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From the Center for Biomedical Research, Population Council, and the Rockefeller University, New York, New York.
| Correspondence to: Dr Matthew P. Hardy, Center for Biomedical Research, Population Council and The Rockefeller University, 1230 York Ave, New York, NY 10021. |
| Received for publication April 27, 2004; accepted for publication May 18, 2004. |
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Abstract |
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Key words: Leydig cell, cyclic AMP, glucocorticoid receptor
During stress, an adaptive response originating in the hypothalamus-pituitary-adrenal (HPA) axis is activated to sustain homeostasis (Moberg, 1987; Xiao et al, 1999). The adaptive response alters the secretion of corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH), as well as adrenal corticosteroids (Moberg, 1987; Rivier and Rivest, 1991). Stress from a variety of stimuli exerts a profound suppression of the reproductive axis (Brann and Mahesh, 1991; Rivier and Rivest, 1991; Chrousos and Gold, 1992; Tilbrook et al, 2000). In males, decreased serum testosterone (T) is one of the first signs of stress (Fenster et al, 1997), and a sharp rise in serum glucocorticoid levels is viewed as a causative factor in the decline of steroidogenesis (Orr and Mann, 1990; Monder et al, 1994b; Gao et al, 1996). Luteinizing hormone (LH), the main tropic stimulus of T production in Leydig cells, may be unchanged (Collu et al, 1979; Charpenet et al, 1981; Srivastava et al, 1993) or lower (Demura et al, 1989; Lopez-Calderon et al, 1991), depending on the duration of the stress being investigated. In rats, acute immobilization (IMO) stress lowers T concentrations primarily at the testicular level with unchanged LH secretion, while chronic IMO stress has inhibitory effects on the hypothalamic-pituitary level and, by lowering serum LH release, decreases serum T concentrations (Maric et al, 1996).
The aims of this study were to define hormonal profiles under acute IMO stress in mice and to elucidate the mechanisms for stress-induced declines in T levels. Since glucocorticoid is thought to inhibit Leydig cell function through a GR-mediated pathway (Orr and Mann, 1992; Monder et al, 1994b; Gao et al, 1996; McEwen, 2000), we employed RU486 (mifepristone), a GR antagonist (Cadepond et al, 1997), to evaluate the effects of GR blockade at the testicular level. We asked whether rapid declines in androgen production are caused by reduced gonadotropic stimulation of Leydig cells by LH, as opposed to increased glucocorticoid activity. The results support the hypothesis that stress-induced increases in serum glucocorticoid levels directly inhibit Leydig cell function and implicate a rapid nongenomic pathway of glucocorticoid action.
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Materials and Methods |
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Immobilization Stress
The animals were placed in wire mesh restrainers (4 x 9 cm in
dimension) as described by McEwen and colleagues
(McEwen and Sapolsky, 1995). The procedure effectively restricted movement. The start of IMO stress began
at 10 AM and the treatment durations were 15 and 30 minutes and 1,
3, and 6 hours (n = 5 per time point). Control animals were left undisturbed
in their cages for the duration of the experiment and sampled at the same time
points (n = 5 per group). At the end of each stress period, trunk blood was
collected after decapitation in tubes containing heparin and centrifuged at
500 x g and the sera were stored at -20°C until assay.
Testes were removed and stored at -70°C. The overall design was replicated
4 times.
GR Blockade
To investigate the involvement of GRs in the glucocorticoid-induced T
decrease during IMO, RU486
[17ß-hydroxy-11ß-(4-dimethy-aminophenyl-1)-17
-(1-prop-1-ynyl)-oestra-4,
9-diene-3-one, Roussel, UCLAF, France] was administered in vivo either by IT
or IP injection using a 29
gauge syringe needle
(Baulieu, 1994) prior to the
stress session. Details of the design for this experiment are presented in
Table 1. The dose of RU486, 16
µg, was selected on the basis of a previous study conducted in rats
(Orr and Mann, 1992). RU486
was first dissolved in absolute ethanol and subsequently diluted with the
vehicle, 45% aqueous 2-hydroxypropyl-ß-cyclodextrin (catalog number 0926,
Sigma Chemical Co, St Louis, Mo), to attain the needed concentrations (the
final concentration of ethanol was 0.8%)
(Roozendaal et al, 2002).
Animals (n = 5 per group) were subjected to IMO stress for 3 hours as
described above. At the end of the stress period, animals were sacrificed by
decapitation and trunk blood was collected for hormonal assays. Leydig cells
were harvested for direct measurement of steroidogenesis in vitro after IT
treatment with RU486 in vivo. The cells were isolated, respectively, from
untreated control, IT vehicle, IMO plus IT RU486, and IMO plus IT vehicle
groups, and then (1 x 104/mL) incubated in a microcentrifuge
tube at 34°C with 100 ng LH in Dulbecco modified Eagle medium (DMEM):F12
culture medium for 3 hours. Spent media were collected for assay of
testosterone concentrations. The overall design was replicated 4 times.
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cAMP Measurement
The ability of Leydig cells to produce cyclic adenosine monophosphate
(cAMP) in response to elevated glucocorticoid during IMO stress was measured.
Briefly, purified Leydig cells (1 x 105/200 µL) were
preincubated in 96-well Falcon culture plates (Becton Dickinson and Co,
Franklin Lakes, NJ) under 5% CO2: 5% O2: 90%
N2 34°C air for 2 hours. The medium was then carefully removed,
and 200 µL fresh phenol-red-free DME/F12 medium was added, buffered with 15
mM HEPES and 26 mM sodium bicarbonate (Leydig cell culture medium [LCM]) and
containing a 1.44 µM final concentration of corticosterone (CORT) (500
ng/mL) that is achieved during stressful conditions in vivo. The cells were
incubated with CORT for 0–60 minutes. After incubation with CORT, media
were moved and cells were then incubated in 200 µL fresh phenol-red-free
LCM with LH 100 ng/mL for 20 minutes. At the end of the incubation, 50 µL
TET buffer (0.05 M Tris, 4 mM EDTA) was added immediately to the plates. The
preparations were frozen in liquid N2 and kept at -80°C until
cAMP and T assay. cAMP assay was assayed with a kit (catalog TRK432, Amersham
Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's directions.
The sensitivity of the assay was 0.05 pmol per assay tube.
Leydig Cell Purification
Leydig cells were purified by the procedure of Klinefelter et al
(Klinefelter et al, 1987) with
modifications. In brief, testes were removed, decapsulated, and dispersed in
10 mL of medium 199 (M-199) with 0.25 mg/mL collagenase in a shaking water
bath at 34°C for 10 minutes. To terminate collagenase dispersion, 1%
bovine serum albumin (BSA) M-199 buffered with 15 mM HEPES and 4 mM sodium
bicarbonate plus 25 µg/mL soybean trypsin inhibitor (SBTI) was added to
dilute the original suspension 1:5. Tubes were then capped and inverted
several times. Seminiferous tubules were allowed to settle, and the
supernatant containing the interstitial cells was collected by aspiration. The
tubes containing the settled seminiferous tubules were refilled with 1% BSA,
and the procedure was repeated several times to further harvest the
interstitial cells. The cells were pelleted in 250-mL tubes by centrifugation
at 800 x g for 20 minutes at 4°C and then fractionated
using a continuous Percoll gradient (55% Percoll in Hanks balanced salt
solution [HBSS] buffered with 15 mM HEPES and SBTI) in a total volume of 35
mL. The gradients were formed in situ by centrifugation in a Beckman JA-20
fixed angle rotor at 22 045 x g for 30 minutes at 10°C. A
tube containing density marker beads and 35 mL of 55% Percoll solution (Sigma)
was used as a reference. Leydig cells were recovered starting at a density of
1.07 gm/mL to the top of the red blood cell layer. HBSS was added to dilute
the Percoll in the resulting cell suspension, and the cells were pelleted at
200 x g for 10 minutes at 4°C. The cell pellets were then
resuspended in 2–4 mL of 1% BSA buffered HBSS and gently layered on top
of a discontinuous BSA gradient, containing 15 mL each of 10%, 5%, and 2.5%
BSA buffered HBSS. The gradient was centrifuged at 60 x g for
10 minutes at 4°C. Supernatants were aspirated until 15 mL of cell
suspension remained at the bottom of the tube. The bottom BSA layer was
transferred to another tube and diluted with HBSS by 1:3. After centrifugation
at 200 x g for 10 minutes at 4°C, the final pellet was
resuspended in LCM. Cells were counted in a hemacytometer (Hausser Scientific,
Horsam, Pa). The Leydig cell purity was approximately 97%, as determined by
histochemical staining for 3ß-hydroxysteroid dehydrogenase (3ß-HSD)
using 0.4 mM etiocholan-3ß-ol-17-one as the enzyme substrate
(Payne et al, 1980). As
expressed in Table 2, fractions
of Leydig cells isolated by this method were comparable to cells obtained by
the earlier unmodified procedure that incorporates an elutriation step
(Klinefelter et al, 1987).
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Intratesticular Testosterone
Intratesticular testosterone concentration was measured using the method of
Knorr et al (Knorr et al,
1970). In brief, testes were homogenized in 5 mL of 70% methanol
using a glass-glass homogenizer. The homogenates were transferred to 15-mL
screw cap test tubes. Tracer steroid (1000 counts per minute [cpm] of
tritiated testosterone) was added to the homogenate to correct for recovery.
The homogenates were left standing overnight at room temperature. The tubes
were centrifuged at 1800 x g, and the supernatant was aspirated
and dried under nitrogen to remove the methanol. The water layer was extracted
twice with high-performance liquid chromatography–grade diethylether.
The ether extracts were then resuspended in 400 µL of radioimmunoassay
(RIA) buffer, and 100 µL was removed for measurement of recovery (the cpm
value in 100 µL x 4 ÷ 1000). The remaining 300 µL was used
for RIA.
Serum T, CORT, and LH Concentrations, and Measurement of T Production
Serum T concentrations were measured using a tritium-based RIA as
previously described (Cochran et al,
1981). Serum CORT was measured by the RIA procedure of Spencer et
al (Spencer et al, 1996), with
an anti-CORT antiserum B3-163 (Endocrine Sciences, Calabasas, Calif). Serum LH
concentrations were assayed by the method of Chandrashekar and Bartke
(Chandrashekar and Bartke,
1988) using rat LH standards, NIDDK-r-LH-19; LH antibody and
National Institute of Diabetes & Digestive & Kidney
Disease-anti-rLH-S-11 from the National Hormone and Pituitary Program; and IgG
antiserum (ICN Pharmaceuticals, Costal Mesa, Calif). Radioactive
125I-rat LH was produced using the Iodogen method (catalog 28601,
Pierce Chemical Co, Rockford, Ill). Values for interassay variation of the T,
LH, and CORT RIAs were between 4% and 8%. The sensitivities of the assays for
LH, CORT, and T were 0.12 ng/mL, 10 ng/mL, and 10 pg/mL, respectively.
Statistics
Data were expressed as the mean plus or minus standard error of mean.
Statistical evaluation of serum and testis parameters was performed by 2-way
analysis of variance (ANOVA) with time and treatment as the subclasses.
Student-Newman-Keuls multiple comparisons testing was applied to identify
significant differences between groups. The Dunn-Sidak method was used to
calculate an experiment-wise error rate for the Leydig cell T production in
vitro after treatment of RU486 in vivo
(Rohlf, 1995). All
calculations were performed using a software package (InStat, San Diego,
Calif). Differences were regarded as significant at P < .05.
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Results |
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CORT
The increase in CORT levels in stressed males was significant by 15
minutes, and then a plateau was reached by 1 hour (492 ± 30 ng/mL) that
was fivefold higher compared with its correspondence control (n = 20
animals/time point, P < .01,
Figure 1). Significant effects
of time and interaction between time and treatment subclasses were detected by
the 2-way ANOVA. This indicated that CORT levels in control and stress males
varied distinctly as a function of time.
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Testosterone
Both serum (Figure 2) and
testicular (Figure 3) T
concentrations were lower starting at 30 minutes in IMO-stressed males and
decreased further to 30% and 8% of control values, respectively, by 6 hours (n
= 20 animals/time point, P < .01). Significant effects of time and
interaction were again recorded by the 2-way ANOVA, suggestive of suppressed
baseline variability of testosterone parameters over time in the stressed
group.
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LH
In contrast to the changes in T and CORT concentrations, serum LH levels
were unchanged by IMO stress (n = 20 animals/time point, P > .05)
(Figure 4). This implied that
decreased gonadotropic stimulation of Leydig cells was unlikely to explain the
suppression of T levels during IMO stress.
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Testicular GR Blockade by RU486
To test the direct inhibitory action of glucocorticoid on T production,
RU486 was administered to the stressed animals with a dose of 16 µg. Serum
CORT levels in all stressed groups went up to 564 ± 23 ng/mL, compared
with 110 ± 14 ng/mL in unstressed controls (n = 20 animals per group,
P < .01), irrespective of the agent delivered (RU486 or vehicle)
or route of injection (systemic or intratesticular, data not shown). This
indicated that administration of RU486 did not block the systemic increases in
serum CORT induced by IMO stress. The average serum T level in all unstressed
animals (including the untreated and vehicle injection groups) was 0.50
± 0.3 ng/mL. T levels among these 3 groups were equivalent, implying
that the injection itself did not disturb hormone levels. IMO stress decreased
serum T concentrations in groups receiving either IT or IP injection of
vehicle to 0.09 ± 0.01 ng/mL, about 20% of the value in unstressed
animals (n = 20 animals per group, P < .01,
Figure 5). Testicular
administration of RU486 increased serum T levels in stressed animals to 0.33
± 0.02 ng/mL compared with vehicle-injected controls (n = 20 animals
per group, P < .01). In contrast, T levels in animals that
received systemic IP injections were unchanged relative to vehicle-treated
controls. LH-stimulated T production (x 103 ng/mL) by Leydig
cells in vitro, after IT administration of RU486 and IMO stress in vivo, was
lower compared with unstressed controls (3.20 ± 0.50 ng/mL) but higher
compared with the IT vehicle plus IMO group (1.40 ± 0.20 ng/mL vs 0.70
± 0.10 ng/mL, Figure 6).
Taken together, the results indicate that RU486 partially blocked the
suppressive effects of CORT on T production and that the blockade was only
effective when testicular, rather than systemic.
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cAMP and T Production
cAMP (Figure 7) and T
(Figure 8) production were
measured using isolated Leydig cells that were cultured with CORT (1.44 µM)
for 0, 15, 30, and 60 minutes and then with LH (100 ng/mL) for 20 minutes.
cAMP levels in CORT-treated groups were significantly lower relative to
control, and rates of T production were also lower, at 30 and 60 minutes. This
indicated that rates of cAMP and T production in Leydig cell are rapidly
suppressed by a direct action of CORT.
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Discussion |
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Stress and other conditions that elevate circulating adrenocorticotropin hormone (ACTH) (Gomez et al, 1996) and CORT levels lead to depressed T levels in all known animal species and in men (Armario and Castellanos, 1984; Bernier et al, 1984; Orr and Mann, 1992; Monder et al, 1994b; Gao et al, 1996). The levels of T in circulation are set by the steroidogenic capacity of individual Leydig cells and the total numbers of Leydig cells per testis. Excessive exposure to CORT initiates apoptosis in rat Leydig cells, potentially contributing to suppression of T levels caused by the decline in steroidogenic capacity (Gao et al, 2002). This process first becomes detectable 12 hours after termination of stressor and is thus not considered a complicating factor in the present 6-hour study design. Leydig cells express GRs and are the primary targets of glucocorticoid action in the testis (Ortlip et al, 1981). The GR is a 94-kd ligand-activated intracellular transcriptional regulator belonging to the nuclear receptor superfamily (Mangelsdorf et al, 1995). The GR mediates glucocorticoid action and thereby modulates metabolism of carbohydrates, proteins, and fats; suppression of the immune/inflammatory response; activation of the central nervous system; and regulation of cardiovascular function (Landers and Spelsberg, 1992; Ge et al, 1997). According to a genomic model of action, glucocorticoid enters the cell and binds to the GR, forming a steroid-receptor complex. Following its formation, the complex enters the nucleus and acts as a transcriptional regulator. Transcriptional and posttranscriptional regulation induced by GRs may cause either positive or negative changes in the steady-state levels of specific mRNAs and proteins modulating cell function (Landers and Spelsberg, 1991; Schmid et al, 1995).
In the Leydig cell, following the above genomic model, glucocorticoid is
thought to directly inhibit processes critical to the biosynthesis of T by
suppressing expression levels of the cholesterol side-chain cleavage enzyme
(P450scc), 3ß-HSD, and 17-hydroxylase/17-20-lyase
(P45017) (Welsh et al,
1982; Hales and Payne,
1989; Payne and Sha,
1991; Srivastava et al,
1993). In our study, an almost fivefold increase in serum CORT
levels was associated with a fall in T levels during IMO stress, whereas LH
concentrations were unchanged. The inverse association between glucocorticoid
and T levels suggests that CORT causes reductions in T levels during IMO
stress.
RU486 (mifepristone), a GR antagonist, is postulated to block the suppressive action of glucocorticoid on T production (Baulieu, 1994). The antiglucocorticoid function of RU486 has been attributed to its high affinity for GR, masking the DNA binding domain of the receptor (Groyer et al, 1987; Lefebvre et al, 1988), although the antagonist can also act at steps subsequent to DNA binding on transcription and/or translation (Guiochon-Mantel et al, 1988; Beck et al, 1993; Edwards et al, 1995). Although the effects of GR blockade on T biosynthesis were analyzed previously in rats (Orr and Mann, 1992; Monder et al, 1994a), the present study was the first to investigate the effects of a testicular blockade. RU486 was administered to IMO-stressed mice and partially prevented the stress-induced decreases in T levels, indicating that blockade of CORT binding to GRs in Leydig cells suppresses the inhibitory effects of glucocorticoid action. The higher efficiency seen with local, IT administration of RU486 compared with systemic is consistent with a direct action of glucocorticoid on the testis. This was further confirmed by the fact that LH-stimulated T production in Leydig cells purified after IMO stress was higher in groups receiving RU486 by IT injection.
To our knowledge there have been no previous studies addressing the time course of stress-mediated inhibition of T levels. The durations of experimentally imposed stress reported in the literature have ranged from 2 hours to over 10 hours (Bernier et al, 1984; Monder et al, 1994a; Orr et al, 1994; Maric et al, 1996), indicating that a period of hours is required for the operation of genomic events initiated by glucocorticoid action. However, the data reported herein showed that CORT levels were sharply increased as early as 15 minutes, and T levels decreased starting at 30 minutes after imposition of IMO stress. The rapidity of the changes in T levels raises the possibility that suppression of T biosynthesis by glucocorticoid occurs through a nongenomic mechanism, not involving decreased expression of steroidogenic enzymes initially. The nongenomic effects of steroids are characterized by a rapid intracellular response to hormone (often within minutes) that does not require protein synthesis. Such rapid nongenomic effects include the activation of protein kinases, opening of ion channels, induction of phospholipid turnover, and increases in intracellular calcium and cAMP levels (Wehling, 1997). Glucocorticoid is reported to have nongenomic effects (Wehling, 1997; Revelli et al, 1998), which might explain the rapid reduction of T induced by CORT. Increased intracellular production of cAMP is part of the LH signaling transduction pathway leading to a rapid increase in Leydig cell steroidogenetic activity. Therefore, a fast acting CORT-mediated decline in intracellular cAMP may account for decreased T levels during acute IMO stress. In our study, CORT reduced the production of the cAMP in Leydig cells from 15 minutes onward and decreased T production from 30 minutes on after incubation with CORT in vitro. This time course is consistent with the rapid changes in serum T observed during IMO stress in vivo. There is a delay in the decline of T levels with respect to the decrease in cAMP formation after CORT exposure. The temporal ordering of the declines may be significant since cyclic AMP is the second messenger involved in the acute regulation of steroidogenesis. Therefore, these results pointed strongly in the direction of a nongenomic action of glucocorticoid. It is reasonable to infer from the present data that both the genomic and nongenomic modes of glucocorticoid action are applicable to the suppression of T levels during stress. However, our results do not exclude the possibility that other mechanisms contribute to the stress-induced declines in T concentrations. For example, catecholamine, opiates, and/or neuroendocrine pathways may also be involved in T suppression (Rivier and Rivest, 1991; Rivier, 2002).
In summary, IMO stress was found to suppress androgen secretion in mice. LH levels were unchanged, but T levels declined in the presence of elevated serum CORT concentrations. Local intratesticular administration of RU486 partially reversed the IMO stress-induced decrease in T levels, confirming that glucocorticoid and its receptor are involved in steroidogenic suppression. This indicates that glucocorticoid-mediated inhibition of Leydig cell steroidogenesis is a direct action at the testicular level. Suppression of intracellular cAMP levels in Leydig cells is implicated in the rapid response pathway induced by IMO stress.
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Acknowledgments |
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Footnotes |
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.05. The T levels in unstressed groups (untreated and
vehicle-injected groups) were equivalent (P > .05). The overall T levels in
stressed animals were significantly lower compared with unstressed males, and
T values in vehicle-injected controls were decreased by 80% to 0.09 ±
0.008 ng/mL (P 
