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The Population Council and Rockefeller University, New York, New York 10021
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-reduced androgens on day 35, and
adult Leydig cells (ALC) that are fully capable of testosterone
biosynthesis on day 90. Numbers of GR, measured by
[3H]dexamethasone binding, in purified cells were
6.34 ± 0.27 (x103 sites/cell; mean ±
SE) for PLC, 30.45 ± 0.74 for ILC, and 32.54 ±
0.84 for ALC. Although GR binding was lower in PLC, steady state levels
for GR mRNA were equivalent at all three stages (P
> 0.05). Oxidative and reductive activities of 11ßHSD were measured
by assaying the conversion of radiolabeled substrates in incubations of
intact Leydig cells. Both oxidative and reductive activities were
barely detectable in PLC, intermediate in ILC, and highest in ALC. The
ratio of the two activities favored reduction in PLC and ILC and
oxidation in ALC (oxidation/reduction, 0.33 ± 0.33 for PLC,
0.43 ± 0.05 for ILC, and 2.12 ± 0.9 for ALC, with a ratio
of 1 indicating equivalent rates for both activities). The mRNA and
protein levels of type I 11ßHSD in Leydig cells changed in parallel
with 11ßHSD reductive activity, which increased gradually during the
transition from PLC to ALC, compared with the sharp rise that was seen
in oxidative activity. We conclude that Leydig cells at all
developmental stages have GR and that their ability to respond to
glucocorticoid diminishes as net 11ßHSD activity switches from
reduction to oxidation. This provides a mechanism for the Leydig cell
to regulate its intracellular concentration of corticosterone, thereby
varying its response to this steroid during pubertal development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid dehydrogenase (3HSD) and insulin-like growth
factor I are expressed in Leydig cell precursors (10, 11); however, the
role of 3HSD in the testis has not been determined. Furthermore, the
presence of GR in Leydig cell precursors has not been directly
established. 11ßHSD, which catalyzes the interconversion of glucocorticoid [corticosterone (CORT) in the rat] and its biologically inert metabolite, 11-dehydrocorticosterone (11DHC), is an important determinant of the intracellular level of bioactive steroid (reviewed in Ref.12). Predominance of the oxidative activity results in glucocorticoid inactivation, whereas the reverse reaction, 11ßHSD reduction, has the opposite effect. In organs such as liver and lung, 11ßHSD acts primarily as a reductase, increasing the availability of active glucocorticoids (13, 14). Recent studies show that reductase activity predominates in both human and rat type I 11ßHSD (15, 16). In contrast, the other 11ßHSD isoform, type II, has been found to be exclusively oxidative in other organs, such as the kidney, where it lowers glucocorticoid concentrations, preventing this steroid from binding nonspecifically to mineralocorticoid receptors (17). In humans, the condition of apparent mineralocorticoid excess results from deficient 11ßHSD oxidation, which is believed to be responsible for the abnormal increase in intracellular glucocorticoid; this is associated with mutations in the gene for type II, not type I (reviewed in Ref.18).
It has been established that 11ßHSD is a predominant oxidase in freshly isolated intact adult Leydig cells (19). However, Phillips et al. (20) observed that type I 11ßHSD is abundantly expressed in Leydig cells only after 4 weeks postpartum near the midpoint of puberty. Therefore, it was unclear whether GR-mediated action and glucocorticoid metabolism by 11ßHSD are involved in Leydig cell development. In the present study, we asked whether GR and 11ßHSD messenger RNA (mRNA) and protein are present in Leydig cells undergoing pubertal differentiation. Postnatal development of Leydig cells can be divided conceptually into three distinct stages of differentiation: initially, they exist as mesenchymal-like progenitors (PLC) by day 21; subsequently, as immature Leydig cells (ILC) by day 35, they acquire steroidogenic organelle structure and enzyme activities, but metabolize most of the testosterone that they synthesize; finally, as adult Leydig cells (ALC) by day 90, they actively produce testosterone (reviewed in Ref.21). Steady state levels of GR mRNA and numbers of glucocorticoid-binding sites were measured at these three distinct stages, as were 11ßHSD mRNA and oxidative and reductive activities. In contrast to the adult, 11ßHSD net reduction prevailed over oxidation in precursor stages of the Leydig cell.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell isolation
PLC from 20 21-day-old rats, ILC from 15 35-day-old rats, and
ALC from six 90-day-old rats were purified as described previously
(22). Purities of Leydig cell fractions were evaluated by histochemical
staining for 3ß-hydroxysteroid dehydrogenase activity, with 0.4
mM etiocholanolone as the steroid substrate (23). In the
PLC fraction, approximately 90% of the cells were lightly stained, and
of the remaining 10%, 6% were intensely stained. More than 95% ILC
and ALC were intensely stained.
[3H]Dexamethasone binding
The measurement of competitive binding to
[3H]dexamethasone was performed using intact Leydig cells
as previously described (24). In brief, Leydig cells were incubated in
suspension (1 x 106 cells in 0.4 ml) at 34 C for
1 h in phenol red-free Leydig cell medium (DMEM-Ham’s F-12),
containing 0.78–50 nM [3H]dexamethasone
(DuPont-New England Nuclear, Boston, MA; 46 Ci/mmol). A preliminary
study showed that incubation for 1 h was sufficient to reach
steady state binding. To correct for nonspecific binding, control tubes
were incubated in the presence of a 200-fold excess of unlabeled
dexamethasone. After incubation, cells were centrifuged and washed
three times with 1.5 ml ice-cold PBS (0.01 M; pH 7.4)
containing 1% BSA and pelleted by centrifugation. The cell pellets
were lysed with 0.5 ml hyamine hydroxide (ICN Radiochemicals, Irvine,
CA), and radioactivity was determined in a ß-counter.
Determination of 11ßHSD oxidative and reductive activities
11ßHSD oxidative and reductive activities were measured in
intact Leydig cells as previously described (25). For the assay of
11ßHSD oxidative activity, isolated intact Leydig cells (0.2 x
106) were incubated with 25 nM
[3H]CORT (DuPont-New England Nuclear; 88 Ci/mmol) in 0.5
ml phenol-red free medium (DMEM) at 34 C for 5 min. For 11ßHSD
reductive activity, 25 nM [3H]11DHC,
synthesized from [3H]CORT according to the technique
described by Lakshmi and Monder (26), was used instead of
[3H]CORT. The reaction was stopped by adding 2 ml
ice-cold ethyl acetate. The steroids were extracted with ethyl acetate,
and the organic layer was dried under nitrogen. The steroids were
separated chromatographically on thin layer plates in
chloroform-methanol (90:10), and the radioactivity was measured with a
scanning radiometer (System 200/AC3000, Bioscan, Washington DC). The
percent conversion of CORT to 11DHC and of 11DHC to CORT was calculated
by dividing the radioactive counts identified as 11DHC (or CORT,
respectively) by the total counts associated with CORT plus 11DHC.
RT-PCR detection of GR and 11ßHSD mRNAs
Rat GR and 11ßHSD complementary DNA (cDNA) fragments were
amplified by RT-PCR using total RNA from Leydig cells according to a
previously described procedure (27). In brief, total RNA was extracted
from isolated Leydig cells by a single step method, using phenol and
guanidinium thiocyanate (Tri-Reagent, Molecular Research Center,
Cincinnati, OH). Leydig cell total RNA (400 ng) was used as the
template for avian myeloblastosis virus reverse transcriptase in the
presence of random hexamers and deoxyribonucleotides at 42 C for 75
min, and the reaction was terminated by heating at 95 C for 5 min. GR
or type I 11ßHSD cDNA was coamplified with ribosomal protein S16
(RPS16) cDNA as an internal standard. PCR was initiated by
Taq DNA polymerase in the presence of
[-32P]deoxy-CTP and was allowed to proceed for 30
cycles with an annealing temperature of 50 C. The sequences of primers
for GR (forward, 5'-TGCAGCAGTGAAATGGGCAA-3'; reverse,
5'-GGGAATTCAATACTCATGGTC-3') were based on the rat GR cDNA sequence
(28). The primer sequences for type I 11ßHSD (forward,
5'-GAAGAAGCATGGAGGTCAAC-3'; reverse, 5'-CTCAAGATTATCCCAGAGGT-3') were
based on the sequence of rat type I 11ßHSD cDNA (29). The GR PCR
product (533 bp) was confirmed by sequence analysis. The identity of
the PCR product for type I (288 bp) 11ßHSD was confirmed by
restriction enzyme analysis after digestion with FokI.
Primer sequences for RPS16 were reported previously (27). Radioactive
PCR bands were visualized on autoradiographic film (Eastman Kodak,
Rochester, NY). Quantitative analysis of mRNA levels was performed
by scanning the films with a densitometer (Ultrascan, LKB, Bromma,
Sweden). The films were exposed and developed so that the optical
densities of bands fell within a linear gray scale on the response
curve. The background signals from the blank areas of the films were
subtracted from the specific, band-associated signals, after which the
signal intensities for GR and type I 11ßHSD were normalized to
RPS16.
Western blot analysis of type I 11ßHSD
Leydig cells were homogenized and boiled in equal volumes of
sample loading buffer, a Tris-Cl buffer (pH 6.8) containing 20%
glycerol, 5% SDS, 3.1% dithiothreitol, and 0.001% bromophenol blue.
Homogenized samples (25 µg protein) of PLC, ILC, and ALC and a
positive control, liver, were electrophoresed on 10% polyacrylamide
gels containing SDS (30). 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
1:2000 dilution of a rabbit polyclonal antitype I 11ßHSD antibody
(no. 56-127) that was generated by immunizing animals (31) with type I
11ßHSD purified from rat liver. The membranes were then washed and
incubated with a 1:2000 dilution of goat antirabbit antiserum that was
conjugated to horseradish peroxidase. The washing step was repeated,
and immunoreactive bands were visualized by chemiluminescence using a
kit (ECL, Amersham, Arlington Heights, IL). Protein levels were
measured by densitometry of the films as described for PCR band
analysis and normalized to liver type I 11ßHSD.
Data analysis
In each experiment, data were obtained from triplicate assays,
and the results are expressed as the mean ± SE. The
dissociation constants (Kd) and the maximal binding
(Bmax) for [3H]dexamethasone in Leydig cells
were estimated by calculation of the Scatchard plots of the ratio of
the amount of ligand bound (B) to the amount of free ligand (F) on the
y-axis against the amount of B on the x-axis
(32). Statistical analysis of the changes in
[3H]dexamethasone-binding sites and 11ßHSD oxidative
and reductive activities was performed by Kruskal-Wallis ANOVA followed
by multiple comparisons testing to identify significant differences
between groups (33). Statistical analysis of the relative levels of GR
mRNA and type I 11ßHSD mRNA and protein was performed after arcsin
transformation of the ratio data (31).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, all three stages of the Leydig cell
possessed [3H]dexamethasone-binding sites, indicating
that they contained GR. The binding affinity of Leydig cell binding
sites was unchanged during pubertal development, with dissociation
constants (Kd) of 5.05 ± 0.91, 4.91 ±
0.53, and 4.64 ± 0.55 nM for PLC, ILC, and ALC,
respectively. However, the numbers of binding sites were higher in ILC
and ALC compared with PLC (Fig. 1b), with 30.45 ± 0.74
(x103 sites/cell; mean ± SE;
P < 0.05) in ILC and 32.54 ± 0.84 in ALC
vs. 6.34 ± 0.27 in PLC. This suggested that GR was
present in each of the three stages of the Leydig cell, and that GR
numbers increase in Leydig cells as they differentiate.
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). It is unlikely, therefore, that
the number of GR is transcriptionally regulated.
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). Although 11ßHSD oxidative and
reductive activities both increased from PLC to ILC, the magnitudes of
the increases were different. 11ßHSD reduction was favored in PLC and
ILC, as demonstrated by ratios of oxidative to reductive activity
(0.33 ± 0.33 in PLC and 0.43 ± 0.05 in ILC; with a ratio of
1 indicating equivalent rates for both activities). In contrast,
11ßHSD oxidation was favored over reduction in ALC by a ratio of
2.13 ± 0.09. These results suggested that 11ßHSD oxidative and
reductive activities varied independently of one another during Leydig
cell differentiation. Moreover, 11ßHSD reductive activity
predominated in Leydig cell precursors, whereas in ALC, the enzyme was
primarily oxidative.
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). Type II 11ßHSD mRNA levels were
undetectable in all stages of Leydig cells (data not shown).
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). As shown in Table 1, the increase in type I mRNA and
protein in Leydig cells corresponded closely to 11ßHSD reductive
activity during the transitions from PLC to ILC and from ILC to ALC.
The same did not hold true for oxidative activity, which increased
disproportionately during the transition from ILC to ALC. This
suggested that type I 11ßHSD contributes both oxidative and reductive
activities in the precursor stages of Leydig cells, with the reductive
activity being predominant. The large increase in 11ßHSD oxidative
activity seen in ALC could either result from a novel isoform expressed
in the mature stage in addition to type I (25) or through
posttranslational modification of the type I protein.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The high levels of GR in ILC on day 35 indicate that ILC can respond to
glucocorticoid at a time when bioactive CORT reaches its highest serum
concentrations (38). Activities of 11ßHSD activities were higher in
ILC than in PLC, and 11ßHSD reductive activity predominated over
oxidation. As with other tissues in which type I 11ßHSD is postulated
to increase concentrations of active glucocorticoid (13, 14), reductive
activity was predominant in prepubertal Leydig cells. The high
intracellular concentration of CORT resulting from 11ßHSD activity in
precursor stages of the Leydig cell is significant in light of studies
showing that glucocorticoids potentiate the sensitivity of prepubertal
Leydig cells to LH (9) and stimulate the expression of 5-reductase
and 3HSD (39, 40), enzymes that are transiently elevated during
Leydig cell differentiation (10, 41). The transient rise in
5-reductase and 3HSD expression leads to secretion of
5-reduced androgens by precursor stages of the Leydig cell (reviewed
in Ref.21). In adult Leydig cells, however, glucocorticoids inhibit
testosterone production. Therefore, the predominance in oxidative
11ßHSD activity, as we have shown, would be required to inactivate
CORT and lower expression of 5-reductase and 3HSD (10, 30).
The present data show that 11ßHSD oxidative and reductive activities have distinct developmental trends in Leydig cells. As type II 11ßHSD is exclusively oxidative, the reductive activity can only originate from type I 11ßHSD. Accordingly, we examined the relationship between 11ßHSD reductive activity and the amount of the type I isoform in differentiating Leydig cells. The trends for 11ßHSD reductive activity and type I mRNA and protein were parallel, indicating that 11ßHSD reductive activity is conferred by this isoform. In contrast, the predominance of 11ßHSD oxidation in adult Leydig cells is difficult to reconcile with the enzymatic properties of the two known isoforms. Previous studies demonstrated that adult Leydig cells contain type I 11ßHSD mRNA and protein (19, 25) and NADPH-dependent 11ßHSD reductive activity (25). If adult Leydig cells contained type I alone, 11ßHSD reductive activity should predominate, as has been shown for liver, lung, uterus (13, 14, 15, 16), and cell lines transfected with type I 11ßHSD cDNA (42, 43). Type II 11ßHSD is a high affinity NAD-dependent oxidase, but is not detected in adult Leydig cells by Northern blot, in situ hybridization (34, 35), or RT-PCR (25). We have recently shown that ALC contain a high affinity (Km = 41 nM) NADP-dependent 11ßHSD in addition to low affinity oxidative and reductive activities that are typical of type I (25). A high affinity NADP-dependent 11ßHSD in adult Leydig cells would account for the predominance of oxidative activity in the enzyme at the physiological levels of glucocorticoids; under conditions of stress CORT levels (>100 nM), this low capacity component becomes saturated, and the high capacity type I 11ßHSD reductase would then predominate (25, 44).
A low Km, NADP-dependent 11ßHSD with exclusively oxidative activity is also present in sheep kidney and the choriocarcinoma cell line, JEG-3 (45, 46). The presence of a high affinity NADP-dependent 11ßHSD oxidative activity could result from the presence of a third isoform or from posttranslational modification of type I 11ßHSD: type I 11ßHSD is a glycosylated protein, and glycosylation of the correct amino acid residues is required for full enzymatic activity. Mutation in the first potential N-linked glycosylation site (asparagine-X-serine, residues 158–160) had a negligible effect on dehydrogenase activity, but caused a 50% decline in reductive activity. Mutations in the second site (asparagine-X-serine, residues 203–205) completely abolished both activities of the enzyme (47).
In conclusion, GR exist in Leydig cells at all three stages of pubertal development. The presence of GR and the predominance of 11ßHSD reductive activity in prepubertal Leydig cells may be important for their differentiation. The switch in the predominant direction of catalysis of 11ßHSD from reduction to oxidation in adult Leydig cells may protect this cell type from glucocorticoid-mediated inhibition of steroidogenesis. In this way, testosterone production is maintained in the presence of normal serum concentrations of corticosterone and is inhibited only if 11ßHSD oxidative capacity in Leydig cells is exceeded by high levels of corticosterone under stress conditions.
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Acknowledgments |
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Footnotes |
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Received July 8, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reductase messenger ribonucleic acid levels and immunocytochemical
localization of the type I protein in the rat testis during postnatal
development. Endocrinology 136:5409–5415[Abstract]
-reductase. Endocrinology 115:2464–2472[Abstract]
-hydroxysteroid dehydrogenase:
three dimensional structure and gene regulation. J Endocrinol
150:S175–S187
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), and adult Leydig
cells (
) was determined in intact cells as described in
Materials and Methods. Binding was determined over a
concentration range of 0.8–50 nM
[3H]dexamethasone in the absence or presence of a
500-fold excess of unlabeled dexamethasone for 1 h at 34 C. The
saturation plot is shown in a. The inset presents the
Scatchard plot of the binding data. The numbers of glucocorticoid
binding sites are calculated for conditions of maximum binding (b).
Each point is the mean of triplicate assays and is
representative of three experiments performed. Shared alphabet
letters refer to groups that were not significantly different
at P < 0.05.
