Neurourology and Urodynamics 17:241–247 (1998)

The Effects of Delayed Hormone

Replacement Therapy on Estrogen
Receptors of the Cynomolgus Monkey
Bladder and Vagina
Deirdre Robinson,1* Thomas C. Register,1 and Laura R. Carter2
1
Departments of Obstetrics and Gynecology, and Comparative Medicine, Bowman Gray
School of Medicine, Wake Forest University, Winston-Salem, North Carolina
2
Michigan State University, East Lansing, Michigan

The aim of this study was to determine cytosolic estrogen receptor content of the cyno-
molgus monkey bladder and vagina after hormone replacement therapy. Animals main-
tained without hormone therapy for 2 years after surgical menopause were randomized to
receive either no hormones (OVX), conjugated equine estrogens (CEE), or estrogen/
medroxyprogesterone acetate (CEE + MPA) treatment for 30 months. Estrogen receptor
content of bladder and vagina cytosolic extracts was determined using a commercial en-
zyme-linked immunosorbent assay. Estrogen receptors were uniformly present, although the
vaginal concentration was 100-fold greater than in the bladder. Estrogen and combination
therapy significantly decreased cytosolic receptor content in both sites compared with
controls. The cynomolgus urogenital tract remains estrogen sensitive 2 years after surgical
menopause. Prolonged exposure to estrogens decreases cytosolic estrogen receptor content
in a manner similar to that described for short-term estrogen therapy. These results suggest
that the effects of hormone replacement on the urinary tract can be identified even if
initiated years after menopause. Neurourol. Urodynam. 17:241–247, 1998.
© 1998 Wiley-Liss, Inc.

Key words: estrogen receptors; bladder; hormone replacement; vagina; cynomolgus monkey

INTRODUCTION
Long-term estrogen replacement therapy (ERT) is now advocated as a preven-
tative measure for atherosclerotic coronary heart disease and osteoporosis [Grady et
al., 1992; Grodstein and Stampfer, 1995]. Concern for possible negative impacts of
ERT, in particular malignancy, has contributed to an overall long-term compliance
rate of <10% in the United States [Bush et al., 1983; Derby et al., 1993; Ravnikar,
1992; Cline et al., 1996; Stanford et al., 1995]. An increased appreciation for the
possible benefits of hormone replacement therapy, especially on secondary sites such

*Correspondence to: Deirdre Robinson, M.D., Assistant Professor, Department of Obstetrics and Gyne-
cology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC
27157.

Received for publication 21 October 1997; Accepted 8 January 1998

© 1998 Wiley-Liss, Inc.

PROD #1020
242 Robinson et al.

as the lower urinary tract, may encourage broader use of estrogen therapy among
eligible women.
The actual impact of postmenopausal estrogen therapy on the female lower
urinary tract continues to be under debate. Although estrogen receptors have been
identified through out the human urogenital tract, the mechanism by which estrogen
can influence the structure and function of the urinary system and the implications of
prolonged hypoestrogenism remain unclear [Batra and Iosif, 1983; Ingelman-
Sundberg et al., 1981; Bussolati et al., 1990; Urner et al., 1983; Blakeman et al.,
1996a].
Clinically, estrogen therapy has been shown to improve symptoms of urinary
urgency, frequency, dysuria, stress and urge incontinence in individual studies and a
meta-analysis of several controlled and uncontrolled trials [Elia and Bergman, 1993;
Walter et al., 1978; Fantl et al., 1988; Faber and Heidenreich, 1977; Fantl et al., 1994].
In contrast, a randomized controlled trial of 3 months of conjugated equine estrogen
therapy did not show either subjective or objective improvement in symptoms of 83
incontinent women who were, on average, 18 years from menopause [Fantl et al.,
1996]. The question of how estrogens affect urinary symptoms and what role the
duration of menopause and its associated estrogen-deficient state plays in the efficacy
of ERT remains unanswered.
Animal models have been used to study the effects of estrogen on the lower
urinary tract particularly at tissue and cellular levels. Studies in the rabbit have
documented the presence of estrogen receptors in the lower urinary tract that are
sensitive to relatively short durations of estradiol therapy [Urner et al., 1983; Batra
and Iosif, 1992; Batra and Iosif, 1983; Rosenzweig et al., 1995]. The cynomolgus
monkey has been extensively used to study the effects of menopause and hormone
replacement on atherosclerosis, osteoporosis, the breast, and brain. This animal has
the advantage of a cyclic monthly menstrual cycle and hormonal patterns that are
similar to women in the reproductive and postmenopausal states [Mahoney, 1970].
The purpose of this study was to determine whether delayed estrogen replacement
therapy, with and without an opposing progestin, initiated 2 years after surgical
menopause modulates the estrogen receptor content of cytosolic extracts of the cy-
nomolgus bladder and vagina.

MATERIALS AND METHODS

The animals used in this study were from a group of 120 feral female adult
cynomolgus monkeys (Macaca fascicularis) imported from Indonesia (Charles River
Primates, Port Washington, NY). All animals were between 5 and 13 years of age, not
pregnant, and quarantined for 2 months after arrival. Bilateral oophorectomies were
then performed and the monkeys were housed in social groups of 4–8 animals for a
2-year period. During this time all animals were fed a moderately atherogenic diet as
part of an atherosclerosis and osteoporosis prevention trial and did not receive hor-
mone replacement.
Seventeen animals died during the initial phase leaving 103 monkeys available
for randomization into four groups using a stratified scheme based on cholesterol
levels during the atherogenic diet period, bone density measurement prior to treat-
ment, and time since ovariectomy. One group was then designated for immediate
necropsy (n 4 20). The remaining groups continued on to a 30-month treatment
 HRT and Urogenital Tract Estrogen Receptors 243

phase during which all animals were fed a plasma lipid–lowering diet with either no
hormone replacement (n 4 28; OVX), conjugated equine estrogens (n 4 28; CEE),
or the diet plus CEE and medroxyprogesterone acetate (n 4 27; CEE/MPA). During
the first 8 months of treatment, the animals in the CEE and CEE/MPA groups
received 7.2 mg of CEE (Premarint, Wyeth-Ayerst, Princeton, NJ) per monkey per
day. For the remaining 22 months, the dose of CEE was increased to 166 mg per
monkey per day to be equivalent to a woman receiving 0.625 mg/day. Monkeys in the
CEE/MPA groups received 650 mg per day of MPA (Cycrint, Wyeth-Ayerst) to be
equivalent to a woman’s dose of 2.5 mg/day. The hormones were administered in the
daily diet and intermittent estradiol levels were used to document ingestion. This
regimen is a well-established technique in our laboratory that mimics a continuous
hormone replacement regimen for a postmenopausal woman.
Fifteen animals died during the treatment phase from causes unrelated to the
experimental manipulation, mostly from gastrointestinal disorders or trauma. The
remaining 68 animals underwent necropsy after sedation with ketamine hydrochloride
and deep anesthesia with sodium pentobarbital. The cardiovascular system was
flushed with normal saline, followed by perfusion with 10% neutral buffered formalin
at a pressure of 100 mm Hg for 1 hr. The urogenital tissues were removed and the
bladder and vagina were separated from surrounding tissues. The urethra and a small
strip of vagina were reserved for use with another protocol, and the remainder of the
bladder and vagina were divided into two halves and each rapidly frozen in liquid
nitrogen for storage at −70°C [Robinson et al., 1996]. All procedures involving
animals were conducted in compliance with state and federal laws and the guidelines
established by the institutional Animal Care and Use Committee.
Tissues from six animals in each of the OVX, CEE, and CEE/MPA groups were
used for receptor analysis. The small sample size was used as a previous study in the
same animals identified significant morphologic changes [Robinson et al., 1996].
Tissue weights from the right halves of the bladders and vaginas were available for
66 animals. No animals were available from the initial baseline group as this phase
was completed before our study was initiated.
The frozen bladder and vagina were weighed and pulverized in a liquid nitro-
gen–cooled mortar and pestle, diluted with cold homogenization buffer and homog-
enized. An aliquot of the homogenate was centrifuged at 40,000 g for 1 hr at 4°C and
the supernatant representing the cytosolic fraction was used for estrogen receptor
determination. Protein content of the fraction was determined by the method of
Lowry.
The estrogen receptor content of each tissue was determined using the protocols
and reagents obtained with a commercially available enzyme-linked immunosorbent
assay (ELISA) (ER-EIA) from Abbott Laboratories (Abbott Park, IL). Estrogen re-
ceptor content was expressed as fmole/mg protein.
Statistical analysis was performed by one-way analysis of variance (ANOVA).
Extreme data values were corrected for by transformation.

RESULTS

Estrogen receptor protein was easily identified in the cynomolgus macaque

bladder and vagina by the Abbott Sandwich ELISA (Table I). The estrogen receptor
244 Robinson et al.

TABLE I. Estrogen Receptor Content (fmole/mg protein), Mean (SD)

OVX CEE CEE + MPA
(n 4 6) (n 4 6) (n 4 6) P value
Bladder 2.18 1.09 1.18 <0.01
Vagina 200.7 64.5 91.5 <0.02

concentration in the cytosolic fraction obtained from bladder homogenates were 1/

100th of the concentration found in the vaginal homogenates.
Comparison between groups revealed a significantly lower number of estrogen
receptors in the cytosolic fraction from both the bladder and the vagina in the CEE
treatment groups compared with controls. Animals that were exposed to the progestin
MPA (CEE + MPA group) had a significantly lower number of receptors than ovari-
ectomized controls (OVX group) but were not significantly different from animals
treated with estrogen alone (CEE group).
In the entire animal group (n 4 66), the mean weight of the vagina significantly
increased with exposure to estrogen (CEE) compared with controls (Table II). The
combination of CEE and MPA therapy did not alter the weights of the vaginal tissue
halves compared with those of the OVX group. Hormonal therapy did not influence
the weight of the bladder.

COMMENT

The cynomolgus monkey has been a useful tool to study the effects of estrogen
on several organ systems. This animal has the onset of menarche at ∼3 years of age
and menopause at 20 years of age [Mahoney, 1970]. Thus the animals used in this
study were in the middle of their reproductive lives when they underwent surgical
menopause. The loss of estrogen for the subsequent 2-year period may reflect the
conditions occurring in a woman during the initial 5–10 years after surgical meno-
pause. Despite the similarities between the cynomolgus and human reproductive lives,
we recognize that factors such as structural differences, surgery, and multiparity
cannot be duplicated in a nonhuman primate model. Earlier work with this group of
animals demonstrated that ERT had a significant impact on the urogenital tract by
increasing the thickness of the vaginal epithelium and the connective tissue layer of
the urethra [Robinson et al., 1996]. The results of this study confirm that estrogen may
have an impact on the bladder after a period of hypoestrogenism by altering receptor
status.
Estradiol receptors have been confirmed in the bladder and urethra of rabbits in
both intact and oophorectomized models and now have been demonstrated in the
cynomolgus monkey model after surgical menopause [Urner et al., 1983; Batra and
Iosif, 1992; Batra and Iosif, 1983]. The relatively low number of bladder estrogen
receptors and higher number of vaginal receptors are consistent with reports from
ovariectomized rabbits, rhesus monkeys, and menopausal women [Batra and Iosif,
1992; Elsner et al., 1977; Wiegerink et al., 1980; Blakeman et al., 1996b]. Reduction
in the number of estrogen receptors after ERT is an effect that has been reported in
tissues from the rabbit urogenital tract with up to 8 weeks of estradiol therapy [Batra
and Iosif, 1992; Batra et al., 1987; Batra et al., 1984]. This response could result from
a decrease in the number of cytosolic receptors either due to receptor translocation to
 HRT and Urogenital Tract Estrogen Receptors 245

TABLE II. Tissue Weights (g), Mean (SD)

OVX CEE CEE + MPA
(n 4 25) (n 4 20) (n 4 21)
Bladder 1.66 1.68 1.78
Vagina 0.45 0.88* 0.63
*P < 0.05.

the nucleus or to downregulation of the cytosolic receptors [Batra, 1979]. The animals
in this study received estrogens for a relatively long time (30 months) and had tissue
levels of estrogen receptors similar to those described after short-term therapy initi-
ated soon after oophorectomy in the rabbit model [Batra and Iosif, 1992; Batra et al.,
1987; Batra et al., 1984]. Thus, these tissues have remained sensitive to estrogen
despite a 2-year hiatus between oophorectomy and onset of ERT.
The addition of MPA to the CEE appeared to dampen the tissue response to
estrogen therapy alone but did not significantly alter the number of receptors mea-
sured in our small study. Short-term studies in the rabbit urogenital tract demonstrated
a progesterone-induced decrease in estrogen receptors in the uterus but not the urethra
or vagina [Batra and Iosif, 1989], whereas a previous study in cynomolgus macaques
suggested that combination therapy may potentiate estrogen-induced structural
changes in both the urethra and vagina [Robinson et al., 1996]. Further research is
needed to determine whether combination therapy has significantly different effects
from estrogen alone on the structure or function of the lower urinary tract.
Increases in bladder and vagina weight after initiation of ERT in the rabbit were
reported to plateau after 1 and 4 weeks of therapy, respectively [Batra and Iosif, 1983;
Batra and Iosif, 1992]. We did not identify a change in tissue weights of the bladder
but did find that estrogen does increase the weight of the cynomolgus vagina if MPA
was not added to the regimen.
The documentation of estrogen-responsive tissues in this model suggests that
there is a substantial period of time during which the urogenital tract remains estrogen
sensitive. Evaluation of tissues from the animals that underwent necropsy prior to
hormonal treatment would have been ideal to establish an untreated control group but,
unfortunately, these tissues were unavailable at the time of our project. Our data
suggest that the urogenital tract is sensitive to prolonged hormonal exposure, even
after a period of hypoestrogenism.

ACKNOWLEDGMENTS
This study was supported in part by Grant No. HL 45666 (TBC), NHLBI, NIH.

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