Yonago Acta medica 2003;46:109–115

Changes in Apoptosis-Related Genes (Bcl-2, Bax) in the
Urethras of Old Female Rats Following Estrogen Replacement
Shinichiro Kinouchi
Division of Urology, Department of Surgery, School of Medicine, Tottori University Faculty of
Medicine, Yonago 683-8504 Japan
Following estrogen replacement in old female rats, changes in apoptosis-related genes(Bcl-2, Bax) in the urethra were investigated by immunohistochemical and RT-PCR
analysis. A total of 40 old (16-month-old) female Wistar rats were divided into the following
5 groups: normal controls; rats replaced with low-dose estradiol (E2) for 2 and 4 weeks,
respectively; and rats replaced with high-dose E2 for 2 and 4 weeks, respectively. After
treatment, the urethras were removed, weighed and stained with hematoxylin and eosin,
and then immunohistochemical stainings of Bcl-2 and Bax were performed. In addition,
the expressions of the apoptosis-related genes bcl-2 and bax were investigated by PCR
analysis, and then evaluated by computerized quantification. In the E2-replaced rats, theimmunostained urethras showed little immunoreactivity against the Bcl-2 protein, and in
the control rats, no reactivity was seen. In contrast, the control rats showed immunoreactivity against the Bax protein, whereas the E2-replaced rats showed little reactivity. UponmRNA analysis, the expression of bcl-2 mRNA was uprisen in rats in the high-dose E2 for
4 weeks group as compared with the controls, but the expression of bax mRNA was suppressed in the E2-replaced rats as compared with the controls. As these results suggest,
estrogen replacement up-regulated the expression of bcl-2 mRNA and down-regulated
the expression of bax mRNA, which might suppress the apoptic action: these changes
might alter lower urinary tract findings.
Key words: apoptosis; estrogen replacement; old rat; urethra

During menopause, the drop in serum levels of estrogen causes metabolic and atrophic alterations inmany organs (Versi, 1990). Postmenopause urogenital atrophy disorder is characterized by dysuria,
urinary urgency, pollakisuria and urinary incontinence (Suguita et al., 2000).
It is known that the urethra, like the vagina, isaffected by estrogen, and that hypoestrogenism atmenopause results in urogenital atrophy in all women, regardless of race, nationality or socioecono-

mic status. With progressing estrogen deficiency
and advancing age, the urethral epithelium becomesthinner, and loses its vascularity, elasticity and regulation (Bachmann, 1998). In addition, estrogen
depletion causes atrophic urethritis, and sometimes
accounts for urinary incontinence (Elia and Bergman,1993). Bhatia et al. (1989) reported that estrogentherapy could improve the proliferation and growthof the urethral mucosa, and that it could have a positive effect on urethral elasticity. In another study,

Abbreviations: E2, estradiol; HE, hematoxylin and eosin; RT, reverse transcription; UIP, universal immuno-enzyme
polymer109

S. Kinouchi

cytological examination revealed that estrogen
treatment for 3 weeks induced proliferation of theatrophic urethral mucosa in postmenopausal women suffering from urinary incontinence (Ulmsten
and Stormby, 1987).
Recent studies have indicated that estrogen
regulates cell survival and cell death factors. In
breast cancer cells (Teixeira et al., 1995; Kandouz
et al., 1996; Huang et al., 1997), the expression of
bcl-2 and other members of the bcl-2 family (bax,bad and bcl-x), by which estrogen regulates, hasbeen shown to inhibit apoptosis. In brain cells(Dubal et al., 1999), estrogen prevented the injuryinduced down regulation of bcl-2 expression.
In the present study, following estrogen replacement therapy, we investigated the changes in
apoptosis-related genes (Bcl-2, Bax) on the urethrasof old female rats.

Serum levels of estradiol
The serum E 2 concentrations were assessed byradioimmunoassay using a kit from Nihon DPC(Tokyo). In the control group, assay was performedon the 14th day after the operation, and in the E2replaced groups, 2 weeks (groups II and IV) or 4
weeks (groups III and V) after hormone replacement.

Histological examination of the rat urethra
After replacement therapy, the animals were allanesthetized by inhalation of diethyl ether and thenweighed. In the control group (group I), this procedure was performed on the 14th day after the operation. The urethras were removed and weighed on a
Mettler Basbal scale (Delta Range, Tokyo). Afterthe procedure, all rats were sacrificed. Four urethras in each group were fixed in 10% formalin andembedded in paraffin, and the remaining 4 werefrozen in liquid nitrogen. Each paraffin block wasserially sectioned and routinely stained with hematoxylin and eosin (HE). For immunohistochemical
staining of Bcl-2 and Bax, we used the monoclonal
antibodies against Bcl-2 (Santa Cruz Biotechnology,
Santa Cruz, CA) and Bax (Santa Cruz Biotechnology).
The serial sections were also immunohistochemically stained with a Histfine Simple Stain Rat MAX
PO (MULTI) kit (Nichirei, Tokyo) according to the
manufacturer’s instructions (UIP method).

Materials and Methods

Animals
The animal experiment was performed at Laboratory Animal Center of Tottori University, in accordance with the guidelines of the University. Estrogen replacement was performed according toFuruta et al. (2002). Female Wistar rats (Shimizu,
Kyoto, Japan) were divided into 5 groups: eachcontained 8 animals. A silastic tube (inside diameter = 1.5 mm, outside diameter = 2.5 mm, length= 30 mm) (Kaneka Medix, Osaka, Japan) containing sesami oil (Kadoya, Tokyo, Japan) was subcutaneously planted into each rat at the age of 16months. Ovariectomy was not performed in anygroups. No additional treatment was performed in
the control group (Group I). Plain sesami oil was
replaced with the one containing 20 µg/mL of estradiol (E2, Sigma, St. Louis, MO), and placed for 2
weeks (Group II) or 4 weeks (Group III), respectively. The higher dose of E2 at 200 µg/mL was administered for 2 weeks (Group IV) or 4 weeks(Group V), respectively.

Reverse transcription-PCR analysis
The remaining 4 urethras per group were frozen inliquid nitrogen. The total RNA was extracted fromthe dissected tissues using TRIzol reagent(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, treated with deoxyribonuclease [reverse transcription (RT) grade] (Nippon
Gene, Tokyo) and then quantified spectrophotometrically. In this experiment, 5 µg of total RNA
was reverse-transcribed in a 20 µL reaction volume.
RT was carried out with Moloney murine leukemia

110Changes in apoptosis-related genes

virus reverse transcriptase (Roche Molecular Systems, Branchburg, NJ), and PCR was performed using AmpliTag gold DNA polymerase (Roche Molecular Systems). The primers used for bcl-2 were5'-CACCCCTGGCATCTTCTCCTT-3' (sense) and
5'-AGCGTCTTCAGAGACAGCCAG-3' (antisense)(Genbank accession number U34964, 519 bp), andthese for bax were 5'-CACCAGCTCTGAACAGATCATGA-3' (sense) and 5'-TCAGCCCATCTTCTTCCAGATGGT-3' (antisense) (Genbank accession
number RRU 49729, 540 bp) (Ananth et al., 2001).
The PCR cycles consisted of denaturation at 94˚C
for 30 s, annealing at 68˚C for 30 s and extension at
72˚C for 1 min for 26 cycles (bcl-2); and denaturation at 94˚C for 30 s, annealing 62˚C for 30 s andextension at 72˚C for 1 min for 26 cycles (bax). As
a control, a 285-bp DNA of rat β-actin was also
amplified using 5'-TCATGAAGT GTGACGTTG
ACATCCGT-3' (sense) and 5'-CCTAGAAGC
ATTTGCGGTGCAGGATG-3' (antisense) primers(Ananth et al., 2001). The temperature cycling
conditions were as follows: 10 min at 94˚C 18
cycles of (94˚C for 30 s, 65˚C for 30 s, 72˚C for 30s) and a final extension at 72˚C for 30 s. The PCR
product was size separated by electrophoresis on a
2% cyber green-containing agarose gel, andphotographed. For quantification, photographs
showing the PCR products were scanned andanalyzed using Densitograph version 4.0 software(ATTO, Tokyo). On each sample, signal strength
was normalized by that of β-actin.

Statistical analysis
All data was analyzed by Student’s t-test. Statistical significance was defined as P < 0.05.

Results

Serum levels of estradiol
The results of serum E2 levels are recorded in Table
1. The serum E2 levels in the replaced groups weresignificantly higher than those in the control group.
Compared in terms of the E2 dose, the serum E2
levels were about 10 times higher in the high-dosegroups (groups IV and V) than in the low-dosegroups (groups II and III) (Table 1).

Body and urethral weight
There were no significant differences amonggroups in body weight. However, urethral weight
was significantly heavier in the E2-replaced groups
than in the control group, although there were no
significant differences among the E 2 -replacedgroups in urethral weight (Table 1).

Table 1. Body weight, urethral weight and serum E2 levels
Body weight(g)

Urethral weight(mg)

Serum E2 levels(pg/mL)Group I353 ± 37.0

23.0 ± 4.6

7.3 ± 2.5

Group II358 ± 28.0**
* 30.7 ± 4.8

25.0 ± 4.8Group III339 ± 24.3*
30.4 ± 6.8**Group IV

332 ± 34.4

29.8 ± 2.8Group V
340 ± 23.2
Data are expressed as mean ± SD.
Statitical analysis by Student’s t-test.
E2, estradiol.
* Significant differences; P < 0.05.
** Significant differences; P < 0.01.

33.7 ± 6.4

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**

**
** 29.4 ± 10.7
*
**
*
*

258.1 ± 55.0
213.6 ± 67.3*

S. Kinouchi

Histological changes in the rat
urethras
There were no significant differences
between the control and E 2-replaced
rats in the HE stains of the urethra.
Figures 1a and b show representative
light microscopic photographs of theurethras immunostained by the
antibody against Bcl-2: Fig. 1a for thecontrol rats, and Fig. 1b for the group V
rats replaced with high-dose E2 for 4
weeks. Bcl-2 was not detected clearlyin the urethra of the control rats.
Fig. 1. Immunohistochemical stains of the rat urethras by an antibody
However, in the mucosal region of the against Bcl-2 protein.
urethra of the E 2-replaced rats, weaka: A control rat (× 200).
immunoreactivity against Bcl-2 wasb: A group-V rat replaced with high-dose E2 for 4 weeks (× 200).
detected.
Figures 2a and b show urethras
similarly stained by the antibody against Bax in thecontrol rats and the group V rats. Bax was detected
strongly in the mucosal region of the urethra of the
RT-PCR analysis of expression of bcl-2 andcontrol rats, but in the urethra of the E2-replaced
bax mRNA
rats, weak immunoreactivity against Bax was deThe expressions of bcl-2 mRNA were significantly
tected.
increased in group V compared withthe control group. In the other E 2 replaced groups, there were no significant differences in the expressions of
bcl-2 mRNA compared with the control
group. However, the expressions ofbax mRNA were significantly decreased in groups III and V comparedwith the control group. In groups II andIV, the expressions of bax mRNA
showed no significant differencescompared with the control group. The
bax/bcl-2 ratio was significantly lower
in groups III and V than in the control
group (Fig. 3).
Fig. 2. Immunohistochemical stains of the rat urethras by an antibody
against Bax protein.
a: A control rat (× 200).
b: A group-V rat replaced with high-dose E2 for 4 weeks (× 200).

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Changes in apoptosis-related genes

Group

a

II

III

IV

Discussion

bp

V

bcl-2

519

bax

540

β-actin285

Relative mRNA abundanceb

I

Rats are fertile until approximately 15 months ofage, and thereafter their estrous cycle stops.
Several studies indicated that the E2 levels are lessthan 10 pg/mL in ovariectomized female rats
(Shulman et al., 1987; Albert et al., 1991). Other
studies have indicated that serum E2 levels of 15 pg/
mL are slightly below the mean level of E 2
throughout the estrous cycle (9–27 pg/mL), or
about 1/3 of the maximum value E2 (35–52 pg/mL)
reached during the estrous cycle (Dupon and Kim,
1973; Overpeck et al., 1978). In the present study,
the E 2 levels were lower than 10 pg/mL in thecontrol group. In the E2-replaced groups, the mean
serum E2 levels reached over 20 pg/mL in the lowdose groups, and 200 pg/mL in the high-dosegroups. Thus, this method of estrogen replacement
was adequate for studying the effect of estrogen on
the rat urethras.
Previous studies showed that estrogen depletion resulted in increased body weight (Eika et al.,1990). In the present study, there was no significant
difference in body weight was observed amonggroups. However, the mean urethral weight of theE2-replaced rats was significantly higher than thatof the control rats. These results suggest that estrogen replacement after menopause increases urethra
weight in rats. No previous studies have shownchanges in female rats’ urethral weight after estrogen replacement. However, a few studies indicatedthat estrogen replacement might raise bladderweight as the collagen content of the bladderincreases and the bladder epithelium thickens(Suguita et al., 2000; Eika et al., 1990). On theother hand, several studies have demonstrated thepresence of estrogen receptors in the human lower
urinary tract. This provides evidence for the direct
actions of estrogens on the bladder and urethra(Batra and Iosif, 1987; Versi, 1990). Estrogentherapy could improve the proliferation and growthof the urethral mucosa (Bhatia et al., 1989), andcause an increase in urethral collagen content andurethral blood flow (Batra et al., 1986). These(% of control)

bcl-2

*

200

100

0

I

II

III

IV

V

Relative mRNA abundanceGroup(% of control)

bax
100

*

*

50

0

I

II

III

IV

V

Group

Relative bax/bcl-2 ratio(% of control)

bax/bcl-2 ratio

100

*

50

0

I

II

III

*
IV

V

Group

Fig. 3.
a: Semiquantitative RT-PCR analysis of mRNA expression of bcl-2, bax and β-actin in rat urethras. RT,
reverse transcription.
b: Relative densities of bcl-2 and bax mRNA signals,
and relative bax/bcl-2 ratio. The signals are normalized with corresponding β-actin signals. Each bar
represent the mean ± SEM. Statistical significanceswere examined in comparison to the control level expressed as 100%; significant difference; P < 0.05.

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S. Kinouchichanges in E2-replaced rats might increase the urethral weight as the bladder weight. However, thepresent study did not show significant differencesin the HE staining of urethras between the control
and E2-replaced rats. The differences in urethralweight between the control and E2-replaced rats
was about 5 to 10 mg. It might be difficult to detect
differences in urethral mucosa or collagen content
by HE staining.
In the present immunohistochemical staining,
immunoreactivity against Bcl-2 was not detected
clearly in the urethras of the control rats, but slightly detected in the mucosal region of the urethras of
the E2-replaced rats. However, immunoreactivity
against Bax was detected more strongly in themucosal region of the urethras of the control rats
than in the E2-replaced rats.
In the RT-PCR analysis, the expressions of
bcl-2 mRNA were significantly increased in group
V (high-dose E2 replaced for 4 weeks) comparedwith the control group. In the other E2-replacedgroups, the mean expressions of bcl-2 mRNA werehigher than in the control group. However, the expressions of bax mRNA were significantly decreased in groups III (low-dose E2 replaced for 4 weeks)
and V (high-dose E2 replaced for 4 weeks) compared with the control group. In the other E2-replacedgroups, the mean expressions of bax mRNA werelower than in the control group. The bax/bcl-2 ratio
was significantly lower in groups III and V than in
the control group. The administration period for 2
weeks might have been too short to cause a difference in gene expression. RT-PCR analysis suggested that estrogen replacement up-regulates the bcl-2
gene expression and down-regulates the bax gene
expression. Considering the histological results,
these changes occured mainly in the mucous membrane of the rat urethra. Thus, it seems that epithelium cells were estrogen dependent rather than submucosal cells.
It is known that Bcl-2 inhibits most types ofcell death, implying a common mechanism of lethality. Bcl-2 is localized to intracellular sites ofoxygen-free radical generation, including mitochondria, endoplasmic reticula and nuclear mem-branes. The mechanism by which Bcl-2 exerts its
antiapoptotic effects is not fully resolved, although
it has been speculated that Bcl-2 acts as either a regulator of an antioxidant pathway that prevents oxidative damage, such as lipid peroxidation caused by
the generation of free radicals (Tsujimoto and
Croce, 1986; Hockenbery et al., 1993), or as aregulator of intracellular Ca2+ compartmentalization (Kane et al., 1993). The antiapoptotic activityof Bcl-2 correlates with its intracellular ratio toanother recently described protein called Bax. Bcl2 heterodimerizes with Bax. High levels of Bax
have been shown to favor apoptosis in cells subjected to growth factor deprivation, whereas highlevels of Bcl-2 prolong cell survival under the same
conditions (Bafy et al., 1993). In other studies, Bcl2, a cell survival factor, has been identified as an
estrogen responsive gene in reproductive tissues.
Estrogen may directly up-regulate this survival
factor through receptor-mediated interactions with
regions of the bcl-2 promotor, which contains several putative estrogen-responsive sites, or by an indirect pathway. Estrogen depletion results in amarked decrease in bcl-2 expression, and in theabsence of estrogen, the bax/bcl-2 ratio is increased. In this type of situation, estrogen-dependent
tissues undergo apoptosis (Teixeira et al., 1995).
In the present study, considering the changes
in apoptosis related genes (Bcl-2, Bax), estrogen
replacement might prevent apoptosis mainly in theurethral mucosa of old female rats. However, it isdifficult to demonstrate the reason why the immunoreactivity was observed mainly in the mucosa.
Further studies need to be performed to assess therelation between the expression of apoptosisrelated genes on the female urethra and estrogen
replacement.
It appears from our study that in the mucosa of
the rat urethra, estrogen replacement up-regulates
the bcl-2 gene expression and down-regulates thebax gene expression. These results indicate that
estrogen replacement may prevent apoptosis in theurethras of old female rats.

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Changes in apoptosis-related genes

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Received September 16, 2003; accepted November 10, 2003
Corresponding author: Shinichiro Kinouchi

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