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Endocrinology Vol. 138, No. 1 156-161
Copyright © 1997 by The Endocrine Society

ARTICLES

Hormonal Regulation of Oxidative and Reductive Activities of 11ß-Hydroxysteroid Dehydrogenase in Rat Leydig Cells1

Hui-Bao Gao, Ren-Shan Ge, Vijaya Lakshmi, Alexandru Marandici and Matthew P. Hardy

The Population Council (H.-B.G., R.-S.G., A.M., M.P.H.) and Rockefeller University (M.P.H.), 1230 York Avenue, New York, New York 10021; and Department of Obstetrics and Gynecology (V.L.), State University of New York, Health Science Center-Brooklyn, Brooklyn, New York 11203

Address all correspondence and requests for reprints to: Matthew P. Hardy, The Population Council, Center for Biomedical Research, 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
 
We have proposed that the 11ß-hydroxysteroid dehydrogenase (11ß-HSD) of Leydig cells protects against glucocorticoid-induced inhibition of testosterone (T) production. However, Leydig cells express type I 11ß-HSD, which has been shown to be reductive in liver parenchymal cells. Because reduction would have the opposite effect of activating glucocorticoid, the present study was designed to determine: 1) whether Leydig cell 11ß-HSD is primarily oxidative or reductive; and 2) whether oxidative and reductive activities are separately modified by known regulators of Leydig cell steroidogenic function. Leydig cells and liver parenchymal cells were purified from mature male Sprague-Dawley rats (250 g BW), and 11ß-HSD oxidative and reductive activities were measured using radiolabeled substrates and TLC of triplicate media samples from 1-h incubations immediately after cell isolation. Enzyme activities also were examined in purified Leydig cells at the end of 3 days of culture in vitro in the presence of LH (10 ng/ml), dexamethasone (DEX, 100 nM), T (50 nM), or epidermal growth factor (EGF, 50 ng/ml). In confirmation of previous reports, the reductive activity of 11ß-HSD was predominant over oxidation in liver parenchymal cells. In contrast, 11ß-HSD oxidative activity prevailed over reduction in Leydig cells by a ratio of 2:1. The activities of 11ß-HSD also were analyzed in Leydig cells that were purified 7 days after endogenous glucocorticoid levels were suppressed by adrenalectomy (ADX). Oxidative activity declined in Leydig cells after ADX (22.53 ± 1.12 pmol/h·106 cells, mean ± SEM vs. 31.47 ± 1.48 pmol/h·106 cells in sham-operated controls, P < 0.05), whereas there was no change in reductive activity. This indicated that physiologically active corticosterone is involved in maintaining the predominance of 11ß-HSD oxidation. When enzyme activities were analyzed in Leydig cells after 3 days of hormonal treatment in vitro, oxidation and reduction were observed to change in opposing directions. Culture of Leydig cells from sham-operated control rats with either LH, T, or EGF resulted in declines in oxidative activity from 33.35 ± 0.77 to 28.24 ± 1.93, 27.30 ± 0.96, and 24.13 ± 1.02 pmol/h·106 cells ( ± SE), respectively. However, EGF stimulated 11ß-HSD reductive activity in cultured Leydig cells from both control (from 18.97 ± 1.10 to 27.16 ± 0.71 pmol/h·106 cells and ADX rats (from 16.51 ± 0.75 to 23.56 ± 0.84 pmol/h·106 cells). Among the hormonal treatments, only DEX increased oxidative activity and simultaneously decreased reductive activity in Leydig cells from ADX rats. This increase accentuated the predominance of oxidative activity in Leydig cells, with a ratio of oxidative to reductive activity of 4:1 after DEX treatment, compared with 2:1 in controls that were untreated. We conclude that 11ß-HSD activity in Leydig cells is primarily oxidative. Moreover, oxidation and reduction are regulated separately by hormones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE 11ß-hydroxysteroid dehydrogenase (11ß-HSD) catalyzes the reversible conversion of physiologically active corticosterone (B) to the biologically inert 11-dehydro form, 11-dehydrocorticosterone (A), thus regulating access of glucocorticoids to mineralocorticoid and glucocorticoid receptors in vivo (1). To date, two distinct forms of 11ß-HSD have been identified: 11ß-HSD type I, which was first purified from rat liver and later cloned, shows both oxidative and reductive activities and has a relatively low affinity for glucocorticoid substrates (2, 3); whereas, 11ß-HSD type II, first identified in kidney and later cloned, is primarily oxidative with a high affinity for glucocorticoids (4, 5, 6, 7).

Glucocorticoids directly inhibit testosterone (T) biosynthesis in Leydig cells via a receptor-mediated mechanism (8). Demonstration that type I 11ß-HSD protein (9) and messenger RNA (mRNA) (10) are present in Leydig cells led to the hypothesis that 11ß-HSD inactivates glucocorticoids within this cell, allowing for normal T production. However, recent studies of rat liver parenchymal cells and transfected cell lines indicate that type I 11ß-HSD is primarily reductive (11, 12, 13). If 11ß-HSD were primarily reductive, glucocorticoid activation would predominate in Leydig cells. The consequent inhibition of T production would inhibit sexual maturation. Because T production and type I 11ß-HSD have parallel increases in Leydig cells during puberty, the resolution of this apparent paradox will be achieved only by determining the predominant direction of the Leydig cell 11ß-HSD reaction.

The hormonal regulation of type I 11ß-HSD activity has been studied extensively, but most reports have demonstrated that oxidative and reductive activities change in parallel in response to hormonal effectors. Thus, in rat liver and cultured human skin fibroblasts, glucocorticoid increases both oxidative and reductive activities, and both increases are antagonized by insulin (13, 14, 15). Glucocorticoids also increase type I 11ß-HSD activities in the hippocampus, potentiating glucocorticoid-mediated neurotoxicity (15). Finally, sex steroids have been found to regulate type I 11ß-HSD activities and mRNA levels in liver, kidney, and hippocampus (16). To date, however, there have been no studies of the hormonal regulation of type I 11ß-HSD in Leydig cells.

The aim of this study was to determine whether Leydig cell 11ß-HSD is primarily oxidative or reductive and to analyze both activities for modulation by known regulators of Leydig cell steroidogenic function: LH, glucocorticoid, T, and epidermal growth factor (EGF).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
[1,2,6,7-N-3H] Corticosterone ([3H]B; specific activity 88 Ci/mmol) was purchased from Dupont-New England Nuclear (Boston, MA). B, A, dexamethasone (DEX), and T were purchased from Steraloids (Wilton, NH). EGF was purchased from Mallinckrodt Co. (Paris, KY). LH was a gift from NIDDK (Bethesda, MD). The oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) and the reduced form (NADPH) were purchased from Sigma Chemical Co. (St. Louis, MO). 11-dehydro[1,2,6,7-N-3H] corticosterone ([3H]A) was prepared from [3H]B as described earlier (17). TLC plates (Polygram Silica Gel/UV254) were obtained from Brinkmann Instruments (Westbury, NY).

Animals
Male Sprague-Dawley rats (200–250 g, Charles River, Wilmington, MA) were subjected to either bilateral adrenalectomy (ADX) or sham-surgery and housed at 26 C in groups of 12. The drinking water of ADX rats was supplemented with 0.9% NaCl to maintain electrolyte balance. Seven days after surgery, the animals were killed by CO2 asphyxiation, following a protocol that was approved by the Institutional Animal Care and Use Committee of the Rockefeller University (protocol 91200).

Isolation and purification of liver parenchymal cells
Male rats were anesthetized with ether, and the liver was perfused in situ with a calcium-free buffer, followed by a solution containing 0.05% collagenase (Sigma Chemical Co.) in 5 mM CaCl2, 30 mM HEPES, 6.75 mM KCl, and 69 mM NaCl, as described by Pertoft and Smedsrød (18). The perfused liver was then removed, and liver cells were isolated by filtration through nylon mesh (60 micron). The parenchymal cells were purified by loading collagenase-dispersed liver cells on the top of Percoll diluted with PBS (pH 7.4) to a density of 1.09 g/ml and an osmolality of 340 mOsm/kg. After centrifugation (800 x g; 10 min; 4 C) intact parenchymal cells were recovered from the pellet. The purity of parenchymal cells in the final suspension was assessed by judging the uniformity of cell size and was typically over 95%.

Isolation and purification of Leydig cells
Leydig cells were isolated from testes by centrifugal elutriation and Percoll density gradient centrifugation, following the method of Klinefelter et al. (19). The purity of cells isolated using this method was evaluated by histochemical staining for 3ß-HSD activity, with 0.4 mM etiocholanolone as the steroid substrate (20). Enrichment of Leydig cells was typically to 95% purity.

Leydig cell Culture and hormonal treatment
Cell culture was conducted as previously described (21). Briefly, Leydig cells were plated at a density of 5 x 106 cells/75-mm flask and cultured for 3 days in 15 ml phenol red-free medium (DMEM-Ham’s F12, D-2906, Sigma Chemical Co.) plus 1 mg/ml bovine lipoprotein at 5% O2, 5% CO2, 34 C. The medium with or without hormones was replaced every 24 h. Hormones were added to Leydig cells at the following concentrations: LH, 10 ng/ml; DEX, 100 nM; T, 50 nM; EGF 50 ng/ml. After 3 days of hormonal treatment, Leydig cells were scraped from the surface with a rubber policeman after 5 min of incubation in a solution containing 0.05% collagenase (Sigma Chemical Co.) in Medium-199 (GIBCO-BRL, Gaithersburg, MD) with 8.45 mM NaHC03, 8.8 mM HEPES, 0.1% BSA, 0.0025% trypsin inhibitor (Sigma Chemical Co.) and assayed for 11ß-HSD enzyme activities and mRNA levels.

11ß-HSD enzyme activities
Determination of 11ß-HSD activities was performed, with slight modification of a previously described procedure (22), on liver parenchymal cells and Leydig cells just after isolation and in Leydig cells after 3 days of culture in vitro. In brief, 0.5 ml reaction mixture was prepared in phenol red-free medium that contained 2 nM [3H]B and 23 nM unlabeled B, or 2 nM [3H]A and 23 nM unlabeled A, giving a final concentration of 25 nM B or A. This concentration was chosen to reflect physiological levels of free steroid. The reaction mixture was maintained at pH 7.2. Oxidation at C-11 was determined by measuring the rate of conversion of B to A in the presence of 200 µM NADP. Reduction at C-11 was determined by measuring the rate of conversion of A to B in the presence of 200 µM NADPH. Reactions were initiated by adding, to the reaction mixture, aliquots of cell suspension containing 0.2–0.3 x 106 liver parenchymal cells or Leydig cells. The reaction mixtures, conducted in triplicate, were maintained at 34 C (the physiological temperature for Leydig cells) in a shaking water bath for 1 h. With appropriate incubation times, the conversion of B to A and A to B was linear with respect to cell number and time of incubation. Reactions were terminated by adding iced ethyl acetate. Steroids were then extracted with ethyl acetate, and the organic layer was dried under nitrogen. The steroid residues were mixed with unlabeled B and A and chromatographed on TLC plates, and radioactivity was measured with a Bioscan System Scanner (Bioscan Inc., Washington, DC). The percentage of the conversion of B to A and A to B was calculated by adding the radioactive counts identified as B and A and expressing radioactive A as a percentage thereof for 11ß-HSD oxidative activity or radioactive B as a percentage thereof for reductive activity.

RT-PCR analysis
Total RNA was extracted from Leydig cells after culture by a single-step method using phenol and guanidinium thiocyanate (TRISOLV, Biotecx Laboratories, Inc., Houston, TX) according to the manufacturer’s instructions. The procedure for RT-PCR was as previously described (10). In brief, Leydig cell total RNA (2.5 µg/sample) was reverse transcribed with avian myeloblastosis virus RT in the presence of random hexamer primers plus dNTPs at 42 C for 75 min, and the reaction was terminated by heating at 95 C for 5 min. The primers for type I 11ß-HSD and the internal control, ribosomal protein S16 (RPS16), were added to the same assay tube, and products were amplified simultaneously by PCR. PCR was initiated by Taq DNA polymerase in the presence of [{alpha}-32P] dCTP and proceeded for 35 cycles with an annealing temperature of 48 C. Finally, the amplified PCR products were separated on the same gel. The sequences of the 11ß-HSD primers were: 5'-TGGAGATCTGTTATGAAAAATACCTCC, corresponding to nucleotides 87 to 103, and 5'-ACTCAAGATTATCCCAG, corresponding to nucleotides 774 to 757 of the rat cDNA (23). The primers for RPS16 were as described previously (24).

Data analysis
Each experiment was repeated at least three times. The data were analyzed by Kruskal-Wallis ANOVA followed by multiple comparisons testing to identify significant differences between groups (25). Differences were regarded as significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oxidative and reductive activities of 11ß-HSD in freshly isolated liver parenchymal cells and Leydig cells
As shown in Fig. 1Go, at physiological levels of B or A, the reductive activity of 11ß-HSD in rat liver parenchymal cells significantly exceeded oxidative activity (P < 0.001). In contrast, the oxidative activity of 11ß-HSD in Leydig cells was higher than reductive activity (P < 0.001). These results indicate that 11ß-HSD activity in freshly isolated Leydig cells is bidirectional, with oxidative activity predominating.



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  		Figure 1. Oxidative and reductive activities of 11ß-HSD in freshly isolated liver parenchymal cells and Leydig cells. Intact liver parenchymal cells (0.5 x 106/ml) or Leydig cells (0.3 x 106/ml) were incubated at 34 C in the presence of 25 nM B with 200 µM NADP or 25 nM A with 200 µM NADPH for 1 h. Values are means ± SEM. The number of experimental replications was n = 3 for liver parenchymal cells and n = 6 for Leydig cells. The level of significance, P < 0.001, is indicated for the comparison between oxidative and reductive activities of 11ß-HSD.

 
Oxidative and reductive activities of 11ß-HSD in freshly isolated Leydig cells from adrenalectomized rats
Physiological levels of glucocorticoid may be involved in regulation of 11ß-HSD activities in Leydig cells. Leydig cells were isolated from animals after 7-day suppression of glucocorticoid levels by ADX, and both oxidative and reductive activities of 11ß-HSD were measured immediately. ADX resulted in a significant decline in oxidative activity, whereas reductive activity was unaltered (Fig. 2Go). This indicated that physiological levels of B are involved in maintaining the predominance of 11ß-HSD oxidation.



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  		Figure 2. Oxidative and reductive activities of 11ß-HSD in freshly isolated Leydig cells from adrenalectomized rats. The cells were isolated 7 days after ADX with Leydig cells from intact sham-operated rats serving as the control (INTACT). Leydig cells (0.3 x 106/ml) were incubated at 34 C in the presence of 25 nM B with 200 µM NADP or 25 nM A with 200 µM NADPH for 1 h. Values are means ± SEM, n = 9. The level of significance, P < 0.001, is indicated for 11ß-HSD oxidative activity in Leydig cells, in the comparison between INTACT and ADX rats.

 
Hormonal regulation of oxidative and reductive activities of 11ß-HSD in cultured Leydig cells
The oxidative activity of 11ß-HSD continued to predominate over reductive activity during 3 days in vitro when the Leydig cells were obtained from intact (sham-operated) rats (Fig. 3Go). In contrast, oxidative activity was initially low in Leydig cells from ADX rats but returned to control values by day 3 in vitro. This suggested that the oxidative activity of 11ß-HSD in Leydig cells from ADX rats is chronically inhibited in vivo and that the inhibition is alleviated during culture in hormone-free medium. Three days of treatment in vitro with either LH, T, or EGF significantly decreased 11ß-HSD oxidative activity in Leydig cells from intact rats (Fig. 4aGo). In Leydig cells from ADX rats, T or EGF in vitro also inhibited the recovery of oxidative activity that occurred during culture in hormone-free medium (Fig. 4bGo).



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  		Figure 3. Oxidative and reductive activities of 11ß-HSD in Leydig cells after culture in hormone-free medium. Leydig cells from intact rats (INTACT) and ADX rats were cultured in hormone-free medium for 3 days and harvested. Leydig cells (0.3x 106/ml) were incubated at 34 C in the presence of 25 nM B with 200 µM NADP or 25 nM A with 200 µM NADPH for 1 h. Values are means ± SEM, n = 9. The level of significance, P < 0.001, is indicated for 11ß-HSD oxidative activity from ADX rats, in the comparison between freshly isolated and 3 days of culture.

 


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  		Figure 4. Hormonal regulation of oxidative activity of 11ß-HSD in cultured Leydig cells. Leydig cells from intact sham-operated rats (INTACT) and ADX rats were cultured for 3 days in hormone-free Leydig cell medium (LCM) or medium containing: LH (10 ng/ml); DEX (100 nM); T (50 nM); EGF (50 ng/ml). Leydig cells were harvested and aliquots of cell suspension (0.3 x 106/ml) were incubated at 34 C in the presence of 25 nM B with 200 µM NADP or 1 h. Values are means ± SEM, n = 9. The level of significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001) is indicated for 11ß-HSD oxidative activity in Leydig cells from INTACT and ADX rats, for comparison between LCM and hormonal treatment.

 
The reductive activity of 11ß-HSD in cultured Leydig cells did not change in parallel with oxidative activity. Reductive activity was unchanged in Leydig cells from either intact or ADX rats after 3 days of hormone-free culture (Fig. 3Go). Moreover, reductive activity in Leydig cells from intact and ADX rats was unchanged after treatment with LH or T in vitro and increased after exposure to EGF (P < 0.01, Fig. 5Go). Taken together, these results indicated that LH, T, or EGF decreased the oxidative activity of Leydig cell 11ß-HSD and did not change or increase reductive activity. A similar contrast in the hormonal modulation of oxidation and reduction was seen after treatment with DEX in vitro. There was no effect of DEX on 11ß-HSD oxidative activity in cultured Leydig cells from intact rats but oxidation was stimulated in cultured Leydig cells from ADX rats (Fig. 4bGo). DEX did not affect the reductive activity of 11ß-HSD in cultured Leydig cells from intact rats but it significantly decreased the reductive activity in Leydig cells from ADX rats (P < 0.01, Fig. 5bGo). This indicated that normal levels of circulating B in intact animals are required to maintain the oxidative activity of 11ß-HSD.



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  		Figure 5. Hormonal regulation of reductive activity of 11ß-HSD in cultured Leydig cells. Leydig cells from intact sham-operated rats (INTACT) and ADX rats were cultured for 3 days in hormone-free LCM or medium containing: LH (10 ng/ml); DEX (100 nM); T (50 nM); EGF (50 ng/ml). Leydig cells were harvested and aliquots of cell suspension (0.3 x 106/ml) were incubated at 34 C in the presence of 25 nM A with 200 µM NADPH for 1 h. Values are means ± SEM, n = 9. The level of significance, P < 0.01, is indicated for 11ß-HSD reductive activity in Leydig cells from INTACT and ADX rats, for comparisons between LCM and hormonal treatment.

 
The results above indicated that the hormones under investigation exerted opposing effects on oxidative and reductive activities of 11ß-HSD in Leydig cells. Treatment with EGF, for example, decreased the net rate of 11ß-HSD oxidation, lowering the ratio of oxidative to reductive activities from approximately 2:1 in Leydig cells from intact and ADX rats to 1:1. Treatment with DEX had the reverse effect, increasing the ratio of oxidative to reductive activities in Leydig cells from ADX rats to approximately 4:1 (compared with 2:1 when these cells were cultured in hormone-free medium). This indicated that endogenous levels of B induce oxidative activity, suppress reductive activity, and thereby maintain a net level of 11ß-HSD oxidation.

Hormonal regulation of type I 11ß-HSD mRNA levels in Leydig cells
Type I 11ß-HSD mRNA levels in Leydig cells, measured after 3 days of culture, are summarized in Fig. 6Go. Type I 11ß-HSD mRNA levels in Leydig cells from both intact and ADX rats were increased after treatment with LH or EGF treatment. DEX had no effect on Leydig cells from intact rats but significantly decreased type I 11ß-HSD mRNA levels in Leydig cells from ADX rats. Changes in steady-state type I 11ß-HSD mRNA levels paralleled the trends that were shown for 11ß-HSD reductive activity after hormonal treatment in vitro. These results indicated that the hormones under investigation regulated 11ß-HSD reductive activity in Leydig cells by modulating the steady-state levels of 11ß-HSD mRNA.



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  		Figure 6. Hormonal regulation of type I 11ß-HSD mRNA levels in Leydig cells. Total RNAs (2 µg) from each group were reverse transcribed. The complementary DNA products were subsequently amplified in the presence of primer pairs for type I 11ß-HSD and the internal control, RPS16, by 35 cycles of PCR, separated in the same gel, detected by autoradiography. The band intensities of type I 11ß-HSD were measured by densitometric scanning and expressed relative to RPS16. Values are means ± SEM, n = 3. The level of significance, P < 0.05, is indicated for type I 11ß-HSD mRNA levels in Leydig cells from INTACT (A) and ADX (B) rats, in the comparison between LCM and hormonal treatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that 11ß-HSD in Leydig cells is primarily oxidative at physiological levels of B. The predominance of oxidative activity in intact Leydig cells agrees with previous results using testicular homogenates (26) and is consistent with the postulated role of 11ß-HSD in the testis. This enzyme probably serves to protect against the suppressive action of B on T production by oxidatively inactivating B to A, which does not bind to glucocorticoid receptors.

The observation that Leydig cell 11ß-HSD is primarily oxidative is difficult to interpret in light of current knowledge regarding the two known isoforms of this enzyme. Leydig cells contain type I 11ß-HSD (8, 9), an oxidoreductase that has been shown to be primarily reductive when its activity was assayed in liver parenchymal cells (11, 13). The type II 11ß-HSD is oxidative but is not detected in Leydig cells (7, 27). The present study confirmed that 11ß-HSD activity in liver parenchymal cells is primarily reductive under the same conditions that showed the predominance of oxidative activity in Leydig cells. It is possible that Leydig cells contain, in addition to type I, a distinct kinetic isoform that has a high affinity for substrate and is responsible for the predominance of 11ß-HSD oxidative activity in this cell. Recently, a high-affinity (Km = 40 nM) NADP-dependent 11ß-HSD with exclusively oxidative activity was reported to be present in the choriocarcinoma cell line JEG-3, and a new isoform of 11ß-HSD was suggested (28).

Leydig cell 11ß-HSD oxidative and reductive activities were regulated separately by LH, T, EGF, and DEX. LH, T, and EGF inhibited Leydig cell 11ß-HSD oxidative activity but stimulated reduction. The reverse was true of DEX, which stimulated Leydig cell 11ß-HSD oxidative activity but inhibited reduction. Changes in steady-state type I 11ß-HSD mRNA levels paralleled the trends shown for 11ß-HSD reductive activity and opposed the trends shown for oxidative activity. Previous studies have shown that a single type I 11ß-HSD mRNA species is translated into a protein radio that expresses both oxidative and reductive activities (11, 12, 13). The oxidative and reductive activities of type I 11ß-HSD have been found to change in parallel (14, 15). The opposing trends of oxidative and reductive activities of 11ß-HSD in response to hormones, seen in the present study, provide further indication of the existence of an unidentified 11ß-HSD isotype in Leydig cells in addition to type I.

Glucocorticoids are known to regulate 11ß-HSD activity in several tissues, including brain and liver (13, 14, 15). The oxidative activity of 11ß-HSD in freshly isolated Leydig cells from ADX rats was lower compared with intact, sham-operated controls. The decline in oxidative activity was reversed in vitro when the Leydig cells were treated with DEX for 3 days. This suggests that endogenous levels of B are involved in maintaining the predominance of 11ß-HSD oxidation. No changes in 11ß-HSD oxidative and reductive activities were observed in Leydig cells from intact rats after 3 days of DEX treatment in vitro. However, DEX strongly increased oxidative and inhibited reductive activities in Leydig cells from ADX rats. This indicated that 11ß-HSD oxidative and reductive activities in Leydig cells from ADX rats were sensitive to the action of glucocorticoids. It is possible that ADX also increased the glucocorticoid receptor levels in Leydig cells, because ADX-induced increases in glucocorticoid receptors have been observed in liver and brain (29, 30).

LH, T, and EGF have been shown to regulate steroidogenic enzyme activities in Leydig cells (31, 32, 33, 34). The results of the present study show LH, T, and EGF regulated 11ß-HSD oxidative and reductive activities in Leydig cells. LH, T, and EGF inhibited 11ß-HSD oxidative activity and either did not change or increased reductive activity, thus lowering the ratio of oxidative to reductive activity down to about 1:1. The likely consequence of this action is to increase the intracellular level of free B, which would then decrease T production.

In summary, 11ß-HSD oxidative activity was predominant over reductive activity in Leydig cells, supporting the hypothesis that 11ß-HSD protects against the suppressive action of glucocorticoids in this cell. Glucocorticoid is involved in maintaining the predominance of 11ß-HSD oxidative activity in Leydig cells. LH and EGF stimulate 11ß-HSD reductive activity and steady-state type I 11ß-HSD mRNA levels and inhibit oxidative activity.


   Acknowledgments
 
The technical assistance of Ms. Chantal Manon Sottas is gratefully acknowledged. Dr. P.C. White generously provided the sequences of 11ß-HSD oligonucleotide primers. We thank Dr. Glen Gunsalus for critical comments on the manuscript.


   Footnotes
 
1 This work was supported in part by funds from the Rockefeller Foundation (to H.-B.G.) and the Contraceptive Research and Development Program of the United States Agency for International Development (to R.-S.G.) and NIH Grant HD-33000 (to M.P.H.) Back

Received July 2, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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