß-hydroxylase catalytic activity in Leydig cells, and CORT formation was further confirmed by GC/MS analysis. These results further support the hypothesis that Leydig cells have the potential to synthesize other 11ß-OH-steroid intermediates via local 11ß-hydroxylation. The presence of 11ß-OH-Prog and 11-keto-Prog in venous drainage from the adrenal gland and in humans under the clinical condition of 17-hydroxylase deficiency provides evidence for synthesis of 11ß-OH-steroid intermediates in vivo (40, 41). The potential for Leydig cells to biosynthesize CORT remains to be established, because a complete synthetic pathway for CORT requires 21-hydroxlase, and expression of this enzyme has not been detected in the testis. It is also possible that adrenal-derived precursors such as DOC will act as substrates for 11ß-hydroxylase in the testis.

In this study we further investigated the effects and relative potencies of several 11ß-OH-steroids and their corresponding 11-keto derivatives on 11ßHSD1 oxidative and reductive activities of Leydig cell homogenates and intact Leydig cells using either CORT or 11-dehydro-CORT as substrates. The 11ß-OH-steroid intermediates, which are logical candidates for locally generated 11ßHSD inhibitors, are capable of inhibiting testicular 11ßHSD1 activity in a directionally specific manner, blocking the 11ßHSD1 dehydrogenase reaction. In contrast, several of the 11-keto compounds inhibited only the oxidoreductase reaction, as expected (18, 19). Similar patterns of directionally specific inhibition and more potent inhibitory activities were observed with several of these substances in whole Leydig cells. At this time, the mechanism of inhibition due to these substances is unknown. The results are consistent with previous studies in vascular tissue, in which 11ß-OH- or 11-ketosteroids have a significant influence on both 11ßHSD1 activity and the resultant vasoconstrictivity in response to catecholamines, which is enhanced by both CORT and 11-dehydro-CORT (32, 33). GA-mediated inhibition of Leydig cell 11ßHSD1 dehydrogenase enhances the ability of CORT to suppress T production (8). These results also suggest the possibility that individual 11ß-OH-steroid derivatives may function as GA does in the testis, with the potential to augment the responses of Leydig cells to CORT through inhibition of testicular 11ßHSD1 dehydrogenase activity. Conversely, certain 11-keto-steroid derivatives may operate in the rat to diminish the responses of Leydig cells to glucocorticoids by lessening the ability to regenerate active glucocorticoids from 11-dehydroglucocorticoids.

11ß-Hydroxylase expression and activity are lower in Leydig cells than in the adrenal cortex. However, small amounts of locally produced inhibitory substances, could exert significant physiological effects on glucocorticoid regulation of T biosynthesis, as Leydig cells are highly sensitive to regulation by CORT. In the present study 11ß-OH-T showed stronger inhibition of 11ßHSD1 oxidative activity compared with 11ß-OH-Prog in whole Leydig cells, indicating that the testicular 11ßHSD1 could be highly sensitive to regulation by locally produced 11ßHSD inhibitors. The present study suggests that T itself may inhibit 11ßHSD oxidative activity due to its high concentration in the testicular interstitium. A local negative feedback system may thereby control T production in the testis through increased inhibitory action of glucocorticoid on T biosynthesis. Local production of 5{alpha}-reduced products of T may play a similar role.

The physiological function of 11ß-hydroxylase in the testis is still unknown. However, local testicular synthesis of 11ßHSD1 inhibitors may provide another control point in stress-induced reproductive dysfunction. The evidence supports the hypothesis that the stress hormones, ACTH and glucocorticoid, attenuate 11ßHSD1 dehydrogenase activity in the testis (5, 42, 43). As adrenal 11ß-hydroxylase is regulated by ACTH, which is increased during stress, it will be of interest to determine whether this hormone stimulates levels of 11ß-hydroxylase in Leydig cells, inhibiting 11ßHSD1 and contributing to stress-mediated declines in T production (6). This may further explain why 11ßHSD1 is unable to fully protect the testis from the deleterious effects of glucocorticoid action in Cushing’s syndrome and during severe stress.

In summary, 11ß-hydroxylase mRNA, protein and activity are expressed in rat Leydig cells. The occurrence of steroid 11ß-hydroxylation in these cells raises the possibility that endogenous inhibitory substances of 11ßHSD are synthesized in the testis, affecting overall CORT action through control of bidirectional 11ßHSD1 activity.


   Acknowledgments
 
We thank Ms. Chantal Manon Sottas for skilled technical assistance, Dr. Cedric Shackleton for GC/MS analysis, and Dr. Dianne Hardy for comments on manuscript.


   Footnotes
 
This work was supported in part by The Population Council and NIH Grant HD-33000.

Abbreviations: CORT, Corticosterone; DHEA, dehydroepiandrosterone; DOC, 11-deoxycorticosterone; EDS, ethane dimethane sulfonate; GA, glycyrrhetinic acid; GC/MS, gas chromatography/mass spectrometry; 11ß-OH, 11ß-hydroxy; 11ßHSD1, 11ß-hydroxysteroid dehydrogenase isoform 1; Prog, progesterone; 3{alpha},5{alpha}-THT, 3{alpha},5{alpha}-metabolite of T; T, testosterone.

Received August 1, 2001.

Accepted for publication October 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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