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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: m-hardy{at}popcbr.rockefeller.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-androstan-3,17ß-diol (3-DIOL)
is the predominant androgen secreted by the testes of immature rats
during days 20–40 postpartum, whereas T is the major androgen by day
56. However, the underlying changes in T biosynthetic and metabolizing
enzymes during Leydig cell development and their magnitudes have
remained unclear. The aim of the present study was to define the
developmental trends for T biosynthetic and metabolizing enzymes in
Leydig cells at three distinct stages of pubertal differentiation:
mesenchymal-like progenitors on day 21, immature Leydig cells on day
35, and adult Leydig cells on day 90. Production rates for precursor
androgen (androstenedione), T, and 5-reduced androgens
[androsterone (AO) and 3-DIOL] were measured in progenitor,
immature, and adult Leydig cells in spent medium after 3 h
in vitro. Steady state messenger RNA (mRNA) levels and
enzyme activities of biosynthetic and metabolizing enzymes were
measured in fractions of freshly isolated cells at each of the three
stages. Unexpectedly, progenitor cells produced significant amounts of
androgen, with basal levels of total androgens (androstenedione, AO, T,
and 3-DIOL) 14 times higher than those of T alone. However, compared
with immature and adult Leydig cells, the capacity for steroidogenesis
was lower in progenitor cells, with a LH-stimulated production rate for
total androgens of 84.33 ± 8.74 ng/106 cells·3 h
(mean ± SE) vs. 330.13 ± 44.19
in immature Leydig cells and 523.23 ± 67.29 in adult Leydig
cells. The predominant androgen produced by progenitor, immature, and
adult Leydig cells differed, with AO being released by progenitor cells
(72.08 ± 9.02% of total androgens), 3-DIOL by immature Leydig
cells (73.33 ± 14.52%), and T by adult Leydig cells (74.38
± 14.73%). Further examination indicated that changes in the
predominant androgen resulted from differential gene expression of T
biosynthetic and metabolizing enzymes. Low levels of type III
17ß-hydroxysteroid dehydrogenase (17ßHSD) mRNA and enzyme activity
were present in progenitor cells compared with immature and adult
Leydig cells. In contrast, levels of type I 5-reductase (5R) and
3-hydroxysteroid dehydrogenase (3HSD) mRNA and enzyme activities
were dramatically lower in adult Leydig cells compared with those in
progenitor and immature Leydig cells. Several T biosynthetic enzymes
attained equivalent levels in immature and adult Leydig cells, but T
was rapidly metabolized in the former to 3-DIOL by high 5R and
3HSD activities, which were greatly reduced in the latter.
Therefore, declines in 5R and 3HSD activities are hypothesized to
be a major cause of the ascendancy of T as the predominant androgen end
product produced by adult Leydig cells. These results indicate that
steroidogenic enzyme gene expression is not induced simultaneously, but
through sequential changes in T biosynthetic and metabolizing enzyme
activities, resulting in different androgen end products being secreted
by Leydig cells during pubertal development. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-androstan-3,17ß-diol (3-DIOL) and
androsterone (AO) are abundant in the circulation between days 20–40
postpartum when T is still low (1). Testicular tissue of rats aged
15–40 days metabolizes radiolabeled progesterone and T to 3-DIOL
and, to a lesser extent, AO (2, 3, 4, 5). Testicular enzyme activities of
3ß-hydroxysteroid dehydrogenase (3ßHSD), 17-hydroxylase/C17–20
lyase (P450c17), and 17ß-hydroxysteroid dehydrogenase
(17ßHSD) increase gradually between the ages of 20 and 60 days and
plateau thereafter (6, 7). The amount of testicular 5-reductase
(5R) activity, on the other hand, sharply increases between days 20
and 40, and then falls between days 40 and 60 (7). Further analysis
demonstrated that type I 5R and 3-hydroxysteroid dehydrogenase
(3HSD) messenger RNA (mRNA) and protein are abundantly present in
progenitor and immature Leydig cells during days 15–35 (8, 9). These
findings suggest that the Leydig cell itself is a metabolizing site for
androgens during puberty, at least via the 5-reduction pathway.
However, technical barriers prevented the testing of this hypothesis,
because steroidogenic enzymes such as 17ßHSD (10, 11, 12), 5R (13, 14), and 3HSD (14, 15) are present in other cell types in the
testis. Therefore, purified Leydig cells were examined in the present
study to delineate T biosynthesis and metabolism occurring in this cell
type during pubertal differentiation.
Androstenedione (DIONE; a precursor of T), T, and the 5-reduced
metabolites, AO and 3-DIOL, were measured in purified progenitor
Leydig cells on day 21, in immature Leydig cells on day 35, and in
adult Leydig cells on day 90. The predominant androgen end product
varied depending on the stage of differentiation; it was AO in
progenitor Leydig cells, 3-DIOL in immature Leydig cells, and T in
adult Leydig cells. These results led to a further examination of
T biosynthetic [cholesterol side-chain cleavage enzyme
(P450scc), 3ßHSD, P450c17, and 17ßHSD] and
metabolizing (5R and 3HSD) enzyme activities in Leydig cells. The
results showed that activities of T biosynthetic and metabolizing
enzymes in developing Leydig cells determine not only their capacity
for T production but also the predominant androgen end product that is
secreted.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxyprogesterone,
[1ß,2ß-N-3H]androst-4-ene-3,17-dione,
[1,2,6,7-N-3H]T,
[1,2-N-3H]dihydrotestosterone,
5-[9,11-N-3H]androstane-3,17ß-diol, and
[9,11-N-3H]AO were purchased from DuPont-New England
Nuclear (Boston, MA). [1,2,6,7-N-3H]Progesterone was
purchased from Amersham International (Aylesbury, UK). Nonradioactive
steroids were purchased from Sigma Chemical Co. (St. Louis, MO) or
Steraloids (Wilton, NH).
4-Methyl-aza-3-oxo-5-pregnan-20(S)-carboxylate, an
inhibitor of 5R, was provided by Merck (Rahway, NJ). The antibodies
for 3-DIOL and AO in RIA were provided by Dr. D. T. Armstrong
(Department of Obstetrics and Gynecology, University of Western
Ontario, London, Ontario, Canada).
Animals
Sprague-Dawley rats (dams with litters of male pups, immature
males, and adult males) were purchased from Charles River Laboratories
(Wilmington, MA). The males rats were 21, 35, and 90 days of age on the
day of Leydig cell isolation. The animals were killed by asphyxiation
with CO2. The animal protocol was approved by the
institutional animal care and use committee of the Rockefeller
University (Protocol 91200). A complete description of the procedure
that was used to isolate each of the three stages of Leydig cell
differentiation has been published (16, 17). The purity of cell
fractions was evaluated by histochemical staining for 3ßHSD activity
with 0.4 mM etiocholanolone as the steroid substrate (18).
Enrichment of the three fractions was typically more than 95%. The
absence of androgen-binding protein mRNA in the cell fractions
demonstrated that there was no appreciable contamination by Sertoli
cells.
Androgen production
Isolated progenitor Leydig cells, immature Leydig cells, and
adult Leydig cells were incubated at a concentration of 0.1–0.25
x 106 cells/ml in Leydig cell culture medium consisting of
DMEM and Ham’s F-12 medium (D2906, Sigma Chemical Co.) buffered with
15 mM HEPES and 14 mM NaHCO3 and
containing 1% BSA for 3 h at 34 C in a shaking water bath.
Incubations of triplicate samples were conducted in medium alone
(basal) or in medium plus a maximally stimulating dose of ovine LH (100
ng/ml). At the end of 3 h, the samples were centrifuged at
500 x g. Supernatants were extracted with 2 ml ethyl
acetate twice, and the organic layer was dried under nitrogen gas.
Steroids in the samples were fractionated using Sephadex LH-20
(Pharmacia Biotech, Uppsala, Sweden) column chromatography as
previously described (19). The elution system was
chloroform-butane-ethanol (50:50:1, vol/vol/vol) saturated with
distilled water. Clear separation of DIONE, AO, T, and 3-DIOL in
this system was confirmed using radiolabeled steroids (19). The
recovery rates following extraction and column separation (DIONE,
90.7 ± 5.8%; AO, 76.2 ± 2.0%; T, 73.9 ± 3.7%;
3-DIOL, 86.0 ± 1.2%) were used to correct the final
concentration measured by RIA. RIAs of DIONE, AO, T, and 3-DIOL were
performed as previously described (20, 21, 22). The results of three
separate experiments were averaged for statistical analysis.
Enzyme assay
With the exception of P450scc, steroidogenic enzyme
activities were measured by incubation of purified Leydig cells with
radiolabeled substrates and separation of products by TLC as previously
described (23, 24, 25). The substrate concentrations used for each enzyme
were maximal to ensure that the concentration of substrate was not rate
limiting. Control samples of culture medium alone were run in parallel
with each enzyme assay. Briefly, reaction mixture (0.5 ml) was prepared
in Leydig cell medium that contained 1 µM substrate (1
µCi) in medium. As T undergoes 5-reduction in immature Leydig
cells, 4-methyl-aza-3-oxo-5-pregnan-20(S)-carboxylate (2
µM) was used to inhibit 5R when P450c17 or
17ßHSD was measured. The reaction mixture was maintained at pH 7.2.
Reactions were initiated by adding to the reaction medium an aliquot of
0.1–0.2 x 106 Leydig cells. The reaction mixtures,
conducted in triplicate, were maintained at 34 C in a shaking water
bath for 10 min. Reactions were terminated by adding ice-cold ethyl
acetate, and steroids were rapidly extracted. The organic layer was
dried under nitrogen. The radioactivity was measured using a
radiometric scanner (System 200/AC3000, Bioscan, Washington DC). The
activity of 3ßHSD was determined by measuring conversion of
pregnenolone to progesterone. P450c17 catalyzes two mixed
function oxidase reactions: 17-hydroxylation and C17–20 cleavage.
The activity of 17-hydroxylation was determined by measuring
conversion of progesterone to 17-hydroxyprogesterone, DIONE, and T.
The activity of C17–20 cleavage was determined by measuring conversion
of 17-hydroxyprogesterone to DIONE and T. The activity of 17ßHSD
was determined by measuring the conversion of DIONE to T. The activity
of 5R was determined by measuring the conversion of T to
dihydrotestosterone, 3-DIOL, and 5-androstane-3ß,17ß-diol.
The activity of 3HSD was determined by measuring the conversion of
dihydrotestosterone to 3-DIOL. The steroids were separated on TLC
plates in chloroform-methanol (97:3) for 3ßHSD, 17ßHSD, and 5R
assays; chloroform-ether (7:1, vol/vol) for P450c17
17-hydroxylation and C17–20 cleavage assays; and diethyl
ether-acetone (98:2) for the 3HSD assay.
Activity of P450scc was determined by measuring the conversion of side-chain labeled 25-[26,27-3H]hydroxycholesterol to radioactive 4- hydroxyl-4-methyl-pentanoic acid as previously described (26). Leydig cells were incubated in a total volume of 0.5 ml medium containing 1 µCi 25-[26,27-3H]hydroxycholesterol (1 µM 25-hydroxycholesterol). Incubations were performed for 30 min at 34 C, and at the end of the incubation 0.5 ml NaOH (0.5 M) was added. The mixture was extracted twice with 2 ml chloroform and mixed with neutral alumna to remove nonmetabolized substrate (26), and an aliquot was removed for measurement by liquid scintillation counting.
RT-PCR
Within the cytochrome P450 enzyme superfamily,
P450scc is encoded by a single P450scc gene
(designated CYP11A1), and P450c17 is encoded by a single
P450c17 gene (designated CYP17). However, the existence of
multiple isoforms of hydroxysteroid dehydrogenases and 5R have been
demonstrated. In the rat, four distinct 3ßHSD complementary DNAs
(cDNAs) have been identified (27). These four isoforms share 76–94%
identity in their amino acid sequences (28, 29). Types I and II are the
only isoforms that are expressed in rat testis (28).
Four distinct 17ßHSD cDNAs have been cloned in several species (reviewed in Ref. 30). Although three isoforms have been identified in the rat (30), it is believed that the type III 17ßHSD isoform is the major protein in the testis responsible for the conversion of DIONE to T (31). In the rat, however, type III 17ßHSD cDNA has not been cloned. Therefore, we designed a pair of primers to amplify the common sequence of human and mouse type III 17ßHSD (31, 32). The primers amplified a 360-bp product from Leydig cell cDNA. The PCR product was partially sequenced and analyzed with respect to the known isoforms (rat types I, II, and IV 17ßHSD, type III human and mouse 17ßHSD) (31, 32, 33, 34, 35) using the LFASTA program (36).
At least three isoforms of 3HSD have been cloned in several species
(37), and the type I isoform has been cloned from rat liver cDNA
(38, 39, 40). This isoform has been demonstrated to be present in Leydig
cells (7). The two isoforms of 5R that have been cloned in the rat
are present in rat testis (41, 42).
Therefore, using the published sequences of the rat P450scc
(43), types I and II 3ßHSD (28), P450c17 (44), types I
and II 5R (41, 42), type I 3HSD (39), and ribosomal protein S16
(RPS16) (45), primers were selected using PRIMER software (Whitehead
Institute of Biomedical Research, Cambridge, MA) and synthesized on an
oligonucleotide synthesizer (Gene Assembler Special, LKB, Rockville,
MD). Table 1
summarizes the properties of
the primers.
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-32P]deoxy-CTP, and
proceeded for 30 cycles at an annealing temperature of 52 C.
Radiolabeled PCR bands were visualized by exposure to autoradiographic
film. Quantitative analysis of mRNA levels was performed by scanning
the films in a laser densitometer (Ultroscan, LKB). The signal
intensities for steroidogenic enzyme mRNAs were normalized to RPS16.
Preliminary studies showed that the targeted cDNAs were amplified
linearly between 15–35 cycles of PCR.
Statistics
All measurements were repeated at least three times. The data
were analyzed by the Kruskal-Wallis ANOVA, followed by multiple
comparisons testing to identify significant differences between groups
(47).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The three Leydig cell preparations
showed, respectively, 2-, 6-, and 6-fold increases in total
androgen production in response to LH stimulation compared with basal
control values (Table 2). These results indicated that progenitor
Leydig cells were less responsive to LH stimulation than immature and
adult Leydig cells.
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-DIOL was the primary androgen
for immature Leydig cells (73.33 ± 14.52% of total androgens),
and T was primary androgen for adult Leydig cells (74.38 ±
14.73%; Fig. 1).
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-DIOL requires 17ßHSD activity, and the low
release rates for these steroids from progenitor cells suggest that the
amount of 17ßHSD is low in these cells. AO and 3-DIOL require the
activities of 5R and 3HSD, and the high release rates of these
steroids by progenitors and immature Leydig cells indicate that
androgen-metabolizing enzymes are most highly expressed in Leydig cells
before the completion of puberty and then decline in adulthood.
Steroidogenic enzyme activities
The underlying basis for the different profiles of androgen
release from three distinct stages of Leydig cell differentiation was
examined further by measuring T biosynthetic and metabolizing enzyme
activities in purified cells. Steroidogenic enzyme activities were
measured in intact Leydig cells, because homogenization can change the
relative rates of oxidative and reductive activities in most
hydroxysteroid dehydrogenases. The first step of T biosynthesis is the
conversion of cholesterol to pregnenolone, which is catalyzed by
P450scc. The conversion of
5-3ß-hydroxysteroids to
4-3-ketosteroids is catalyzed by 3ßHSD.
P450c17 catalyzes both the 17-hydroxylase and C17–20
lyase reactions to produce DIONE (48). The final step of T biosynthesis
is catalyzed by 17ßHSD reductive activity. DIONE and T can be
metabolized by 5R and 3HSD into AO and 3-DIOL, respectively.
As shown in Fig. 2, the level of
P450scc was low in progenitor Leydig cells and increased
during Leydig cell differentiation. 3ßHSD and P450c17
(17-hydroxylation and C17–20 cleavage) attained half of their
mature activity levels in progenitor cells, and levels in immature and
adult Leydig cells were statistically equivalent. Progenitor cells had
negligible 17ßHSD reductive activity, which greatly increased in
immature and adult Leydig cells.
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R activity during the
transition from progenitor to immature Leydig cell, followed by a sharp
decline to almost undetectable levels in adult Leydig cells. The levels
of 3HSD activity were highest in progenitor Leydig cells and lowest
in adult Leydig cells (Fig. 2
). These results indicate that changes in
the activities of both T biosynthetic and metabolizing enzymes catalyze
the formation of distinct androgen end products during Leydig cell
development.
Steady state mRNA levels of steroidogenic enzymes
Differential changes in T biosynthetic and metabolizing enzyme
activities could result from variation in the steady state levels of
mRNAs that encode these enzymes. Therefore, the steady state mRNA
levels of Leydig cell steroidogenic enzymes were evaluated. With the
exception of P450scc and P450c17, multiple
isoforms of hydroxysteroid dehydrogenases and 5R have been
identified. Four distinct 3ßHSD cDNAs have been found in the rat
(27), and rat Leydig cells are known to express 3ßHSD isoforms I and
II (28). Five distinct isoforms of 17ßHSD have been identified in
several species. Type III 17ßHSD is thought to be responsible for
catalyzing the conversion of DIONE to T in Leydig cells (31). Although
it has not been cloned in the rat, type III 17ßHSD is the predicted
isoform for rat Leydig cells. A pair of primers was designed to
identify sequences common to human and mouse type III 17ßHSD cDNAs
(31, 32). A 0.36-kb PCR product was identified in immature Leydig cells
and adult Leydig cells (Fig. 3). Partial
sequence analysis of the first 200 bp of the 0.36-kb PCR product showed
that it had a 75% sequence similarity with mouse type III 17ßHSD
compared with only 35%, 40%, and 35% similarity with rat type I, II,
and IV isoforms. Accordingly, this cDNA fragment was identified as type
III. Two distinct isoforms of 5R have been identified in the rat
testis (9, 41). In the testis, type I is present exclusively in Leydig
cells (9). Type I 5R mRNA was abundant in progenitor and immature
Leydig cells and almost undetectable in adult Leydig cells. No type II
5R mRNA was detected in any stage of Leydig cell despite a clear
signal in the positive control, the prostate (data not shown). Of the
three isoforms of 3HSD that have been identified, type I is known to
be expressed in the testis (8). As shown in Fig. 3, when the steady
state mRNA levels of these genes were evaluated, the trends for mRNAs
corresponded closely with the trends defined for enzyme activities,
suggesting that these genes transcriptionally regulate the levels of
steroidogenic enzyme activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-reduced androgens are high in immature males (1, 2, 3, 4, 5). In the
rat, 3-DIOL is the predominant androgen in circulation during days
20–40 (1). A distinction must therefore be drawn between steroidogenic
ability and the capacity for T production. Steroidogenic ability is
defined as the ability of cells to produce steroids, whereas the
capacity for T production reflects the balance of both T biosynthetic
and metabolizing enzyme activities. In the present study, using
purified intact Leydig cells, we measured a 46-fold increase in basal T
production during the transition from 21-day-old progenitor to
90-day-old adult Leydig cell under LH-stimulated conditions. In
contrast, when the release rate was totaled for the four androgens,
DIONE, T, AO, and 3-DIOL, it only increased by 5-fold during the
transition between progenitor and adult Leydig cell. This indicated
that steroidogenic ability and capacity for T production are distinct
measures of Leydig cell function. The steroidogenic ability of the
progenitor Leydig cells, although lower than that of the more mature
Leydig cell stages, is notable because these cells are devoid of smooth
endoplasmic reticulum membranes that are the sites of steroidogenic
enzyme localization. It is possible, therefore, that the steroidogenic
ability of progenitor Leydig cells results from cytosolic, as opposed
to membrane-bound, isoforms of steroidogenic enzymes. This is supported
by the presence of type I 3HSD, a cytosolic
isoform, which attained its highest levels in progenitor Leydig cells.
Another possible explanation is that mitochondrial isoforms of
steroidogenic enzymes may be present in progenitor Leydig cells before
the development of membrane-bound isoforms in the smooth endoplasmic
reticulum. In this regard, a mitochondrial form of 3ßHSD has been
found to exist in bovine adrenal cortical cells (49). The primary
androgen end product was different for each of the three stages of
Leydig cell differentiation, with AO being the primary androgen
released by progenitor Leydig cells, and 3-DIOL and T,
respectively, the primary androgens for immature and adult Leydig
cells.
Synthesis of T and 3-DIOL require 17ßHSD reductive activity, and
their low release rates from progenitor cells suggest that 17ßHSD
activity is low in these cells. AO and 3-DIOL require the activities
of 5R and 3HSD, and the high release rates for these steroids by
progenitors and immature Leydig cells indicate that
androgen-metabolizing enzymes are abundantly expressed in Leydig cells
before the completion of puberty, with declining levels in adulthood.
Direct measurements of enzyme activities in purified cells explained
the changes in end products released during Leydig cell
differentiation. In progenitor cells, several androgen biosynthetic
enzyme activities attained half of the more mature values, with the
exception of 17ßHSD, which was low, resulting in negligible
conversion of DIONE to T. However, progenitor Leydig cells readily
converted DIONE to 5- androstanedione by 5R and ultimately to
the androgen end product, AO, through 3HSD, because the activities
of both 5R and 3HSD were high. Although T biosynthetic
enzymes nearly all attained adult values in immature
Leydig cells, T formed in these cells was rapidly metabolized to
another androgen end product, 3-DIOL. Adult Leydig cells contain
significant amounts of 3HSD activity. However, the absence of
5-reductase in adult Leydig cells eliminates the catabolism of T,
making this steroid the primary androgen end product, as T is not a
substrate for 3HSD. The 3HSD activity present in adult Leydig
cells may act to metabolize dihydrotestosterone, which is still
produced at trace levels in the mature testis.
Steroidogenic enzyme activities have been measured in the developing
testis (5, 6, 7). However, these studies report that 3-DIOL is the
primary androgen secreted from the immature testis on day 21
postpartum, when progenitor Leydig cells are present (2, 3, 4, 5). Given the
negligible 3-DIOL production that was observed in progenitor Leydig
cells, the predominance of this steroid in immature rat testis
indicates that high levels of 17ßHSD are expressed by other cell
types in the testis. The seminiferous tubules and Sertoli cells in
particular contain 17ßHSD activity (10, 11). Indeed, the testis
remains capable of 17ßHSD activity when Leydig cells are completely
destroyed by the Leydig cell toxicant ethylene-1,2-dimethanesulfonate,
with only a 50% decline in conversion of DIONE to T (12).
The present data indicate that the development of steroidogenic enzyme
activities results from sequential, rather than simultaneous, induction
of steroidogenic enzyme gene transcription. The steady state mRNA
levels of steroidogenic enzymes were measured in Leydig cells to
evaluate the relationship between mRNA levels and enzyme activity. We
demonstrated that mRNA levels for T biosynthetic enzymes were
significantly lower in progenitor Leydig cells than in immature and
adult Leydig cells. Of the T biosynthetic enzymes, type III 17ßHSD
was lowest in progenitor cells, consistent with the enzyme activity
data. The presence of type III 17ßHSD mRNA in rat Leydig cells
indicated that this isoform encodes Leydig cell-specific 17ßHSD,
which is also expressed in human and mouse testes (31, 32). Levels of
type I 5R were highest in immature Leydig cells, followed by
progenitor and adult Leydig cells, confirming the transitory expression
of this enzyme activity during pubertal development. Although type II
5R is reported to be in rat testis (8, 42), this isoform
was undetectable in Leydig cells.
In summary, these data demonstrate that biosynthetic and
androgen-metabolizing capacities are separately modulated in Leydig
cells during pubertal differentiation and that this has significant
consequences for the overall rate of T production. Progenitor Leydig
cells primarily produce AO, with 3-DIOL predominant in immature
Leydig cells and T predominant in adult Leydig cells. During the
transition from progenitor to immature Leydig cells, increased type III
17ßHSD mRNA and enzyme activities confer adult levels of T
biosynthetic capacity. During the later transition from immature to
adult Leydig cell, loss of type I 5R and decline of type I 3HSD
lower androgen metabolism, making T the primary androgen end
product.
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Acknowledgments |
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-DIOL, and Dr. James Catterall for
comments on the manuscript. |
Footnotes |
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Received January 20, 1998.
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 E. Rijntjes, H. J. M. Swarts, R. Anand-Ivell, and K. J. Teerds Prenatal induced chronic dietary hypothyroidism delays but does not block adult-type Leydig cell development Am J Physiol Endocrinol Metab, February 1, 2009; 296(2): E305 - E314. [Abstract] [Full Text] [PDF]
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 H. Lin, R.-S. Ge, G.-R. Chen, G.-X. Hu, L. Dong, Q.-Q. Lian, D. O. Hardy, C. M. Sottas, X.-K. Li, and M. P. Hardy Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero PNAS, May 20, 2008; 105(20): 7218 - 7222. [Abstract] [Full Text] [PDF]
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 K. J Teerds, E. Rijntjes, M. B Veldhuizen-Tsoerkan, F. F G Rommerts, and M. de Boer-Brouwer The development of rat Leydig cell progenitors in vitro: how essential is luteinising hormone? J. Endocrinol., September 1, 2007; 194(3): 579 - 593. [Abstract] [Full Text] [PDF]
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 B. T. Akingbemi, T. D. Braden, B. W. Kemppainen, K. D. Hancock, J. D. Sherrill, S. J. Cook, X. He, and J. G. Supko Exposure to Phytoestrogens in the Perinatal Period Affects Androgen Secretion by Testicular Leydig Cells in the Adult Rat Endocrinology, September 1, 2007; 148(9): 4475 - 4488. [Abstract] [Full Text] [PDF]
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 G.-R. Chen, R.-S. Ge, H. Lin, L. Dong, C. M. Sottas, and M. P. Hardy Development of a cryopreservation protocol for Leydig cells Hum. Reprod., August 1, 2007; 22(8): 2160 - 2168. [Abstract] [Full Text] [PDF]
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 R.-S. Ge, G.-R. Chen, Q. Dong, B. Akingbemi, C. M. Sottas, M. Santos, S. C. Sealfon, D. J. Bernard, and M. P. Hardy Biphasic Effects of Postnatal Exposure to Diethylhexylphthalate on the Timing of Puberty in Male Rats J Androl, July 1, 2007; 28(4): 513 - 520. [Abstract] [Full Text] [PDF]
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 C. N Kahiri, M W. Khalil, F. Tekpetey, and G. M Kidder Leydig cell function in mice lacking connexin43. Reproduction, October 1, 2006; 132(4): 607 - 616. [Abstract] [Full Text] [PDF]
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G. Shaw, J. Fenelon, M. Sichlau, R. J. Auchus, J. D. Wilson, and M. B. Renfree Role of the Alternate Pathway of Dihydrotestosterone Formation in Virilization of the Wolffian Ducts of the Tammar Wallaby, Macropus eugenii Endocrinology, May 1, 2006; 147(5): 2368 - 2373. [Abstract] [Full Text] [PDF]
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R.-S. Ge, Q. Dong, C. M. Sottas, V. Papadopoulos, B. R. Zirkin, and M. P. Hardy In search of rat stem Leydig cells: Identification, isolation, and lineage-specific development PNAS, February 21, 2006; 103(8): 2719 - 2724. [Abstract] [Full Text] [PDF]
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K. De Gendt, N. Atanassova, K. A. L. Tan, L. R. de Franca, G. G. Parreira, C. McKinnell, R. M. Sharpe, P. T. K. Saunders, J. I. Mason, S. Hartung, et al. Development and Function of the Adult Generation of Leydig Cells in Mice with Sertoli Cell-Selective or Total Ablation of the Androgen Receptor Endocrinology, September 1, 2005; 146(9): 4117 - 4126. [Abstract] [Full Text] [PDF]
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R.-S. Ge, Q. Dong, C. M. Sottas, H. Chen, B. R. Zirkin, and M. P. Hardy Gene Expression in Rat Leydig Cells During Development from the Progenitor to Adult Stage: A Cluster Analysis Biol Reprod, June 1, 2005; 72(6): 1405 - 1415. [Abstract] [Full Text] [PDF]
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X. Wu, R. Arumugam, S. P. Baker, and M. M. Lee Pubertal and Adult Leydig Cell Function in Mullerian Inhibiting Substance-Deficient Mice Endocrinology, February 1, 2005; 146(2): 589 - 595. [Abstract] [Full Text] [PDF]
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S Ramaswamy, C R Pohl, G R Marshall, and T M Plant A switch from continuous to episodic testicular testosterone release in response to pulsatile LH stimulation in juvenile rhesus monkeys (Macaca mulatta) J. Endocrinol., October 1, 2004; 183(1): 61 - 68. [Abstract] [Full Text] [PDF]
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B. T. Akingbemi, C. M. Sottas, A. I. Koulova, G. R. Klinefelter, and M. P. Hardy Inhibition of Testicular Steroidogenesis by the Xenoestrogen Bisphenol A Is Associated with Reduced Pituitary Luteinizing Hormone Secretion and Decreased Steroidogenic Enzyme Gene Expression in Rat Leydig Cells Endocrinology, February 1, 2004; 145(2): 592 - 603. [Abstract] [Full Text] [PDF]
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G.-M. Wang, P. J. O'Shaughnessy, C. Chubb, B. Robaire, and M. P. Hardy Effects of Insulin-Like Growth Factor I on Steroidogenic Enzyme Expression Levels in Mouse Leydig Cells Endocrinology, November 1, 2003; 144(11): 5058 - 5064. [Abstract] [Full Text] [PDF]
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J. Killian, K. Pratis, R. J. Clifton, P. G. Stanton, D. M. Robertson, and L. O'Donnell 5{alpha}-Reductase Isoenzymes 1 and 2 in the Rat Testis During Postnatal Development Biol Reprod, May 1, 2003; 68(5): 1711 - 1718. [Abstract] [Full Text] [PDF]
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J. D. Wilson, R. J. Auchus, M. W. Leihy, O. L. Guryev, R. W. Estabrook, S. M. Osborn, G. Shaw, and M. B. Renfree 5{alpha}-Androstane-3{alpha},17{beta}-Diol Is Formed in Tammar Wallaby Pouch Young Testes by a Pathway Involving 5{alpha}-Pregnane-3{alpha},17{alpha}-Diol-20-One as a Key Intermediate Endocrinology, February 1, 2003; 144(2): 575 - 580. [Abstract] [Full Text] [PDF]
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B. T. Akingbemi, R. Ge, C. S. Rosenfeld, L. G. Newton, D. O. Hardy, J. F. Catterall, D. B. Lubahn, K. S. Korach, and M. P. Hardy Estrogen Receptor-{alpha} Gene Deficiency Enhances Androgen Biosynthesis in the Mouse Leydig Cell Endocrinology, January 1, 2003; 144(1): 84 - 93. [Abstract] [Full Text] [PDF]
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D. O. Hardy, R.-S. Ge, J. F. Catterall, Y.-t. Hou, T. M. Penning, and M. P. Hardy Identification of the Oxidative 3{alpha}-Hydroxysteroid Dehydrogenase Activity of Rat Leydig Cells as Type II Retinol Dehydrogenase Endocrinology, May 1, 2000; 141(5): 1608 - 1617. [Abstract] [Full Text] [PDF]
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R.-S. Ge, D. O. Hardy, J. F. Catterall, and M. P. Hardy Opposing Changes in 3{alpha}-Hydroxysteroid Dehydrogenase Oxidative and Reductive Activities in Rat Leydig Cells during Pubertal Development Biol Reprod, April 1, 1999; 60(4): 855 - 860. [Abstract] [Full Text]
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