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REPRODUCTION-DEVELOPMENT |
Laboratory of Reproductive Biology, Shanghai Second Medical University (H.-B.G., M.-H.T., Y.-Q.H., Q.-S.G.), Shanghai, People’s Republic of China; and The Population Council (R.G., M.P.H.) and Rockefeller University (M.P.H.), 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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The levels of T in the circulation are set by the steroidogenic capacities of individual Leydig cells and the total numbers of Leydig cells per testis. Glucocorticoid action inhibits the expression of T-biosynthetic enzymes (9, 10) and thereby affects the steroidogenic capacities of Leydig cells. Glucocorticoids also control mitosis and induction of apoptosis (11, 12) in a variety of target cells. Glucocorticoid-mediated changes in the dynamic balance between mitosis and apoptosis may be one mechanism for the control of total cell numbers in tissues and organs (12, 13). It is not known, however, whether increases in circulating CORT induce apoptosis of Leydig cells.
Apoptosis of Leydig cells has been observed under experimental conditions in which the cells were treated in vivo with a toxicant, ethylene dimethanesulfonate (EDS), or in vitro with a synthetic glucocorticoid, methylprednisone, at a dose of 20 µM (14). Methylprednisone, like dexamethasone, is not subject to oxidative inactivation at the 11 carbon position and is consequently a more potent glucocorticoid than endogenous adrenal steroids such as CORT (15).
The glucocorticoid-metabolizing enzyme type 1 11ß-hydroxysteroid dehydrogenase (11ßHSD-1) catalyzes the reversible conversion of physiologically active CORT to the biologically inert 11-dehydrocorticosterone in rat testes. This enzyme is hypothesized to modulate Leydig cell steroidogenesis by controlling the intracellular concentration of glucocorticoid. By doing so, 11ßHSD-1 can protect the Leydig cell against the suppressive effects of glucocorticoids (15). In the testis 11ßHSD-1 is localized exclusively within Leydig cells and is not detectable until d 25 postpartum (16). In the present study, anti-11ßHSD-1, an antibody conjugated to fluorescein isothiocyanate (FITC) was used as a marker to identify immature and adult Leydig cells after CORT administration in vivo. As Leydig cells express 11ßHSD-1 and may consequently be protected from excessive glucocorticoid action (16), the aim of the present study was to test whether an exogenously administered glucocorticoid induces apoptosis in Leydig cells, thereby contributing to glucocorticoid-mediated declines in T production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal procedures
Male Sprague Dawley rats (21, 35, and 90 d of age) were
purchased from the animal center of the Academy of Sciences of China
(Shanghai, China). For the in vivo study, rats were
subjected to either sham surgery (sham) or bilateral adrenalectomy
(ADX) and housed at 26 C in groups of six. The drinking water supplied
to ADX rats was supplemented with 0.9% NaCl to maintain electrolyte
balance. In one set of experiments, rats were treated with CORT by ip
administration of 2.5, 5.0, or 7.5 mg/100 g BW·d twice daily,
starting on d 3 postsurgery using 5% dimethylsulfoxide in DMEM/F-12 as
the vehicle. At 3, 4, 5, and 6 d after surgery, the animals were
killed by CO2 asphyxiation. In another set of
experiments, rats (35 and 90 d old) were treated with CORT
in vivo as described above. To detect phagocytosis of dying
Leydig cells by macrophages, the animals also received an ip injection
of trypan blue (2 ml, 3% concentration in 0.9% NaCl) 48 h before
removal of the testes (18) 3, 4, 5, or 6 d after
surgery. As a negative control for the phagocytosis of dying cells by
neighboring macrophages, control animals received ip injections of the
vehicle, 0.9% NaCl. Trunk blood was collected for RIA of serum CORT
and LH concentrations (19). All animal procedures were
carried out in accordance with standards for humane care and use of
laboratory, which has been approved by the Rockefeller University
animal care and use committee (Protocol 91200).
Isolation of Leydig cells
Mesenchymal-like progenitors (PLC), immature Leydig cells (ILC),
and adult Leydig cells (ALC) were obtained from rat testes on d 21, 35,
and 90 postpartum, respectively. Leydig cells were purified by Percoll
density gradient centrifugation as described previously
(20), preceded by centrifugal elutriation in the case of
ALC. The purities of isolated cell fractions were evaluated by
histochemical staining for 3ßHSD activity, with 0.4 nM
etiocholanolone as the steroid substrate (21). Enrichment
of PLC, ILC, and ALC was typically to 90%, 94%, and 94%,
respectively.
Cell culture
Cell culture was conducted as previously described
(22). Briefly, PLC, ILC, and ALC were seeded in 50-mm
flasks, at a density of 4 x 106
cells/flask. In some cases, aliquots of cell suspension were seeded at
a density of 1 x 106 cells on a 24 x
24-mm coverslip lying within a 35-mm culture dish and cultured for 4 or
24 h in phenol red-free medium (DMEM/Ham’s F-12, D-2906,
Sigma) supplemented with 1 mg/ml bovine lipoprotein at 5%
O2/5% CO2 at 34 C. CORT
was added to the cultures at a concentration of 50, 100, 200, or 400
nM. After the hormonal treatments for varying periods
in vitro, Leydig cells were scraped from the surface of the
flasks with a rubber policeman after a 5-min incubation in a solution
containing 0.05% collagenase (Sigma) in medium 199
(Life Technologies, Inc.) buffered with 8.45
mM NaHCO3, 8.8
mM HEPES, 0.1 BSA, and 0.0025% trypsin inhibitor
(Sigma).
Detection and analysis of apoptotic cells
As there is no single feature that can be used to discriminate
between cell death occurring by apoptosis vs. necrosis, and
not every cell type will display all of the classic features of
apoptosis, multiple end points were evaluated. Apoptosis was identified
by morphological and biochemical criteria, including cell shrinkage,
changes in organelles, chromatin condensation (11, 23)
detected by electron microscopy; doublestranded DNA cleavage in
chromatin between nucleosomes producing fragments of approximately 185
bp or multiples of 185 bp in length detected by the TUNEL and DNA
ladder electrophoresis assays (11, 24, 25); and the
translocation of the membrane phospholipid phosphatidylserine by
annexin V staining (26).
Electron microscopic analysis
Leydig cells were cultured in vitro in the presence
or absence of 100 nM CORT and harvested after
24 h. The cell pellets were immersed in 1.5% glutaraldehyde
buffered in 0.1 M sodium cacodylate containing
0.05% calcium chloride (pH 7.4) and fixed for 1.5 h on ice. The
cells were then postfixed for 1.5 h in 1% osmium tetroxide and
embedded in Epon resin.
The testes of the rats treated with CORT in vivo and the controls were fixed by perfusion through the abdominal aorta with 1.5% glutaraldehyde buffered in 0.1 M sodium cacodylate containing 0.05% calcium chloride (pH 7.4). The testes were removed after 5 min of perfusion, trimmed into 1-mm3 pieces, and immersed in the same fixative for 1 h at 4 C. The pieces of testis were postfixed in 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in Epon.
Before ultrathin sectioning, semithin sections were examined under the light microscope, and the areas representing testicular interstitial regions were selected. Ultrathin sections were cut on an LKB-V ultratome, stained with uranyl acetate followed by lead citrate, and examined in a transmission electron microscope (Hitachi 500).
TUNEL assay
Leydig cell apoptosis was evaluated by in situ TUNEL
labeling. This method detects extensive DNA degradation, a
characteristic event that often occurs in the early stages of apoptosis
(11, 24, 25). The TUNEL assay was carried out on
paraffin-embedded sections of testis. Briefly, sections were
deparaffinized and dehydrated in graded concentrations of xylene and
ethanol. The sections were digested with 20 µg/ml proteinase K for 15
min at room temperature. The sections were then washed and incubated
with the TUNEL reaction mixture (enzyme solution and labeling solution)
for 60 min at 37 C in a humidified atmosphere. After washing, the
sections were analyzed by confocal imaging (LSM 50, Carl Zeiss, Jena, Germany).
DNA extraction and agarose electrophoresis
Agarose gel electrophoresis was performed to detect damage to
nuclear chromatin, a characteristic biochemical feature of apoptosis.
Electrophoresis of extracted DNA produces a characteristic ladder of
oligonucleosomal fragments (180–200 bp). DNA was extracted and
analyzed by agarose gel electrophoresis as described by Wilson et
al. (27). Briefly, aliquots of 1 x
106 ILC and ALC were collected 24 and 72 h,
respectively, after treatment either in vitro or in
vivo. The samples were centrifuged at 500 x g for
5 min at 4 C, the supernatant was discarded, and the pellet was
resuspended in 20 µM lysis buffer [50
mM Tris-HCl (pH 8.0), containing 10
mM EDTA, 0.5% sodium lauryl sarcosinate and 0.5
µg/ml proteinase K] and incubated for 1 h at 50 C. Ribonuclease
A (10 µl, 0.5 mg/ml) was added and incubated for an additional 1
h at 50 C. Low melting temperature agarose (10 µl, 1%) was added to
the sample, and 40 µl of each sample were placed into wells of 2%
agarose gel (containing 10 µg/ml ethidium bromide), which was
electrophoresed at 40 V for 2 h. DNA bands were visualized by UV
fluorescence.
Annexin V-FITC labeling
Annexin V has been used as a marker for apoptosis
(26) due to its high affinity for phosphatidylserine, a
phospholipid located on the cytoplasmic face of the cell membrane.
Annexin V does not bind to living cells but, rather, to
phosphatidylserine exposed on the surface of apoptotic cells. Apoptotic
cells therefore show a characteristic fluorescent index (bright green
staining) after labeling with annexin V-FITC. Annexin V is not able to
bind to nonapoptotic cells, as it does not penetrate the phospholipid
bilayer. In dead cells, however, the inner leaflet of the membrane is
available for binding of extrinsically applied annexin V, because the
integrity of the plasma membrane is lost. Therefore, to discriminate
between dead and apoptotic Leydig cells, the membrane-impermeable DNA
stain, propidium iodide (PI) was added simultaneously to the cell
suspension. Necrotic Leydig cells take up PI and are stained orange and
green, whereas apoptotic Leydig cells are stained green alone. In this
way, intact cells undergoing apoptosis and dead cells were identified,
and automatically analyzed by confocal laser scanning microscopy, as
described by Akner (28). The cell suspension on the
coverslips was analyzed by confocal laser scanning microscopy with an
excitation wavelength of 488 nm. In addition, flow cytometric analysis
(FACS) was used to distinguish apoptotic cells with specific DNA
fragmentation and maintained membrane integrity from necrotic cells
(29, 30). After labeling with annexin V-FITC and PI,
samples were analyzed in a flow cytometer (FACScan, Becton Dickinson and Co., Mountain View, CA) with argon laser
excitation set at 488 nm (emitting power = 15 mw), the emission of
the two fluorochromes was recorded through specific bandpass filters:
530 ± 30 nm for FITC and 675 ± 22 nm for PI. The LYSIS2
software program was used for acquisition and analysis of data.
Aliquots of 1 x 106 cells were analyzed in
triplicate for each experiment.
11ßHSD immunohistochemistry
11ßHSD-1 was used as a marker to identity Leydig cells
in situ based on the results of Phillips et al.
(16). 11ßHSD-1 immunohistochemistry was performed on
paraffin-embedded testis sections that were deparaffinized and
rehydrated in graded concentrations of xylene and ethanol. Slides were
washed (twice, 10 min each time) in PBS, buffer A (PBS containing 0.1%
Triton X-100; twice, 5 min each time), and buffer B (PBS containing
0.1% Triton X-100 and 2% BSA; twice, 5 min each time). The sections
were incubated overnight at 4 C in buffer B containing anti-11ßHSD-1
antibody at a dilution of 1:500, washed in buffer B (twice, 5 min each
time), and incubated for 2 h at room temperature in
FITC-conjugated antirabbit IgG. After further washing in buffer B
twice, 5 min each time) and PBS (twice, 10 min each time), sections
were dehydrated stepwise through xylene and ethanol. From each of 4
animals in each group, 1 testis was selected randomly. Two sections
were prepared per testis and assessed by the TUNEL and 11ßHSD-1
staining procedures. Randomly chosen interstitial spaces (20/section),
defined as the space bounded by at least 3 seminiferous tubule
profiles, were evaluated by counting cells identified by green
fluorescing nuclei (TUNEL assay) or green fluorescing cytosol with an
unstained nuclear profile (11ßHSD-1 assay). All counting was
performed blind on a Nikon Optiphot-2
microscope (Tokyo, Japan) using a x40 objective. The testis
weights did not significantly change over the period studied, which
allowed for a direct comparison of cell counts between treatments.
Statistics
The data were analyzed by Kruskal-Wallis ANOVA, followed by
multiple comparison testing to identify significant differences between
groups (31). Differences were regarded as significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of the in vivo study was to determine whether excessive CORT exposure induces apoptosis in Leydig cells. Male rats at 35 and 90 d of age were subjected to bilateral adrenalectomy and subsequently treated with a high dose of CORT (2.5 mg/100 g BW, administered by ip injection twice daily) starting 3 d after surgery. Normal and stress values for CORT reported in the literature are 60.6 ± 7.6 and 427.1 ± 99.0 nM, respectively (6). As expected, serum concentrations of CORT declined to undetectable levels after ADX and were maintained at 5–10 times the average normal concentration by CORT replacement (sham, 52.1 ± 3.2 nM; ADX, 0.2 ± 0.005 nM; ADX + CORT, 397.5 ± 80.4 nM; at the 2.5-mg dose). Leydig cells were treated with three different doses of CORT in vivo (2.5, 5.0, or 7.5 mg/100 g BW). Increased apoptotic frequencies were found in both ILC and ALC treated with all three doses of CORT. In contrast, apoptosis was seen in germ cells in neighboring seminiferous tubules only at the two higher doses of CORT (5.0 and 7.5 mg). Therefore, the focus of the in vivo studies was on the 2.5-mg dose, at which the effects were confined to the interstitium.
Ultrastructural analysis in vitro and in vivo
Ultrastructural analysis showed that the chromatin in CORT-treated
ALC in vitro was highly condensed and massed at the center
of the nucleus as a "cat eye." The nuclear membrane was shrunken
and formed a deeply convoluted profile. The cytoplasmic organelles had
degenerative changes, including dilation of the endoplasmic reticulum
and mitochondrial swelling (Fig. 1B
).
These features were not observed in control Leydig cells (Fig. 1A) and
were typical of apoptosis.
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), occasionally forming
crescent-shaped masses of condensed chromatin underlying nuclear
membrane. The cytoplasmic organelles had degenerative changes not seen
in control Leydig cells (Fig. 1C), such as dilatation of the
endoplasmic reticulum and mitochondrial swelling.
FACS analysis
Increased labeling of ALC was detectable by FACS after exposure to
100 nM CORT in vitro, with an incidence of
22 ± 2% after 4 h (Fig. 3) and 30 ± 2% after 24
h (Figs. 2B and 3) compared with
7 ± 1% at both 4 h (Fig. 3)
and 24 h (Figs. 2A and 3) in untreated controls (P
< 0.05). These results suggest that CORT initiates apoptosis in ALC.
The higher dose of 400 nM CORT was associated
with necrotic changes in Leydig cells, as shown in Fig. 2C.
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Figure 4A shows ALC counts within the interstitial spaces that stained
positively for apoptotic DNA. The controls had negligible numbers of
positively labeled cells in the interstitial spaces, whereas
significant numbers of labeled cells were identifiable at 24 h
after CORT administration (P < 0.05). The frequency of
labeling was lower at 72 h (compared with control,
P < 0.05). The numbers of 11ß-HSD1-positive ALC in
Figure 4B show that the relative numbers of ALC per interstitial area
decreased starting at 24 h and fell to a minimum by 72 h
(P < 0.05). This indicated that dead Leydig cells were
eliminated from the interstitium during the 24- to 72-h period
following the start of CORT replacement.
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). Similar results were obtained after
ALC were subjected to CORT treatment in vivo (data not
shown).
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, the
numbers of trypan blue-labeled macrophages in interstitial spaces of
testes from 90-d-old rats increased at 24 and 72 h after CORT
administration compared with controls (P < 0.05). The
rise in the number of apoptotic Leydig cells was coincident with an
increase in the number of phagocytic macrophages after CORT treatment
in vivo. This indicated that dead Leydig cells were
phagocytized during the 24- to 72-h period after the start of CORT
administration.
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, the levels of
circulating LH in 90-d-old rats were not altered by the high
concentrations of CORT seen in treated animals (P <
0.05). This indicated that the mechanism of glucocorticoid induction of
Leydig cell apoptosis was independent of changes in serum LH levels.
Serum T levels decreased starting at 24 h and fell to a minimum by
72 h (P < 0.05), suggesting that the declines in
T production are due to a direct effect of excessive CORT on Leydig
cells.
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). These results indicated
that CORT does not initiate apoptosis in PLC, which is consistent with
the low numbers of GR measured at this stage of Leydig cell
differentiation.
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). These results suggest that CORT
initiates apoptosis in ILC. Other parameters of the apoptotic process,
including TUNEL and 11ßHSD1 labeling, formation of DNA ladders, and
clearance of apoptotic Leydig cells by macrophages (Table 3), indicate that immature and adult
Leydig cells are equivalently sensitive to glucocorticoid action.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The lowest dose in vivo (2.5 mg) resulted in serum concentrations of nearly 400 nM in adrenalectomized rats. Unlike the in vitro situation, the 400-nM CORT level in vivo was associated with Leydig cell apoptosis, not necrosis. Our interpretation is that corticosteroid-binding globulin associates with a significant proportion of the free CORT in vivo (32), and that the concentration of bioavailable CORT in rats receiving the 2.5-mg dose is considerably lower than 400 nM. Subordination stress in male subordinate rats, housed in a visible burrow system that models normal psychosocial interactions, results in serum CORT levels as high as 450 nM (33). Therefore, it is not unlikely that the serum CORT concentrations achieved in our study have application to the natural environment, in which case the frequency of CORT-mediated Leydig cell apoptosis would be significant.
High levels of glucocorticoids have been shown to cause a decrease in serum T levels in both humans and rats under a variety of stressful conditions (4, 5, 6, 7, 8). A number of studies in vitro have shown that glucocorticoid inhibits T production in Leydig cells (3, 34). Moreover, it is known that glucocorticoid directly inhibits the transcription of genes encoding T biosynthetic enzymes (3, 9, 10, 35). Despite evidence that steroidogenic enzymes are specifically targeted for glucocorticoid action, it remains unclear whether decreased numbers of Leydig cells in the testis also contribute to glucocorticoid-mediated declines in serum T concentrations. If glucocorticoids influence Leydig cell numbers, they could do so through a reduction in the rate of mitosis and/or differentiation of Leydig cell precursors or by increasing the rate of Leydig cell apoptosis. The present study points to the possibility that glucocorticoids also lower T levels by decreasing the number of Leydig cells through induction of apoptosis.
The results showed that TUNEL-positive interstitial cells were detectable in histological sections as soon as 12 h after the start of CORT administration. The most dramatic increase in TUNEL labeling of Leydig cells was seen at 24 h after CORT administration in vivo. Positively labeled Leydig cells were detected in the sections prepared from control animals, consistent with the low turnover rate reported for Leydig cells (36). Taken together with the Leydig cell counts, our results indicate that there was a reciprocal change between the incidence of apoptotic Leydig cells and decreased Leydig cell numbers. The longer Leydig cells were exposed to CORT, the lower the numbers of Leydig cells that were detectable in situ by 11ßHSD-1 immunoreactivity. The largest decreases were observed at 72 h. TUNEL labeling does not distinguish between apoptotic internucleosomal DNA damage vs. random necrotic degradation of DNA into fragments that are irregular in size (29, 30). However, the data on DNA ladder formation after CORT exposure supported the hypothesis that the DNA damage was associated with apoptosis.
The observed increase in the frequency of apoptosis within the interstitium preceded the decrease in 11ßHSD-positive cell numbers by 24 h. This pattern of staining suggests that Leydig cells undergo apoptosis, with DNA fragmentation occurring before removal of the apoptotic cells from the interstitium. These observations are in agreement with previous studies, in which apoptotic changes in cell morphology were noted, followed by phagocytosis of the dying cells by neighboring macrophages (37). Macrophages are not the only phagocytic cell type, even in the testis; Sertoli cells, for example, phagocytose the cytoplasmic droplets of elongating spermatids (38). However, uptake of trypan blue administered ip has remained a reliable method for labeling testicular macrophages. The method appears to be specific for phagocytic cells in the testicular interstitium and does not label Sertoli cells (39). We propose that dying Leydig cells are cleared by macrophages after excessive exposure to CORT, a process that is also observed after cytotoxic destruction of Leydig cells by ethane dimethanesulfonate (37).
Many hormonally responsive tissues and organs are subject to apoptosis upon addition or removal of an appropriate regulatory factor. For example, the withdrawal of T after castration induces apoptosis and involution of the prostate. Few studies are available concerning the regulation of Leydig cell apoptosis (14). The Leydig cell-specific toxicant, EDS is known to induce apoptosis after EDS administration. The present findings are the first to show induction of Leydig cell apoptosis by an exogenously administered natural glucocorticoid. It had been observed in an earlier study that Leydig cells undergo apoptosis when exposed to a 10-µM concentration of the synthetic glucocorticoid, methylprednisone (MP) in vitro. MP is not as readily metabolized as CORT (15, 34) and is therefore expected to be more potent. However, a pharmacological dose of MP (100 mg/kg BW) appears to have no effect on Leydig cells in vivo (14), pointing to the need for further testing of endogenously secreted glucocorticoids in the in vivo setting. Our observations show that exogenous CORT induced Leydig cell apoptosis in vivo and in vitro, whereas serum LH concentrations were unaffected by CORT administration. We can infer that the induction of apoptosis occurred through a direct mechanism at the level of the Leydig cell, rather than indirectly through suppression of LH. The present results indicate that apoptosis of Leydig cells follows excessive exposure to glucocorticoid, as can occur during stressful conditions. The results also suggest that decreases in Leydig cell numbers are involved in stress-induced declines in T levels.
Immature thymocytes must be selected for maturation before the circadian peak of cortisol reaches the thymus. If not, they are eliminated by glucocorticoid action, and a new cohort of thymocytes takes their place (40). Similarly, the role of glucocorticoid, which has been chiefly defined with respect to inhibition of T biosynthesis (41, 42), may be to regulate Leydig cell development. The present experiments showed that CORT induces apoptosis of ILC and ALC at an equivalent rate. As high levels of GR are present in ILC on d 35 (43), this stage of Leydig cell development is expected to be sensitive to glucocorticoid action. As Leydig cell proliferative activity is limited to the prepubertal period, the total number of Leydig cells is determined during pubertal development (44). Thus, regulation by CORT potentially has a role in limiting growth of Leydig cell numbers during pubertal development.
CORT initiated apoptosis of ILC and ALC, whereas PLC were unaffected at the 100-nM dose. The relative insensitivity of PLC to CORT could be attributed to the lower numbers of GR measured at this stage of Leydig cell differentiation (43). As glucocorticoid levels remain stable during pubertal development, GR and 11ßHSD-1 in Leydig cells are the primary determinants of intracellular levels of bioactive steroid (15, 43). Due to the negligible expression of 11ßHSD-1 in PLC, GR numbers are probably the sole determinant of bioactive CORT levels at this stage of Leydig cell differentiation. Accordingly, it can be inferred that CORT induction of apoptosis in ILC and ALC apoptosis is a GR-mediated process, as has been shown in the case of thymocytes. Glucocorticoid activation of apoptosis in thymocytes is a steroid receptor-mediated process that can be blocked by the GR antagonist RU-486 and cannot be induced in cells devoid of GR (45, 46). In previously published studies, we observed that blockade of GR by RU486 alleviated CORT-mediated suppression of T production (6), whereas inhibition of 11ßHSD1 in Leydig cells accentuated this suppressive effect of CORT (47). It will be necessary to perform similar studies with RU486, synthetic glucocorticoids, and inhibitors of 11ßHSD1 to further analyze the roles of GR and enzymatic protection in Leydig cell apoptosis. We tentatively propose that a GR-mediated mechanism of glucocorticoid-activated apoptosis is applicable to Leydig cells and is a naturally occurring phenomenon in this cell type.
In conclusion, the data reported herein are the first to demonstrate that exposure of Leydig cells to high concentrations of CORT, levels that might occur during stress, reduced their numbers through induction of cell apoptosis. The induction process is most likely a direct GR-mediated effect at the level of the Leydig cell, rather than through suppression of LH. CORT induced apoptosis in both ILC and ALC, whereas PLC were insensitive to CORT at the dose used. A developmental difference in glucocorticoid sensitivity therefore exists in this process. Our study indicates that excessive exposure to CORT initiates apoptosis in rat Leydig cells, potentially contributing to suppression of circulating T levels during stress and other conditions in which glucocorticoid concentrations are elevated.
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Acknowledgments |
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Footnotes |
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Abbreviations: ADX, Adrenalectomy; ALC, adult Leydig cells; BW, body weight; CORT, corticosterone; EDS, ethylene dimethanesulfonate; FITC, fluorescein isothiocyanate; 11ßHSD-1, type 1 11ß-hydroxysteroid dehydrogenase; ILC, immature Leydig cells; MP, methylprednisone; PI, propidium iodide; PLC, mesenchymal-like progenitors; TUNEL, terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling.
Received May 25, 2001.
Accepted for publication September 25, 2001.
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 V. G. Martinez, E. H. Pellizzari, E. S. Diaz, S. B. Cigorraga, L. Lustig, B. Denduchis, C. Wolfenstein-Todel, and M. M. Iglesias Galectin-1, a cell adhesion modulator, induces apoptosis of rat Leydig cells in vitro Glycobiology, February 1, 2004; 14(2): 127 - 137. [Abstract] [Full Text] [PDF]
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M. Kusakabe, I. Nakamura, and G. Young 11{beta}-Hydroxysteroid Dehydrogenase Complementary Deoxyribonucleic Acid in Rainbow Trout: Cloning, Sites of Expression, and Seasonal Changes in Gonads Endocrinology, June 1, 2003; 144(6): 2534 - 2545. [Abstract] [Full Text] [PDF]
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Y. Lukyanenko, J.-J. Chen, and J. C. Hutson Testosterone Regulates 25-Hydroxycholesterol Production in Testicular Macrophages Biol Reprod, November 1, 2002; 67(5): 1435 - 1438. [Abstract] [Full Text] [PDF]
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