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From the Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland.
| Correspondence to: Dr Lindi Luo, Department of Biochemistry and Molecular Biology, Division of Reproductive Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205 (e-mail: lluo{at}hsph.edu). |
| Received for publication May 17, 2004; accepted for publication July 16, 2004. |
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Abstract |
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Key words: Leydig cell, cyclic AMP, steroidogenesis, Brown Norway rat
As yet, we do not know which among the Leydig cell deficits that accompany Leydig cell aging in fact is responsible for reduced steroidogenesis. It seems unlikely that each of the deficits associated with Leydig cell aging occur independently of one another, but rather that an initiating event occurs that leads to coordinate downstream changes in cholesterol import and/or the steroidogenic machinery.
It has long been known that cyclic AMP, produced in response to LH, phosphorylates a number of proteins and, in doing so, directly or indirectly causes increased synthesis of androgen (Stocco, 1992; Clark and Stocco, 1996). Under normal circumstances, the rate-limiting step of steroidogenesis is cholesterol transport to the inner mitochondrial membrane, a step in which StAR (Stocco, 1996) and PBR (Culty et al, 2002; Liu et al, 2003) play important roles. This is followed by the important first step of converting cholesterol to pregnenolone by the mitochondrial P450scc. Herein we examine cAMP production, StAR, and P450scc in relationship to testosterone production by Leydig cells isolated from the testes of Brown Norway rats of increasing ages (4, 9, 15, and 20 months). The major objectives of the study were to determine the age(s) at which changes in cAMP, StAR, and P450scc occur, and the temporal relationship of such changes to age-related reductions in Leydig cell testosterone production.
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Materials and Methods |
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Isolation and Culture of Leydig Cells
Leydig cells were isolated and purified by centrifugal elutriation and
Percoll density gradient centrifugation, as previously described
(Klinefelter et al, 1987). The
purity of the Leydig cell preparations in this study consistently was greater
than 93%, as assessed by determining the percentage of cells that stained
histochemically for 3ß-HSD
(Klinefelter et al, 1987). Leydig cells (2 x 105) were placed in tissue culture dishes
without or with maximally stimulating ovine LH (100 ng/ml, National Hormone
and Pituitary Program, National Institute of Diabetes and Kidney Disease),
dibutyryl cyclic adenosine monophosphate (1 mM), 22R-hydroxycholesterol (20
µM), or pregnenolone (2.5 µM). All treatments were performed at 34°C
for 2 hours.
Testosterone Radioimmunoassay (RIA)
Testosterone concentrations were determined in aliquots of culture medium
or serum by RIA, according to methods described previously
(Schanbacher and Ewing, 1975).
The assay sensitivity was 10 pg/tube, with intra-assay and interassay
coefficients of variation of 11.2% and 9.6%, respectively.
cAMP Production
Leydig cells were incubated with maximally stimulating LH (100 ng/ml) for
2.5, 5, 10, 15, or 20 minutes. Immediately afterward, cells were extracted and
cAMP was assayed with a cAMP [3H] assay system (Amersham Pharmacia
Biotech, Piscataway, NJ), according to the manufacturer's directions. The
sensitivity of the assay was 0.05 pmol per assay tube.
Isolation of RNA and Northern Blot Analysis
Total RNA was extracted from freshly isolated Leydig cells by a single-step
method (Chomczynski and Sacchi,
1987; Kedzierski and Porter,
1991). Prior to RNA extraction, 35S-labeled RNA (10 000
counts per minute [cpm]) was added to each sample in order to determine the
percent recovery of total RNA (Wright et
al, 1993). Total RNA from equal numbers of Leydig cells was
analyzed.
Northern blot analysis was carried out as described previously
(Sambrook et al, 1989). Total
RNA from 1 x 106 cells was loaded per lane and
electrophoresed through a denaturing 1.2% agarose gel. The gels subsequently
were blotted by capillary action to a nylon filter and were further hybridized
with a 32P-labeled complementary DNA (cDNA) probe for a 1.5 Kb
mouse StAR cDNA (Clark et al,
1994) and a 1.2-Kb rat P450scc cDNA
(Oonk et al, 1989). cDNA was
labeled with [
-32P] deoxycytidine triphosphate to
approximately 1 x 108 cpm/µg DNA, using a random primer
synthesis method (Megaprime DNA labeling system, Amersham, Piscataway, NJ).
Nylon membranes were probed sequentially for StAR, P450scc, and 18S rRNA.
Immunoblot Analysis
Purified Leydig cells from 4-, 9-, 15-, and 20-month-old rats were
solubilized in sample buffer (50 mM Tris, pH 6.8, 5% ß-mercaptoethanol,
2% sodium dodecyl sulfate, 10% glycerol, and 0.001% bromophenol blue).
Proteins were separated by 10% polyacrylamide gel electrophoresis, with each
lane containing protein from equal numbers of cells (2 x
105), and then electro-transferred onto a nitrocellulose filter.
Blots were incubated with rabbit antibodies to StAR
(Clark et al, 1994) and
P450scc (Chemicon International Inc, Temecula, Calif). Subsequently, blots
were incubated with a 1:5000 dilution of horseradish peroxidase-conjugated
anti-rabbit IgG (donkey IgG, Amersham Pharmacia Biotech, Piscataway, NJ), and
an enhanced chemiluminescence (ECL) kit was used to detect the
horseradish-peroxidase-labeled protein, according to the manufacturer's
instructions (Amersham). The x-ray films were quantified by densitometry.
Statistical Analyses
Data are expressed as the mean ± standard error of the mean.
Statistical differences involving multiple group comparisons were determined
by 1-way analysis of variance (ANOVA). If group differences were revealed by
ANOVA (P < .05), differences between individual groups were
determined with the Scheffe F test (P < .05).
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Results |
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With Leydig cells from each of 4-, 9-, 15-, and 20-month-old rats, peak intracellular cAMP levels were reached by 5-10 minutes of incubation with LH (Figure 2A). Age-related decreases in cAMP production were seen; cAMP production, greatest in cells from 4-month-old rats, was reduced by 9 months, and reduced further in cells from 15- and 20-month-old rats. At 20 minutes of incubation with LH, cells from 20-month-old rats produced about half the cAMP of cells from 4-month-old rats.
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Figure 2B shows testosterone production by the same Leydig cells that were assessed for cAMP production, in this case from rats of 4-15 months of age. As expected, age-related decreases were seen in the ability of the Leydig cells to produce testosterone. In striking contrast to cAMP production, which peaked at 5-10 minutes of incubation with LH, testosterone production by these cells was still rising at the end of a 20-minute incubation period.
StAR protein is involved in the movement of cholesterol to the inner mitochondrial membrane, the step in the steroidogenic pathway that is considered to be rate limiting. We examined the effect of aging on StAR as well as on P450scc, the mitochondrial enzyme responsible for converting cholesterol to pregnenolone. Leydig cells were isolated from 4-, 9-, 15-, and 20-month-old rats and assessed for steady-state mRNA (Figure 3) and immunoreactive protein (Figure 4) for StAR and P450scc. Northern blot analysis revealed that StAR mRNA was expressed as 2 major transcripts, of 3.8 Kb and 1.7 Kb, and 1 minor transcript, of 1.2 Kb. The 1.7 Kb transcript was reduced in Leydig cells from testes of 9-, 15-, and 20-month-old rats compared to cells from 4-month-old rats (Figure 3), although not significantly at 9 months. P450scc mRNA also was reduced at each age, although not significantly until 15-20 months. Western blot analysis (Figure 4 above and below) similarly revealed decreases in StAR and P450scc proteins; StAR was reduced by 60% from the young value in Leydig cells from 15-month-old rats, and P450scc protein by 40%.
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Figure 5 shows testosterone production by Leydig cells cultured short term, for 2 hours, in the absence (basal) or presence of maximally stimulating LH (100 ng/ml), dbcAMP (1 mM), 22-hydroxycholesterol (20 µM), or pregnenolone (2.5 µM). The rationale for this study was that dbcAMP bypasses LH receptor-G protein coupling; 22-hydroxycholesterol enters mitochondria directly, without the need for functioning transport proteins (StAR, PBR); and pregnenolone bypasses P450scc. With each of dbcAMP, 22-hydroxycholesterol, and pregnenolone, significant age-related declines in testosterone production were seen at 15 months, with further declines at 20 months. As with LH, reductions were seen at 9 months that were not significant.
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Discussion |
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A major objective of the present study was to determine the ages at which critical changes related to steroidogenesis are initiated in aging Leydig cells in relationship to reduced steroidogenesis. We focused on cAMP production, StAR, and P450scc. Significant reduction in the ability of the Leydig cells to produce testosterone, as assessed by their response to short-term (2 hour) exposure to maximally stimulating LH, was first seen in cells isolated from rats of 15 months of age. LH-stimulated intracellular cAMP levels were found to decline significantly by 9 months, before significant reductions in testosterone production were seen. In part, these results support the contention (Catt et al, 1980; Liu et al, 2003) that testosterone production can be maximal in the face of reduced cAMP. Further reductions in cAMP were seen through 15 and 20 months, along with decreases in testosterone. The observation that aging Leydig cells produce less cAMP than young cells suggests that the old cells have defects in the LH-cAMP signaling cascade.
Short-term (2 hours) exposure of Leydig cells to dbcAMP, which bypasses LH receptor-G protein coupling, is used to assess steps of the steroidogenic pathway beyond this point. Likewise, culturing the cells with 22-hydroxycholesterol, which enters mitochondria directly and thus obviates the need for functioning transport proteins (StAR, PBR), or pregnenolone, which bypasses P450scc, is used to examine the possibility of downstream deficits. Significant age-related declines in testosterone production were seen in each case. The results indicate that, as expected, aging cells have deficits at multiple steps along the steroidogenic pathway. It should be pointed out that exposure of the cells for 2 hours to LH, dbcAMP, 22-hydroxycholesterol, or pregnenolone is too short a time for steroidogenic function to be restored, and therefore this study was conducted to describe existing defects.
Given the critical role played by LH-stimulated cAMP in steroidogenesis (Dufau et al, 1980), it seemed reasonable to hypothesize that if cAMP production was reduced to sufficiently low levels, the result would be reduced testosterone production. In fact, significant decreases were seen in cAMP production at age 9 months, preceding decreases in StAR and P450scc mRNA, StAR and P450scc protein levels, and testosterone production. These observations suggested that changes in StAR and P450scc, and thus in testosterone production, might occur as a consequence of reduced cAMP. This hypothesis is supported by a recently completed study in our lab in which aged Leydig cells were cultured with dbcAMP, a membrane-permeable cAMP agonist that bypasses the LH receptor-adenlyly cyclase cascade (Chen et al, 2004). In that study, long-term (3 day) culture restored the ability of Leydig cells from the testes of aged (21-24-month-old) rats to produce testosterone at the high level of cells from 4-month-old rats, and thus reversed the steroidogenic deficits of aging Leydig cells. Accompanying the restoration of testosterone production, StAR and P450scc also were restored to the levels of young cells. These results are consistent with the hypothesis that the many changes in the steroidogenic pathway that accompany aging probably do not occur independently of one another, but rather that there is an initiating, early event (or events), perhaps reduced cAMP production, that ultimately leads to coordination of downstream changes in the remainder of the steroidogenic machinery, and thereby to reduced testosterone production.
The mechanism by which changes occur in aging Leydig cells remains uncertain. Reduced LH levels essentially have been ruled out (Chen et al, 1994, 1996; Gruenewald et al, 1994; Bonavera et al, 1997, 1998; Grzywacz et al, 1998). Although aged cells have fewer LH receptors than young cells, this is unlikely to explain the decreased cAMP production (Chen et al, 2002). If LH receptor coupling to adenylyl cyclase through G proteins was compromised, reduced cAMP would result (Dufau et al, 1980), and this certainly is a possibility. There is extensive evidence that free radical damage may contribute to cell aging (Pacifici and Davies, 1991; Knight, 2000). We have reported that aged Leydig cells produce more reactive oxygen than young cells (Chen et al, 2001), and unpublished studies from our lab indicate that the major scavengers of reactive oxygen (superoxide dismutase [SOD] I and II, glutathione peroxidase, and glutathione) all are reduced in aged cells. In combination, increased reactive oxygen production and a decreased ability of the cells to rid themselves of potentially damaging reactive oxygen species could lead to free radical damage. If free radical damage affected membrane fluidity (Vlasova, 2000; Karbownik et at, 2001), the LH receptor-G protein-adenylyl cyclase coupling cascades probably would become less efficient (Kolena et al, 1986), and this could disrupt LH-stimulated cAMP production (Wu et al, 1993). This is an appealing hypothesis to explain how aging might result in reduced intracellular cAMP, which, in turn, could result in reductions in cholesterol transport and/or metabolism and thus in reduced testosterone production.
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Footnotes |
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