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Department of Physiology and Biophysics (K.H.H., T.D., S.G., B.K.S., M.R., H.B.B., D.B.H.), University of Illinois at Chicago, Chicago, Illinois 60612-7342; and Department of Urology (T.D.), University Hospital of the Justus-Liebig-University, 35392 Giessen, Germany
Address all correspondence and requests for reprints to: Dale B. Hales, Department of Physiology and Biophysics, University of Illinois at Chicago, 835 South Wolcott Avenue, Chicago, Illinois 60612-7342. E-mail: dbhale{at}uic.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4-5-isomerase, or P450c17. In the
adrenal, StAR protein was increased, as was StAR protein mRNA. No
changes were observed in the levels of mRNA for P450scc,
3ß-hydroxysteroid dehydrogenase
4-5-isomerase, or P450c21. Thus, although
the mechanisms of regulation differ, changes in the levels of StAR
protein are a sensitive indicator of the steroidogenic capacity of
these two tissues. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4-5-isomerase
(3ß-HSD) to progesterone. Progesterone, in turn, is converted, by a
two-step process, to androstenedione via the action of
17
-hydroxylase/C17–20 lyase (P450c17). The
conversion of androstenedione to testosterone is catalyzed by
17ß-hydroxysteroid dehydrogenase (17ß-HSD) (for reviews see Refs.
3, 5). The biosynthesis of corticosteroids in the mouse
adrenal is regulated by ACTH and angiotensin II. Analogous to the
action of LH on Leydig cells, ACTH, via the production of cAMP,
stimulates StAR, P450scc, and 3ßHSD expression and activity. However,
the mouse does not express P450c17 in the adrenal cortex and
consequently does not generate 17-hydroxysteroids. Thus, progesterone
is converted to deoxycorticosterone by 21-hydroxylase cytochrome P450
(P450c21) and to corticosterone by 11ß-hydroxylase cytochrome P450
(P45011ß) or to aldosterone by the further action of aldosterone
synthase (P450aldo). Immune activation of the adrenal is thought to
occur by cytokine stimulation of CRF production in the median eminence
and/or cell bodies in the hypothalamus, which, in turn, stimulates the
secretion of ACTH from the pituitary. In parallel, immune activation
has been shown to inhibit GnRH and LH secretion, resulting in
inhibition of gonadal steroidogenesis (1). However,
inflammatory mediators also act directly on the gonad to inhibit
steroidogenesis. We have observed direct perturbation of Leydig cell
steroidogenesis in three models of immune activation.
Intracerebroventricular injection of interleukin (IL)-1ß in
male rats, in addition to decreasing secretion of GnRH and LH, also
causes a blunting of the Leydig cell response to human CG (hCG)
(6). Experimental sepsis, induced in male rats by cecal
slurry, results in a significant decrease in serum testosterone
(7). After a single ip injection of the gram negative
endotoxin lipopolysaccharide (LPS) into male mice, serum testosterone
levels are decreased by 80% within 2 h and are still completely
inhibited at 24 h (8). In all three of these models,
we have shown that the acute inhibition of testosterone production is
correlated to the inhibition of steroidogenic acute regulatory (StAR)
protein expression in Leydig cells (6, 8, 9). During LPS
endotoxemia, Leydig cell StAR protein, but not messenger RNA (mRNA), is
decreased. In contrast, the expression of the steroidogenic enzymes
P450scc, 3ß-HSD, and P450c17 did not decrease appreciably until at
least 6–8 h after LPS (10). The purpose of the present
study was to determine whether testosterone inhibition during
LPS-induced endotoxemia is correlated to a decrease in serum LH, Leydig
cell LH receptor number or affinity, or cAMP production. A further
objective was to examine the effects of LPS injection on adrenal
steroidogenesis. We find that, in contrast to the inhibitory effects
observed in the Leydig cell, adrenal steroidogenesis is markedly
elevated. The diametric effects of LPS on adrenal vs.
testicular steroidogenesis include a 1.5- to 2-fold increase in StAR
protein and mRNA levels in the adrenal within 2 h, concomitant
with increased production of adrenal corticosteroids. |
Materials and Methods |
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-32P]deoxycytidine triphosphate and
Na125I were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). A Random Primed
Labeling kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Metrizamide was purchased from
Accurate Chemical and Scientific Corp. (Westbury, NY); collagenase was
purchased from Worthington Biochemical Corp. (Lakewood,
NJ). (22R)-hydroxycholesterol, HEPES, BSA (fraction V), bovine insulin,
EDTA, and sodium bicarbonate were purchased from Sigma
(St. Louis, MO). Medium 199, DME/F12, Waymouth’s MB752/1, penicillin,
streptomycin, and phenol were obtained from Life Technologies (Gaithersburg, MD). Mouse P450scc and P450c21 complementary DNA (cDNA) were a gift from Dr. Keith Parker (University of Texas South Western Medical Center, Dallas, TX). Mouse P450c17 and 3ß-HSD type I cDNA were a gift from Dr. Anita H. Payne (Stanford University, Palo Alto, CA). Mouse StAR cDNA was a gift from Dr. Douglas M. Stocco (Texas Tech University, Lubbock, TX).
Animals
Mice were housed for at least 1 week in groups of five per cage.
They were given food and water ad libitum and maintained on
a 14-h light,10-h dark schedule. Adult (60–70 days old) male outbred
pathogen-free CD-1 mice (Charles River Laboratories, Inc.
Portage, MI), averaging 33 g, were injected ip with LPS
[Escherichia coli strain JS (Re mutant),
Sigma] or vehicle alone (PBS); and blood, testes, and
adrenals were collected at the times indicated. The mice were procured,
maintained, and used in accordance with the Animal Welfare Act and were
killed by CO2 asphyxiation before
exsanguination.
Isolation of Leydig cells
Testes from each treatment group were collagenase-dispersed, and
Leydig cells were isolated on Metrizamide gradients, as described
previously (11). For the (22R)-hydroxycholesterol
experiments, Leydig cells were cultured in serum-free DME/F12 culture
medium [a 1:1 mixture of DMEM and Ham’s nutrient mixture F-12
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] and incubated in a humidified
atmosphere of 95% air-5% CO2 at 32 C.
Steroid RIAs
Blood was collected by cardiac puncture. After clotting at room
temperature for 1 h, the clot was loosened from the edge of the
tube and centrifuged at 1000 x g for 20 min at 4 C.
The serum was removed to a new tube and stored at -20 C before
assaying. Culture media were boiled for 5 min and centrifuged at
2,000 x g for 20 min, at 4 C, before assaying.
Testosterone, corticosterone, and progesterone concentrations were
determined with Coat-a-Count RIA kits (Diagnostic Products, Los Angeles, CA).
LH RIA
LH RIAs were performed by Brigitte G. Mann, as described
(12, 13, 14).
hCG binding assay
Highly purified hCG (batch CR127) was obtained from the National
Hormone and Pituitary Program, NIDDK (Rockville, MD) and was
radiolabeled with carrier-free Na125I using the
chloramine T method, as described by Dufau et al.
(15). Briefly, 25 µg hCG and 12.5 µg chloramine T were
added to 1 mCi Na125I, vortexed, and incubated on
ice for 30 sec. After termination of the reaction by adding 0.125 mg
sodium metabisulfite containing 1.0% KI, the purified iodinated hCG
was obtained by gel-filtration on a Sephadex G-50 column that had been
equilibrated with 50 mM Tris buffer containing 1
mM EDTA and 0.1% BSA. The specific activity, as
determined by self-displacement analysis, was 3 µCi/µg protein from
mouse testis homogenate.
After being thawed on ice, cells were dispersed in 0.25 ml PBS
containing 0.1% BSA and filtered through three layers of organza. The
resulting suspension was diluted to a final vol of 6.5 ml and used for
binding studies. Incubation of 300-µl cell suspensions with
125I-hCG (100,000 cpm in 750 µl), in the
presence of increasing concentrations of unlabeled hCG, was performed
in polystyrene tubes at 27 C. After 18 h, the suspensions were
washed twice with cold PBS-BSA and centrifuged at 20,000 x
g for 20 min. After aspiration of the second supernatant,
radioactivity was determined by counting for 1 min in a 
-counter.
Specific binding was determined by subtracting the total amount of
radioactivity bound from radioactivity present when excess unlabeled
hCG was added. Binding data were analyzed by the method of Scatchard
(16).
cAMP assay
Leydig cells, purified from vehicle- or LPS-injected animals,
were extracted immediately after isolation, for cAMP determination, or
incubated at 37 C with 0.125 mM IBMX and ± 2
nM hCG in 95% O2-5%
CO2 for 3 h with shaking. cAMP was extracted
from the cells with 65% ethanol and quantitated using the BioTRak cAMP
RIA assay (Amersham Pharmacia Biotech, Arlington Heights,
IL), according to the manufacturer’s instructions for the
nonacetylation assay.
Northern analysis
RNA was extracted from isolated Leydig cells using RNeasy spin
columns (QIAGEN, Chatsworth, CA). RNA was extracted from
adrenals by acid guanidine phenol chloroform (17)
after grinding the tissue to a powder on dry ice. Northern blots were
performed as described (11) and quantitated by
phosphorimage analysis (Molecular Dynamics, Inc.,
Sunnyvale, CA).
Western analysis of StAR
Pelleted Leydig cells were resuspended in lysis buffer (0.1%
SDS in PBS), and dissected adrenals were ground to a powder in a mortar
and pestle on dry ice and resuspended in lysis buffer. Samples were
subjected to brief sonication. Protein concentrations were determined
by microBCA protein assay (Pierce Chemical Co., Rockford
IL). Equal amounts of protein were analyzed as described
(10). Blots were incubated with antisera raised in rabbits
against a GST-StAR fusion protein. Briefly, the mouse StAR cDNA
(18) was subcloned as a SmaI-BamHI fragment using a BamHI
linker to convert the blunt end to a compatible site that would
preserve the reading frame when inserted into the BamHI site of pGEX-2T
(Pharmacia, Piscataway, NJ). The protein was expressed by
isopropyl ß-D-thiogalactopyranoside induction in
DH5 cells and solubilized, as described (19), using 1.5%
sarkosyl. The fusion protein was bound to glutathione agarose beads
(Sigma) in the presence of 4% TritonX-100 and eluted by
boiling in 0.75 M HEPES (pH 7.4) with 1% SDS. Before
injection into rabbits, the protein was extensively dialyzed against
PBS. Western blot detection was performed using ECL (Amersham Pharmacia Biotech, Piscataway, NJ), and the signal was
quantitated, after densitometry, using Imagequant software
(Molecular Dynamics, Inc., Sunnyvale, CA).
35S-labeling and immunoprecipitation
Leydig cells were isolated from control or LPS-injected mice and
cultured in methionine and cysteine-free medium [DMEM;
ICN, Irvine, CA) 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] for 30 min. Cells were then metabolically labeled with 250
µCi/ml [35S]-methionine
(35S-Translabel, ICN) in methionine and
cysteine-free media for 2 h. Lysates were flash-frozen on dry ice,
then subjected to immunoprecipitation, as described (20).
Lysates were divided, and StAR and P450scc were immunoprecipitated in
parallel from equal amounts of protein, subjected to SDS-PAGE, and
analyzed by PhosphorImaging. To determine the effect of LPS injection
on total protein synthesis, an aliquot of lysate from
35S-labeled Leydig cells from control and
LPS-injected mice, was TCA-precipitated onto GF/A glass fiber filters,
washed extensively with cold TCA and ethanol, and counted. In parallel,
total protein in the sample was quantitated, and results are expressed
as cpm/µg protein.
Statistical analysis
Data were presented as means ± SEM of three or
more independent experiments. For group comparison, one-way ANOVA
followed by a Student-Newmann-Keuls multiple-range test were performed
using the InStat, version 3.0, statistical software package
(GraphPad Software, Inc., San Diego, CA). Differences were
considered as significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Inhibition of LH release has been
observed after LPS injection. To determine whether this could account
for the rapid inhibition of steroidogenesis in the testes, LH levels
were measured at 2 h after animals were injected with LPS. As
shown in Fig. 1B, we did not observe any change in serum LH that could
account for the decrease in serum testosterone. LH levels also remained
unchanged between 15 min and 2 h and at 24 h post LPS
injection (data not shown). Rapid loss of LH receptors on the Leydig
cells might also account for the inhibition of steroidogenesis.
However, there was no significant difference in LH receptor number or
binding affinity in Leydig cells from vehicle or LPS-injected animals
2 h after treatment (Fig. 2). The
results indicate affinity constants of 1.58 x
108
M-1 for
vehicle and 1.2 x 108
M-1 for LPS; r
values were 0.969 and 0.860, respectively. Furthermore, as shown in
Fig. 3, no difference was observed in the
amounts of cAMP in freshly isolated cells from control or LPS-injected
mice, 2 h post injection (95 ± 22 and 120 ± 35
fmol/µg protein, respectively). In addition, there was no difference
in basal or hCG-stimulated cAMP levels in Leydig cells isolated from
control or LPS-injected animals after 3 h incubation with or
without hCG (39 ±13 vs. 28 ±14 fmol/µg protein for
basal, and 195 ± 29 vs. 208 ±18 fmol/µg protein for
hCG-stimulated, for control and LPS-injected, respectively).
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. By 2 h, we observed a 30%
decrease in the level of the mature 30-kDa StAR protein, a greater than
75% decrease by 4 h, and greater than 90% by 8 h.
This loss of the 30-kDa form of StAR indicates that the protein is not
accumulating in the mitochondria and could account for the rapid loss
of serum testosterone. One possible explanation for this decrease in
StAR might be a decrease in StAR mRNA. To address this possibility,
Leydig cells were purified from animals, 2 and 24 h after LPS
injection; and mRNA levels for StAR, P450scc, 3ß-HSD, and P450c17
were quantitated by Northern hybridization. No changes in the amounts
of mRNA for these four proteins were observed 2 h after injection
(Fig. 5, A and B), indicating that
transcriptional repression of neither StAR nor the three steroidogenic
enzymes could account for the rapid decrease in testosterone synthesis.
There was no apparent difference in any of the StAR transcripts in
control vs. LPS, and all three transcripts were included in
the quantitation. At 24 h, we did observe decreases in the mRNAs
for the steroidogenic enzymes (Fig. 5C and Ref. 10), which
likely contributes to the chronic inhibition of testicular
steroidogenesis, but found no change in the level of StAR mRNA.
These data suggest that StAR protein synthesis has been uncoupled from
StAR transcription. To assess the effect of LPS on StAR protein
synthesis, Leydig cells were isolated from mice, 2 h after LPS
injection, and incubated with 35S-methionine; and
newly synthesized StAR protein was immunoprecipitated. The results,
shown in Fig. 6A, indicate that LPS
treatment caused a marked inhibition of
35S-incorporation into newly synthesized 30-kDa
StAR. However, as shown in Fig. 6B, there was an equivalent amount of
label incorporated into 37-kDa StAR in both groups of Leydig cells. To
determine whether the inhibition was restricted to StAR, or whether
other mitochondrial proteins were affected,
35S-incorporation into newly synthesized P450scc
was assessed in parallel. As shown in Fig. 6C, LPS injection had no
effect on P450scc de novo synthesis. Moreover, there was no
effect on overall protein synthesis, as determined by incorporation of
radioactivity into TCAprecipitable protein (Fig. 6D).
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, the production of testosterone from
LPS-treated animals after cAMP treatment was inhibited by more than
60%, compared with vehicle-injected animals, whereas there was no
difference in testosterone synthesis when the cells were incubated with
(22R)-hydroxycholesterol. However, this was not the case 24 h
after LPS injection. Figure 7B shows testosterone production is
inhibited in cells from LPSinjected animals after incubation with
either cAMP or (22R)-hydroxycholesterol, indicating a block in
steroidogenesis downstream of cholesterol transport. This likely
reflects a decrease in translation of the steroidogenic enzymes
resulting from the decrease in mRNAs at 24 h.
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, animals receiving LPS had serum
corticosterone levels 40-fold greater than naïve animals and
4-fold greater than vehicle-injected animals. By 24 h, serum
corticosterone in LPS-injected animals had fallen to a level 16-fold
greater than naïve animals but were 7-fold greater than
vehicle-injected animals. Similarly, progesterone was increased more
than 13-fold over naïve animals 2 h after LPS injection
(Fig. 8B). By 24 h, this level had decreased to less than 2-fold
over naïve and vehicle-injected animals. Thus, adrenal
steroidogenesis is acutely activated after LPS injection, returning
toward more normal levels by 24 h. As expected, adrenal
steroidogenesis in vehicle-injected animals returned to uninduced
levels more rapidly. To determine whether transcriptional activation
was involved in the response to LPS, we quantitated mRNAs for StAR,
P450scc, 3ß-HSD, and P450c21. Analysis of adrenal mRNAs indicated
that, at 2 h after injection, StAR mRNA was increased by 50%,
whereas there was no observable difference in mRNAs for the three
steroidogenic enzymes (Fig. 9). We then
determined whether the increase in StAR mRNA resulted in an increase in
StAR protein. A representative Western blot is shown in Fig. 10A. The 30-kDa StAR protein was
quantitated by Western blot at 2, 4, 8, and 24 h after LPS
injection. As shown in Fig. 10B, StAR protein increased more than
2-fold during the first 8 h after injection and remained high at
24 h. It is likely that the acute activation of adrenal
steroidogenesis is mediated at the level of StAR synthesis and that the
stimulus is not of sufficient duration to result in the induction of
the steroidogenic enzymes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, adrenal steroidogenesis was markedly, but transiently, stimulated after LPS injection. Serum corticosterone and progesterone levels were significantly increased 2 h after LPS injection but decreased toward control levels at 24 h. The mRNA for StAR was markedly increased at 2 h in adrenals from LPS injected mice, but mRNAs for steroidogenic enzymes remained at control levels. Finally, the 30-kDa form of StAR protein was increased by 2 h, peaking at 8 h, and remained elevated at 24 h.
It is now well established that inflammation and activation of the immune system inhibit male reproductive function [in particular, Leydig cell steroidogenesis (21)]. We have shown previously, in three different experimental paradigms, that immune activation inhibits Leydig cell steroidogenesis and that inhibition of testosterone production is correlated with decreased StAR protein expression (6, 8, 9). Initially, we observed that the decrease in serum testosterone 2 h after LPS injection was accompanied by a decrease in the 30-kDa form of StAR protein. More recently, we have observed a decrease in StAR protein in rat Leydig cells as early as 30 min after ICV injection of IL1ß, which parallels a decrease in serum testosterone. In addition, StAR levels are reduced after induction of septic shock in rats. In the present study, we extend these observations and now report that LPS injection results in a rapid cessation of testosterone production, that there is a time-dependent decrease in the mature 30-kDa form of StAR protein, and that the inhibition of steroid production is at the level of cholesterol transfer, as shown by the (22R)-hydroxycholesterol experiment. Finally, the decrease in StAR protein is posttranscriptional, as shown by a lack of change in StAR mRNA but a marked decrease in StAR protein synthesis and expression.
Our observations indicate that the inhibition of steroidogenesis is distal to LH receptor activation. As shown here, there is no significant change in serum LH levels after LPS injection. Because of the pulsatile nature of LH secretion, we did observe a large interanimal variation in LH levels (22, 23). Another consequence of the pulsatile release of LH is the large interanimal variation in serum testosterone levels in mice, requiring a large number of mice to obtain statistically significant data. Further evidence that a decrease in LH is not responsible for inhibition is the observation that steroidogenesis cannot be rescued by treatment with hCG (24, 25, 26). A loss of cAMP and/or protein kinase A (PKA) signaling is also not a likely explanation, because we do not observe a change in cAMP production or a change in StAR mRNA, which has been determined to have a half-life of about 3 h and is regulated by LH (27). We examined StAR mRNA levels ex vivo after cAMP or R22 stimulation of testosterone production, and we observed that cAMP caused an increase in the amount of StAR mRNA in Leydig cells from LPS-injected mice, which indicated that LPS did not prevent the cells from responding to cAMP, suggesting that PKA was not inhibited (data not shown). This observation supports a more distal site of action in the LH signaling pathway than inhibition of PKA by LPS. Moreover, there was no change in Leydig cell LH receptor number or binding after LPS injection at any time point examined. These results are similar to those observed after ICV injection of IL-1ß in rats. The loss of testicular responsiveness to hCG stimulation was not attribu-table to alteration of Leydig cell LH receptor number or binding affinity (6). We do observe a decrease in steroidogenic enzyme mRNA expression in Leydig cells after 24 h that we have shown is caused by transcriptional repression by cytokines induced by LPS. That StAR mRNA remains unaffected further suggests that this repression is occurring downstream of the initial signaling pathway for LH.
In models of immune-activation that involve central administration of cytokines or LPS, LH levels are decreased. ICV administration of LPS or IL-1ß to male rats results in a measurable decrease in serum LH, by 30 min, in both intact or gonadectomized animals (25, 28). This does not hold true for systemic injection (ip) of LPS (29), which does not alter LH, as we have also shown. In contrast, iv administration of tumor necrosis factor (TNF) to rats results in the inhibition of testosterone accompanied by the expected increase in LH (30). Interestingly, we do not observe a rebound in LH levels in the absence of testosterone, suggesting that there is a secondary inhibition of LH release. In this way, our data are reminiscent of the effects of dioxin on testosterone production (31, 32). Thus, the effect of immune-activation on LH is dependent on the particular inflammatory mediator and the manner of its administration.
Diametric response: increase in adrenal steroidogenesis
LPS endotoxemia does not result in a global or systemic
steroidogenic failure but is restricted to the gonads [i.e.
LPS also inhibits ovarian steroidogenesis (33)]. Indeed,
activation of adrenal steroidogenesis modulates the deleterious effects
of global immune activation. In the present study, we show that LPS
causes a rapid and marked increase in adrenal steroidogenesis. Serum
corticosterone levels increased by 4-fold within 2 h (compared
with vehicle-injected controls) and 40-fold [compared with
naïve (uninjected) mice]. The elevation of corticosterone by
injection of vehicle alone is likely caused by the stress of needle
penetration. Serum progesterone levels are also markedly elevated
within 2 h. Elevated progesterone production by the adrenal has
previously been reported (34), in particular, in
association with immune activation of the
hypothalamic-pituitary-adrenal axis (25). Elevated ACTH
levels are also associated with increased progesterone production by
the adrenal gland in the female (35). Furthermore, the
rise and fall of serum progesterone parallels changes in serum
corticosterone, supporting the adrenal source. In contrast, serum
testosterone levels decrease rapidly and remain depressed, whereas
progesterone rises and falls again. Moreover, because Leydig cell
steroid production is inhibited at the level of cholesterol transfer,
it stands to reason that the progesterone must be coming from the
adrenal.
Our data indicate that elevation of adrenal steroid production is caused by increased StAR protein expression. Both ACTH and angiotensin II transcriptionally regulate adrenal expression of StAR (36). An increase in adrenal StAR mRNA, followed by an increase in StAR protein, is observed after injection of animals with ACTH or treatment of adrenal cells in culture with cAMP analogues or angiotensin II (36, 37, 38). We also observed a significant and proportional increase in the level of all three StAR mRNA transcripts in the adrenals within 2 h of LPS injection, supporting the conclusion that transcriptional regulation of StAR underlies the observed increase in steroid production in the adrenals.
Elevated glucocorticoids are associated with decreased male reproductive function. Glucocorticoids are known to inhibit the hypothalamic-pituitary-gonadal axis at multiple sites, including GnRH and gonadotropin secretion, and to exert direct inhibitory effects on Leydig cells (1). Indeed, we have shown previously that glucocorticoids inhibit P450scc transcription and de novo synthesis in isolated mouse Leydig cells (39). Elevated serum corticosterone, therefore, may contribute to the long-term inhibition of Leydig cell function at the level of steroidogenic enzyme expression (40). It is possible that elevated glucocorticoids are causative in the lack of rebound in serum LH levels after decreased serum testosterone. In castrated males, or males whose testosterone is inhibited pharmacologically, there is a reciprocal and pronounced elevation in LH. The lack of a secondary elevation in LH in the presence of markedly reduced serum testosterone suggests that other factors, i.e. glucocorticoids, may be inhibiting LH secretion. It is unlikely that glucocorticoid-mediated inhibition of Leydig cell function contributes to the immediate cessation of testosterone production, because the effects of glucocorticoids on Leydig cells have been shown to be at the level of transcriptional repression, and our data demonstrate that the immediate inhibition of steroid production was independent of changes in mRNA levels.
The present study presents data that demonstrate that serum
testosterone levels decrease rapidly after LPS injection. The serum
half-life of testosterone in rodents is reported to be approximately
4–5 min (41, 42). Testicular interstitial macrophages and
Leydig cells are intimately associated, suggesting a functional
interaction between these cells (for review, see Ref. 43).
Our studies have established that macrophage-secreted proinflammatory
cytokines, such as IL-1, TNF, and IL-6, are potent repressors of
LH/hCG or cAMP-stimulated testosterone production and steroidogenic
enzyme gene expression, both in vitro and in
vivo, and have demonstrated that the interstitial testicular
macrophages express and produce IL-1 and TNF (10, 26, 44). We have demonstrated that LPS, acting via the elaboration
of proinflammatory cytokines, causes a profound inhibition of
steroidogenic enzyme gene expression, analogous to what we observed
in vitro when we supplied exogenous recombinant cytokines to
primary cultures of Leydig cells. However, testosterone inhibition
after LPS injection occurs faster than can be accounted for by the
cytokine-mediated inhibition of steroidogenesis (8, 10).
Moreover, in vitro treatment of Leydig cells with cytokines
or LPS does not reproduce the acute inhibition of StAR protein
expression, cholesterol transfer activity, or immediate cessation of
testosterone production. LPS is known to activate the respiratory burst
and production of reactive oxygen species from testicular macrophages.
Treatment of corpora luteal cells or MA-10 tumor Leydig cells, in
primary culture, with exogenous reactive oxygen donors (such as
hydrogen peroxide) inhibits steroid hormone production by blocking
cholesterol transfer (45, 46). Together, these
observations suggest that LPS may be acting via the rapid production of
reactive oxygen by testicular interstitial macrophages to perturb
Leydig cell steroidogenesis. It is possible that the adrenal cortex
escapes oxidative stress after LPS injection because of the elevated
concentrations of the antioxidants ascorbic acid and -tocopherol,
reported to be among the highest of all tissues (Refs. 47, 48) and references therein). Current studies in our laboratory
are examining these possibilities.
StAR protein is essential for gonadal and adrenal steroidogenesis. It
is a nuclear-encoded protein targeted to the mitochondria by
aminoterminal signal peptides. Controversy exists about the mechanism
through which StAR facilitates cholesterol transfer to the inner
mitochondrial matrix. It is clear, though, that StAR is synthesized as
a larger molecular mass protein (37 kDa in the mouse) that is
proteolytically processed to the mature 30-kDa form in the mitochondria
(for recent reviews, see Refs. 4, 49). The
inner-mitochondrial 30-kDa form of StAR protein represents the
inactive, postfunctional form of the protein. It has a half-life of
approximately 2 h (K. Held Hales and D. B. Hales, unpublished
observation). The transiently expressed 37-kDa form, thought to have a
half-life of minutes, is now recognized to be the active form of StAR.
Therefore, changes in the 30-kDa form are an indirect measure of
changes in the active form. The function of StAR in steroidogenesis
depends on new protein synthesis, yet the degradation of the StAR
protein is much slower than its functional inactivation (4, 50, 51). Therefore, that we see a slower time-dependent decrease in
30-kDa StAR, even though steroidogenesis has been rapidly inhibited, is
to be expected. Furthermore, mitochondrial transport and processing of
active StAR, as well as steroidogenesis, are dependent upon an intact
mitochondrial electrochemical gradient (52). The
intriguing observation, shown in Fig. 6
, A and B, that there is a
greater decrease in the accumulation of newly synthesized 30-kDa form
of StAR, relative to the 37-kDa form after LPS injection, suggests that
StAR processing and/or mitochondrial import, as well as de
novo synthesis, may have been inhibited. This observation suggests
a perturbation of the mitochondrial electrochemical gradient, which is
known to be required for mitochondrial import of StAR (52, 53).
The present study demonstrates that a single ip injection of a sublethal dose of LPS causes an almost immediate decrease in serum testosterone levels. At 2 h after LPS, the mature 30-kDa form of StAR protein is markedly reduced and continues to decrease over the next several hours. The inhibition of testosterone production was at the level of cholesterol transfer, as shown by the ability of R22-hydroxycholesterol to restore testosterone production. The inhibition was distal to serum LH or Leydig cell LH receptor binding on Leydig cells. In contrast, there was a marked increase in adrenal steroid production, an elevation in glucocorticoid and progesterone serum levels, and concomitant increase in StAR mRNA and protein levels. Thus, our observation of decreased accumulation of 30-kDa StAR in the Leydig cell, and increased accumulation in the adrenal, are a sensitive read-out of changes in the steroidogenic potential of the cell.
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Acknowledgments |
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Received March 21, 2000.
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  A. K. Pandey, X. Yin, R. B. Schiffer, J. C. Hutson, D. M. Stocco, P. Grammas, and X. Wang Involvement of the Thromboxane A2 Receptor in the Regulation of Steroidogenic Acute Regulatory Gene Expression in Murine Leydig Cells Endocrinology, July 1, 2009; 150(7): 3267 - 3273. [Abstract] [Full Text] [PDF]
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 F. Favreau, L. Rossard, K. Zhang, T. Desurmont, E. Manguy, A. Belliard, S. Fabre, J. Liu, Z. Han, R. Thuillier, et al. Expression and modulation of translocator protein and its partners by hypoxia reoxygenation or ischemia and reperfusion in porcine renal models Am J Physiol Renal Physiol, July 1, 2009; 297(1): F177 - F190. [Abstract] [Full Text] [PDF]
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 J. J Boonekamp, A. H.F Ros, and S. Verhulst Immune activation suppresses plasma testosterone level: a meta-analysis Biol Lett, December 23, 2008; 4(6): 741 - 744. [Abstract] [Full Text] [PDF]
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 D. Gunnarsson, P. Leffler, E. Ekwurtzel, G. Martinsson, K. Liu, and G. Selstam Mono-(2-ethylhexyl) phthalate stimulates basal steroidogenesis by a cAMP-independent mechanism in mouse gonadal cells of both sexes Reproduction, May 1, 2008; 135(5): 693 - 703. [Abstract] [Full Text] [PDF]
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 S. A. Andric, M. M. Janjic, N. J. Stojkov, and T. S. Kostic Protein kinase G-mediated stimulation of basal Leydig cell steroidogenesis Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1399 - E1408. [Abstract] [Full Text] [PDF]
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S.-Y. Zhang, Y. Ito, O. Yamanoshita, Y. Yanagiba, M. Kobayashi, K. Taya, C. Li, A. Okamura, M. Miyata, J. Ueyama, et al. Permethrin May Disrupt Testosterone Biosynthesis via Mitochondrial Membrane Damage of Leydig Cells in Adult Male Mouse Endocrinology, August 1, 2007; 148(8): 3941 - 3949. [Abstract] [Full Text] [PDF]
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J. A. Allen, T. Shankara, P. Janus, S. Buck, T. Diemer, K. Held Hales, and D. B. Hales Energized, Polarized, and Actively Respiring Mitochondria Are Required for Acute Leydig Cell Steroidogenesis Endocrinology, August 1, 2006; 147(8): 3924 - 3935. [Abstract] [Full Text] [PDF]
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M. Herman and C. Rivier Activation of a Neural Brain-Testicular Pathway Rapidly Lowers Leydig Cell Levels of the Steroidogenic Acute Regulatory Protein and the Peripheral-Type Benzodiazepine Receptor while Increasing Levels of Neuronal Nitric Oxide Synthase Endocrinology, January 1, 2006; 147(1): 624 - 633. [Abstract] [Full Text] [PDF]
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 M. Hedger, J. Klug, S. Frohlich, R. Muller, and A. Meinhardt Regulatory Cytokine Expression and Interstitial Fluid Formation in the Normal and Inflamed Rat Testis Are Under Leydig Cell Control J Androl, May 1, 2005; 26(3): 379 - 386. [Abstract] [Full Text] [PDF]
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S. R. King, S. D. Ginsberg, T. Ishii, R. G. Smith, K. L. Parker, and D. J. Lamb The Steroidogenic Acute Regulatory Protein Is Expressed in Steroidogenic Cells of the Day-Old Brain Endocrinology, October 1, 2004; 145(10): 4775 - 4780. [Abstract] [Full Text] [PDF]
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 Z. Granot, R. Geiss-Friedlander, N. Melamed-Book, S. Eimerl, R. Timberg, A. M. Weiss, K. H. Hales, D. B. Hales, D. M. Stocco, and J. Orly Proteolysis of Normal and Mutated Steroidogenic Acute Regulatory Proteins in the Mitochondria: the Fate of Unwanted Proteins Mol. Endocrinol., December 1, 2003; 17(12): 2461 - 2476. [Abstract] [Full Text] [PDF]
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V. Morales, P. Santana, R. Diaz, C. Tabraue, G. Gallardo, F. L. Blanco, I. Hernandez, L. F. Fanjul, and C. M. Ruiz de Galarreta Intratesticular Delivery of Tumor Necrosis Factor-{alpha} and Ceramide Directly Abrogates Steroidogenic Acute Regulatory Protein Expression and Leydig Cell Steroidogenesis in Adult Rats Endocrinology, November 1, 2003; 144(11): 4763 - 4772. [Abstract] [Full Text] [PDF]
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T. Diemer, J. A. Allen, K. H. Hales, and D. B. Hales Reactive Oxygen Disrupts Mitochondria in MA-10 Tumor Leydig Cells and Inhibits Steroidogenic Acute Regulatory (StAR) Protein and Steroidogenesis Endocrinology, July 1, 2003; 144(7): 2882 - 2891. [Abstract] [Full Text] [PDF]
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 J. Cottom, L. M. Salvador, E. T. Maizels, S. Reierstad, Y. Park, D. W. Carr, M. A. Davare, J. W. Hell, S. S. Palmer, P. Dent, et al. Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase J. Biol. Chem., February 21, 2003; 278(9): 7167 - 7179. [Abstract] [Full Text] [PDF]
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 S. R. King, P. R. Manna, T. Ishii, P. J. Syapin, S. D. Ginsberg, K. Wilson, L. P. Walsh, K. L. Parker, D. M. Stocco, R. G. Smith, et al. An Essential Component in Steroid Synthesis, the Steroidogenic Acute Regulatory Protein, Is Expressed in Discrete Regions of the Brain J. Neurosci., December 15, 2002; 22(24): 10613 - 10620. [Abstract] [Full Text] [PDF]
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 A.L. Johnson, E.V. Solovieva, and J.T. Bridgham Relationship Between Steroidogenic Acute Regulatory Protein Expression and Progesterone Production in Hen Granulosa Cells During Follicle Development Biol Reprod, October 1, 2002; 67(4): 1313 - 1320. [Abstract] [Full Text] [PDF]
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S. Eimerl and J. Orly Regulation of Steroidogenic Genes by Insulin-Like Growth Factor-1 and Follicle-Stimulating Hormone: Differential Responses of Cytochrome P450 Side-Chain Cleavage, Steroidogenic Acute Regulatory Protein, and 3{beta}-Hydroxysteroid Dehydrogenase/Isomerase in Rat Granulosa Cells Biol Reprod, September 1, 2002; 67(3): 900 - 910. [Abstract] [Full Text] [PDF]
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L. M. Salvador, E. Maizels, D. B. Hales, E. Miyamoto, H. Yamamoto, and M. Hunzicker-Dunn Acute Signaling by the LH Receptor Is Independent of Protein Kinase C Activation Endocrinology, August 1, 2002; 143(8): 2986 - 2994. [Abstract] [Full Text] [PDF]
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A. L. Johnson and J. T. Bridgham Regulation of Steroidogenic Acute Regulatory Protein and Luteinizing Hormone Receptor Messenger Ribonucleic Acid in Hen Granulosa Cells Endocrinology, July 1, 2001; 142(7): 3116 - 3124. [Abstract] [Full Text] [PDF]
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C. Mauduit, I. Goddard, V. Besset, E. Tabone, C. Rey, F. Gasnier, F. Dacheux, and M. Benahmed Leukemia Inhibitory Factor Antagonizes Gonadotropin Induced-Testosterone Synthesis in Cultured Porcine Leydig Cells: Sites of Action Endocrinology, June 1, 2001; 142(6): 2509 - 2520. [Abstract] [Full Text] [PDF]
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