Yonago Acta medica 2002;45:95–102

Effects of Estrogen Replacement Therapy on the Expression of
Apoptosis-Related Genes in Old Female Rats
Ryo Ohata
Division of Urology, Department of Surgery, School of Medicine, Tottori University Faculty of
Medicine, Yonago 683-8504 Japan
Effects of estradiol (E2) on the urinary bladder of old female rats were investigated bymorphological, immunohistochemical and apoptosis-related gene analysis. Sixteenmonth-old female Wistar rats were divided into the following 5 groups: normal controls;
rats replaced with low-dose E2 for 2 and 4 weeks, respectively; and rats replaced withhigh-dose E2 for 2 and 4 weeks, respectively. After treatment, the bladders were removed, weighed and stained with hematoxylin and eosin, and immunostained by monoclonal
antibodies against Bcl-2 and Bax proteins. Further, the expression of apoptosis-related
genes bcl-2 and bax were investigated by PCR analysis, and evaluated by computerized
quantification. Immunostained bladders showed no difference between control andE2-replaced rats in reactivity against Bcl-2 protein. In contrast, against Bax protein,
control rats showed immunoreactivity, whereas E2-replaced rats showed little reactivity.
Upon mRNA analysis, the expression of bax mRNA was suppressed in the E2-replaced
rats as compared with the controls, though the expression of bcl-2 mRNA showed nodifference among groups. These results suggest that E2 administration affects the expression of apoptosis-related genes and suppressees apoptotic action, and that thesechanges could consequently alter voiding behavior.
Key words: apoptosis; bladder; estrogen; rat

spaces between the muscle cells of the bladderand smooth muscles with hypertrophy were observed in old female rats. The interspaces were
occupied by collagen fibrils. The collagen content of the detrusor muscle, which could be affected by estrogen concentration in the serum,
has been found to be significantly increased inwomen after the age of 50 years as compared toyounger women and to men of the same age(Eika et al., 1990). Increased collagen deposits
between the smooth muscle cells of the detrusor
and the urethra might affect the functional properties of the lower urinary tract, and estrogen isconsidered to influence the lower urinary tract.
On the other hand, several studies haveindicated that estrogen regulates cell survival
and death factors. Estrogen administration
decreases apoptosis. In breast cancer cells

The decrease in serum level of estrogen that
occurs in menopause causes metabolic andatrophic alterations in many organs. Postmenopausal urogenital atrophy causes pollakiuria,
urinary urgency, stress and urge incontinence,dysuria and so on (Versi, 1990). The symptoms
show increasing incidence with age (Eika et al.,1990).
Previous investigations have demonstrated
the presence of estrogen receptors in the humanlower urinary tract. This supports the practiceof giving estrogen treatment to postmenopausal
women with lower urinary tract dysfunction.
Estrogen has been found to improve lower urinary tract function in postmenopausal women(Batra et al., 1987; Iosif et al., 1981).
A decrease in serum estrogen results in an
increase of collagen in the bladder. Large inter-

Abbreviations: E2, estradiol; HE,hematoxylin and eosin; RT, reverse transcription

95

R. Ohata(Teixeria et al., 1995; Kandouz et al., 1996; Huang
et al., 1997) and brain cells (Dubal et al., 1999),
respectively, estrogen was shown to have aneffect on mRNA expression of bcl-2, bad andbax, and, ultimately, to prevent the preapoptotic
actions of the members of the bcl-2 family suchas bax and bad.
In the present study which uses female rats
after menopause, the effects of estrogen replacement therapy on their bladders were investigated, including the effects on the expressionsof bcl-2 and bax.

(groups II and V) or 4 weeks (groups III and V)
after hormone replacement.

Histological examination of the rat bladder and reverse transcription-PCR analysis
Animals at the age of 16 months or after replacement therapy were all anesthetized by an
intraperitoneal injection of sodium pentobarbital and then weighed. The bladder was removed and weighed after emptying the urine. Four
bladders per group were placed in 10% formalin and embedded in paraffin. Each paraffinblock was serially sectioned and routinely
stained with hematoxylin and eosin (HE). The
serial sections were also immunohistochemically stained by the monoclonal antibody
against Bcl-2 (Santa Cruz Biotechnology, Santa
Cruz, CA) or Bax (Santa Cruz Biotechnology)
in a buffer diluted 1:100 with phosphate-buffered saline. Diaminobenzidine tetrahydrochloride was used as the final chromogen.
After weighing, 4 bladders per group werefrozen in liquid nitrogen. Total RNA was extracted from the dissected tissues using TRIzol
reagent (Invitrogen Corporation, Carlsbad, CA)according to the manufacturer’s instructions
and then quantified spectrophotometrically.
Reverse transcription (RT) was carried out with
Moloney murine leukemia virus reverse transcriptase (Roche Molecular Systems, Branchburg,
NJ), and PCR was performed using AmpliTaggold DNA polymerase (Roche Molecular Systems). For bcl-2 and bax cDNA amplification,
the primers were 5'-CACCCCTGGCATCTT
CTCCTT-3' (sense) and 5'-AGCGTCTTCAGAGACAGCCAG-3' (antisense) for bcl-2 (Genbank
accession number U34964, 519-bp DNA fragment), and 5'-CACCAGCTCTGAACAGATCATGA-3' (sense) and 5'-TCAGCCCATCTTCTTCCAGATGGT-3' (antisense) for bax(Genbank accession number RRU 49729, 540bp DNA fragment) according to the method ofAnanth et al. (2001). The PCR cycles consistedof denaturation at 94˚C for 30 s, annealing at
68˚C for 30 s and extension at 72˚C for 1 minfor 25 cycles (bcl-2); and denaturation at 94˚C

Materials and Methods

Animals
All animal experiments were performed inaccordance with the guidelines set out by the
Tottori University Committee for Animal Experimentation. Sixteen-month-old female
Wistar rats were divided into 5 groups. Ingroup I, no hormonal treatment was carried out(control group; n = 8). In group II, a silastic
tube containing estradiol (E 2 ) dissolved insesame oil at 10 µg was subcutaneously
placed for 2 weeks from the age of 16 months,
without an ovariectomy (low-dose E2 replacedfor 2 weeks; n = 8). In group III, E2 was administered for 4 weeks in the same manner as ingroup II (low-dose E2 replaced for 4 weeks; n =8). In group IV, E2 was replaced in the form ofsilastic capsules containing 2.5 mg β-E2 for 2
weeks (high-dose E2 replaced for 2 weeks; n =8), and in group V, the same capsules as ingroup IV were given for 4 weeks (high-dose E2
replaced for 4 weeks; n = 8). Rats in the control
group received placebos by the same methods
as E2-replaced rats. All animals were kept in an
air-conditioned room lighted 12 h a day, andallowed access to food and water ad libitum.

Measurement of plasma E2
Plasma E2 concentrations were measured when
the rats were 16 months old in the control
group, and in E 2 -replaced groups, 2 weeks

96Effects of E2 on apoptosis-related genes

Table 1. Body weight, bladder weight and serum E2 levels
Serum E2 levels(pg/mL)

Body weight(g)

Bladder weight(mg)

I: control

353.5 ± 37.0

110.7 ± 10.4

7.3 ± 2.5

II: low-dose E2 replaced for 2 wk

358.7 ± 28.0

116.1 ± 10.6

25.0 ± 4.8

III: low-dose E2 replaced for 4 wkGroup

339.0 ± 24.3

**
**
**
136.5 ± 24.8** **29.4 ± 10.7

IV: high-dose E2 replaced for 2 wk

332.2 ± 34.4

124.2 ± 9.3

258.1 ± 55.0

V: high-dose E2 replaced for 4 wk

340.0 ± 23.2

128.3 ± 10.5

*

**

**

**
*

213.6 ± 67.3

E2, estradiol; wk, weeks.
* Significant difference; P < 0.05.
** Significant difference; P < 0.01.

for 30 s, annealing 62˚C for 30 s and extension
at 72˚C for 1 min for 25 cycles (bax). As aninternal control, a 285-bp DNA fragment of rat
β-actin was also amplified using the following
primers: 5'-TCATGAAGTGTGACGTTG
ACATCCGT-3' (sense) and 5'-CCTAGAAGCATTTGCGGTGCAGGATG-3' (antisense).
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 30 s) and a final
extension at 72˚C for 30 s.
In this experiment, 5 µg of total RNA wasreverse-transcribed in a 15-µL reaction volume.
An 8-µL aliquot of each PCR product was sizeseparated by electrophoresis on a 2% cybergreen-containing agarose gel and photographed. For quantification, photographs showing
PCR products were scanned and analyzed using Densitograph version 4.0 software (ATTO
Corp., Tokyo, Japan). For each sample, thePCR product values were normalized to β-actin
PCR product values. Data were analyzed byStudent’s t-test.

III), from 100 to 330 pg/mL in high-dose E2replaced groups (groups IV and V) and less than
10 pg/mL in the control group.
Serum E2 levels in the replaced groups weresignificantly higher than those in the control
group. Compared in terms of the E 2 dose,
serum E2 levels were significantly higher in thehigh-dose groups (groups IV and V) than in the
low-dose groups (groups II and III) (Table 1).

Body and bladder weight
There were no significant differences amonggroups in body weight. However, E2-replacedgroups showed a lower tendency in mean bodyweight than the control group: body weighttended to be higher as the E2 dose was increased.
On the other hand, bladder weight was significantly heavier in the E 2-replaced groups
than in the control group, except for group II
replaced with low-dose E2 for 2 weeks. The
mean bladder weight showed no significant
differences among the low- and high-dose
replaced groups (Table 1).

Results

Histological changes in the rat bladders
Plasma E2 levels

There were no significant differences between
control and E2-replaced rats in the HE stains of
the bladder.

Plasma E2 levels ranged from 15 to 45 pg/mL in
low-dose E 2-replaced groups (groups II and97

R. Ohata

Figs. 1ab. Immunohistochemical stains of rat bladders by an antibody against Bcl-2 protein. a: A control rat(× 100). b: A group-V rat replaced with high-dose E2 for 4 weeks (× 100).

Figs. 2ab. Immunohistochemical stains of rat bladders by an antibody against Bax protein. a: A control rat(× 100). b: A group-V rat replaced with high-dose E2 for 4 weeks (× 100).

98

Effects of E2 on apoptosis-related genes

I

Group
III

II

IV

V
bp

bcl-2

519

bax

540

β-actin285

a(% of control)

bcl-2

150

Relative mRNA abundanceb

Relative mRNA abundance(% of control)

100

50

0

I

II

III
IV
Group

V

bax

150

100

** ** **

50

0

I

II

III
IV
Group

V

Figs. 3ab.
a: Semiquantitative RT-PCR analysis of mRNA expression of bcl-2, bax and β-actin in rat bladders. RT,
reverse transcription.
b: Relative densities of bcl-2 and bax mRNA signals. The signals are normalized with corresponding β-actin
signals. Each bar represents the mean ± SEM. Statistical significances were examined in comparison to
the control level expressed as 100%: **Significant difference; P < 0.01. Group I, control; group II,
replaced with low-dose E2 for 2 weeks; group III, replaced with low-dose E2 for 4 weeks; group IV,
replaced with high-dose E2 for 2 weeks; group V, replaced with high-dose E2 for 4 weeks; E2, estratiol.

Figures 1a and b show representative lightmicroscopic photographs of the bladders immunostained by the antibody against Bcl-2: Fig.
1a for a control rat and Fig. 1b for a group-V rat
replaced with high-dose E2 for 4 weeks. Bothstains of the bladders demonstrate low immunoreactivity against Bcl-2, not different in appearance.
Figures 2a and b show bladders similarly
stained by the antibody against Bax in a control
rat and a group-V rat. Bax was detected in themucosal region of the bladder of the control rat(Fig. 2a), but not in the bladder of the E2-replacedrat (Fig. 2b).

RT-PCR analysis of expression of bcl-2
and bax mRNA
The expressions of bcl-2 mRNA were not significantly different among groups. In contrast,
the expressions of bax mRNA were significantly decreased in groups III to V as comparedwith the control group. In group II, the expression of bax mRNA was almost the same level asthat in the control group (Fig. 3).
99

R. Ohata

Discussion
In the present study, plasma E 2 levels werelower than 10 pg/mL in the control group. It is
well known that rats have a fertility period thatlasts until the age of approximately 15 months,
at which time the estrous cycle ceases. The
values of plasma E 2 in the control group reflected this time frame, and the estrous cycle
disappeared in control rats. In the E2-replacedgroups, the mean plasma E2 levels reached over20 pg/mL in low-dose groups and 200 pg/mL in
high-dose groups. In high-dose groups, theplasma E2 level was quite over the peak level ofE2 throughout the usual estrous cycle (Handa etal., 1987; Shulman et al., 1987; Albert et al.,1991). Albert et al. (1991) found that a plasma
E 2 level of 15 pg/mL was slightly below themean level of E2 present throughout the estrous
cycle in rats maintaining normal body weight.
Therefore, the method of E2 replacement in thisexperiment is adequate in studying the effect of
E2 on the rat bladder.
Previous studies demonstrated that ovariectomized rats showed an increase in body weight
and a decrease in bladder weight as comparedwith control rats (Lonhurst et al., 1992). In thepresent study, no significant difference in mean
body weight was observed among groups;
however, there was a linear correlation between
the E2 dose and rat body weight. When the E2
dose was increased, body weight tended to behigher. Further, mean bladder weight was significantly lower in control rats than E2-replaced
rats, except for those receiving low-dose E2 for
2 weeks (group II). These results suggest thatE2 replacement after menopause increases bladder weight while decreasing body weight inrats. The explanation for these changes is notclear, but it is suspected that the lack of E 2
causes a reduction in physical activity and E2
replacement abolishes this reduction.
On the other hand, in E2-replaced rats, thebladder might gain weight as the collagen content of the bladder increases and the bladderepithelium thickens (Suguita et al., 2000; Eikaet al., 1990). Suguita et al. (2000) performed E2
replacement in female rats: they compared theobtained results with the parameters in theprogesterone-replaced group, and found an increase in thickness of the mucosa of the bladderand the proximal urethra. The present study,
however, showed no significant difference inthe HE stains of bladders between control andE2-replaced rats. It might have been difficult to
detect differences in the bladder mucosa or collagen content by HE staining due to the fact thatthe bladder weight increased 10% to 20%.
In the present histological study, immunoreactivity with the Bcl-2 antibody was weak inthe bladder of control and E 2-replaced rats.
However, the immunoreactivity with the Bax
antibody was stronger in the control rats than inE2-replaced rats.
RT-PCR analysis for bcl-2 showed no significant difference among the groups. Upon bax
analysis, however, the expression was stronger
in the control group than in the E2-replacedgroups, except for group II (low-dose E2 replacedfor 2 weeks). Considering the histological results, it seems that E2 replacement suppresses
the bax gene expression in the mucosa of the rat
bladder. In group II replaced with low-dose E2
for 2 weeks, the E2 dose might have been too
low or the administration period might havebeen too short to cause a difference in gene expression.
It is clear from previous studies that the bcl2 gene prolongs cell survival without affecting
proliferation and that the bcl-2 gene is able toprevent apoptosis induced by growth factor
deprivation. The mechanism by which bcl-2
exerts its antiapoptotic effect is not fully clear,although it has been speculated that bcl-2 acts
either as a regulator of an antioxidant pathwaythat prevents oxidative damage such as lipid
peroxidation caused by the generation of free
radicals (Tsujimoto et al., 1986; Hockenbery etal., 1993) or as a regulator of intracellular Ca2+
compartmentalization (Kane et al., 1993). bcl-2
is a member of a large family of proteins, called
the bcl-2 family, which is involved in the regulation of apoptosis. Among its members, bax
has a unique effect and plays an important role.
It is able to heterodimerize with bcl-2 and alter
the latter’s antiapoptotic action (Garcia et al.,1992). bcl-2 blocks the preapoptotic actions of100

Effects of E2 on apoptosis-related genes

other members of the bcl-2 family such as bax
and bad (Dubal et al., 1999). bcl-2 and othermembers of the bcl-2 gene family have been
identified, in addition to being regulators of cell
death, as estrogen-responsive genes (Teixeira etal., 1995; Kandouz et al., 1996). E2 may directly
upregulate the survival factor through receptormediated interactions with regions of the bcl-2
promoter or by indirect pathways (Teixeira etal., 1995). The antiapoptotic activity of bcl-2
correlates with its intracellular ratio to anotherprotein called Bax. High levels of Bax havebeen shown to favor apoptosis in cells subjected
to growth factor deprivation, whereas highlevels of Bcl-2 prolong cell survival under thesame conditions (Bafy et al., 1993). Teixeria etal. (1995) suggested that in the absence of E2,
the bax/bcl-2 ratio was increased and the cellswith estrogen receptors underwent apoptosis.
In the present study, it was suggested thatE 2-replacement therapy might prevent apoptosis mainly in the bladder mucosa through its
effect on the bcl-2 family in the bladder of old
female rats. Although the reason why the activity was demonstrated chiefly in the mucosa is
not clear, one explanation might be the regulation of the apoptotic reaction in the sensory receptors or nerve fibers in the bladder. In fact,
recent studies have allowed conjecture thatthe sensory nerve receptors against capsaicin(Lecci et al., 1998; Shea et al., 1999), bradykinin(Wotherspoon and Winter, 2000) and ATP(Cockayne et al., 2000; Vlaskovska et al., 2001)
exist in the epithelial cells, and that the activation of the sensory nerves is related to urinary
incontinence, voiding and the sensations of
bladder fullness, discomfort and pain.
In this study, findings demonstrated that
estrogen administration to menopausal rats suppresses the expression of apoptosis-related
genes; bax mRNA was suppressed in the bladder of old female rats by estrogen administration, especially in the bladder mucosa, while theaddition of estrogen prevented a decrease incontractile response induced by the absence of
estrogen (Longhurst et al., 1992; Diep andConatantinou, 1999). Sensory nerve receptors
exist in the mucosa, and it might follow thatsuppression of bax mRNA in the mucosa would
influence voiding behavior.

References
1 Albert DJ, Jonik RH, Gorzalka BB, Newlove T,
Webb B, Walsh ML. Serum estradiol concentration required to maintain body weight, attractivity, proceptivity, and receptivity in the ovariectomized female rat. Physiol Behav 1991;49:225–
231.
2 Ananth C, Thameem DS, Gopalakrishnakone P,
Kaur C. Domoic acid-induced neuronal damage
in the rat hippocampus: change in apoptosisrelated genes (Bcl-2, Bax, Caspase-3) and microglial response. J Neurosci Res 2001;66:177–190.
3 Batra SC, Iosif CS. Progesterone receptors in thefemale lower urinary tract. J Urol 1987;138:
1301–1304.
4 Bafy G, Miyashita T, Williamson JR, Reed JC.
Apoptosis induced by withdrawal of interleukin3 (IL-3) from an IL-3-dependent hematopoetic
cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2
oncoprotein production. J Biol Chem 1993;268:
6511–6519.
5 Cockayne AD, Hamilton GS, Zhu MQ, Dunn
MP, Zhong Y, Novakovic S, et al. Urinary bladder hyporeflexia and reduced pain-related behavior in P2X3-deficient mice. Nature 2000;407:
1011–1015.
6 Diep N, Conatantinou EC. Age dependent response to exogenous estrogen on micturition,
contractility and cholinergic receptors of the rat
bladder. Life Sci 1999;64:279–289.
7 Dubal BD, Shughrue JP, Wilson EM,
Merchenthaler L, Wise MP. Estradiol modulates
bcl-2 in cerebral ischemia: a potential role for
estrogen receptors. J Neurosci 1999;19:6385–
6393.
8 Eika B, Salling LN, Christensen LL, Andersen A,
Laurberg S, Danielsen CC. Long-term observation of the detrusor smooth muscle in rats. UrolRes 1990;18:439–442.
9 Garcia I, Martinou I, Tsujimoto Y, Martinou J-C.
Prevention of programmed cell death of sympathetic neurons by the Bcl-2 proto-oncogene.
Science 1992;258:302–304.
10 Handa RJ, Stadelman HL, Resko JA. Effect of
estrogen on androgen receptor dynamic in female
rat pituitary. Endocrinology 1987;121:84–89.
11 Hockenbery DM, Oltvai ZN, Yin XM, Milliman
CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993;
75:241–251.

101

R. Ohata

12 Huang Y, Ray S, Reed JC, Ibrado AM, Tang C,
Nawabi A, et al. Estrogen increases intracellular
p26Bcl-2 to p21Bax ratios and inhibits taxolinduced apoptosis of human breast cancer MCF-7
cells. Breast Cancer Res Treat 1997;42:73–81.
13 Iosif CS, Batra S, Ek A, Astedt B. Estrogen
receptors in the human female lower urinary
tract. Am J Obstet Gynecol 1981;414:817–820.
14 Kandouz M, Siromachkova M, Jacob D, Marquet
CB, Therwath A, Gompel A. Antagonism between estradiol and progestin on bcl-2 expressionin breast-cancer cells. Int J Cancer 1996;68:120–
125.
15 Kane DJ, Sarafin TA, Auton S, Hahn H, Gralla
FB, Valentine JC, et al. Bcl-2 inhibition of neuralcell death: decreased generation of reactive
oxygen species. Science 1993;262:1274–1276.
16 Lecci A, Giuliani S, Tramontana M, Santicioli P,
Criscuoli M, Dion S, et al. Bladder distention andactivation of the efferent function of sensory fibers: similarities with the effect of capsaicin. Br
J Pharmacol 1998;124:259–266.
17 Longhurst AP, Kauer J, Leggett ER, Levin MR.
The influence of ovariectomy and estradiol replacement on urinary bladder function in rats. J
Urol 1992;148:915–919.
18 Shea VK, Cai R, Crepps B, Mason L, Prel ER.
Sensory fibers of the pelvic nerve innervating the
rat’s urinary bladder. J Neurophysiol 2000;84:
1924–1933.
19 Shulman DI, Sweetland M, Duckett G, Root AW.
Effect of estrogen on the growth hormone (GH)secretory response to GH-releasing factor in the

castrated adult female rat in vivo. Endocrinology
1987;120:1047–1051.
20 Suguita M, Girão MJBC, Simões MJ, Sartori
MGF, Baracat EC, Rodrigues G. Amorphologic
and morphometric study of the vesical mucosa
and urethra of castrated female rats following
estrogen and/or progestogen replacement. Clin
Exp Obstet Gynecol 2000:27:176–178.
21 Teixeira C, Reed CJ, Pratt CAM. Estrogenpromotes chemotherapeutic drug resistance by amechanism involving Bcl-2 proto-oncogene
expression in human breast cancer cells. Cancer
Res 1995;55:3902–3907.
22 Tsujimoto Y, Croce CM. Analysis of thestructure, transcripts and protein products of Bcl2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA 1986;83:52145218.
23 Versi E. Incontinence in the climacteric. Clin
Obstet Gynecol 1990;33:392–398.
24 Vlaskovska M, Kasakov L, Rong W, Bodin P,
Bardini M, Cockayne AD, et al. P2X3 knock-out
mice reveal a major sensory role for ureotheliallyreleased ATP. J Neurosci 2001;15:5670–5677.
25 Wotherspoon G, Winter J. Bradykinin B1 receptor is constitutively expressed in the rat sensory
nervous system. Neurosci Lett 2000;294:175–
178.

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Received October 8, 2002; accepted October 24, 2002
Corresponding author: R. Ohata