(enclomiphene) isomers of clomiphene citrate, and a fixed-ring antiestrogen, nafoxidine, only. ZUC was able to induce gap junctions in myometrial and serosal.
OF REPRODUCTION
BIOLOGY
36,
741-75
1 (1987)
Gap Junction Modulation in Rat Uterus Ill. Structure-Activity Relationships of Estrogen Receptor-Binding L igands on Myometrial and Serosal Cells’ ROBERT
C. BURGHARDT,2
DANA
REBECCA LAWRENCE C. KURTEN,3 and
RICHARD
Department
GADDY-KURTEN,3
BURGHARDT, PHILIP A. MITCHELL4
of Biology
Texas A&M College Station,
University Texas 77843
ABSTRACT A
number
affect serosal
of
steroidal
the formation cells. Potent
estrogens,
estriol
and
and
estrone,
fashion when serosal epithelium
lower
of hormone
levels
myometrial longitudinal
to
highest isomers
clomiphene myometrial
dosage tested. of tam oxifen
with EB. However, progesterone-to-estrogen
responses
(Adiol) 5 days,
a fixed-ring cells. These
and L whereas
the
over
5 days. pattern
tested
gap
junctions
Induction of estrogen
differential
rodent
that
course
growth reflects locally
and differentiation the action of produced agents,
of the
uterine
wall
these
factors
in
(Fuchs, specific
of
and related ZUC) and
nafoxidine, only that induction
requires
higher
the
gap
in a
junctions in but requires
of circular
and
longi-
to induce myometrial junctions in the serosal
compounds evaluated, trans (enclomiphene)
was able junctions
of estrogen
than to EB
of myometrial cell estrogen receptor-binding
to induce gap in rat uterine
than
other
gap gap cells
including isomers of junctions myometrial
in
estrogen-dependent
ill-defined, and they complicate actions of estrogens and progesterone, regulatory agents of uterine function. progesterone are initiators of the
dramatic
of the mammalian uterus a number of humoral and as well as physical stretching uterine
levels
ZUC
of gap
failed gap
to and weak
detected in the circular gap junction response
appearance 100:1. The
derivatives (zuclomiphene,
ability
in myometrium
of annular stimulation
stimulation
3, 1 7(3-diol induce annular
their
myometrial 7j3, and the
uterus.
pregnancy,
1978).
EB-stimulated ratio exceeded
for
rat uterine estradiol-1
(EB),
annular
-androstene-3 Adiol did
antiestrogen, studies indicate
response
in the
the
INTRODUCTION During
and
In myometrium,
Of the triphenylethylene and CI 628, the cis
estrogen-dependent
cell
of macular
response.
were
cell layers was observed, with 3 to 5 times more gap junctions layer. Progesterone, estriol, or estrone suppress the myometrial
citrate, and and serosal
is an
target
the
ligands
in hypophysectomized estradiol benzoate
in daily injections a similar dose-dependent
5a-androstane-3 j3, 1 7j3-diol at doses up to 5 mg/day for
at the mixed
cells
formation
administered follows initiate
receptor-binding
of gap junctions diethyistilbestrol,
stimulate
when administered concurrently junctions was blocked hen the androgens, junctions
estrogen
and internalization estrogens, including
dose-dependent the uterine tudinal in the
nonsteroidal
The
interactions
changes required for implantation, maintenance of the conceptus, the timely alterations in the contractile state of uterine smooth muscle, and myometrial sensitivity to factors that play an integrated
of
compartments
are
role
Accepted September 29, 1986. Received June 5, 1986. ‘Supported by NIH Grant HD-14781 and an NIH BRSG 2 Reprint requests: Robert C. Burghardt, Electron Center, Texas A & M University, College Station, TX 77843. Present address: Department of Cell Biology, Baylor Medicine, Houston, TX 77030. address: Department of Pathology, University Health Science Center at Houston, Houston, TX 77030.
to R.C.B. Microscopy College of
analysis of the the primary Estrogens and serial functional
in the
Texas
741
of labor relevant is that from
activity to coordinated tion (Krishnamurti
et
transition numbers (reviewed
of
initiation
A steroid response in the present report undergoes a transition
(Sakamoto
et al.,
1986).
to the studies described prepartum myometrium asynchronous electrical
activity just al., 1982).
prior This
to parturielectrical
is associated with the development of large of gap junctions between myometrial cells by Cole and Garfield, 1985). In small
BURGHARDT
742 laboratory exist good terone
animals and correlations
levels
within
the
1984a)
or
and
large domestic animals, between estrogen and
the
appearance
myometrium. ovary
of
Pituitary
ablation
(MacKenzie
(EB), estradiol-17j3, 5a-androstane-3(3,
junctions
(Burghardt
1985) and steroid-replacement-therapy rat uteri support the idea that terone occupied
gap
there progeset al.,
and
Garfield,
studies of and proges-
estrogen
modulate the amount of plasma membrane by gap junctions within the myometrium.
In the present investigations, we studied induction of the appearance of myometrial junctions in uteri from hypophysectomized (HX)
the gap rats
as a specific assumption
the pre-
cursors tion ability
end that
point the
of estrogen action, making synthesis of gap junction
is a direct “late” response to estrogen stimula(Katzenellenbogen and Gorski, 1975). The of a number of anti-estrogens to suppress
estrogen-directed these ability reduce
development
of
gap
junctions
second
formed in vitro (Garfield with this assumption.
end
point
was
gap
junction
response
the cell
serosal cell covering type was previously
also
et al., 1980b), Evaluation of a
conducted,
utilizing
in another
uterine
of the found
uterus. This to respond
cell
the type-
epithelial to much
lower levels of exogenous estrogen by increasing the number of gap junctions as well as inducing their internalization (Burghardt et al., 1984a). The action of estrogens on the status of junctional membrane was investigated relationships of “impeded” androgens, referred
by a
evaluating number of
structure-activity ligands, including
or “short-acting” estrogens, several and a group of compounds collectively to as nonsteroidal antiestrogens, all of which
bind
the estrogen receptor. The estrogenic properties the latter compounds, which include triphenylethylene derivatives, vary widely, depending upon chemical structure, geometric configuration, and the species, tissue, and parameter of estrogen action
of
studied Therefore, ethylene whether agonism junctions.
(reviewed by Sutherland and Murphy, 1982). the estrogenicity of several triphenylderivatives was evaluated to determine any of these compounds exhibit sufficient to
cause
the
appearance
of myometrial
AND METHODS
Reagents Diethylstilbestrol
3(3,17(3-diol (5-Adiol) were purchased Chemical Co. (St. Louis, MO). Synthetic were obtained as gifts from a number Enclomiphene citrate (ZUC), citrate ceuticals isomers)
(DES),
j3-estradiol-3
citrate the trans
(ENC) and and cis isomers
(CLOM), were gifts Inc., (Cincinnati, from Warner-Lambert
tamoxifen Americas, chloride
from
progesterone, -androstenefrom Sigma antiestrogens of sources. zuclomiphene of clomiphene
Merrell Dow PharmaOH); CI-628, (mixed Co., (Ann Arbor, MI);
citrate (TAM), (mixed isomers) from ICI (Wilmington, DE); and nafoxidine hydro(NAF), from Upjohn Inc., (Kalamazoo, MI).
Culture saline
media and Dulbecco’s (PBS) were obtained
phosphate-buffered Irvine Scientific
from
(Irvine, Electron
CA), and fixatives and embedding media Microscopy Sciences (Fort Washington,
Animal
Treatments
gap
Immature female
-benzoate
and
Tissue
from PA).
Preparation
hypophysectomized Sprague-Dawley
tained (North
(HX) (CD
strain)
animals
were
used
the possible contributions (Quarmby et al., 1984) to HX was performed at 21
in these of
uterine days
and rats
from the Charles River Breeding Wilmington, MA). HX rather
tomized
were
intact ob-
Laboratories than ovariec-
studies
to reduce
adrenal steroids cellular responses. of age and animals
were kept 3 per cage with HX animals receiving 5% glucose water ad libitum in addition to standard laboratory diet. Intact animals of the same age were used
of
as HX
controls according
for to
verification criteria
of detailed
the
efficacy previously
(Burghardt and Matheson, 1982). Animals were divided into groups consisting of 3 animals per treatment group and each experiment was conducted in duplicate. Exogenous hormone treatments were initiated 60 days subsequent to pituitary ablation unless otherwise noted. Steroids and nonsteroidal estrogen receptorbinding ligands were dissolved in sesame oil with up to 5% absolute ethanol, followed by stirring oil at 50#{176}C
tions mone series, lowing
MATERIALS
estrone, estriol, 1 7f3-diol (Adiol), and
in
animals (Burghardt et al., 1984b), as well as the of actinomycin D and cyclohexamide to the number of estrogen-stimulated myometrial
gap junctions is compatible
ET AL.
for 2 h to drive off the ethanol. Animal injecwere delivered i.p. in 200-pl volumes of horcarrier. Subsequent to each hormone injection rats were killed by cervical dislocation folmild
ether
anesthesia.
Uteri
were
excised
and
immediately immersed in 2% paraformaldehyde, 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3). The middle one-third segment of each uterine horn was dissected out after 5 mm in fixative.
GAP
Following
fixation
for
2
h,
tissues
JUNCTION
were
MODULATION
washed
ment
several times in 0.1 M cacodylate buffer, postfixed for 1 h in 1% 0504 in 0.1 M cacodylate and washed in the
same
times
in
buffer.
distilled
Tissues water
were and
then
stained
washed en
bloc
electron
Organ
Culture
microscope
1%
of to
formation responses
the
effects
estrogen
on
was from
obtained
NJ).
of
HX
prior
by strips
and
and
myometrial
conducted cultured
from
concurrent gap
junction
comparing of uterine
intact,
immature
cellular tissue (30-60
g)
animals. Uteri from each animal were obtained rapidly after death, cut longitudinally, and strips measuring at least 1 cm were pinned onto stainless steel organ culture supports covered with Nucleopore filters
(Pleasanton,
animal
were
described Treated Essential
CA).
Control
processed
above tissues Medium
10-tl
replaced rinsed
for
tissues
electron
from
each
microscopy
as
without incubation in culture. were cultured in 2 ml Minimum (MEM) without serum and main-
aliquots. after in PBS
Media
and
24 h. After and processed
organ for
supplements
were
culture, tissues were microscopic analysis
as above.
mately
2500
smooth
muscle
were
made
bundles. epithelial
Measurements Observations of sections containing
im
Light
uterine cells were the entire uterine
microscopy
was
used
to
made wall in
from cross
evaluate
the
from
number
of gap
junctions
in a given area of myometrial tissue, or direct measurement of junctional and nonjunctional membrane length was obtained using an electronic digitizer (Numonics 1224, Data Aids Associates, Houston, Relative generated
measurements when the
of gap junction numbers objective of the experi-
the
receptor-
formation
of
number of gap junctions in in grid squares (approxi-
area)
uniformly numbers
longitudinal
wide). was by to
covered
with
of measurements
and
circular
muscle
The goniometer stage control used to facilitate identification orienting a plane of section
the
studied herent
for each limitation
membrane
for
The number was counted
verification
of
of gap junctions in with 50 grid squares
cell type and treatment of this procedure
for
group. An inquantifying
gap junctional membrane is that treatments causing increases in cell size would change the fraction of open grid square occupied by cytoplasm. However,
values junctional
cell size and to estrogenicity
obtained response
therefore During junctional
myometrial by exogenous
layers
and axis. of
underestimate potent estrogens
interpretation of data, it was of longitudinal To
were
circular Regardless myometrial evaluated
stimulation the possible the longitudinal with each of the
reoriented
muscle of
the and
of data. noted that the and circular
differential reduce
of only surveying from uteri treated
estrogens
tudinal posite
number of gap junctions of compounds tested, the
the
exhibited estrogen.
sampling problem axis, tissue blocks steroidal
the
would simply for the more
do not alter the collection responses
to
bundles tissue
cut
along orientation,
the
longithe
opthe
gap junction induction and by the procedures outlined
above remained consistent. Direct measurements of junctional and nonjunctional membrane were obtained with an electronic digitizer when the objective of the experiment was to
magnification).
the
estrogen
cells were evaluated if continuous traversed the width of the grid square
morphology. grid squares
quantified
counting
open
junction eligible
evaluate
by
The counted
cells. Equal
Serosal sheets
extent of hypertrophy of uterine luminal epithelium following hormone treatments. In these investigations, either a relative measure of gap junction numbers as a function of exogenous stimulation was
TX). were
stimulate
(at least 50 zm of the microscope of gap junctions
pattern modulation
section.
whether
could
membrane. was
since both are related
tained for 48 h in a CO2 incubator at 37#{176}C.Culture medium was supplemented with 0, 125, or 250 ng/ml estradiol-17(3 dissolved in absolute ethanol and added in
determine
perpendicular
Evaluation exposure
(Mahwah,
to ligands
junctional myometrium
aqueous uranyl acetate, dehydrated in ethanol series and embedded in Embed-Araldite. Light microscopy was conducted using a Zeiss IM-35 microscope (Thornwood, NY). Thin sections were mounted on 300-mesh copper grids and examined with a Philips 400T
was
binding
several with
743
relative
estrogenicity
of
compounds
lating gap micrographs
junction of both
formation. longitudinal
metrial
bundles
were
taken
at
length
of
myometrial
brane from light
cell
The
stimu-
Nonoverlapping and circular myo6000X
(original cell
mem-
and the length of gap junctions were recorded negatives projected on a frosted-glass-surfaced box at a final magnification of 50,000X. At least
5000 jim of myometrial cell membrane was measured from each treatment group, with equal lengths of membrane evaluated from each muscle layer. Analysis
BURGHARDT
744
ET AL.
cells
accompanies
the
uterotrophic
from daily administration of Estrogen-stimulated junctional serosal cells include increases size of macular gap junctions myometrial cells, are present hormone
administration.
effects estrogen responses
resulting
to in
HX rats. uterine
in both the number and that, in contrast to at basal levels prior to Further,
induction
of
annular gap junctions in both cell types appears to be a consequence of the amplification of junctional membrane by estrogen. Annular gap junctions are spherical membrane
cytoplasmic that have
inclusions lost continuity
membrane
during
internalization.
effects of and serosa
estrogen of HX
administration rats is shown
gap junctional in Figure 2. Quantitative
membranes
(initiated
days
pearance serosal the
fact
that
in HX estrogen
gap junctions
in the
than
the
by
to
and
inner
this
therapy
HX)
on
circular
circular
the
treatment
the
ap-
myometrial 3. Noteworthy
rats respond stimulation.
number
uterotrophic
EB-replacement in uterine in Figure
longitudinal
junctional the plasma
on myometrium in Figure 1 and
subsequent
of gap junctions cells are illustrated
myometrium exogenous greater
of
gap with
The
induced
effects
60
of
and is
layers
of
the
differentially The number
is 3 to 5 times
layer
observed
to of
in the
outer
longi-
tudinal layer. These results were obtained regardless of whether muscle layers were sectioned longitudinally or in cross section. Furthermore, at the lowest dosages studied capable of inducing myometrial gap junctions, only cells in the circular layer found to be morphologically coupled. Significant is the fact detected uterus that received 5 daily injections of oil carrier hypophysectomy. Peritoneal mesothelium or serosa (S), myometrium (M), uterine epithelium (E), and uterine glands (G) appear undifferentiated. b) Estradiol benzoate (50 pg/day for 5 days) replacement therapy initiated 60 days after hypophysectomy. FIG.
1. a) Control 60 days after
Uterine epithelium and associated gland exhibit hypertrophic stimulation. Myometrial and serosal cell hypertrophy is accompanied by the induction of gap junctional membranes shown in Figure 2. X 600.
of variance of sample Duncan’s multiple range
means was conducted, test at a significance
using level of
p
