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Endocrinology 145(10):4441– 4446
Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2004-0639
Dibutyryl Cyclic Adenosine Monophosphate Restores the
Ability of Aged Leydig Cells to Produce Testosterone at
the High Levels Characteristic of Young Cells
HAOLIN CHEN, JUNE LIU, LINDI LUO,
AND
BARRY R. ZIRKIN
Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg
School of Public Health, Baltimore, Maryland 21205
The wealth of knowledge about the function and regulation of
adult Leydig cells, the cells within the mammalian testis that
produce testosterone, make these cells ideal for studying principles and mechanisms of aging. A hallmark of mammalian
aging is decreased serum testosterone concentration. In the
Brown Norway rat, this has been shown to be associated with
the reduced ability of aged Leydig cells to produce testosterone in response to LH. Herein, we demonstrate that culturing
the aged cells with dibutyryl cAMP, a membrane-permeable
I
N RATS, AS IN MEN, reduced serum testosterone concentration is a hallmark of aging (1, 2). The decreases in
serum testosterone may be accompanied by a constellation
of symptoms, sometimes termed andropause, that include
sexual dysfunction, lack of energy, loss of muscle and bone
mass, increased frailty, loss of balance, cognitive impairment,
and decreased general well-being (3). We have shown in the
Brown Norway rat, which is now a well-established model
for human male reproductive aging, that age-related reduction in serum testosterone results from the reduced production of testosterone by individual Leydig cells (4). The aged
cells are characterized by a number of deficits in the steroidogenic pathway, including reductions in LH receptor number, cAMP production (5), the cholesterol transport proteins
steroidogenic acute regulatory protein (StAR) (6) and peripheral benzodiazepine receptor (7), and the steroidogenic
enzymes of the mitochondria and smooth endoplasmic reticulum that convert cholesterol to testosterone (8). As yet,
the roles played by particular deficits in the reduced steroidogenic capacity of aging Leydig cells and the mechanism by
which these deficits arise are uncertain.
In young rats, experimental suppression of serum LH levels results in decreases in Leydig cell volume and testosterone production (9), qualities that also characterize aged Leydig cells (4). However, serum LH concentrations in Brown
Norway rats do not decline significantly with age (4, 6), and
the administration of exogenous LH to old rats in vivo or the
incubation of purified, aged Leydig cells with LH in vitro
failed to increase testosterone production by old Leydig cells
cAMP agonist that bypasses the LH receptor-adenlyly cyclase
cascade, restores testosterone production to levels comparable to those of young cells and also restores steroidogenic
acute regulatory protein and P450scc, the proteins involved in
the rate-limiting steps of steroidogenesis. These results
strongly suggest that signal transduction deficits are responsible for reduced steroidogenesis by aged Leydig cells and
that bypassing signal transduction reverses the steroidogenic
decline by the aged cells. (Endocrinology 145: 4441– 4446, 2004)
(5, 10), suggesting that deficits in LH are unlikely to represent
the underlying cause of age-related decline in Leydig cell
steroidogenesis.
The observation that LH-stimulated old Leydig cells produce far less cAMP than young cells (5) suggests that the old
cells have defects in the LH-cAMP signaling cascade. Recognizing the critical role played by LH-stimulated cAMP in
steroidogenesis (11), we hypothesized that reduced cAMP
production might cause age-related reductions in steroidogenesis. To test this hypothesis, we reasoned that if the steroidogenically hypofunctional aged cells could be induced to
produce testosterone at the high levels of young adult cells
by incubating the cells with dibutyryl cAMP (dbcAMP), then
we would conclude that aged-related reductions in the steroidogenesis most likely result from defects in the LH signaling pathway leading to reduced cAMP.
Materials and Methods
Animals
Male Brown Norway rats at ages 4 – 6 months (young) and 21–24
months (old) were obtained through the National Institute on Aging and
supplied by Harlan Sprague Dawley Inc. (Indianapolis, IN). Rats were
housed in controlled light (14 h light and 10 h dark) and temperature (22
C), with free access to rat chow and water. All procedures were in accord
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals, with protocols approved by the Johns Hopkins
Animal Care and Use Committee.
Suppression of LH
Abbreviations: dbcAMP, Dibutyryl cAMP; Gi, inhibitory G; Gs, stimulatory G; IBMX, isobutyl-methylxanthine; StAR, steroidogenic acute
regulatory protein.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
To experimentally suppress LH in young rats, the rats were administered testosterone via sc polydimethylsiloxane (SILASTIC brand; Dow
Corning, Midland, MI) implants. Details of the fabrication of the implants have been described previously (12). Briefly, rats received implants of 3 cm placed sc into the interscapular region for 5 d to suppress
Leydig cell steroidogenesis through feedback suppression of serum LH.
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Endocrinology, October 2004, 145(10):4441– 4446
Leydig cell purification
Leydig cells were isolated as previously described (13). In brief, the
testicular artery was cannulated and perfused with collagenase (1 mg/
ml, Type 3; Worthington, Freehold, NJ) in dissociation buffer (M-199
medium with 2.2 g/liter HEPES, 1.0 g/liter BSA, 25 mg/liter trypsin
inhibitor, 0.7 g/liter sodium bicarbonate, pH 7.4) to clear blood from the
testes. Testes were decapsulated and digested in collagenase (0.25 mg/
ml, 34 C) with shaking (90 cycles/min, 30 min). The dissociated cells
were then subjected to centrifugal elutriation and Percoll gradient centrifugation purification, as previously described (13). The final purity of
the Leydig cells obtained this way, determined by staining the cells for
3␤-hydroxysteroid dehydrogenase activity, was consistently about 95%.
Cell viability, assessed by trypan blue exclusion, was over 95%.
Culture of isolated Leydig cells with LH and dbcAMP
in vitro
Leydig cells were cultured according to the procedure described by
Klinefelter and Ewing (14). Briefly, purified Leydig cells were resuspended (106/ml) in M-199 medium (GIBCO, Grand Island, NY) supplemented with 2.2 g/liter NaHCO3, 2.4 g/liter HEPES, 0.1% BSA, and
12.5 mg/liter gentamicin sulfate (pH 7.4). One milliliter of cell suspension (1.0 ⫻ 106) was then added to a low attachment 24-well culture plate
(catalog no. 3473; Corning Inc., Acton, MA) containing 0.2 ml of Cytodex
3 beads (Sigma, St. Louis, MO). Bovine lipoprotein (Sigma) was added
to provide a final concentration of 0.5 mg/ml. For cells that were cultured for more than 24 h, either LH (final concentration, 0.5 ng/ml;
USDA-bLH-B-6, United States Department of Agriculture Animal Hormone Program, Beltsville, MD) or dbcAMP (final concentration, 1 mm)
was added. In preliminary studies, we demonstrated that these LH and
dbcAMP concentrations maintained testosterone production at about
50% of the steroidogenic capacity of the cells. To determine the maximal
24-h testosterone production, cells were stimulated with either 100
ng/ml LH or 5 mm dbcAMP for the final 24 h of culture. These concentrations of LH and dbcAMP were selected based on the results of
previous studies (14) and from preliminary experiments. The final culture volume was adjusted to 2.0 ml with M-199 culture medium, and the
cultures were maintained at 34 C in 5% CO2-5% O2-90% N2.
Media were changed every 24 h. For this purpose, the Cytodex 3
beads with Leydig cells attached were allowed to settle to the bottom of
the culture wells, and the supernatants were collected and frozen for
testosterone assay. The beads, with Leydig cells attached, were washed
once with fresh culture medium and then resuspended in fresh culture
medium and placed in the reduced-oxygen environment. At the end of
culture, media were collected and stored at ⫺80 C. Testosterone in the
medium was assayed by RIA (testosterone antibody from ICN, Costa
Mesa, CA; and 3H-testosterone from NEN Life Science Products, Boston,
MA). The sensitivity and intraassay and interassay coefficients of variation of the RIA were 13 pg/tube and 8.9 and 13.6%, respectively. Leydig
cells attached to the beads were lysed with TES buffer (10 mm Tris, pH
8.0; 1 mm EDTA, 1% sodium dodecyl sulfate, and 100 mm KCl) at 50 C
for 30 min. DNA was assayed fluorometrically with 4⬘,6-diamidino-2phenylindole (15). Testosterone production was normalized by the
amounts of DNA recovered from the beads.
cAMP production
Purified Leydig cells were resuspended (5 ⫻ 105/ml) in M-199 medium supplemented with 2.2 g/liter NaHCO3, 2.4 g/liter HEPES, and
0.1% BSA (pH 7.4). The cells (1 ⫻ 105/200 ␮l) were preincubated in
96-well Falcon culture plates (Becton Dickinson, Franklin Lakes, NJ)
under 5% CO2-95% air at 34 C for 4 h. The medium was then carefully
removed, and 50 ␮l fresh phenol red-free M-199 medium, containing LH
(100 ng/ml) or forskolin (500 ␮m; Sigma), was added to the plates. These
concentrations of LH and forskolin stimulate cAMP production maximally (5). The medium also contained 100 ␮m isobutyl-methylxanthine
(IBMX; Sigma) to inhibit phosphodiesterase activity. After 15 min of
incubation, 50 ␮l Tris buffer (0.05 m, pH 7.5; containing 4 mm EDTA and
2 mg/ml theophylline) was added to the culture medium, and the plates
were frozen by dry ice and stored at ⫺80 C until cAMP assay. In some
experiments, 500 ng/ml cholera toxin (Calbiochem, La Jolla, CA) or 100
ng/ml pertussis toxin (Sigma) were included in the medium during the
Chen et al. • cAMP and Aged Leydig Cells
preincubation period. We found that these concentrations of toxins
maximally inhibited G protein function or stimulated G protein function
without affecting overall cell viability. cAMP was assayed with a cAMP
[3H] assay system (Amersham, Piscataway, NJ) according to the manufacturer’s directions. The sensitivity of the assay was 0.05 pmoles per
assay tube.
Adenylyl cyclase activity
Adenylyl cyclase activity was assayed by incubating Leydig cell membrane fractions at 34 C for 15 min and monitoring the conversion of ATP
to cAMP (16). In brief, about 5 ⫻ 106 cells were homogenized in 200 ␮l of
HEPES buffer (50 mm, pH 8) containing 1 mm EDTA and protease inhibitor
cocktail (Sigma) on ice. After centrifugation at 400 ⫻ g for 2 min, the
supernatant was centrifuged at 100,000 ⫻ g for 15 min at 4 C. To assay the
adenylyl cyclase activity, 10 ␮g of membrane protein were incubated in the
100 ␮l reaction solution (50 mm HEPES, pH 8; 1 mm EDTA, 10 mm MgCl2,
0.5 mm ATP, 1.5 mm K⫹phosphoenolpyruvate, 10 ␮g/ml pyruvate kinase,
0.1 mg/ml BSA, 100 ␮m IBMX, and 100 ␮m forskolin) at 34 C for 15 min.
cAMP in the final reaction solution was assayed with a cAMP [3H] assay
system (Amersham) according to the manufacturer’s directions. Enzyme
activity was proportional to the concentration of membranes and the time
of incubation.
Western blot analysis
To analyze the stimulatory G (Gs) protein, the cell membrane fractions were lysed in solubilization buffer (100 mm Tris-HCl, pH 6.8; 1%
Triton X-100, 100 mm dithiothreitol, and 10% protease inhibitor cocktail;
Sigma) at 4 C for 30 min. To analyze the StAR and P450scc proteins, the
purified cells were lysed in the same solubilization buffer at 4 C for 60
min. After centrifugation (15,000 ⫻ g for 15 min at 4 C), the supernatant
was mixed with 3⫻ SDS sample buffer (New England BioLabs Inc.,
Beverly, MA). Twenty micrograms of protein from each sample were
separated on a 10% polyacrylamide gel and then transferred onto a
nitrocellulose membrane. After incubation with primary antibodies (1:
1000) and horseradish peroxidase-conjugated secondary antibody (1:
5000; Amersham), antibodies bound to the proteins were detected with
the SuperSignal West Pico Luminescence kit (Pierce, Rockford, IL). For
the Gs ␣-subunit, membranes were analyzed with antibody (catalog no.
371731) from Calbiochem (La Jolla, CA), and the result was confirmed
with antibody (SA-131) from Biomol (Plymouth Meeting, PA). StAR
antibody was a gift from Dr. Douglas M. Stocco (Texas Tech University
Health Sciences Center, Lubbock, TX). Cytochrome P450 cholesterol
side-chain cleavage (P450scc) antibody (AB1294) was purchased from
Chemicon International (Temecula, CA).
Statistical analyses
Data are expressed as the mean ⫾ sem. Statistical differences were
determined by one-way ANOVA. If group differences were revealed by
ANOVA (P ⬍ 0.05), then differences between individual groups were
determined using the Student-Newman-Keuls test (P ⬍ 0.05) by using
SigmaStat software (Systat Software Inc., Richmond, CA). Values were
considered significant at P ⬍ 0.05.
Results
Figure 1 shows the capacity of isolated Leydig cells to
produce testosterone when stimulated maximally with LH or
dbcAMP for 24 h in vitro (d 1) or in the last 24 h of a 3-d culture
period (d 3). Leydig cells were isolated from young control
and old control rats and from young rats that had received
LH-suppressive testosterone implants for 5 d. The latter cells
served as a positive control because high levels of testosterone production would be expected to be restored in these
cells with LH stimulation (17). Young Leydig cells from untreated rats produced significantly more testosterone than
old cells or cells from young LH-suppressed rats, whether the
cells were incubated with LH or dbcAMP for 24 h (d 1). With
Chen et al. • cAMP and Aged Leydig Cells
Endocrinology, October 2004, 145(10):4441– 4446 4443
FIG. 1. Testosterone-producing capacity of Leydig cells in vitro in
response to LH or dbcAMP for 1 or 3 d. Leydig cells were isolated from
young rats (YC), young rats that received LH-suppressive testosterone for 5 d before isolation (YT), and old rats (OC). The cells were
cultured for 1 (Day 1) or 3 (Day 3) d with either LH (100 ng/ml) or
dbcAMP (5 mM). For cells cultured for 3 d, lower concentrations of LH
(0.5 ng/ml) or dbcAMP (1 mM) were used for the first 2 d. Each bar
represents the mean ⫾ SEM of four experiments. a, Significant differences from the same cells treated with LH at d 1. b, Significant
differences from the same cells treated with dbcAMP at d 1.
3 d of culture with either LH or dbcAMP, testosterone production by cells from young rats maintained their ability to
produce testosterone at high levels. With LH for 3 d, the cells
from young LH-suppressed rats, which produced very low
levels of testosterone on d 1, had a significantly increased
ability to produce testosterone; and with dbcAMP for 3 d, the
ability of these cells to produce testosterone increased even
more. With 3 d of culture with LH, the old cells did not
increase in their ability to produce testosterone. In striking
contrast, the ability of old Leydig cells to produce testosterone increased to close to young levels when the cells were
incubated with dbcAMP for 3 d. Note that the old cells
cultured with dbcAMP for 3 d produced twice the testosterone of cells cultured with LH.
Two of the key proteins in the steroidogenic pathway,
StAR and P450scc, were reduced in response to LH suppression and age (Fig. 2A). The high levels of production of both
proteins in young control cells were maintained after 3 d of
culture with either LH or dbcAMP. The cells from young
LH-suppressed rats contained low levels of StAR and
P450scc at d 1. After 3 d of treatment, both proteins increased
significantly with either LH or dbcAMP. With LH treatment
of the old cells, however, the two proteins did not increase
over the 3-d culture period. In contrast, with dbcAMP treatment of the old cells, both proteins increased significantly
by d 3.
The individual activities of 17␣-hydroxylase and C17-20
lyase, which convert progesterone to 17␣-hydroxyprogesterone and androstenedione, respectively, were reduced in
response to LH suppression of the young cells and by age
FIG. 2. A, Representative Western blots of P450scc and StAR protein.
Leydig cells isolated from young rats (YC), young rats that received
LH-suppressive testosterone for 5 d (YT), and old rats (OC) were
incubated with LH for 1 d or with LH or dbcAMP (DC) for 3 d. B,
Activities of 17␣-hydroxylase and C17-20 lyase, which convert progesterone to 17␣-hydroxyprogesterone and androstenedione, respectively. Each bar represents the mean ⫾ SEM of four experiments.
(Fig. 2B). Interestingly, neither activity was restored to young
levels by LH or dbcAMP, although dbcAMP restored testosterone production to these cells.
These results suggest that reduced cAMP plays a central
role in the reduced steroidogenic ability of old Leydig cells.
Consequently, we wished to determine the mechanism by
which aging results in reduced cAMP production. Figure 3A
confirms that, with maximally stimulating LH, Leydig cells
isolated from aged rats produced about half the cAMP of
cells isolated from young rats. To determine whether im-
4444
Endocrinology, October 2004, 145(10):4441– 4446
Chen et al. • cAMP and Aged Leydig Cells
the young and aged cells were stimulated with forskolin,
cAMP production was equivalent between the two groups of
cells (Fig. 3A), suggesting that adenylyl cyclase is maintained
in old Leydig cells. To determine whether the inhibitory G
(Gi) proteins were responsible for the reduced cAMP production by aged cells, cells were cultured with pertussis
toxin, which inhibits Gi proteins in Leydig cells. The difference between cAMP production of young and aged cells
persisted, suggesting that changes in Gi protein are unlikely
to be responsible for the reduced cAMP production in aged
cells. To determine whether the Gs proteins were responsible
for the reduced cAMP production, cells were cultured with
cholera toxin, which activates Gs proteins. Under this condition, the ability of the aged cells to produce cAMP significantly increased. Indeed, cAMP production was equivalent
between young and aged cells after cholera toxin treatment.
To further examine whether Gs protein content changes with
age, the ␣-subunits of the Gs protein of young and aged cells
were analyzed by Western blots (Fig. 3B). No differences
were seen between the two ages, confirming that Gs proteins
do not themselves change with age. These results suggest
that Gs protein is maintained in old cells but that, in contrast
to young cells, it may not be activated efficiently by LH in the
membranes of aged cells.
Next, forskolin effects on adenylyl cyclase activity were
assayed directly in cell membrane fractions isolated from
young and old cells (Fig. 3C). In membranes of both young
and aged cells, adenylyl cyclase activity was low in its unstimulated (basal) state. Incubation of the cells with forskolin
resulted in a dramatic increase in activity in the membrane
fractions of both young and old cells, with no differences
between the two ages. This result supports the contention
that adenylyl cyclase does not itself change with age and,
therefore, is unlikely to be responsible for the decreases in
cAMP production in aged Leydig cells.
Discussion
FIG. 3. A, cAMP production by Leydig cells isolated from young and
old rats in response to LH, forskolin (F), pertussis toxin (P⫹LH), or
cholera toxin (C⫹LH). Purified Leydig cells were incubated for 15 min
with LH (100 ng/ml) or forskolin (500 ␮M) in the presence of IBMX
(100 ␮M) after a 4-h preincubation period with or without pertussis
toxin (100 ng/ml) or cholera toxin (500 ng/ml). Values represent the
mean ⫾ SEM of four experiments. B, Western blot of Gs protein ␣subunit from young (Y1, Y2, and Y3) and old (O1, O2, and O3) cells.
C, cAMP production by cell membranes isolated from young and old
Leydig cells and incubated without (basal) or with forskolin and with
IBMX (100 ␮M). *, Significant differences from young and old cells
treated with LH alone.
peded function of adenylyl cyclase in old cells was responsible for the reduced cAMP production, cells were incubated
with forskolin, an agent known to increase cAMP production
by directly activating adenylyl cyclase in Leydig cells. When
Although aging male Brown Norway rats exhibit defects
on both testicular and hypothalamic/pituitary levels, as is
the case in humans (18, 19), the primary cause of reduced
testosterone production by aged Leydig cells resides in deficits at the testicular level (5, 10, 18). Previous studies have
shown that under LH stimulation, old Leydig cells produce
far less cAMP than young cells (5). These differences in cAMP
production persist when cells are cultured with the phosphodiesterase inhibitor, IBMX, suggesting that old Leydig
cells have deficits in cAMP production and not in its metabolism. The first question we addressed was whether the
defect in LH signaling pathway is responsible for the agerelated reduction in Leydig cell steroidogenesis. The ability
of young Leydig cells to produce testosterone was sustained
over a 3-d culture period with either LH or dbcAMP stimulation. Testosterone production by cells isolated from the
LH-suppressed young rats increased with both LH and
dbcAMP, indicating that the in vitro system was capable of
restoring steroidogenic function. Culturing the old Leydig
cells with LH failed to increase testosterone production and
thus was unable to reverse the steroidogenic deficits of old
Leydig cells. In striking contrast, culturing these cells with
Chen et al. • cAMP and Aged Leydig Cells
dbcAMP, which bypasses the defect in the LH signaling
pathway, resulted in the restoration of testosterone production to young levels thus, in effect, reversing aging effects.
These results provide strong evidence that inefficient LH
signal transduction, leading to reduced cAMP production, is
responsible for the reduced steroidogenesis that characterizes aged Leydig cells.
Accompanying the restoration of testosterone production
by old cells, two of the key proteins in the steroidogenic
pathway, StAR and P450scc, were restored to the levels of
young cells. Interestingly, however, the individual activities
of P450c17, namely 17␣-hydroxylase and C17-20 lyase activities, which convert progesterone to 17␣-hydroxyprogesterone and androstenedione, respectively, and which were
reduced by LH suppression of the young cells and by age,
were not restored to young levels by LH or dbcAMP, despite
the fact that dbcAMP restored testosterone production to
these cells. These results indicate that testosterone production can be maximal even in the face of reductions in some
of the steroidogenic enzymes or, alternatively, that under
some circumstances, testosterone production can be regulated by P450c17-independent pathways (20).
The results with dbcAMP clearly suggest that changes in
the signal transduction pathway of old Leydig cells cause
reductions in cAMP production and, in turn, in testosterone
production. Therefore, we addressed the issue of which
changes in signal transduction might be responsible for reduced cAMP. LH receptors are coupled to adenylyl cyclase
through G proteins (11). Forskolin can activate adenylyl cyclase by directly binding to the enzyme, thus bypassing the
hormone receptor-G protein signal transduction pathway
(21). When stimulated with forskolin, old cells produced the
same amount of cAMP as young cells, suggesting that adenylyl cyclase is maintained in the old cells. This was confirmed by direct assay of adenylyl cyclase activity in the cell
membrane fractions. As in other cells, LH receptors are coupled to adenylyl cyclase mainly through the Gs protein in
Leydig cells (11). In addition to Gs protein, a group of Gi
proteins may also be involved in the negative modulation of
adenylyl cyclase activity in rat Leydig cells (22). We asked
whether the reduced cAMP production in old cells results
from changes in Gs and/or Gi proteins. Pretreatment of old
cells with pertussis toxin, which inhibits Gi proteins, did not
restore the LH-stimulated cAMP production to the level of
young cells, suggesting that changes in Gi protein are unlikely to be the cause of old cells producing less cAMP than
young cells. Pretreatment with cholera toxin, which bypasses
the LH receptor and activates Gs proteins directly, increased
the ability of old cells to produce cAMP almost to the levels
of young cells, suggesting that Gs protein itself is well maintained in old cells. This was confirmed by Western blot analysis of Gs protein ␣-subunit, which indicated that Gs protein
does not change quantitatively with age.
We have reported previously (5) that, although LH receptor number decreases in old Leydig cells, such changes in LH
receptor number are unlikely to account for decrease in
cAMP production. This and the observations herein that the
Gs and Gi proteins and adenylyl cyclase are well maintained
in old cells suggest that deficits in these proteins are not
responsible for reduced cAMP production in old cells.
Endocrinology, October 2004, 145(10):4441– 4446 4445
Rather, we suggest that reduced cAMP production results
from defects in the LH receptor itself or in coupling of the LH
receptor to adenylyl cyclase through Gs proteins. What
would cause such defects? During normal metabolism, cells
produce reactive oxygen species that can damage DNA, protein, and lipids (23). There is extensive evidence that free
radical damage may contribute to cell aging (24, 25). We have
reported that aged Leydig cells produce more reactive oxygen than young cells (26), and unpublished studies from our
lab indicate that the major scavengers of reactive oxygen
(superoxide dismutase 1 and 2, glutathione peroxidase, catalase, and glutathione) are reduced in aged cells. If free radical
damage affected membrane fluidity (27, 28), the LH receptor-G protein-adenylyl cyclase coupling cascades probably
would become less efficient (29). Indeed, it has been shown
that rat aortic endothelial cell membrane fluidity decreases
with aging (30) and that changes in rat corpus luteum cell
membrane fluidity in response to oxygen radicals disrupt
LH-stimulated cAMP production (31). Whether or not this is
the mechanism by which signal transduction is made inefficient in old cells and, if so, whether age-related deficits
result from extrinsic changes that impinge upon the Leydig
cells or from changes intrinsic to the cells themselves remain
uncertain.
Acknowledgments
Received May 19, 2004. Accepted June 22, 2004.
Address all correspondence and requests for reprints to: Dr. Haolin
Chen, Department of Biochemistry and Molecular Biology, Division of
Reproductive Biology, Johns Hopkins University Bloomberg School of
Public Health, Baltimore, Maryland 21205. E-mail: [email protected].
This work was supported by National Institutes of Health Grant
AG21092 from the National Institute on Aging.
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