Yonago Acta medica 1998;41:17–22

Morphological Effects of Estradiol on the Neurons of the Major Pelvic
Ganglia and the Urinary Bladders of Female Rats
Hiroshi Yamaguchi
Department of Urology, Faculty of Medicine, Tottori University, Yonago 683-0826, JapanTo elucidate the effect of estradiol (E2) on the morphology of the neurons of the lower
urinary tract during puberty, we carried out morphometric and immunohistochemical
analyses of the major pelvic ganglia (MPG) in female rats. Four-week-old female Wistar
rats were divided into the following groups: normal controls, ovariectomies, and ovariectomies receiving E2 replacement. At the age of 12 weeks the MPG were stained withhematoxylin and eosin (H&E), and immunostained by the antibody against rat tyrosinehydroxylase (TH). Each section was evaluated by an image analysis system. In theovariectomized rats, the size of neurons in the MPG was significantly reduced. Immunohistochemical analysis revealed that the size of TH-positive neurons was significantly
increased compared with TH-negative neurons in the E2-replaced rats. The results
suggest that pubertal E2 deficit reduces neuron size, yet induces a notable increase in
the size of TH-positive (adrenergic) neurons in the MPG.
Key words: estradiol; major pelvic ganglia; ovariectomy; puberty

The sympathetic nervous system is thought toplay a role in the control of bladder and urethralfunction, and estrogen has been used clinicallyfor many years in the treatment of stress urinary
incontinence. Based on studies of intraurethralpressure profiles of women, Schreiter and colleagues (1976) suggested that estrogen has atonicising effect on urethral musculature, butthey did not present any quantitative or doseresponse data. Downie and coworkers (1975)
showed that alpha-adrenoceptors of the bladderneck of estrous rabbits are more sensitive toadrenergic agonists than those of the detrusor.
The major pelvic ganglia (MPG) are located in
the lateral ligament of the bladder, lateral to theprostate in male, and the vagina in female rats, andinnervate the internal reproductive organs, lower
bowel and lower urinary tract (Langworthy, 1965;
Purinton et al., 1973; Tabatabai et al., 1986; Keastet al., 1989). Greenwood and colleagues (1985)
showed that the significant differences in thenumber of neurons in the MPG correlated withthe sex in rats. However, there have been no reports on the morphological effects of estradiol(E2) on the lower urinary tract during puberty. In
the present study, morphological effects of the
MPG were investigated by observing changes
in the E2 level of rats.
Materials and Methods

Animals
Four-week-old female Wistar rats were divided
into 3 groups. Group 1 remained intact and received no hormonal manipulation (control group;n = 15). Group 2 underwent bilateral ovariectomy
at the age of 4 weeks and received no hormonal
replacement (ovariectomized group, n = 10).
Group 3 underwent bilateral ovariectomy at theage of 4 weeks, and daily from the age of 8 weeks
subcutaneous replacement of 50 μg/body E2
benzoate dissolved corn oil (E2-replaced group, n= 5). Ovariectomies were performed using standard surgical procedures with the aid of ether
anesthesia.

Abbreviations: ABC, avidin-biotin-peroxidase comples; DAB, 3,3-diaminobenzidine tetrahydrochloride; E2,
estradiol; H&E, hematoxylin and eosin; MPG, major pelvic ganglia; PBS, phosphate buffered saline; TH,tyrosine hydroxylase

17

Hiroshi Yamaguchi

IgG (Vectastein ABC kit, Burlingame, CA) wasused to detect bound antibodies. 3,3-Diaminobenzidine tetrahydrochloride (DAB) was usedas the final chromogen.

Measurement of plasma E2
Plasma E2 concentrations were measured at 4
weeks in the control group, 8 weeks in thecontrol and ovariectomized groups, and 12
weeks in each group. Since the highest valuesof plasma E 2 concentration were found insamples collected on the morning of pro-estrus,
blood samples were collected in the control
group in the morning of pro-estrus during therats’ estrous cycles, as determinated by vaginal
smears.

Morphometry of MPG
Each section was viewed under a light microscope, and the MPG’s TH-positive and THnegative neurons were counted in the 3 different sections from each rat. The size of theneurons in the sections stained with H&E wasdetermined by means of an image analysis system. After routine light microscopic study,
photomicrographs (× 400) were taken randomly
and scanned into the National Institutes of
Health image analysis system (Macintosh,
Cupertino, CA). Outlines of neuron profiles
were traced by a digital mouse and the outlined
areas were then analyzed. Only neurons showing nuclei were counted into the calculation,and neuron profiles with an area < 50 mm2 were
excluded. For each group, the MPG neurons
were counted in each section from each of 10
animals.

Histopathology
Five rats at the age of 4 weeks from the control
group, 5 rats at the age of 8 weeks from each of
the control and ovariectomized groups, and 5
rats at the age of 12 weeks from all groups wereanesthetized with an intraperitoneal injection of
sodium pentobarbital (20 mg/kg), weighed andthen perfused through the heart with physiological saline. The bladder of each rat in the 12week age group was removed and weighed, andits MPG was removed and immediately frozen
in an acetate/dry-ice bath. Using a serial section technique (10 μm section), hematoxylin
and eosin (H&E) stain was used for routine observation and morphometric study, and tyrosinehydroxylase (TH) stain was used for immunohistochemical analysis.

Statistical analysis
All results were expressed as the mean ± SD
and analyzed using the Mann-Whitney test.

Control
group(pg/mL)

Plasma E2 level

Immunohistochemistry of MPG
The avidin-biotin-peroxidase complex (ABC)method was employed for TH detection(Klimaschewski et al., 1994). Cryostat sections
were hydrated, and endogenous peroxidase
activity was quenched by incubation for 30 min
with 0.3% H2O2, followed by washing in a phosphate buffered saline (PBS, pH 7.2). Normal
goat serum served as blocking agent. A rabbit
antiserum against rat TH (Chemicon, Temecula,CA) was used at the recommended dilution of
1:250 in bovine serum albumin (1%)-containing
PBS. The sections were incubated with the antiserum for 18 h at 4 ˚C. PBS was used to replace
the antiserum in the controls. Goat anti-rabbit

Ovariectomized groupE2-replaced
group

*

50

*
40

**

30
20
10
0

4

8

12

(week)

Age
Fig. 1. Plasma E2 levels (pg/mL). A striking rise ofplasma E2 levels is observed at the age of 8 weeks inthe control rats. E 2 replacement rises plasma E2level 10 times that of the subjects with the ovariectomized group. *Significant differences fromvalues at 4 weeks for the control group; P < 0.01.
**Significant differences from values for the ovariectomized group; P < 0.01.

18

Estradiol’s effect on the major pelvic ganglia

Table 1. Body weight and bladder weight in
12-week-old rats

Results

Group

Plasma E2 levels
In the control rats, a striking rise of plasma E2levels was observed at the age of 8 weeks,reaching a plateau that lasted until week 12.
The ovariectomized group exhibited no rise inplasma E2 levels. In the E2-replaced group at
the age of 12 weeks, the mean plasma E2 levelrose to 32.5 ± 5.1 pg/mL, a level significantly
higher than that of the ovariectomized group(Fig. 1).

Body weight(g)

Bladder weight(mg)

Control
212.7 ± 17.9
167.3 ± 19.2****
Ovariectomized 241.6 ± 14.6
130.0 ± 21.5**
E2-replaced
218.3 ± 21.6
185.6 ± 43.0 ****Significant differences; P < 0.001.

The pattern of TH-immunostaining was heterogeneous in the MPG neurons. Positive
neurons showed a homogeneous cytoplasmic
staining (Fig. 2).
The size distributions of the neurons in the
MPG were shown in Fig. 3. Neuron size wasmeasured in 1,167 to 1,361 cells in each group.
The percentage of neurons more than 300 μm2
were 4.5, 27.5 and 23.2 in 4-, 8- and 12-weekold control rats, respectively, and 3.9 and 4.5 in
8- and 12-week-old ovariectomized rats, and29.0 in 12-week-old E2-replaced rats. The size
distributions in the 8- and 12-week-old ovariectomized rats did not disclose an increase in
the percentage of large neurons, but in the E2replaced rats the distribution was similar to thatof the control rats.
The number of counted immunostained
neurons ranged from 1,235 to 1,648. The mean
areas of TH-positive neurons (adrenergic
neurons) and TH-negative neurons (nonadrenergic neurons) significantly increased in
8- and 12-week-old rats compared with those ofBladder and body weights
Body weights were significantly heavier in theovariectomized group than in the control andE 2-replaced groups. On the other hand, thebladder weights were significantly lower in theovariectomized group than in the control andE2-replaced groups. No significant differenceswere observed between the control and E 2replaced groups in regard to both body weightand bladder weight (Table 1).

Morphological study of the MPG
In the 4-, 8- and 12-week-old control rats, mostneurons in the MPG stained with H&E were
either round or slightly ovoid in shape andvaried in size.

A

B

Fig. 2. Morphological study of MPG. A: H&E stain of MPG. The neuron cell bodies are round or oval andcontain a prominent pale nucleus (× 200). B: TH stain of MPG. TH-positive neurons are stained brown. THnegative neurons are not stained (× 200).

19

Hiroshi Yamaguchi(%)

4-week-old rats

(%)

50

8-week-old rats

(%)

50

50

40

40

40

30

30

30

20

20

20

10

10

0

0

10
300

(μm 2)

300

Neuron size

Upper: control group
Middle: ovariectomized group12-week-old rats

0

2

(μm )

50

50

40

40

30

30

20

20

10

10

300

(μm 2)

300

(μm2)

300

(μm2)

0

0
300

(μm2 )
50

Neuron size

Lower: E2-replaced group

40
30
20

Fig. 3. Neuron size in the MPG. In 8- and 12-week-old rats of thecontrol group, large neurons (300 μm2 < ) increase in number comparedto the 4-week-old group. But such an increase of neuron size is not seenin the ovariectomized group. In the E2-replaced group, an increase inneuron size is comparable to the control group at the age of 12 weeks.

the 4-week-old rats in the control group. However, such an increase of mean areas of both
TH-positive and TH-negative neurons was notfound in the ovariectomized group. In the 12week-old E2-replaced rats, the mean areas ofboth TH-positive and TH-negative neurons

100

Neuron size

were significantly larger than that of 8- and 12week-old ovariectomized rats, with a significantly larger increase in TH-positive neurons
than in TH-negative neurons. The ratios of THpositive neurons to all neurons obtained showed
no differences between each group (Table 2).

Table 2. The mean areas (μm2) of TH-positive neurons and TH-negative neurons, and thepercentage of TH-positive neurons to all neurons
Group

Rats
8-week-old

4-week-old
Control group
TH-positive neurons
TH-negative neurons
TH+%

138.4 ± 102.6
136.9 ± 52.3
27.3

**
**
**

259.5 ± 72.5
229.9 ± 42.7
29.0

**

12-week-old****

258.7 ± 109.6
243.6 ± 74.8
31.3

**
**

Ovariectomized group
TH-positive neurons
TH-negative neurons
TH+%

150.9 ± 55.5
166.5 ± 67.5
30.5

E2-replaced group
TH-positive neurons
TH-negative neurons
TH+%
TH+%, percentage of TH-positive neurons to all neurons.
* Significant differences; P < 0.01.
**Significant differences; P < 0.001.

20

**
**

159.6 ± 56.8
176.5 ± 71.7
28.3

303.3 ± 123.9
233.7 ± 128.1
29.6*

Estradiol’s effect on the major pelvic ganglia

the size of adrenergic neurons showed a significant increase in comparison with that of nonadrenergic neurons. This result suggests thatadrenergic neurons are more sensitive to E2 replacement than are non-adrenergic neurons.
Levin and colleagues (1980, 1981) showedthat estrogen induced a marked increase in the
response to alpha-adrenergic and muscarinic
cholinergic agonists in immature rabbit urinary
bladders, but no alterations were noted in their
response to beta-adrenergic agonists. In addition, it was observed that estrogen administration induced a moderate increase in the adrenergic innervation of the immature rabbit bladderdetrusor, whereas no changes could be observed in cholinergic innervation. Hodgson andcoworkers (1978) found that in mature female
rabbits, the annular urethral alpha-adrenergic
response was decreased by ovariectomy, but the
bladder alpha-adrenergic response was not
influenced. Miodrag and others (1988) reported that pretreatment of immature female rabbitswith E2 significantly increased the density of
the alpha-adrenoceptors in the bladder body but
not in the bladder base, but showed no significant changes in beta-adrenoceptor density inany part of the bladder. Shapiro (1986) has
shown that estrogens increase the weight andmodulate the muscarinic cholinergic receptor
density of the mature female rabbit bladder body,and this may result in a reduction of detrusor
tone and an increase in bladder capacity. Batra
and Andersson (1989) showed that estrogen
treatment of mature ovariectomized rabbits
caused a marked reduction in the density of
muscarinic receptors. These studies suggestthat in the bladders of immature animals
estrogen administration induces a significant
increase in adrenergic and cholinergic responses, which results in an increase in adrenergic innervation and an increase in adrenergicand muscarinic receptor densities. Yet in the
bladders of mature animals, estrogen administration results in no such increase in adrenergic
response. Our study also showed that the sizeof both adrenergic and non-adrenergic neurons
in the MPG of rats increased the percentage ofthe small neurons and decreased the percentageof large neurons by ovariectomy at 4 weeks,

Discussion
The present study demonstrated a rapid increaseof plasma E2 levels between the ages of 4 weeks
and 8 weeks in the control rats. Due to theovariectomies, plasma E2 levels were kept low
until the age of 4 weeks, but after E2 replacement, plasma E2 levels rose about 10 times thatof the subjects with no E2 replacement. In theE 2 -replaced group, mean plasma E 2 levels
reached 32.5 ± 5.1 pg/mL at the age of 12
weeks. This level was over the mean level of E2
through the estrous cycle, or about two-thirds ofthe maximum value reported during the estrous
cycle (Yoshinaga et al., 1969; Brown-Grant etal., 1970; Dupon and Kim, 1973; Handa et al.,
1987; Shulman et al., 1987; Albert et al., 1991).
Albert and workers (1991) showed that aplasma E2 level of 15 pg/mL was slightly below
the mean level of E2 present through the estrous
cycle in rats maintaining normal body weights.
Therefore, our method of E2 replacement wasthought to be sufficient for the study of theeffect of E2 on the MPG of rats.
A previous study demonstrated that ovariectomized rats showed an increase in bodyweight and a decrease in bladder weight in comparison to control rats (Tanaka, 1959). In thepresent study, the body weights of the ovariectomized rats significantly increased in comparison with those of the normal and the E2replaced rats, while the bladder weights of theovariectomized rats significantly decreased incomparison with those of the normal and the E2replaced rats. These results suggest that E 2
increases bladder weight while decreasing bodyweight, and that an E2 deficit causes the opposite reaction. The reason for this is unclear, but
it is suspected that E2 may increase the bladder’s cellular mass and capacity (Shapiro,1986).
Our morphometric and immunohistochemical studies have demonstrated that prepubertal
ovariectomy reduces the size of both TH-positive(adrenergic) and TH-negative (non-adrenergic)
neurons in the MPG, and E 2 replacement recovered the size of both types of neurons in the
MPG following prepubertal ovariectomy, yet
21

Hiroshi Yamaguchi

7 Handa RJ, Stadelman HL, Resko JA. Effect of
estrogen on androgen receptor dynamics in fe
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and the size of the neurons, particulary adrenergic neurons, increased by E 2 administrationfrom 8 weeks. These results suggest that thegrowth of both adrenergic and non-adrenergic
neurons in the MPG is stimulated by estrogen in
immature rats, but adrenergic neurons are more
susceptible to later estrogen stimulation. Postganglionic branches from the MPG reach not
only to the bladder but also to the rectum,
clitoris, proximal urethra, and the dorsal surfaceof the bladder (Langworthy, 1965; Purinton etal., 1973). Therefore our study may show that
estrogen administration influences the responseof the urinary bladder by stimulating the growthof the MPG surrounding it.
In summary, the present study suggests thatpubertal estrogen deficit reduces the size ofneurons in the MPG and that estrogen replacement increases neuron size, especially theadrenergic neurons in the MPG.

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(Received December 22, Accepted December 25, 1997)22