International Journal of Impotence Research (2003) 15, 391–396
& 2003 Nature Publishing Group All rights reserved 0955-9930/03 $25.00
www.nature.com/ijir
Downregulation of androgen, estrogen and progesterone receptor
genes and protein is involved in aging-related erectile dysfunction
M Shirai1, M Yamanaka1, H Shiina1, M Igawa2, M Fujime3, TF Lue4 and R Dahiya1*
1
VA Medical Center and UCSF, Urology, San Francisco, California USA; 2Shimane Medical University, Izumo, Shimane,
Japan; 3Juntendo University, Bunkyo-ku, Tokyo, Japan; and 4UCSF/Mt. Zion Medical Center, Department of Urology,
San Francisco, California, USA
We hypothesize that downregulation of sex hormone receptors (androgen, estrogen and
progesterone receptors) is involved in aging-related erectile dysfunction. To test this hypothesis,
we investigated the expression of sex hormone receptors in penile crura of aging rats. A total of 40
rats were divided into four groups based on age (6, 12, 18 and 24 months), and the erectile function
was analyzed by the measurement of intracavernous pressure. Gene and protein expressions of sex
hormone receptors were analyzed by RT-PCR and immunostaining, respectively. The mean
intracavernous pressures of 6-, 12-, 18- and 24-month-old rats were 110.1, 89.6, 73.5 and
42.7 cmH2O, respectively. Gene and protein expressions for androgen receptor, estrogen receptorbeta and progesterone receptor were present in similar levels in 6-, 12- and 18-month-old rat crura,
but significantly lower or absent in 24-month-old crura. This is the first study to demonstrate that
downregulation of sex hormone receptors in aging rat crura is associated with erectile dysfunction.
International Journal of Impotence Research (2003) 15, 391–396. doi:10.1038/sj.ijir.3901050
Keywords: steroid hormone receptor genes; erectile dysfunction; aging
Introduction
Erectile dysfunction (ED) is common in elderly
men.1,2 The Massachusetts Male Aging Study
reported that the combined prevalence of minimal,
moderate and complete ED reached up to 52% in
aging men. The prevalence of complete ED tripled
from 5 to 15% in subjects aged 40–70 y old.1 Older
men are more frequently affected by systemic
diseases and often take a lot of medication.
Although it might be possible that these factors are
inter-related with the higher incidence of ED, the
aging process itself undoubtedly plays a significant
role in the development of ED.
Circulating androgen in serum is considered to be
one of the principal factors that affect male sexual
function. It has been shown in rats that an adequate
testosterone level is essential to maintain the
availability of nitric oxide in the cavernous
compartment through stimulation of expression
and activity of penile endothelial and neuronal
nitric oxide synthase (NOS) isoforms.3 Estrogens
and progesterone are present in the circulation in
men, and it may be possible that the altered balance
between sex hormones and their receptors exerts an
influence on male sexual function. In the central
nervous system, progesterone could mediate male
sexual behavior by interacting with progesterone
receptor (PR) located in the olfactory bulb.4
Although male organs produce estrogens and progesterone, their effects on penis are not clear. In a
previous study, we have demonstrated that the
estrogen receptor-beta (ER-b) gene expression was
significantly reduced in diabetic rat penis as
compared to controls.5 However, there have been
few reports on the relationship of sex hormone
receptors (SHR) with aging-related ED. We hypothesize that SHR (androgen receptor (AR), ER and PR)
play a significant role in maintaining the male
sexual function and altered levels of SHR expression
are involved in the etiology of aging-related ED. To
test this hypothesis, we analyzed gene and protein
expressions of SHR in aging rat crura and correlated
them with erectile function.
Materials and methods
*Correspondence: R Dahiya, VA Medical Center and
UCSF, Urology, 112F, 4150 Clement Street, San Francisco,
CA 94121, USA.
E-mail: [email protected]
Received 26 October 2002; revised 7 March 2003; accepted
11 March 2003
Experimental animals
A total of 40 male Fisher rats, divided into four
groups (n ¼ 10) based on age (6, 12, 18 and 24
Downregulation of sex hormone receptor genes
M Shirai et al
392
months) were used in this study. These rats
were purchased from the National Institute of
Aging, Washington, D.C. and/or Simonson, Gilroy,
California.
Electrostimulation
After anesthetizing by intraperitoneal injection of
pentobarbital (40 mg/kg), a lower abdominal incision was made and the cavernous nerve was
exposed. Electrostimulation was performed using a
delicate stainless-steel bipolar hook electrode. The
diameter of each pole was 0.2 mm and the two poles
were separated by 1-mm distance. Monophasic
rectangular voltage pulses were generated by a
computer and converted to current pulses by a
computer-assisted stimulator. The stimulus parameters were 1.5 mA, 20 Hz frequency, pulse width
0.2 ms and duration 60 s. Each cavernous nerve was
stimulated. For monitoring intracavernous pressure
(ICP), the skin overlying the penis was incised and
the penile crura were exposed. A 25-gauge needle
filled with heparin solution (1000 U/ml) was inserted
into the right crus and connected to a pressure
monitor with polyethylene tubing.6 ICP was measured and recorded using a computer with Lab View
5.01 software (National Instruments, Austin, TX,
USA). The ICP was defined as the maximum
pressure obtained by the stimulation minus the
basal pressure before the stimulation.
Tissue preparation
Penile crura of each rat were collected immediately
after completing electrostimulation. Half of the
sample was fixed in 10% buffered formalin (pH
7.0) and embedded in paraffin wax, and used for
immunostaining. The remaining half of each sample
was immediately frozen and stored at –801C until
analyzed.
Reverse transcription-polymerase chain reaction
(RT-PCR)
Total RNA was extracted from the samples of penile
crura using Tri-Reagent (Molecular Research Center,
Cincinnati, OH, USA). Complementary DNA (cDNA)
was constructed by reverse transcription (Promega
Corp., Madison, WI, USA) using the total RNA as a
template. The samples then underwent PCR amplification, cDNA samples were diluted in 20 ml of
solution containing 200 mM of dNTP, 500 nM of each
primer, 0.5 U of Red Taq DNA polymerase (Sigma, St
International Journal of Impotence Research
Louis, MO, USA) and PCR reaction buffer provided
by the manufacturer. The primers used for rat AR,
ER-a, ER-b, PR and glyceraldehyde 3-phosphate
dehydrogenase (G3PDH) as a reference gene in
RT-PCR were as follows: AR forward, 50 -TGTTATC
TAGTCTCAACGAGC-30 ; AR reverse, 50 -CATCATTT
CAGGAAAGTCC-30 ; ER-a forward, 50 -GTCCAATTC
TGACAATCG-30 ; ER-a reverse, 50 -CTTCAACATTC
TCCCTCC-30 ; ER-b forward, 50 -TGTACCATAGACA
AGAACC-3; ER-b reverse, 50 -GGAGTATCTCTGTGT
GAAG-30 ; PR forward, 50 -CTCCTGGATGAGCCTGA
TG-30 ; PR reverse, 50 -CCCGAATATAGCTTGACCTC30 , G3PDH forward, 50 -TCCCATCACCATCTTCCA-30
and G3PDH reverse, 50 -CATCACGCCACAGTTTCC30 . PCR reactions were performed in a PTC-200
thermal cycler (MJ Research, Watertown, MA, USA)
at 941C for 3 min; 32 cycles at 941C for 1 min, 521C
for 1 min, 721C for 1 min; followed by final extension
at 721C for 5 min. For semiquantitative analysis,
three other dilution sets of each sample (original, 1/
2, 1/4 and 1/8 dilution) underwent PCR. Annealing
temperature was 521C for all RT-PCR reactions. The
PCR products were electrophoresed on 2.0% agarose
gel and the expression level of these genes was
evaluated by an ImageJ software (http://rsb.info.
nih.gov/ij), and the areas under the curves were
calculated and analyzed. PCR cycles were adjusted
until these four preparations in each sample were
within linear range. Between 28 and 32 cycles all
SHR PCR reactions were within linear range. The
expression of each gene was quantified relative to
G3PDH expression and expressed as arbitrary units
(AU). The expected sizes of PCR products were 587
base pair (bp), 346, 633, 293 and 380 bp in AR, ER-a,
ER-b, PR and G3PDH, respectively.
Immunohistochemistry
Tissue sections were cut (4 mm) and mounted on
a slide. Tissue block sections were dewaxed,
rehydrated, incubated with 0.3%(v/v) hydrogen
peroxide for 20 min, and autoclaved at 1211C for
15 min in 10 mM citrate buffer (pH 6.0). The samples
were incubated with serum block (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 20 min at
room temperature followed by overnight at 41C with
the following antibodies: 1:150 dilution of a monoclonal antibody against mouse ER-a (Ab-10) (Lab
Vision Corp., Fremont, CA, USA), 1:400 dilution of a
polyclonal antibody against rabbit AR (C-19), 1:400
dilution of a polyclonal antibody against rabbit ER-b
(H-150) and 1:100 dilution of a polyclonal antibody
against rabbit PR (H-190) (Santa Cruz Biotechnology,
Inc.). After the slides were washed, they were
incubated with a biotinylated goat anti-mouse antibody and streptavidin-peroxidase (Lab Vision Corp.)
for ER-a; or biotinylated goat anti-rabbit antibody
and HRP-streptavidin (Santa Cruz Biotechnology,
Downregulation of sex hormone receptor genes
M Shirai et al
Inc.) for AR, ER-b and PR at room temperature for
30 min. Following incubation with the avidin–biotin
complex, the antigen–antibody complexes were
visualized with diaminobenzidine (Lab Vision
Corp.). The sections were counterstained using
hematoxylin. As a negative control, normal goat
IgG was substituted for the specific antibody at
the same dilution. Paraffin sections of a normal
rat uterus and prostate were used as a positive
control.
A Leitz LaboLux S-microscope and a Nikon digital
camera were used to determine each SHR immunoreactivity within the cavernous tissue. The immunostaining pictures were analyzed on a PC with Image
Pro software. The nucleus in the cavernous tissue
was identified at 400. The pixels in the color
range that corresponded to the stain of each SHR
and negative nucleus were counted and expressed
as the percentage of the total number of pixels in the
field. The number of pixels that corresponded to all
nucleus (positive and negative nuclei) was counted,
and defined it as 100% in each area. Three different
sections in each animal were used for each SHR
immunostaining, and 10 different fields were analyzed in each specimen.
Gene expression of AR, ER-a, ER-b and PR in aging
rat crura (Figures 1 and 2).
393
Typical results and summarized data of each RTPCR are shown in Figures 1 and 2, respectively.
There was no significant difference in AR mRNA
expression between 6-, 12- and 18-month-old rats.
In 24-month-old rats, expression of AR mRNA
transcript was significantly reduced (Po0.01,
respectively).
For ER-a mRNA, there were two splicing isoforms
within the primer spanning region, and a strong
band corresponded to 346 bp (Figure 1). There was
no significant difference in the expression of ER-a
mRNA among rats of different ages. As shown in
Figure 1, expression level of the splicing variant of
ER-a mRNA was decreased with advancing age.
Statistical analysis
Parametric data were expressed as mean7s.e. Data
were analyzed by one-way analysis of variance
(ANOVA) with Fisher’s protected least-significance
t post hoc test using StatView 5.01 software
(SAS Institute Inc., Cary, NC, USA). The P-value of
less than 0.05 was considered to be statistically
significant.
Results
Functional study of aging rat penis
The ICP of rats after electrostimulation was reduced
with advancing age. In 6-month-old rat, the pattern
of ICP curve was sharp and the peak pressure was
the highest. On the other hand, in 24-month-old rat,
the peak pressure was the lowest. The mean ICP
was 110.178.3, 89.6712.1, 73.5713.9 and 42.77
13.3 cm H2O in 6-, 12-, 18- and 24-month-old rats,
respectively, indicating that ICP was decreased with
advancing age. Significant differences in ICP were
observed between 6- and 12-month-old rats, or
between 18- and 24-month-old rats (Po0.05, respectively). The difference in ICP between 12- and 18month-old rats was close to statistical significance
(P ¼ 0.05).
Figure 1 Typical RT-PCR results of SHR in aging rats are shown.
Note that in 24-month-old rat, expression level of AR and ER-b
messenger RNA transcript is weak but preserved, while that of PR
is lost (* ¼ 100 bp DNA ladder). Relative mRNA value is shown on
each band.
International Journal of Impotence Research
Downregulation of sex hormone receptor genes
M Shirai et al
394
Figure 3 Typical immunostainings of AR (a and c), ER-a (b and
d). Note that protein expression of AR was decreased in 24-monthold rat, whereas that ER-a was not significantly different: (a and b)
6-month ( 400); (c and d): 24-month ( 400).
Figure 2 Alterations of SHR mRNA expression with aging are
shown. Expression of AR, ER-b and PR mRNA was decreased
along with aging, whereas that of ER-a was not significantly
different among all aging groups. Values are expressed as
means7s.e. (AU ¼ arbitrary units).
ER-b mRNA expression among 6-, 12- and 18month-old rats did not differ, but was significantly
reduced in 24-month-old rats (Po0.01, respectively).
The expression of PR mRNA transcript was the
highest in 6-month and was lost in 24-month-old
rats. The difference in PR mRNA expression
between 6- and 24-month-old rats reached statistical
significance (Po0.01), but no significant difference
was observed between 12- and 18-month-old rats.
Protein expression of AR, ER-a, ER-b and PR
in aging rat crura (Figures 3–5).
Typical result of each immunostaining is shown in
Figures 3 and 4, and summarized data are shown in
Figure 5.
AR protein expression was the highest in 6month-old rat. The immunoreactivity was observed
in the nuclei of smooth muscle cells of small artery
as well as smooth muscle fibers within the crura
(Figure 3a). In 24-month-old rat crura, the number of
AR-positive cells was the lowest as compared to 6month-old rats (Figure 3c). A significant difference
was observed between 12- and 18-month-old rats or
18- and 24-month-old rats (Po0.05); however, the
difference between 6- and 12-month-old rats did not
reach statistical significance.
International Journal of Impotence Research
Figure 4 Typical immunostainings of ER-b (a and c) and PR (b
and d). Note that protein expression of ER-b and PR was
decreased and lost in 24-month-old rat, respectively: (a and b)
6-month ( 400); (c and d) 24-month ( 400).
ER-a-positive cells were distributed in the nuclei
of smooth muscle cells in 6-month-old rat crura
(Figure 3b). The number of the positive cells was
similar among 6- and 24-month-old groups (Figures
3b and d) and no significant difference in ER-a
immunoreactivity was observed among rats of
different ages.
ER-b immunoreactivity was reduced with advancing age of rats (Figure 4a and c). The difference in
the immunoreactivity reached statistical significance between 6- and 12-month as well as between
18- and 24-month-old rats (both Po0.05).
PR immunoreactivity was significantly reduced
with advancing age (Figure 4b and d). Interestingly
in 24-month-old rat crura, the PR immunoreactivity
was completely lost (Figure 4d).
Downregulation of sex hormone receptor genes
M Shirai et al
Figure 5 Alterations of SHR immunoreactivity with aging.
Reduction of immunoreactivity of AR, ER-b and PR was
associated with aging, while ER-a immunoreactivity was not
significantly changed among all age groups. In 24-month-old rats,
AR and ER-b protein expressions were significantly lower than 6-,
12- and 18-month-old rats (Po0.05). PR protein expression was
lost in 24-month-old rats. Values are expressed as means7s.e.
Discussion
Previous studies in our laboratory and in others
have shown that aging is associated with ED.7,8 The
molecular mechanisms of aging-related ED are not
fully understood. We hypothesized that alterations
in SHR are involved in the pathogenesis of ED in
aging rats. To test this hypothesis, we analyzed gene
and protein expression of AR, ER and PR in aging rat
crura. First, we measured ICP to verify the presence
of ED in the aging rat. ICP was lowered with
advanced age, which is consistent with the finding
observed in aging ED.9
Several lines of studies have demonstrated that
androgen deprivation is related to apoptosis of
smooth muscle cells in addition to an increase in
connective tissue contents.10–12 Penile eNOS and
nNOS are two major contributors to maintain
erectile function, of which activity is restored after
androgen stimulation in castrated rats. In the
present study, both levels of protein and mRNA
transcript of AR were significantly decreased with
advancing age in rat crura. In addition, this agingrelated alteration of AR expression was well correlated with reduction of ICP. Circulating level of
serum testosterone has been reported to be reduced
with advancing age in rat.13 Even if sufficient
amount of androgen is present in the older rats,
significant reduction of AR expression itself might
cause relative androgen deprivation status in aging
rat crura. These alterations are probably associated
with the enhancement of degenerative processes of
smooth muscle cells and/or reduction in eNOS and
nNOS.3,14
Estrogen functions as regulator of growth and
differentiation in various target tissues. Its receptors,
ER-a and ER-b mediate the functional role of
estrogen in target organs, but the distribution of
ERs is quite different among rat tissues. The
pituitary, kidney, epididymis and adrenal are predominant for ER-a, whereas prostate, ovary, lung,
bladder, brain and testis are higher in ER-b.15 In
rat crura, gene and protein expression of ER-b
was reduced with advancing age and markedly
decreased in 24-month-old rats, whereas ER-a gene
and protein expressions were not significantly
changed among 6-, 12-, 18- and 24-month-old rats.
In addition, the age-related ICP alteration correlated
with the change of ER-b expression, but not with
that of ER-a expression. These findings indicate that
the functionally predominant form of ER in rat crura
is ER-b and age-related alteration of ER-b is probably
related to the pathogenesis of ED in older rats. The
mechanism of ER-b involved in aging-related ED
still remains to be elucidated. Estrogen exerts direct
vascular protection effect on endothelial and/or
smooth muscle cells, and this effect is exclusively
mediated by ER-b, but not by ER-a.16,17 Estrogen also
exerts an inhibitory effect on apoptosis in human
endothelial cells with upregulation of antiapoptotic
Bcl-2.18,19 Loss of cytoprotective role of ER-b with
aging might be one of the mechanisms of development and/or progression of aging-related ED. Alternatively, potential antiapoptotic effect by estrogen
might be impaired by reduced expression of ER-b
with advancing age. In fact, a recent publication
clearly demonstrated that loss of antiapoptotic genes
in rat crura is associated with the pathognesis of
aging-related ED.20
There are no reports of the relationship between
development and/or progression of ED and PR
expression in the aging rat crura. Progesterone has
been shown to be involved in the modulation of
NOS activity21 and may be due to PR. The most
interesting finding was that PR gene and protein
expression were almost lost in 24-month-old rat
crura. In addition, the mean ICP drop between 18and 24-month-old rats (about 31 cm H2O) is greater
than those observed between 6- and 12-month
(about 20 cm H2O) or between 12- and 18-month
(about 16 cm H2O). Taken together, it might be
plausible that the impaired functional role of PR as
modulator of NOS activity reflects the lowest level
of ICP in 24-month-old rats.
In summary, ICP alteration in relation to aging
crura is well correlated with AR, ER-b and PR
expression. Even between 18- and 24-month of
age, the abrupt reduction in AR, ER-b and PR
still displayed significant correlation with ICP
395
International Journal of Impotence Research
Downregulation of sex hormone receptor genes
M Shirai et al
396
reduction. These findings strongly suggest that AR,
ER-b and PR are involved in the pathogenesis of ED
with advancing age.
Conclusions
To our knowledge, this is the first study demonstrating that downregulation of gene and protein expression of ER-b and PR is associated with ED of aging
rats. These results may be important in understanding the pathogenesis of aging-related ED and
also in providing better strategies for the management of aging-related ED.
Acknowledgements
This research was supported by the National
Institutes of Health Grants RO1DK055040 and
RO1AG016870.
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