Yonago Acta medica 2005;48:93–99

Effects of Testosterone Replacement on Lower Urinary
Tract Functions in Elderly Male Rats
Shuichi Morizane, Sumiyo Toji, Mayuko Matsumoto and Ikuo Miyagawa
Division of Urology, Department of Surgery, School of Medicine, Tottori University Faculty of
Medicine, Yonago 683-8504 Japan

Testosterone has been clinically used to improve hormone deﬁciency in the aging male;
however, investigations how testosterone exerts its effects on lower urinary tract functions
are not many. In order to shed light on the efﬁcacy of testosterone on the functions, we
replaced teststerone in elderly male Wistar rats aged 19 months. A relevant dose (120mg) of testosterone was subcutaneously replaced through an implanted silastic tube into
6 rats for 4 weeks (treated group). Another 6 rats received no treatment for 4 weeks (control group). After the end of the 4-week period, we measured plasma testesterone, weightof bladder, prostate and body, bladder muscle content, spontaneous micturition behavior
and cystometric parameters, and compared the results between the 2 groups. The daily
micturition frequency (18.8 ± 1.5 times/day versus 15.5 ± 1.9 times/day), volume of residual urine at cystometry (0.66 ± 0.10 mL versus 0.24 ± 0.03 mL), bladder capacity (1.03 ±0.06 mL versus 0.65 ± 0.05 mL), bladder weight (258 ± 9 mg versus 198 ± 19 mg), prostate
weight (2.08 ± 0.22 g versus 1.29 ± 0.22 g) and ratio of smooth muscle area/connective
tissue area (3.59 ± 0.13 versus 2.59 ± 0.36) were signiﬁcantly higher in the treated group(P < 0.05). In contrast, the average volume of spontaneous micturition was signiﬁcantly
lower in the treated group (0.84 ± 0.07 mL versus 0.98 ± 0.09 mL). Differences in bodyweight and volume of 24-h urine were not signiﬁcant between groups. Daily micturition frequency and volume of residual urine at cystometry were increased in the treatedgroup. Testosterone replacement exerted unfavorable effects on the lower urinary functions of elderly rats, including prostatic hypertrophy.
Key words: lower urinary tract function; prostate; rat; testosterone

nents of bladder outlet are regulated by complex
neural mechanisms. During micturition reﬂexes,
the nervous system coordinates the muscles ofthe detrusor, bladder neck and urethra to promoteurine flow (Lluel et al., 2003), modulating theabilities of the lower urinary tract to store and release urine.
Micturition behavior changes with age (Chunet al., 1988), and aging males’ alpha adrenergic
receptor density is reduced. If chronic testosterone deficiency is prolonged, it might ultimately
affect their bladder outlet resistance (Anderson etMale hormone deﬁciency in the aging male is a
popular topic among andrologists, endocrinologists and urologists (Lunenfeld, 2003). Comparedwith normal ranges for young male adults, total
serum testosterone levels are lower in 19% of men
in their 60s, 28% of men in their 70s and 49% of
men in their 80s (Harman, 2001). Their bladderfunctions are frequently disrupted mostly by thesecondary effects of central neural pathologies(senile dementia, cerebral vascular accident), aging and/or bladder outlet obstruction (Lluel et al.,2003). The bladder activity and various compo93

S. Morizane et al.

The studied animals were 12 male Wistar rats
aged 19 months (SLC, Shizuoka, Japan), rearedin an air-conditioned room under a 12/12 h light/
dark cycle and allowed access to food and water
ad libitum. Of the 12, 6 received no treatment for
4 weeks (control group). The remaining 6 had
subcutaneous implants for 4 weeks via a silastic
tube (inside diameter, 2.5 mm; length, 30 mm)(Kaneda Medix, Osaka, Japan) containing 120
mg of testosterone (Wako, Osaka, Japan) in sesame oil (Kadoya, Tokyo, Japan) (the treated group)
according the dose and method reported by Satoet al. (1998).

al., 1988) and eventually cause functional disorders of the lower urinary tract. Male- and femalehormone receptors are co-localized in the urothelia, bladder smooth muscle cells, proximal urethra
striated muscle cells and neurons in the autonomic ganglia of the prostatic plexus, and together
exert their effects directly on the lower urinary
tract of aged males (Salmi et al., 2001; Keast etal., 1998). Conditions induced by co-localization
have recently drawn attention such as partial androgen deﬁciency in aging males, andropause or
male climacteric. Patients are clinically treatedwith testosterone replacement via injection, orally
or percutaneously (Jockenhovel, 2003) in order
to alleviate subjective symptoms such as feebleness with loss of concentration, decreased sexualdesire, impotence and/or muscle amount degradation (Lunenfeld, 2003).
Generally, testosterone replacement isthought to have little influence on the lower urinary tract, serum prostate-specific antigen andprostate volume (Holmang, 1993; Cooper, 1998;
Leder et al., 2004). However, in some animal experiments, testosterone replacement has resulted
in prostatic hypertrophy and lower urinary tract
passage disorder (Constantinou, 1996), despite favorable results in the bladder functions of female
hormone-replaced elderly female rats (Longhearst
et al., 1992). To study the effects of testosterone
replacement on lower urinary tract functions inelderly male rats, we measured plasma testesterone, bladder, prostate and body weight, bladdermuscle content, spontaneous micturition behavior
and cystometric parameters using testosteronereplaced and non-treated rats.

Micturition behavior
After treatment, micturition behavior was monitored for 24 h in a metabolic cage containing aurine collection funnel. Urine ﬂowed through aduct into a 250-mL plastic beaker placed on anelectronic balance (HF200, A.N.D., Tokyo), which
was connected to a personal computer (Macintosh
iBook G3, Apple Computer, Cupertino, CA) via
a multi-port controller (Maclab/400, AD Instruments, Castle Hill, Australia). Volumes of spontaneous micturition were automatically sampled
every 150 s for 24 h, and the data including micturition frequency were stored on the compunter’s
hard disk. Monitoring per each group started at
10:00. The rats were allowed to take water but nofood during the observation period.
Cystometry
Cystometry was performed under subcutaneous
urethane anesthesia (1.0 g/kg) with a 24-gauge
catheter inserted into the apex of the bladderdome to record pressure. On day 31 of the experiment, the bladder was filled with physiologicalsaline by using an infusion pump (5200, TOP,Tokyo) at a constant rate of 0.4 mL/min until
micturition was detected. A cystostomy catheter
was connected to an external pressure transducer(P2310, Gould, Eastlake, OH) to measure theintravesical pressure, which was recorded on a
Macintosh Powerbook G3 via a bridge ampliﬁer

Materials and Methods
Animals
Experiments were performed in accordance with
the Guidelines of Tottori University Committee for Animal Experimentation at the Divisionof Laboratory Animal Science, Research Center
for Biosience and Technology, Tottori University.
94Testosterone on lower urinary tract

Table 1. Plasma testosterone levels and weightof the rat bladder, prostate and body

(ML112, AD Instruments) and a multi-port controller Maclab/400. During micturition, ﬁlter paper was carefully placed at the meatus to absorb
excreted urine without spillage, and the excreted
volume was calculated by the difference of theweight. The following parameters were measured: bladder capacity (mL), volume of excreted
urine (mL), volume of residual urine (mL) andmaximal detrusor pressure (cmH2O). Maximal
detrusor pressure was defined as (instantaneouspressure of the detrusor muscle) minus (restingpressure of the detrusor muscle after contraction).
The measurements were performed for 24 h pereach rat.

Controlgroup [6]Treatedgroup [6]Testosterone (ng/mL)

0.83 ± 0.41

2.61 ± 0.69*

Bladder weight (mg)

198 ± 19258 ± 9*Prostate weight(g)

1.29 ± 0.22

2.08 ± 0.22*

Body weight(g)

575 ± 33598 ± 26

Values are mean ± SD.
[ ], number of animals.
* Signiﬁcant difference, P < 0.05.

Macintosh Powerbook G3 (Apple Computer), andquantiﬁed by using National Institutes of Health
Image 1.55 software (Research Service Branch,
National Institutes of Health, Bethesda, MD).
We then calculated the components of smoothmuscles and connective tissues per full screen.
At least 10 ﬁelds were examined from each tissue
section.

Measurement of plasma testosterone andhistological examination of the rat bladder
Blood was drawn by a heart puncture and a volume of 2.5 mL was collected to assess the plasma
testosterone level by radioimmunoassay kit (SRL,
Tokyo). After blood sampling, the bladder andprostate were surgically removed from the surrounding tissues and weighed. Bladders transected at the urethra level were sectioned into sagittal
slices, fixed in 10% formalin and embedded inparaffin, from which 10 μm-thick vertical crosssections were made. The sections were deparafﬁnized with xylene, rehydrated with a graded series of ethanol and stained with hematoxylin andeosin and the Elastica-van Gieson method whichstains smooth muscles yellow and collagen inconnective tissues red.

Statistical analysis
Experimental values are expressed as mean ± SD.
Statistic probabilities were calculated with thenon-parametric t-test between 2 groups, and P <0.05 was considered signiﬁcant.

Results
Plasma testosterone levels

Color-assisted quantitative image analysis

The treated group showed signiﬁcantly (P < 0.05)
higher plasma testosterone levels than the control
group (Table 1).

The stained smooth muscles were quantified byusing color-assisted quantitative image analysis. Sections of stained tissues were observed
at a magniﬁcation of 400 times by light microscopy (BX50, Olympus, Tokyo), displayed on acolor monitor (PVM-20M4V, Sony, Tokyo) andprinted with a digital color printer (CP700DSA,
Mitsubishi, Tokyo). Prints were digitized with ascanner (GT-8000, Epson, Tokyo) operated on a

Bladder, prostate and body weight
The bladder and prostate were significantly (P< 0.05) heavier in the treated group than in thecontrol (Table 1). The difference in body weight
between groups was not signiﬁcant (Table 1).
95

S. Morizane et al.

A

B
Fig. 1. Light micrographic presentations of sections of the bladder body in elderly male rats (Elastica-van Gieson
stain; original magniﬁcation, × 400). A: an untreated rat (control group). B: a testosterone-replaced rat (treatedgroup).
Table 2. Ratio of smooth muscle area/connective tissue area

Smooth muscle area/connective tissue area

Control group [6]Treated group [6]2.59 ± 0.36

3.59 ± 0.13*

Values are mean ± SD of 10 times or more measurements per each rat.
[ ], number of animals.
* Signiﬁcant difference, P < 0.05.

96Testosterone on lower urinary tract

Table 3. Comparison of micturition behavior
Measurement

Control group [6]Treated group [6]24-h volume of urine(mL/day)

15.0 ± 0.8

15.8 ± 1.1

Daily micturition frequency(time/day)

15.5 ± 1.9

18.8 ± 1.5*

0.98 ± 0.09

0.84 ± 0.07*

Volume of spontaneous micturition (mL)
Values are mean ± SD.
[ ], number of animals.
* Signiﬁcant difference, P < 0.05.

Table 4. Comparison of urodynamic parameters

Bladder capacity
Maximal detrusor pressure

Control group [6]Treated group [6]0.65 ± 0.05

1.03 ± 0.06*

29.8 ± 1.3

34.5 ± 1.3*

(mL)
(cmH2O)

Excreted urine†(mL)

0.42 ± 0.06

0.37 ± 0.07

Residual urine †(mL)

0.24 ± 0.03

0.66 ± 0.10*

Values are mean ± SD.
* Signiﬁcant difference, P < 0.05.
† During cystometry to measure intravesical pressures.

Histological examination of the rat bladder
Discussion

The smooth muscle area was significantly (P< 0.05) smaller in the control group than in thetreated group (Fig. 1 and Table 2)

The influence of sex hormones upon the lower
urinary tract is of interest. Male and female
hormones together regulate the development andfunctions of lower urinary tract muscles in malerats (Saija et al., 2001). In humans, total plasma
testosterone declines by approximately 1% peryear on an average from the age of around 40
years (Gray et al., 1991). Due to the concomitant
increase of sex hormone binding globulin, freetestosterone levels decrease even more steeply
from this age. Nonbinding testosterone also decreases with aging (Feldman et al., 2002).
Testosterone exerts a powerful inﬂuence onmale reproductive organs, including spermatogenesis and mounting behavior (Lunenfeld, 2003);
however, its effects on lower urinary tract functions have not been fully characterized. In thepresent study on its effects on elderly male rats,
the level of testosterone we replaced was accord-

Micturition behavior
Micturition frequency was signiﬁcantly (P < 0.05)
higher in the treated group than in the control
group, but the difference in 24-h volume of urine
was not signiﬁcant (Table 3). The average volumeof spontaneous micturition was signiﬁcantly (P <0.05) lower in the treated group than the control
group (Table 3).
Urodynamic study
Bladder capacity and volume of residual urine
at cystometry were also significantly larger inthe treated group. The difference in volume of
excreted urine at cystometry was not significant
between groups (Table 4).
97

S. Morizane et al.

obstruction caused an increase in residual urine
volume. This disorder of the lower urinary tract
passage caused the smooth muscle of the bladderto swell, and increased the ratio of smooth muscle
area/connective tissue area, leading to an increasein detrusor pressure. More studies will be necessry to ﬁnd out if these changes in the bladderare due to the direct effect of testosterone or thesecondary effect of increased urethra resistance.
In humans, testosterone replacement does
not signiﬁcantly increase serum levels of prostatespeciﬁc antigen or prostate volume (Ebert, 2004),
and the lower urinary tract does not appear to besignificantly influenced. However, testosterone
replacement caused prostatic hypertrophy and inﬂuenced lower urinary tract function in the present study with rats. The prostate maintains theability to respond to androgens throughout life
and the proliferative response of prostatic cells
to androgens depends on expression of the intracellular androgen receptor. Following prostate
growth to its adult size, the rates of cell proliferation and cell death reach equilibrium, preventing
further growth.
However, cellular hyperplasia occurs laterin the lives of several species, including humans,
dogs and some rat strains despite a decrease intesticular androgen production and a concomitant
fall in the peripheral levels of androgen reaching
the prostate (Banerjee et al., 2001). The imbalance in cell death and cell proliferation that leads
to age-dependent prostatic hyperplasia might berelated to increased prostatic sensitivity to androgen. Nuclear androgen receptor expression would
be changed with cellular aging, and contributeto the evolution of cellular hyperplasia. In fact,
nuclear androgen receptor levels are higher in hyperplastic prostate tissues than in normal tissues(Barrack et al., 1983). Age-related increases in
the relative estrogen level, as well as other factors,
might increase androgen receptor expression in
the aging prostate. Further growth or decrease incell death results, although androgen is decreasedin the peripheral circulation with normal dihydrotestosterone levels in the prostate.
In the present testosterone-replaced elderly

ing to Sato et al. (1981). They reported averageplasma testosterone levels as 2.34 ± 0.21 ng/mL
in 12-month-old rats and 0.78 ± 0.17 ng/mL in
24-month-old rats. The corresponding levels inour study were 2.61 ± 0.69 ng/mL in treated rats,
almost the same as in their reproductive rats, and0.83 ± 0.41 ng/mL in control rats, almost the sameas in their elderly rats.
The difference in mean body weight was not
signiﬁcant between both groups. The mean bladder weight was signiﬁcantly greater in the treatedgroup than the control group. Histologically, theamount of muscle tissues signiﬁcantly increasedin the treated group, and the ratio of smooth muscle area/connective tissue area was significantly
higher in the treated group than in the control
group. The increase in muscle tissues caused anincrease in bladder weight. Prostate weight signiﬁcantly increased in the treated rats as well.
In our observations of rat micturition behavior, differences between the 2 groups were notsigniﬁcant in 24-h volume of urine. However, thetreated group showed significantly higher daily
micturition frequency and signiﬁcantly lower average spontaneous micturition. Through cystometry, the bladder capacity, the volume of residual
urine and the maximal detrusor pressure weresignificantly higher in the treated group than in
the control group.
The increase in the ratio of smooth muscle
area/connective tissue area appeared to raise thedetrusor pressure, and bladder capacity then increased with bladder compliance, as we observed.
Usually, average micturition volume increases
and daily micturition frequency decrease; however, we observed different results in the present
experiments, presumably because residual urine
increased due to an increase in prostate weight.
As the structures of the prostate of the rat
and the human are different, particularly in theway the ventral and dorsolateral prostate loops
around the urethra without compression, the rat
model could be considered to be non-obstructive.
However, in the present study, testosterone replacement enlarged the prostate, which then compressed the urethra. Thus, lower urinary tract98

Testosterone on lower urinary tract

male rats, the amount of bladder smooth muscles,
the ratio of smooth muscle area/connective tissue area, bladder capacity and detrusor pressure
were increased. However, the prostates wereswollen and lower urinary tract passage disorder
was induced. Average micturition volume wasdecreased, and daily micturition frequency increased. Testosterone replacement exerted unfavorable effects on the lower urinary functions ofelderly rats, including prostatic hypertrophy.

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Received October 14, 2005;accepted November 24, 2005
Corresponding author: Shuichi Morizane

99