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Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Dr. Wen-Chao Song, Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 905 Stellar-Chance Laboratories, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: song{at}spirit.gcrc.upenn.edu
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Abstract |
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-dihydrotestosterone (5-DHT). The
activity of 5-DHT in stimulating EST expression was blocked by the
androgen receptor antagonist, hydroxyflutamide. These data suggested
the involvement of androgen in (Bu)2cAMP-induced EST
expression. Further evidence came from the study with interleukin-1ß,
another agent known to suppress Leydig cell steroidogenesis by
down-regulating P450c17 gene expression. Treatment of Leydig cells with
0.2 ng/ml interleukin-1ß inhibited (Bu)2cAMP-induced EST
expression, which was overcome by the addition of 5-DHT. Finally, in
the testis-feminized mouse (Tfm) in which the androgen
receptor is nonfunctional due to a frameshift mutation, testicular EST
expression is completely absent, whereas messenger RNAs of
steroidogenic enzymes such as P450c17 and 3ß-hydroxysteroid
dehydrogenase are relatively abundant. We conclude that, by acting as
an autocrine or paracrine factor, androgen plays an essential role in
the regulation of estrogen sulfotransferase expression in Leydig cell
by LH and cAMP. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In earlier investigations, we have found and reported that EST is
expressed prominently in testicular Leydig cells of male animals and
man (9, 10). In many respects, the testis may be regarded as an
estrogen target as well as an estrogen-secreting tissue. It is well
recognized that the estrogen biosynthetic enzyme P450arom is expressed
(11, 12, 13), and a significant amount of estrogens is produced, in the
testes of rodents and man (14, 15). A physiological role for estrogen
in testicular biology is further supported by the detection of estrogen
receptors, both the and the newly discovered ß form, in the
testis (16, 17, 18) and by the unexpected phenotypes displayed by male
estrogen receptor knockout mice (19, 20). It is possible that
Leydig cell EST offers a protective role, preventing testicular cells
from excessive stimulation by the locally synthesized estrogen.
We have shown in our previous studies that expression of EST in mouse Leydig cells was correlated with sexual maturity and was under the sole control of LH (10). Thus, EST was not expressed in Leydig cells from prepubertal or hypophysectomized adult mice (10). Administration of hCG to hypophysectomized mice was sufficient to restore the testicular expression of EST (10). In this study, we sought to determine the downstream mechanisms by which LH/hCG exerts its regulatory effect on Leydig cell EST. Using primary cultures of mouse Leydig cells, we show that the in vivo stimulating effect of LH/hCG on EST expression could be mimicked in vitro by (Bu)2cAMP, and that the effect of (Bu)2cAMP required the participation of androgen as an autocrine or paracrine factor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone (5-DHT), and
interleukin-1ß (IL-1ß) were obtained from Sigma Chemical Co. (St. Louis, MO). Hydroxyflutamide was a gift from Dr. R.
Neri (Schering Corp., Kennilworth, NJ).
Isolation and purification of mouse Leydig cells
Mouse Leydig cells were isolated and cultured by a procedure
modified from methods described previously (21, 22). Briefly, mice were
killed by cervical dislocation, and testes were removed aseptically.
After decapsulation, tubules and interstitial cells from 6–10 mice
were dispersed by shaking for 12 min at 37 C in 10 ml medium 199
(Life Technologies, Grand Island, NY) containing
collagenase (0.4 mg/ml, type I-S, Sigma Chemical Co.).
Intact tubules were left to settle under gravity for 2 min on ice, and
the medium, containing dissociated interstitial cells, was collected by
centrifugation at 1000 x g for 5 min. The cell pellet
was resuspended in 6 ml medium 199 supplemented with 2.2 µg/liter
sodium bicarbonate, 10 mM HEPES (pH 7.4), 500 ng/ml insulin
(Sigma Chemical Co.), 100 IU/ml penicillin, 100 µg/ml
streptomycin, and 1 mg/ml BSA. Three milliliters of the resuspended
cells were loaded onto a five-layer discontinuous Percoll gradients
(21, 26, 34, 40, and 60% Percoll in PBS) and centrifuged at 800
x g for 30 min.
The location of Leydig cells in the discontinuous Percoll gradients was determined in pilot experiments by Western blot detection of EST. The percentage of Leydig cells in the identified fraction was quantified by staining 3ß-hydroxysteroid dehydrogenase (3ßHSD) as a further Leydig cell marker (22). The histochemical staining of 3ßHSD was performed on paraformaldehyde-fixed cells as described using NAD+, nitro blue tetrazolium, and 3ß-hydroxyandrostan-17-one (22). After staining, cells that contained dark blue formazan deposits of reduced-nitroblue tetrazolium were counted as Leydig cells (22). By this standard, highly purified Leydig cells (>90%) were recovered from the 40% layer of the Percoll gradient. These cells were used in all of the experiments described below.
Culture of mouse Leydig cells
Purified Leydig cells were plated on day 1 in Waymouth medium
(Life Technologies) containing 15% horse serum at a
density of 0.5–1 x 106 cells/60-mm dish and
incubated in a humidified atmosphere of 95% air-5% CO2 at
32 C. On the following day (day 2), cells were changed to serum-free
medium consisting of a 1:1 mixture of Ham’s F-12 and DMEM (Life Technologies) supplemented with 2.2 g/liter sodium bicarbonate,
10 mM HEPES (pH 7.4), 500 ng/ml insulin, 100 IU/ml
penicillin, 100 µg/ml streptomycin, and 1 mg/ml BSA. They were
maintained under this culture condition for the remainder of the
experiment with daily medium change. Cells were treated on day 4 with
various agents or combinations of them as specified
[(Bu)2cAMP, aminoglutethimide, 5-DHT,
hydroxyflutamide, and IL-1ß] for 24 or 48 h. Control cells were
treated with equivalent amounts of solvent vehicles (ethanol or water).
At the end of the treatment, cells were harvested for total RNA
extraction.
Northern blot analysis
Total RNAs from cultured mouse Leydig cells or testes were
isolated using the Trizol reagent (Life Technologies). RNA
samples (5 or 10 µg in each lane) were separated on a 1.0%
formaldehyde-agarose gel and transferred onto a nylon membrane
(Hybond-N, Amersham, Arlington Heights, IL) via capillary action
overnight in 5 x SSC. Membranes were cross-linked under UV and
hybridized first with a 32P-labeled full-length mouse EST
complementary DNA (cDNA) probe (9, 10) synthesized with random primers.
In some experiments, membranes were stripped after detection with the
EST probe and rehybridized with a cDNA probe for the mouse P450c17 or
3ßHSD. To generate a probe for the mouse Cyp17 messenger RNA (mRNA),
the following primers, 5'-GCC-TGA-CAG-ACA-TTC-TG-3' (upstream) and
5'-TCG-TGA-TGC-AGT-GCC-CAG-3' (downstream), were used in a RT-PCR to
amplify a 420-bp cDNA fragment (23). Similarly, the following two
primers, 5'-TGG-TGA-CAG-GAG-CAG-GA-3' (upstream) and
5'-AGG-AAG-CTC-ACA-GTT-TCC-A-3' (downstream), were used to generate a
890-bp 3ßHSD cDNA by RT-PCR (23). All RNA hybridizations were carried
out in QuikHyb solution (Stratagene, La Jolla, CA) at 68 C
for 1 h. The membranes were washed, first in 2 x SSC-0.1%
SDS at 55 C for 15 min and then in 0.1 x SSC-0.1% SDS at 55 C,
and exposed to x-ray film.
Assay of EST activity
Sulfotransferase activity of cultured Leydig cells was measured
with 3H-labeled estradiol
([2,4,6,7-N-3H]estradiol; 87.6 Ci/mmol; DuPont New England Nuclear; final concentration, 1.2 nM) in 200
µl 200 mM Tris-acetate buffer, pH 7.9, containing 10
mM Mg acetate, 1.25% Triton X-100, and 100
µM 3'-phosphoadenosine-5'-phosphosulfate. Cells
were scraped off the plate, washed with PBS, and sonicated after
resuspension in PBS. The cell lysate was then centrifuged (10,000
x g), and the supernatant was collected. The protein
concentration of the supernatant was determined by the Bradford method
with a colorimetric assay kit from Bio-Rad Laboratories, Inc. (Richmond, CA). The activity assay was started by adding
the substrate to the incubation mixture that contained Leydig cell
protein extract. The reaction was continued for 30 min at 37 C, and the
mixture was subsequently extracted with 2 vol dichloromethane. An
aliquot of the aqueous phase from each sample was then counted and
taken as a measure of the amount of sulfated products.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Newly isolated Leydig cells contained
high amounts of EST and Cyp17 mRNAs (Fig. 1, day 1). When plated and
kept in serum-containing medium, levels of Cyp17 and EST mRNAs
decreased rapidly with time. The time-dependent decline in EST
expression appeared to lag somewhat behind that of Cyp17. Nevertheless,
by day 4, mRNAs for both enzymes dropped to undetectable levels (Fig. 1). Omission of serum from the culture medium has previously been found
to prolong Cyp17 expression in primary cultures of mouse Leydig cells
(Hales, D. B., personal communication). This was found to be true
as well for the EST enzyme. When the cells were changed to serum-free
medium after overnight attachment, a moderate amount of EST mRNA could
be detected for several days (up to a week; data not shown).
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shows that
treatment of cultured Leydig cells with 100 µM
(Bu)2cAMP time dependently increased EST mRNA
expression (Fig. 2A) and enzyme activity (Fig. 2B). The stimulating
effect of (Bu)2cAMP was evident at 24 h.
After 48 h of treatment, the level of EST mRNA in the treated
cells increased 3- to 5-fold compared with the control value. This
up-regulation of Leydig cell EST by (Bu)2cAMP was
again correlated with concomitant induction of Cyp17 expression (Fig. 2).
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shows that aminoglutethimide
attenuated the stimulating effect of (Bu)2cAMP on
Leydig cell EST expression. We next investigated whether exogenous
androgen could directly induce EST expression in the cultured Leydig
cells. Figure 3B shows that addition of 0.1 µM 5-DHT to
the cells induced EST expression. Furthermore, this stimulating effect
of androgen could be blocked by the androgen receptor antagonist,
hydroxyflutamide, added at a final concentration of 0.1
µM (Fig. 3B). To further confirm the up-regulation of EST
expression by androgen, the EST enzyme activity in Leydig cells treated
with increasing concentrations of 5-DHT was determined. Figure 3C shows
that 5-DHT increased EST activity in the treated cells in a
dose-dependent manner.
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shows that
addition of 0.2 ng/ml IL-1ß to the culture medium essentially
abolished the cAMP-induced EST expression. However, this inhibitory
effect of IL-1ß could be overcome in large part by addition of 5-DHT
to the culture medium (Fig. 4). Thus, the negative regulatory activity
of IL-1ß on EST did not appear to be direct and most likely involved
inhibition of Cyp17 gene expression and steroidogenesis (22).
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shows
that testicular EST is not expressed in Tfm mice. In
contrast, both Cyp17 (Fig. 5) and 3ßHSD (not shown) are expressed in
relatively abundant amounts.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous in vivo study in mice has shown that Leydig cell EST expression was correlated with the sexual maturity of the animal, supporting the idea that EST plays a role in male reproduction (10). Additionally, we have established that LH was necessary and sufficient for testicular expression of EST in adult mice (10). In the present study, we have used primary cultures of mouse Leydig cells and defined further details in the regulation of EST by LH. We demonstrate that the stimulative effect of LH could be mimicked by (Bu)2cAMP in cultured Leydig cells. We also present evidence indicating that the effects of LH and cAMP on EST gene expression are mediated by receptor-dependent androgen action.
That androgen plays an obligatory role in Leydig cell EST expression is supported by several lines of evidence. First, aminoglutethimide, a steroidogenesis inhibitor, attenuated the cAMP-induced EST expression. Secondly, exogenously added 5-DHT was shown to stimulate EST expression in a dose-dependent manner, and this stimulation could be blocked by the androgen receptor antagonist, hydroxyflutamide. Thirdly, the cytokine IL-1ß, which is known to inhibit Leydig cell steroidogenesis by negatively regulating P450c17 gene expression, blocked cAMP-induced EST expression. Furthermore, the inhibitory effect of IL-1ß could be overcome by the addition of exogenous androgen, 5-DHT. Finally, EST is not expressed in the androgen receptor-insensitive Tfm mice, providing more direct evidence that receptor-dependent androgen action is required for Leydig cell EST expression.
The Tfm mouse is a particularly useful model system to differentiate the direct vs. the indirect effect of LH on EST gene expression. Studies by other investigators have found that although there is an impairment in Leydig cell development at puberty, total Leydig cell numbers in adult Tfm mice are more or less the same as those in normal mice (28). On the other hand, the lack of androgen activity impairs the negative feedback mechanism of the hypothalamus-pituitary-testis axis, resulting in elevated serum LH level in adult Tfm mice (28). The fact that P450c17, a sensitive marker gene for LH activity in Leydig cells, is still expressed prominently in the Tfm mouse testis suggests that the coupling of LH to its receptor is not blocked. Thus, the lack of EST expression in Tfm mice cannot be attributed to Leydig cell atrophy or lack of coupling between LH and its receptor. Additionally, the expression of both P450c17 and 3ßHSD (data not shown) in Tfm mouse testis suggests that the lack of EST expression is not a nonspecific phenomenon related to cryptorchidism.
The stimulation of Leydig cell EST expression appeared to be specific
to androgen, as estrogen did show the same effect (result not shown).
Also, unlike in the androgen receptor-defective Tfm mice
(Fig. 5
), EST was shown to be expressed abundantly in the Leydig cells
of estrogen receptor knockout mice (unpublished result, tissue
slides kindly provided by Dr. K. Korach). It is of interest to relate
the result of this study to the earlier finding that androgen plays a
role in the regulation of hepatic EST (4, 29). Expression of EST in the
mouse (10) and rat (4) liver is male specific, and in the case of the
rat, it was shown to correlate with markers of androgen sensitivity in
the liver (4, 29). Thus, EST was expressed in the androgen-sensitive
liver of mature adult rats but not in the androgen-insensitive liver of
prepubertal or senile rats (29). Furthermore, treatment with 5-DHT
induced abnormal hepatic EST expression in ovariectomized female rats
(29). Together, these findings indicate that the stimulating effect of
androgen on the EST gene is not limited to Leydig cells or hepatocytes,
and that androgen can function as both an endocrine and an autocrine
factor in this regard. The molecular mechanism by which androgen
regulates the EST gene, e.g. whether there are
androgen-responsive elements in the EST gene promoter, remains to be
determined. However, there must be other factors, in addition to the
androgen receptor, that participate in this process, as EST is not
expressed in all androgen target tissues or in fetal Leydig cells where
androgen biosynthesis also takes place (10).
As androgen serves as a precursor for estrogen biosynthesis, induction of EST by androgen constitutes a self-triggered regulatory mechanism that ensures that the activity of estrogen in the testis is properly controlled. There is now mounting evidence to suggest that estrogen is essential for testicular function (19, 20, 30). On the other hand, excessive estrogen activity could become detrimental to steroidogenesis and spermatogenesis (31, 32, 33, 34, 35). The androgen-responsive estrogen sulfotransferase may be one of the mechanisms that keep in balance the desirable and unwanted actions of estrogen in the testis.
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Acknowledgments |
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Footnotes |
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Received July 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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knock-out and wild-type mice. Endocrinology 139:982–2987-hydroxylase/C17–20 lyase
cytochrome P450 expression. Endocrinology 131:2165–2172-hydroxylase/C17–20 lyase, and P450 aromatase in
fetal mouse gonads. Endocrinology 135:262–268[Abstract]
-hydroxylase/C17–20
lyase in the fetal rat testis is reduced by maternal exposure to
exogenous estrogens. Endocrinology 137:1063–1070[Abstract]
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