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Biology of Reproduction 60, 855-860 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Opposing Changes in 3{alpha}-Hydroxysteroid Dehydrogenase Oxidative and Reductive Activities in Rat Leydig Cells during Pubertal Development1

Ren-Shan Gea, Dianne O. Hardya, James F. Catteralla, and Matthew P. Hardy2,a

a The Population Council and Rockefeller University, New York, New York 10021


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The enzyme 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD) has an important role in androgen metabolism, catalyzing the interconversion of dihydrotestosterone (DHT) and 5{alpha}-androstane-3{alpha},17ß-diol (3{alpha}-DIOL). The net direction of this interconversion will affect the amount of biologically active ligand available for androgen receptor binding. We hypothesize that in Leydig cells, differential expression of 3{alpha}-HSD enzymes favoring one of the two directions is a mechanism by which DHT levels are controlled. In order to characterize 3{alpha}-HSD in rat Leydig cells, the following properties were analyzed: rates of oxidation (3{alpha}-DIOL to DHT) and reduction (DHT to 3{alpha}-DIOL) and preference for the cofactors NADP(H) and NAD(H) (i.e., the oxidized and reduced forms of both pyridine nucleotides) in Leydig cells isolated on Days 21, 35, and 90 postpartum. Levels of 3{alpha}-HSD protein were measured by immunoblotting using an antibody directed against the liver type of the enzyme. Levels of 3{alpha}-HSD protein and rates of reduction were highest on Day 21 and lowest on Day 90. The opposite was true for the rate of 3{alpha}-HSD oxidation, which was barely detectable on Day 21 and highest on Day 90 (59.08 ± 6.35 pmol/min per 106 cells, mean ± SE). Therefore, the level of 3{alpha}-HSD protein detectable by liver enzyme was consistent with reduction but not with oxidation. There was a clear partitioning of NADP(H)-dependent activity into the cytosolic fraction of Leydig cells, whereas on Days 35 and 90, Leydig cells also contained a microsomal NAD(H)-activated 3{alpha}-HSD. We conclude that 1) the cytosolic 3{alpha}-HSD in Leydig cells on Day 21 behaves as a unidirectional NADPH-dependent reductase; 2) by Day 35, a microsomal NAD(H)-dependent enzyme activity is present and may account for predominance of 3{alpha}-HSD oxidation over reduction and the resultant high capacity of Leydig cells on Day 90 to synthesize DHT from 3{alpha}-DIOL.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The function of fully differentiated Leydig cells is to produce testosterone. Androgen receptors are present in Leydig cells [1–3], and it has been shown that androgen has autocrine effects on developing and mature Leydig cells [4, 5]. Leydig cells are the primary site of androgenic steroid synthesis, and multiple androgens with various potencies and intracellular concentrations are ultimately secreted. Dihydrotestosterone (DHT) is the most potent, binding to the androgen receptor with a dissociation constant (Kd) of 2.0 x 10-10 M, compared to 4.0 x 10-10 M for testosterone and 10-6 M for 5{alpha}-androstane-3{alpha},17ß-diol (3{alpha}-DIOL) [6,7]. In the Leydig cell, 5{alpha}-reductase catalyzes the irreversible conversion of testosterone to DHT. Another Leydig cell enzyme, 3{alpha}-hydroxysteroid dehydrogenase (3{alpha}-HSD), catalyzes the reductive breakdown of DHT to 3{alpha}-DIOL, but it can also catalyze, in the opposite direction, the oxidative conversion of 3{alpha}-DIOL to DHT. Therefore, the relative rates of 3{alpha}-HSD oxidation and reduction reactions will have important consequences for the steady-state concentration of DHT.

Messenger RNA analysis indicates that rat Leydig cells express a 3{alpha}-HSD that is identical to the one first cloned from rat liver [8–11]. This 3{alpha}-HSD, a member of the short-chain alcohol dehydrogenase family, is a cytosolic protein catalyzing both NADP-dependent oxidation and NADPH-dependent reduction in cell homogenates [12–15]. However, some of the hydroxysteroid dehydrogenases in the short-chain alcohol dehydrogenase family, such as 11ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase, catalyze oxidation or reduction, but not both, when assayed in intact cells [16–19]. In these instances, different enzymes encoded by separate genes catalyze unidirectional reactions, regulating active steroids in a cell-specific manner [16–19]. Whether 3{alpha}-HSD in Leydig cells catalyzes oxidation or reduction when assayed in intact cells has not been established. Moreover, it has not been determined whether 3{alpha}-HSD has different isoforms, frequently observed for other hydroxysteroid dehydrogenases. In this regard, liver 3{alpha}-HSD is NADP(H)-dependent and cytosolic [12–15], while the brain contains an NAD(H)-dependent and microsomal form of the enzyme [20, 21]. The goal of the present study was to test the hypothesis that different 3{alpha}-HSD enzymes catalyze oxidation and reduction thereby controlling androgenic composition during Leydig cell development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Radiolabeled steroids, [1,2-3H(N)]dihydrotestosterone and 5{alpha}-[9,11-3H(N)]androstane-3{alpha},17ß-diol, each with measured purities of 99%, were purchased from DuPont New England Nuclear (Boston, MA). Nonradioactive steroids were purchased from Sigma Chemical Co. (St. Louis, MO) or Steraloids (Wilton, NH). The antibody for rat liver 3{alpha}-HSD was provided by Dr. Trevor Penning (Department of Pharmacology, University of Pennsylvania, Philadelphia, PA).

Animals

Sprague-Dawley rats (dams with litters of male pups, immature males, and adult males) were purchased from Charles River Laboratories (Wilmington, MA). The male rats were 21, 35, and 90 days of age on the day of cell isolation. The animals were killed by asphyxiation with CO2 according to an animal protocol that was approved by the Institutional Animal Care and Use Committee of the Rockefeller University (Protocol 91200).

Cell Isolation and Preparation of Subcellular Fractions

Progenitor, immature, and adult Leydig cells from 21-, 35-, and 90-day-old rats, respectively, were purified as described previously [1, 4, 22]. Purity of Leydig cell fractions was evaluated by histochemical staining for 3ß-hydroxysteroid dehydrogenase activity, with 0.4 mM etiocholanolone as the steroid substrate [23]. In the progenitor Leydig cell fraction, approximately 90% of the cells were lightly stained, and of the remaining 10%, 6% were intensely stained. More than 95% of the immature and adult Leydig cells were intensely stained.

Aliquots of isolated cells were homogenized in 5 volumes of ice-cold 20 mM phosphate buffer, pH 7.4, containing 250 mM sucrose. Cell homogenates were centrifuged at 750 x g for 30 min, and resulting supernatants were collected as the homogenate fraction. The homogenates were centrifuged at 12 000 x g for 30 min, and the postmitochondrial supernatants were obtained by ultracentrifugation at 105 000 x g for 60 min, twice, to collect the microsomal pellets. The microsomal pellets were resuspended in 25 mM phosphate buffer (pH 7.4). The cytosolic fractions were obtained by collecting supernatants. Both microsomal and cytosolic fractions were stored at -80°C until assay.

Enzyme Assay for 3{alpha}-HSD

For measurements of 3{alpha}-HSD activity, 3H-labeled 3{alpha}-DIOL was provided as a substrate to calculate oxidation rates, and 3H-labeled DHT was used for reduction rates. Measurement of 3{alpha}-HSD activities in intact Leydig cells was performed by incubating intact Leydig cells (0.2 x 106) with radiolabeled substrates in phenol red-free Dulbecco's Modified Eagle's medium/F12 medium (pH 7.2). Enzyme activities in homogenates, microsomes, or cytosols were assayed in 25 mM phosphate buffer (pH 7.4), with or without 200 µM pyridine nucleotide cofactors. A preliminary study showed that 3{alpha}-HSD activities were linear with respect to cell number or protein amount and time of incubation (3–30 min). Therefore, the samples were incubated at 34°C for 5–30 min, depending on best conditions with linear velocity. The steroids were extracted with 2 ml of ice-cold ethyl acetate, and the organic layer was evaporated under nitrogen gas. The steroids were separated chromatographically on thin-layer plates (Baker-flex, Phillipsburg, NJ) in diethyl ether:acetone (v:v, 98:2), in which DHT, 3{alpha}-DIOL, and androstane-3ß,17ß-diol (a possible metabolite of DHT) were separated. The Rfs (mobility ratios) for DHT, 3{alpha}-DIOL, and androstane-3ß,17ß-diol were 0.86, 0.75, and 0.64, respectively. The radioactivity was measured with a scanning radiometer (System 200/AC3000; Bioscan Inc., Washington, DC). The percentage of conversion of oxidation and reduction of 3{alpha}-HSD was calculated by dividing the radioactive counts identified as DHT or 3{alpha}-DIOL by the total counts associated with 3{alpha}-DIOL plus DHT.

Western Blot Analysis of 3{alpha}-HSD

Leydig cell homogenates were boiled in sample buffer containing SDS and dithiothreitol. Homogenized samples (50 µg protein) of progenitor, immature, and adult Leydig cells were electrophoresed on 10% polyacrylamide gels containing SDS [24]. Proteins were electrophoretically transferred onto nitrocellulose membranes, and, after 30-min exposure to 10% nonfat milk to block nonspecific binding, the membranes were incubated with a 1:1500 dilution of a rabbit polyclonal antibody [25] against 3{alpha}-HSD that was generated by immunization of animals with 3{alpha}-HSD purified from rat liver [15]. The membranes were then washed and incubated with a 1:2000 dilution of goat anti-rabbit antiserum that was conjugated to horseradish peroxidase. The washing step was repeated, and immunoreactive bands were detected by chemiluminescence using a kit (ECL; Amersham, Arlington Heights, IL). Relative protein levels were measured by densitometry of the films.

Data Analysis

In each experiment, data were obtained from triplicate assays, and the results were expressed as the mean ± SEM. The dissociation constants (Km) and the maximal velocity (Vmax) for 3{alpha}-HSD oxidative and reductive activities in Leydig cells were determined using the Michaelis-Menten equation and derived from Lineweaver-Burk plots. Statistical analysis of the changes in 3{alpha}-HSD oxidative and reductive activities was performed by Kruskal-Wallis ANOVA followed by multiple-comparisons testing to identify significant differences between groups [26]. Statistical analysis of the relative levels of 3{alpha}-HSD protein was performed after arcsin transformation of the ratio data [26].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
3{alpha}-HSD Protein Levels during Leydig Cell Development

3{alpha}-HSD protein was measured by Western blot using an antibody directed against rat liver 3{alpha}-HSD in three stages of differentiating Leydig cells: progenitor, immature, and adult Leydig cells isolated on Days 21, 35, and 90 postpartum. Protein levels for 3{alpha}-HSD decreased during Leydig cell differentiation, with values for adult Leydig cells being about 60% reduced compared to those for progenitor Leydig cells (Fig. 1).



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  		FIG. 1. Immunoblot analysis of 3{alpha}-HSD protein levels in Leydig cells. 3{alpha}-HSD protein levels in progenitor (PLC), immature (ILC), and adult (ALC) Leydig cell homogenates (50 µg total protein) were measured using antiserum raised against rat liver 3{alpha}-HSD. 3{alpha}-HSD-immunoreactive bands were visualized by chemiluminescence (left). 3{alpha}-HSD levels in Leydig cells were normalized to progenitor Leydig cells after densitometric analysis (right). Values represent means ± SE for 3 independent experiments. The asterisk indicates a significant difference at p < 0.05 compared to PLC.

3{alpha}-HSD Oxidative and Reductive Activities in Intact Leydig Cells during Development

In order to study enzyme activities in the presence of endogenous pyridine nucleotide cofactors, 3{alpha}-HSD oxidation and reduction rates were measured in intact Leydig cells at three stages of differentiation. The oxidation rates were calculated as the percentage of conversion of 3{alpha}-DIOL to DHT when 3H-labeled 3{alpha}-DIOL was provided as the substrate. This rate was 1.2 ± 0.7 pmol/min per 106 cells for progenitor Leydig cells, 33.8 ± 3.7 for immature Leydig cells, and 59.1 ± 6.4 for adult Leydig cells (Fig. 2). Rates of reductive activity were calculated as the percentage conversion for DHT to 3{alpha}-DIOL when 3H-labeled DHT was provided as the substrate. The rates of reduction were 51.75 ± 4.85 pmol/min per 106 cells for progenitor Leydig cells, 44.62 ± 2.87 for immature Leydig cells, and 34.31 ± 1.95 for adult Leydig cells. Therefore, in adult Leydig cells, the reduction rate was 35% lower relative to progenitor Leydig cells, whereas the oxidation rate was more than 100 times higher. As shown in Table 1, decreases in 3{alpha}-HSD reductive activity in the three stages of differentiating Leydig cells corresponded closely to 3{alpha}-HSD mRNA (data from [8] and [27]) and protein levels. This did not hold true for oxidative activity, which increased during Leydig cell differentiation.



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  		FIG. 2. 3{alpha}-HSD oxidation and reduction in intact Leydig cells. 3{alpha}-HSD oxidative and reductive activities were measured in 0.2 x 106 progenitor (PLC), immature (ILC), and adult (ALC) Leydig cells after 10-min incubations with 1 µM radiolabeled substrate. Values represent means ± SE from 9 different assays in three independent experiments. Shared alphabet letters refer to groups that were not significantly different at p < 0.05.


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  		TABLE 1. Comparative analysis of 3{alpha}-HSD mRNA and protein, and oxidative and reductive activities measured in intact Leydig cells.

Effects of Pyridine Nucleotides on 3{alpha}-HSD Oxidation and Reduction Rates

The effects of NADP+ and NAD+ on 3{alpha}-HSD oxidative activity, and of NADPH and NADH on reductive activity, were tested in Leydig cell homogenates at 4 µM substrate concentration, using 3H-labeled 3{alpha}-DIOL or 3H-labeled DHT. As shown in Figure 3, 3{alpha}-HSD oxidative activity in progenitor cell homogenates was activated by NADP+, and reductive activity by NADPH. No 3{alpha}-HSD activity was observed with either NAD+ or NADH. This showed that the 3{alpha}-HSD in progenitor Leydig cells was NADP(H)-dependent. The ratio of NADPH to NADP+ is known to be high in cells [28, 29]; therefore, NADP(H)-dependent 3{alpha}-HSD activities in intact Leydig cells were expected to be exclusively reductive, and this was confirmed by the data. Rates of NADP(H)-dependent oxidoreductase activity were equivalent in progenitor and immature Leydig cells, and the rates for both were higher than in adult Leydig cells.



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  		FIG. 3. Cofactor preference of 3{alpha}-HSD oxidation and reduction in Leydig cell homogenates. 3{alpha}-HSD oxidative and reductive activities were measured in progenitor (PLC), immature (ILC), and adult (ALC) Leydig cell homogenates (50 µg protein) after 15-min incubations with 4 µM radiolabeled substrate; A shows NADP(H)-dependent activity and B shows NAD(H)-dependent activity. Values represent means ± SE from 9 different assays in three independent experiments. Shared alphabet letters refer to groups that were not significantly different at p < 0.05.

In addition to NADP(H)-dependent activities, homogenates prepared from immature and adult Leydig cells had NAD(H)-dependent 3{alpha}-HSD oxidoreductase activities. The NAD(H)-dependent oxidoreductase activity was high in immature and adult Leydig cells and undetectable in progenitor Leydig cells. These results showed that NADP(H)-dependent and NAD(H)-dependent 3{alpha}-HSD oxidoreductase activities were present in immature and adult Leydig cells but that progenitor cells contained only one form, an NADP(H)-dependent 3{alpha}-HSD.

Subcellular Location of 3{alpha}-HSD Activities

Leydig cell 3{alpha}-HSD oxidative and reductive activities were measured in cytosolic and microsomal fractions in the presence of pyridine nucleotide cofactors. In immature and adult Leydig cells, an NADP(H)-dependent oxidoreductase was primarily present in cytosolic fractions, whereas an NAD(H)-dependent oxidoreductase was primarily present in microsomal fractions (Fig. 4). In progenitor Leydig cells, no 3{alpha}-HSD activity was found in microsomal fractions, and NADP(H)-dependent activities were detected exclusively in cytosols (data not shown). This corroborated the hypothesis that NADP(H)-dependent and NAD(H)-dependent activities were catalyzed by separate isoforms.



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  		FIG. 4. Subcellular localization of NADP(H)-dependent and NAD(H)-dependent 3{alpha}-HSD activity in immature Leydig cells. 3{alpha}-HSD oxidation (left) and reduction (right) were measured in immature Leydig cell cytosolic fractions (25 µg protein) and microsomal fractions (12.5 µg protein) after 20-min incubations with 4 µM radiolabeled substrate. Values represent means ± SE from 9 different assays in three independent experiments. The asterisk indicates a significant difference at p < 0.05 compared to the no-cofactor control.

As shown in Table 2, Km values of for NADP(H)-dependent oxidation and reduction in the cytosolic fractions were similar among the various stages of Leydig cell. The Km in Leydig cells is comparable to that of liver cytosolic NADP(H)-dependent 3{alpha}-HSD. Comparison of the estimates of Vmax showed that adult Leydig cells had lower NADP(H)-dependent 3{alpha}-HSD activities than progenitor and immature Leydig cells. This suggests that NADP(H)-dependent activities were reduced in adult Leydig cells. In microsomal fractions, Km values associated with NAD(H)-dependent 3{alpha}-HSD oxidation and reduction were similar in immature and adult Leydig cells. In contrast, the Km values for the NAD(H)-dependent activities were different in comparison to cytosolic NADP(H)-dependent activities, indicating that NADP(H)- and NAD(H)-dependent 3{alpha}-HSD activities reside in different isoforms. Comparison of the estimates for Vmax showed that adult Leydig cells had higher NAD(H)-dependent 3{alpha}-HSD activities than immature Leydig cells. This suggests that NAD(H)-dependent 3{alpha}-HSD activities increased developmentally.


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  		TABLE 2. Kinetic parameters for 3{alpha}-HSD oxidation and reduction in cytosols and microsomes of Leydig cells: progenitor (PLC), immature (ILC) and adult (ALC) Leydig cells (mean ± SE).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Biochemically, both cytosolic NADP(H)-dependent and microsomal NAD(H)-dependent isoforms of 3{alpha}-HSD have been shown to be present in several rat tissues including liver and brain [12–15, 20, 21]. In the present study, 3{alpha}-HSD activities were assessed in purified, intact cells within a specific lineage. Data were thus obtained on the activity of the enzyme in a milieu that approximated in vivo conditions. This is an important consideration for hydroxysteroid dehydrogenases, which have been found to have altered rates of activity when assayed in vitro. Secondly, 3{alpha}-HSD activity was analyzed in subcellular fractions prepared from Leydig cells. Rat liver 3{alpha}-HSD is a cytosolic NADP(H)-dependent enzyme [12–15]. An antibody directed against this enzyme detected a 34-kDa (molecular mass) immunoreactive protein in Leydig cells, in amounts that were consistent with NADPH-dependent 3{alpha}-HSD activity (Fig. 1). Northern blotting and reverse transcription-polymerase chain reaction analysis confirmed the expression of the mRNA encoding rat liver 3{alpha}-HSD in Leydig cells [8,29]. Therefore, these studies indicate that the NADP(H)-dependent 3{alpha}-HSD in Leydig cells is similar to the enzyme in rat liver. The presence of this enzyme accounts for all of the measurable 3{alpha}-HSD activity in progenitor Leydig cells and for most of the cytosolic 3{alpha}-HSD activity in immature and adult Leydig cells.

In intact progenitor cells, the NADP(H)-dependent enzyme was exclusively reductive. Previous studies of 3{alpha}-HSD were performed using cell homogenates, cytosols, or preparations of purified enzyme [12–15, 20, 21], and those conditions might have altered 3{alpha}-HSD activities because exogenous pyridine nucleotide cofactors were added. The NADP(H)-dependent 3{alpha}-HSD in Leydig cells was equally oxidative and reductive when measured in all homogenates, which demonstrated that cofactor availability affected the prevailing direction of catalysis in 3{alpha}-HSD. Immature and adult Leydig cells had another 3{alpha}-HSD activity that was NAD(H)-dependent and localized in the microsomal fraction. The presence of NAD(H)-dependent activities in immature and adult, but not progenitor, Leydig cells may result from changes in the relative abundance of smooth endoplasmic reticulum (SER), a major constituent in microsomes. In progenitor Leydig cells, the SER is absent; and an abundant SER is not formed until the differentiation of immature Leydig cells during Days 28–35 postpartum in the rat [1]. The occurrence of NAD-dependent oxidation by a microsomally located 3{alpha}-HSD could account for the 30-fold higher oxidation rate in intact adult Leydig cells compared to progenitor cells, and it is probable that this NAD-dependent 3{alpha}-HSD is associated with the SER.

The results of the present study are consistent with earlier data on the heterogeneity of 3{alpha}-HSDs. Although different forms of 3{alpha}-HSD have been cloned in several species [30–32], it is unclear which gene encodes an NAD(H)-dependent 3{alpha}-HSD. All three isoforms of 3{alpha}-HSD cloned in humans are NADP(H)-dependent [30–32]. The heterogeneity of 3{alpha}-HSD is also complicated by the fact that other dehydrogenases such as several of the retinol dehydrogenase isoforms possess NAD(H)-dependent 3{alpha}-HSD activities [33–36].

The direction of the reaction catalyzed by the putative NAD(H)-dependent microsomal 3{alpha}-HSD could not be determined directly because NADP(H)-dependent activities were also present in Leydig cells. However, the previously described NADPH-dependent 3{alpha}-HSD is exclusively reductive in intact cells (in the present paper); this could account for all of the reductive activity observed in adult Leydig cells. Since the NAD/NADH ratio is normally high in intact cells [28, 29], NADH-dependent 3{alpha}-HSD reductive activity may not convert DHT to 3{alpha}-DIOL in intact Leydig cells. We hypothesize that a microsomal NAD(H)-dependent 3{alpha}-HSD catalyzes oxidative activity in vivo, which amplifies androgen action by generating the potent androgen, DHT, from the weak androgen, 3{alpha}-DIOL. This is corroborated by other studies showing that the NAD-dependent type II 11ß-hydroxysteroid dehydrogenase [17, 18] and type II 17ß-hydroxysteroid dehydrogenase [19] are exclusively oxidative in intact cells but catalyze NADH-dependent reduction in homogenates and subcellular fractions.

The present results help to resolve a discrepancy between the level of testicular 3{alpha}-HSD activity, thought to be high only before puberty, and testicular levels of DHT, which are high during puberty and low in adulthood. Leydig cells have an NADPH-dependent, cytosolic 3{alpha}-HSD that is exclusively reductive in progenitor cells. The unidirectional 3{alpha}-HSD accounts for metabolism of DHT to 3{alpha}-DIOL during Days 14–40 postpartum. The primary androgen is known to be 3{alpha}-DIOL, both in the testis and in circulation, during the period when progenitor and immature Leydig cells are the predominant stages of Leydig cell in the testis [37–39]. Progenitor and immature Leydig cells possess high 5{alpha}-reductase activity and produce DHT from testosterone [40]. In contrast, adult Leydig cells do not express 5{alpha}-reductase [40] and do not biosynthesize DHT from testosterone. This developmental shift in 5{alpha}-reductase and 3{alpha}-HSD activities suggests that the active androgen, DHT, is developmentally regulated by these two enzymes. The NAD-dependent 3{alpha}-HSD oxidase of immature and adult Leydig cells is capable of generating DHT from 3{alpha}-DIOL obtained from the circulation. DHT remains detectable in adult rat testis [39] probably as a result of 3{alpha}-HSD oxidative activation of 3{alpha}-DIOL in circulation. The production of DHT from 3{alpha}-DIOL could provide a more potent androgen to testicular cells, which may be important for spermatogenesis [41].

In conclusion, Leydig cells express the cytosolic liver type 3{alpha}-HSD, which behaves as a unidirectional NADPH-dependent reductase in intact cells. By Day 35, Leydig cells also express NAD(H)-dependent activity. We infer that a second microsomal 3{alpha}-HSD enzyme is responsible for the predominance of oxidation over reduction in adult Leydig cells on Day 90.


   ACKNOWLEDGMENTS
 
The technical assistance of Ms. Chantal Manon Sottas is gratefully acknowledged. We also thank Dr. Trevor M. Penning (Department of Pharmacology, University of Pennsylvania, Philadelphia, PA) for discussions of the preliminary results and for providing the 3{alpha}-HSD antibody.


   FOOTNOTES
 
1 Supported by NIH grant R29-HD32588. Back

2 Correspondence: Matthew P. Hardy, The Population Council, 1230 York Avenue, New York, NY 10021. FAX: 212 327 7678; m-hardy{at}popcbr.rockefeller.edu Back

Accepted: November 11, 1998.

Received: August 7, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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