Yonago Acta medica 2001;44:45–53

The Effect of Aging and Exogenous Testosterone Replacement on
Nitric Oxide Concentration and Activity of Nitric Oxide Synthase in the
Rat Corpus Cavernosum
Manabu Shiono
Department of Urology, Tottori University Faculty of Medicine, Yonago 683-0826 JapanThe effects of testosterone replacement on the nitric oxide (NO) concentration andactivity of NO synthase (NOS) in the penis were investigated. Male Wistar rats (n = 39)
were divided into 5 groups: 14-week-old males, 13-month-old males, 15-month-old
males, 15-month-old males treated with low-dose testosterone replacement and 15month-old males treated with high-dose testosterone replacement. The testosterone
concentration in serum and the NO concentration in penile tissue were measured, andthe endothelial NOS (eNOS) and neuronal NOS (nNOS) expressions were examined
immunohistochemically. The testosterone concentration in serum tended to decrease
with aging, but the 15-month-old testosterone-replaced rats maintained almost the samelevel as the 14-week-old rats. Nitrite and nitrite/nitrate concentrations in penile tissuetended to decrease with aging. Nitrite concentrations in the 15-month-old rats weresignificantly higher in the testosterone-replaced groups than in the non-replaced group,
but no significant difference in nitrite/nitrate concentration was recognized between
the 15-month-old rats not treated and treated with testosterone replacement. Immunohistochemical staining for eNOS and nNOS demonstrated a decreasing expression of
the 2 NOSs with aging and recovering of the NOSs by testosterone replacement. The
results of this study suggest that NO plays a major role in the mediation of penile erection,and testosterone replacement may favorably alter age-related erectile dysfunction.
Key words: aging; immunohistochemical staining; nitric oxide; testosterone replacement

muscle relaxation, in part through changes inpotassium conductance (Seftel et al., 1996;
Burnett, 1997).
The production of NO is mediated by afamily of NO synthase (NOS) that all represent
distinct gene products. These enzymes produce
NO through a complex set of redox reactions
that result in the conversion of L-arginine to Lcitrulline. The isoforms of NOS have been
categorized as being either inducible or constitutive (Forstermann et al., 1995). The inducible
isoform of NOS (iNOS) is associated primarily
with macrophages and is activated by specificcytokines as part of the immune response. The
endothelial (eNOS) and neuronal (nNOS) isoforms of NOS are constitutive and are activated,

Recently, several studies have revealed thatnitric oxide (NO) is an important neural messenger which mediates penile erection (Ignarro
et al., 1990; Holmquist et al., 1991; Kim et al.,
1991; Burnett et al., 1992; Rajifer et al., 1992).
Erection is mediated by the release of NO from
non-adrenergic non-cholinergic nerve terminals, the endothelium of penile blood vessels,
and corporal smooth muscle, producing smoothmuscle relaxation and vasodilation (Burnett,
1997). NO stimulates the formation of guanylate cyclase in smooth muscle cells, converting
GTP to 3'5'-cyclic GMP (cGMP) (Burnett,
1997). A cascade of cGMP-dependent intracellular events then leads to a decrease in intracellular calcium, ultimately causing smoothAbbreviations: eNOS, endothelial NOS; iNOS, inducible NOS; mRNA, messenger RNA; nNOS, neuronal
NOS; NO, nitric oxide; NOS, NO synthase; PBS, phosphate-buffered saline45

M. Shiono

in part, by an increased concentration of
intracellular calcium and calmodulin binding to
the enzyme. Experimental evidence suggeststhat the constitutive isoforms of NOS may be
responsible for NO production in penile erection (Bush et al., 1992; Burnett et al., 1993).
Recent evidence suggests that eNOS and its
subsequent production of NO may be a significant route by which NO-mediated cavernosal
relaxation is brought about because transgenic
mice lacking nNOS are still capable of erectile
activity with pelvic nerve stimulation (Burnett
et al., 1996).
A decrease in the serum testosterone levelwith aging may contribute to a reduction of sexual potency (Meisel and Sachs, 1994; Garban etal., 1995), although this reduction with aging isa multi-factorial phenomenon.
A recent study also hypothesized that androgens maintained and facilitated male sexual
potency through enhancement or maintenanceof NOS activity in the corpus cavernous tissue
in the penis (Mills et al., 1996). In addition,
several studies have shown that androgen replacement facilitated neural activities in someareas of the brain which mediate sexual function (Okamura et al., 1994a, 1994b; Pu et al.,1996).
In this article, the changes in the serum tes
tosterone concentration, NO concentration and
NOS protein expression in penile tissue were
examined by using the rat as our model of aging, and the changes brought about by testosterone replacement, as well. The goal in thisstudy was to evaluate whether testosterone replacement helps to resolve age-related erectile
dysfunction.

dose testosterone replacement group (G4, n = 8)
and high-dose testosterone replacement group(G5, n = 8). In the replacement groups, Silastic
tubes (Dow Corning, Midland, MI; outer diameter, 3.17 mm; inner diameter, 1.57 mm) whichwere 3 cm in length, and contained 40 mg of testosterone powder (Sigma, St. Louis, MO) were
subcutaneously implanted in the backs of the rats
when they were anesthetized with pentobarbital(30 mg/kg) at the age of 13 months, and thereplacement was continued for 2 months. In
G4, 4 tubes were implanted and in G5, 8 tubes
were implanted. As each group reached the age
desired for the experiment as mentioned above,
blood (7–8 mL) was collected through the inferior vena cava of each rat under pentobarbital(30 mg/kg) anesthesia, and the serum wasseparated by centrifugation and stored at –80˚Cuntil it was assayed. Total testosterone and freetestosterone concentrations in serum were determined by radioimmunoassay (Total Testosterone Kit and Free Testosterone Kit, DPC Corp.,
Tokyo, Japan). Rats were killed in succession
by means of additional pentobarbital (30 mg/kg), and the penis was removed and weighed.
The penis was divided into 2 pieces, and one
was used for measurement of NO concentrationand the other was used for immunohistochemistry.

Measurement of NO concentration in penile tissue
The final products of NO in vivo are nitrite andnitrate. The relative proportion of nitrite andnitrate is variable and cannot be predicted withcertainty. Thus, the best index of total NO production is the sum of both nitrite and nitrate.
NO was measured by means of the Griess
method. The penile tissue was weighed andhomogenized in phosphate-buffered saline(PBS) (pH 7.4) and centrifuged at 10,000 × g
for 20 min. The supernatant was ultracentrifuged at 100,000 × g for 15 min, and then the supernatant was ultrafiltered using a 30 kDa molecular weight cut-off filter. The sample was assayed by means of a nitrate/nitrite colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI).
The values were estimated per tissue weight andMaterials and Methods
Subjects used were 14-week-old (G1, n = 10),13-month-old (G2, n = 5) and 15-month-old
male Wistar rats (n = 24) (SLC, Shizuoka,
Japan). All rats were housed in a room with
controlled lighting and allowed access to foodand water ad libitum. The 15-month-old rats
were divided into 3 groups as follows: no testosterone replacement group (G3, n = 8), low46

Erectile dysfunction with aging in rats

Table 1. Body and penile weight, and testosterone concentration in serum
Young adult rats

Middle-aged rats

14-week-old 13-month-old
G1
G2
[10]
[5]

G3
[8]

Body weight(g)
367 ± 14596 ± 59562 ± 39*
Penis weight(mg)
394 ± 40659 ± 73596 ± 42*
Total testosterone (ng/mL)
2.0 ± 0.4
1.0 ± 0.2
0.8 ± 0.1*
Free testosterone (pg/mL)
8.3 ± 1.9
5.2 ± 0.8
4.2 ± 1.2*
Values represent mean ± SEM.
[ ], number of animals.
Significant difference (P < 0.05): *G3 versus G1; **G4 or G5 versus G3.

per amount of protein in the tissue. Protein wasdetermined using a commercial kit (BCA Protein
Assay Reagent Kit, Pierce, Rockford, IL).

15-month-old
Testosterone-replaced
Low-dose
High-dose
G4
G5
[8]
[8]
554 ± 19587 ± 47621 ± 45621 ± 383.7 ± 0.4**
5.0 ± 1.1**
14.5 ± 3.6** 20.0 ± 6.7**

ped by washing the sections in water. Mayer’s
hematoxylin was used for counterstaining.

Statistical analysis
eNOS and nNOS immunohistochemistryin penile tissue

Experimental values were expressed as mean ±SEM for the number of separate determinations
indicated in each case. The non-parametric ttest was used for calculating probabilities whencomparing 2 groups independent from the others, and those with P values less than 5% (P <0.05) were considered significant.

The penis was immediately fixed with neutral
buffered 15% formaldehyde-saline. The tissues
were embedded in paraffin after the fixation.
Sections (3 µm) were subjected to immunohistochemical stains for eNOS and nNOS. The
sections were retrieved by micro-wave andtreated with 3% methanol/hydrogen peroxidefor 15 min at room temperature to reduce background staining. They were then reacted with
200 µg/mL of rabbit anti-eNOS or nNOS polyclonal antibody (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) at a 1:5000 dilution in Dako
antibody diluent (Dako, Carpenteria, CA) overnight at 4˚C. Sections were washed with PBS
and the specifically bound first antibodies werevisualized by means of biotinylated anti-rabbit
secondary antibody and with a Vectastain ABC
kit (Vector Laboratories, Burlingame, CA) for 30
min at room temperature. Again, sections were
washed with PBS and incubated for 30 min withhorseradish peroxidase-labeled streptavidin atroom temperature. After the incubation period,
tissue sections were washed and a diaminobenzidine peroxidase substrate solution (Vector)
was applied for 5 min. The reaction was stop-

Results

Body weight and penile weight
Among the group of 15-month-old rats, bodyweight and penile weight of rats treated without
testosterone replacement (G3) were not significantly different from those treated withtestosterone replacement groups (G4 and G5)(Table 1).

Serum testosterone concentration
The total testosterone level tended to decrease
with aging, but the level in the 15-month-old
rats without testosterone replacement (G3) was
significantly lower than that in 14-week-old rats
(G1). Among 15-month-old rats, the rats with47

M. Shiono

Table 2. Nitrite and nitrite/nitrate concentration in penile tissue
Young adult rats
14-week-old
G1[10]

Middle-aged rats
13-month-old
G2[5]

Nitrite†(µmol/g wt) 17.3 ± 3.4
16.2 ± 3.4
Nitrite‡(nmol/mg pro) 1.11 ± 0.24
1.05 ± 0.22
Nitrite/nitrate† (µmol/g wt) 48.4 ± 7.1
42.1 ± 5.2
Nitrite/nitrate‡ (nmol/mg pro) 3.28 ± 0.53
2.47 ± 0.33
† Per tissue weight (µmol/g weight).
‡ Per amount of protein in the tissue (nmol/mg protein).
Values represent mean ± SEM.
[ ], number of animals.
pro, protein; wt, weight.
Significant difference (P < 0.05): *G4 or G5 versus G3.

low- and high-dose testosterone
replacement (G4 and G5) showedsignificantly higher levels of total
testosterone than the rats without replacement (G3), but they did nothave significantly higher levels than
14-week-old rats (Table 1). The freetestosterone level in 15-month-old
rats without testosterone replacement was significantly lower thanthat in 14-week-old rats. Among 15month-old rats, the free testosterone
level in the low- and high-dose testosterone replacement groups was
significantly higher than that in the
non-replacement group (Table 1).

G3[8]11.0 ± 1.9
0.54 ± 0.05
40.6 ± 4.3
2.32 ± 0.23

15-month-old
Testosterone-replaced
Low-dose
High-dose
G4
G5
[8]
[8]
31.5 ± 3.6* 27.0 ± 6.6*
2.59 ± 0.73* 1.61 ± 0.50*
43.4 ± 6.7
49.8 ± 10.4
3.27 ± 0.54
3.10 ± 0.66

A

B
A: G1, young adult rat (14-week-old)
B: G2, middle-aged rat (13-month-old)

Fig. 1. Sections of rat corpus cavernosum tissue immunostained by ant-endothelial nitric oxide synthase(eNOS) antibodies (original magnification × 200). Sections are counterstained with hematoxylin.
A: Immunohistochemical localization of eNOS shows strong staining of the vascular endothelium.
C: Aged rats without replacement have weak staining.
D and E: Testosterone replacement restored the same level of staining as in the young adult group.
[Figs. 1A and B on p. 48 and Figs. 1C–E on p. 49]

48

Erectile dysfunction with aging in rats

Nitrite and nitrite/nitrate concentration in penile tissue

Nitrite level
This level tended to decrease with aging, butthere was no significant difference between 14week-old rats (G1) and 15-month-old rats (G3).
Among 15-month-old rats, the low- and highdose testosterone replacement rats showed asignificantly higher nitrite level per amount ofprotein in the tissue and per tissue weight thanrats without replacement. There was no significant difference in nitrite levels between thehigh- and the low-dose replacement rats (Table
2).

Nitrite/nitrate level
This level tended to decrease withaging, but there was no significant difference between 14-week-old rats and
15-month-old rats without testosterone
replacement. The nitrite/nitrate level
increased with testosterone replacement; however, the difference was not
significant between 15-month-old rats
treated with and without testosterone
replacement (Table 2).

C

eNOS and nNOS immunohistochemistry in penile tissue
Figures 1 and 2 show light microscopic
photographs of the corpus cavernosum
from G1 to G5. Immunohistochemical
localization of eNOS showed strong
staining of the vascular endothelium in
14-week-old rats (G1) (Fig. 1A).
Thirteen-month-old (G2) and 15month-old rats without testosterone replacement (G3) had weak staining(Figs. 1B and C), and testosterone replacement restored the level of staining(Figs. 1D and E). Immunohistochemical stainings in 14-week-old rats, 15month-old rats with low- and high-dose
testosterone replacement rats were almost at the same level. Immunohistochemical staining with anti-nNOS

DE
C: G3, middle-aged rat
D: G4, low-dose testosterone replaced
middle-aged rat
E: G5, high-dose testosterone replaced
middle-aged rat(G3–G5: 15-month-old)

Figs. 1C–E. Continued from the previous page.

49