Endocrinology Vol. 138, No. 1 344-350
Copyright © 1997 by The Endocrine Society
‘Distinct Cellular Localization of the Messenger Ribonucleic Acid for Prostaglandin E Receptor Subtypes in the Mouse Uterus during Pseudopregnancy1
Masato Katsuyama2,
Yukihiko Sugimoto,
Kimiko Morimoto,
Ken-yuh Hasumoto,
Manabu Fukumoto,
Manabu Negishi and
Atsushi Ichikawa
Department of Physiological Chemistry, Faculty of Pharmaceutical
Sciences, and Department of Pathology, Faculty of Medicine (M.F.),
Kyoto University, Kyoto, Japan
Address all correspondence and requests for reprints to: Atsushi Ichikawa, Ph.D., Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan.
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Abstract
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As an initial step to clarify the mechanisms of various uterine actions
of PGE2, expression patterns of the messenger RNAs (mRNAs)
for four subtypes of PGE receptors, EP1, EP2,
EP3, and EP4, were investigated in the mouse
uterus during pseudopregnancy. Relative expression levels were
investigated by Northern blot analysis of mRNA levels in uteri obtained
on days 0, 1, 3, 5, 7, and 9 of pseudopregnancy (day 0 = 48 h
after PMSG injection), and cellular localization was determined by
in situ hybridization in uteri obtained on days 0 and 5.
EP2 mRNA was specifically expressed on day 5, and its
expression was confined to the luminal epithelium. On the other hand,
the level of the EP3 mRNA expression progressively
increased until day 5. Cell populations expressing the EP3
mRNA were confined to the longitudinal smooth muscle on day 0, but they
changed to the circular smooth muscle on day 5. The expression level of
EP4 mRNA was low on days 0 and 1, but it became high on
days 3 and 5. On day 0, EP4 mRNA was localized to the
luminal epithelium. On day 5, diffuse, but significant, EP4
expression was observed over the endometrial stroma and epithelium. No
EP1 mRNA signals were observed. Transient expression of
EP2 on day 5 of pseudopregnancy in the luminal epithelium
suggests its involvement in blastocyst implantation signaling.
EP4 in the endometrial stroma is suggested to be involved
in decidual transformation of the stromal cells, whereas
EP3 in the myometrium is believed to be involved in
regulation of myometrial activity.
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Introduction
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PGE2 IS A MAJOR metabolite of
arachidonic acid synthesized by cyclooxygenase pathways in the uterus,
which is demonstrated to be produced in the implantation sites or the
amnion and decidua at the onset of labor (1, 2). PGE2
regulates various uterine functions, such as contraction and relaxation
of the uterine smooth muscles, cervical ripening and labor induction,
elevation of endometrial vascular permeability, and induction of
decidualization (1, 2, 3, 4, 5). These actions of PGE2 are exerted
through its binding to specific receptors on plasma membranes.
PGE2-binding activity has been determined in the myometrium
in various species (5, 6, 7, 8), and changes in the number of binding sites,
depending on the state of pseudopregnancy or the estrous cycle, were
reported in the endometrium (9, 10, 11). The PGE receptor is
pharmacologically divided into four subtypes, EP1,
EP2, EP3, and EP4 (5, 12), but the
contributions of the four receptor subtypes to PGE2-induced
uterine actions have not yet been well established, except for the
contractile action on the myometrium, which is probably mediated by
EP3 (6, 7, 8). We recently isolated complementary DNAs
encoding these four PGE receptor subtypes (13, 14, 15, 16, 17), which enabled us to
investigate their cellular distribution in pseudopregnant mouse uterus
by Northern blot and in situ hybridization analyses. We
report here distinct cellular localization of the messenger RNAs
(mRNAs) encoding mouse PGE receptor subtypes in the uterus during
pseudopregnancy.
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Materials and Methods
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Animals
ddY mice were purchased from Japan SLC (Hamamatsu, Japan).
Pseudopregnancy was achieved in virgin mice as follows. Immature
(21-day-old) female mice were obtained and housed under a 12-h light,
12-h dark photoperiod, with lights on at 0800 h. The mice received
a single ip injection of 5 IU PMSG. After 48 h, mice were injected
ip with 5 IU hCG and paired overnight with a vasectomized male of the
same inbred line. The morning on which a vaginal plug was detected was
taken as day 0.5 of pseudopregnancy. Mice were killed at different
intervals after mating, and the uteri were isolated, frozen immediately
in liquid nitrogen (for RNA isolation) or in 2-methylbutane at -50 C
(for preparation of uterine sections), and stored at -80 C until use.
For RNA isolation, uteri were collected from three mice for each day of
pseudopregnancy studied, and Northern blot experiments were
independently repeated three times. In situ hybridizations
were repeated twice or three times for each day of pseudopregnancy
studied.
Hybridization probes
Mouse complementary DNAs for EP1, EP2,
EP3, and EP4 were subcloned into pBluescript II
(Stratagene, La Jolla, CA) for synthesis of both sense and antisense
complementary RNA (cRNA) probes (13, 14, 15, 17). For Northern
hybridization, antisense 32P-labeled cRNA probes were
generated, whereas for in situ hybridization, sense and
antisense 35S-labeled cRNA probes were generated using the
appropriate polymerases. Cold antisense riboprobes were also
synthesized by the same procedure with unlabeled nucleotides.
Northern blot analysis
Uteri were collected from three mice for each day of
pseudopregnancy studied. Uterine total RNAs were isolated by the acid
guanidinium thiocyanate-phenol-chloroform method (18). Total RNA (15
µg) was separated by electrophoresis on a 1.5% agarose gel and
transferred onto a nylon membrane (Hybond-N, Amersham, Arlington
Heights, IL). Four different blots were prepared and separately
hybridized with 32P-labeled probes for EP1,
EP2, EP3, and EP4. Hybridization
was carried out essentially as described previously (17). After
hybridization, the blots were washed under stringent conditions, and
the hybrids were detected by autoradiography. Then, the blots were
stripped and rehybridized with 32P-labeled DNA probe for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described
previously (14). Autoradiograms were subjected to densitometric
scanning (AE-6900M, ATTO, Tokyo, Japan) for quantitation of PGE
receptor mRNA levels relative to GAPDH mRNA levels. Northern blot
experiments were independently repeated three times, and similar
results were obtained. Representative results are shown in Fig. 1
.
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Figure 1. a, Time-dependent expression of the mRNAs for PGE
receptor subtypes in the mouse uterus during pseudopregnancy. Uteri
were collected from three mice for each day of pseudopregnancy studied,
and total RNAs were isolated. mRNA values were determined by Northern
blot analysis using 32P-labeled antisense riboprobes as
described in Materials and Methods. The same blots were
rehybridized with 32P-labeled DNA probe for GAPDH, and the
representative results are shown. The day of pseudopregnancy is shown
at the top. Day 0 represents 48 h after PMSG
injection. The positions of the major bands are indicated by
arrowheads. b, Quantitation of the signals for the PGE
receptor (EP) mRNAs in a. The autoradiograms were subjected to
densitometric scanning, and EP mRNA levels were normalized to GAPDH
mRNA levels. The increase in EP mRNA levels is expressed as the fold
increase over the level on the control day (days 3, 1, and, 0 for
EP2, EP3, and EP4, respectively).
Northern blot analyses were independently repeated three times, and
similar results were obtained. Representative results are shown.
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Preparation of uterine sections
Uterine sections 10 µm thick were cut on a Jung Frigocut 2800E
cryostat and mounted onto poly-L-lysine-coated glass
slides. The sections were fixed in 4% formalin in PBS for 10 min,
rinsed in PBS, and acetylated with 0.25% acetic anhydride in 0.1
M triethanolamine-0.9% NaCl for 10 min at room
temperature. After dehydration sequentially in 70%, 95%, and 100%
ethanol, the sections were air-dried and stored at -80 C until
use.
In situ hybridization
Riboprobes were prepared essentially as described above by
transcription in the presence of [
-35S]CTP to specific
activities of 1.0 x 109 cpm/µg. After removing
unincorporated nucleotides, riboprobes were degraded to about 150 bases
by alkaline hydrolysis. Hybridization was carried out essentially as
described previously (19). The specificity of the signal with each
antisense probe was verified either by its disappearance with the
addition of an excess of unlabeled probe (see an example in Fig. 4f for
EP3, not shown for others) or by no specific hybridization
with sense probe (data not shown). Hybridized sections were dipped in
nuclear track emulsion (NTB3, Eastman Kodak, Rochester, NY), diluted
1:1 with water. After exposure for about 4 weeks at 4 C, the dipped
slides were developed and fixed, then counterstained with hematoxylin
and eosin. These experiments were repeated twice or three times on each
day of pseudopregnancy studied.
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Figure 4. Regional distribution of the mRNAs for PGE
receptor subtypes in the mouse uterus on day 5 of pseudopregnancy. a,
Brightfield transverse section through the uterus. b–e, Darkfield
photomicrographs of transverse sections hybridized with
35S-labeled antisense riboprobe for the EP1
(b), EP2 (c), EP3 (d), and EP4 mRNA
(e). Hybridization signals for EP3 shown in d were
abolished in the presence of excess unlabeled probe (f). Scale
bar = 300 µm.
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Results
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Changes in the expression levels of the PGE receptor mRNAs in the
uterus during pseudopregnancy
In the uteri from pseudopregnant mice, the mRNAs encoding three
PGE receptor subtypes, EP2, EP3, and
EP4, were detected by Northern blot analyses (Fig. 1a
); the
major transcripts were 2.8, 2.3, and 3.9 kilobases for EP2,
EP3, and EP4, respectively. Minor bands for the
EP2 and EP3 mRNAs were also observed, as
reported previously (13, 17). No expression of the EP1
transcript was detected (data not shown). The level of expression of
the EP2 transcript showed marked variations during the
period examined. The transcript was barely detectable on days 0 and 1
of pseudopregnancy. On day 3, EP2 mRNA was detected at a
very low level, and the increase was evident on day 5: the mRNA level
was 11-fold the level on day 3. The EP2 mRNA thereafter
sharply decreased to that seen on day 3. EP3 mRNA was
hardly detected on day 0. Beginning on day 1, the transcript
progressively increased in abundance to reach the maximum on day 5,
which was about 13-fold the level observed on day 1 (Fig. 1b). On days
7 and 9, expression decreased to lower levels than those on day 3. The
expression of EP4 mRNA, which was low on days 0 and 1,
progressively increased and was maintained at a high level after day 5,
which was approximately 10- to 15-fold the level on day 0 (Fig. 1b).
Cellular localization of PGE receptor mRNAs in the pseudopregnant
uterus
To define the cell populations expressing the PGE receptor subtype
mRNAs, in situ hybridization analysis was performed on uteri
on days 0 and 5 of pseudopregnancy. In situ hybridization
using 35S-labeled riboprobes revealed distinct regional
distributions of EP2, EP3, and EP4
mRNAs in the uterus (
Figs. 2–5). No signals for the
EP1 mRNA were detected in uteri on days 0 and 5 (Fig. 4b
for day 5).
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Figure 2. Regional distribution of the mRNAs for PGE
receptor subtypes in the mouse uterus on day 0 of pseudopregnancy (48 h
after PMSG injection). a, Brightfield transverse section through the
uterus. b–d, Darkfield photomicrographs of transverse sections
hybridized with 35S-labeled antisense riboprobe for the
EP2 (b), EP3 (c), and EP4 mRNA (d).
Scale bar = 300 µm.
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Figure 3. Cellular localization of the mRNAs for PGE
receptor subtypes in the mouse uterus on day 0 of pseudopregnancy. a
and b, Bright- and darkfield photomicrographs of the myometrium,
respectively, showing hybridization signals for the EP3
mRNA. c and d, Bright- and darkfield photomicrographs of the luminal
epithelium, respectively, showing hybridization signals for the
EP4 mRNA. Arrowheads, Cells labeled with
each probe. CM, Circular smooth muscle; LM, longitudinal smooth muscle;
L, uterine lumen; LE, luminal epithelium; S, stroma. Scale
bar = 50 µm.
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Figure 5. Cellular localization of the mRNAs for PGE
receptor subtypes in the mouse uterus on day 5 of pseudopregnancy. a
and b, Bright- and darkfield photomicrographs of the luminal
epithelium, respectively, showing hybridization signals for the
EP2 mRNA. c and d, Bright- and darkfield photomicrographs
of the myometrium, respectively, showing hybridization signals for the
EP3 mRNA. e and f, Bright- and darkfield photomicrographs
of the endometrium, respectively, showing hybridization signals for the
EP4 mRNA. Arrowheads, Cells labeled with
each probe. L, uterine lumen; LE, luminal epithelium; S, stroma; G,
glandular epithelium; CM, circular smooth muscle; LM, longitudinal
smooth muscle. Scale bar = 50 µm.
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On day 0 of pseudopregnancy, EP3 and EP4 mRNAs
were detected (Fig. 2, c and d), but no significant signals for
EP2 were seen (Fig. 2b). Hybridization signals for the
EP3 mRNA were found in the myometrium (Fig. 2c).
Microscopic examination of the myometrium exhibited weak, but
significant, labeling of the longitudinal smooth muscle cells, but the
circular smooth muscle cells were hardly labeled with the
EP3 probe (Fig. 3, a and b). As shown in
Fig. 3, c and d, signals for the EP4 mRNA were localized in
the luminal epithelium.
On day 5 of pseudopregnancy, strong hybridization signals for
EP2 mRNA were observed in the luminal epithelial cells
(Figs. 4c and 5, a and b). As shown in
Fig. 4d, strong hybridization signals for EP3 mRNA were
seen in the myometrium. Microscopic examination of the myometrium
exhibited intense labeling of the circular smooth muscle cells, but the
longitudinal smooth muscle cells were not labeled (Fig. 5, c and d).
Diffuse, but significant, EP4 expression was observed in
the endometrium (Fig. 4e). Microscopic examination of the endometrium
revealed significant labeling of the luminal epithelial cells like that
on day 0 of pseudopregnancy. In addition, endometrial stromal cells and
glandular epithelium were labeled (Fig. 5, e and f).
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Discussion
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In the present study, we demonstrated time-dependent changes in
expression level and distinct cellular localization of the mRNAs
encoding three subtypes of PGE receptors, EP2,
EP3, and EP4, in the mouse uterus during
pseudopregnancy. Such time-dependent fluctuation of PGE receptor mRNAs
has not been identified in the other tissues or systems, except the
uterus. In this study, mice were administered PMSG; on day 0, defined
as 48 h after injection, mice were regarded as being in proestrous
by analysis of vaginal smears or by confirmation of ovulation on the
following morning. On the other hand, day 5 was chosen as a time of
maximal uterine sensitivity to the presence of blastocysts (20, 21). We
used superovulation of immature mice and induction of pseudopregnancy
as a model system to examine the changes in PGE receptor expression.
Although this system is known to include nonphysiological hormone
stimuli, we preliminary performed in situ hybridization on
pseudopregnant day 5 mice that were generated from mature and cycling
females, and consequently obtained almost the same results as those
achieved using superovulated day 5 mice (data not shown).
The mRNA encoding EP2, which is coupled to the stimulation
of adenylate cyclase, was expressed almost exclusively on day 5 of
pseudopregnancy, and its expression was confined to the luminal
epithelium. This observation is inconsistent with the previous report
that no [3H]PGE2-binding sites were detected
in the luminal epithelium (22). In this previous study, however,
binding experiments were performed using the uterine epithelium of
progesterone-treated ovariectomized rats, not that of rats on day 5 of
pseudopregnancy. The high level selective expression of EP2
on day 5 suggests the involvement of PGE2 in
blastocyst implantation that occurs during days 4 and 5 of pregnancy in
the mouse (21, 23, 24). It was noted that cyclooxygenase-2 (COX-2) is
transiently expressed on day 4.5 of pregnancy in the luminal epithelium
(25). Considering the similarity in fluctuation between EP2
and COX-2 expression, PGE2 synthesized in the luminal
epithelium may exert its effect through EP2 in an autocrine
manner. EP2 might mediate signals for morphological changes
in the luminal epithelium essential for blastocyst implantation (1). In
fact, treatment with indomethacin, a COX inhibitor, has been shown to
elicit ultrastructural changes in the luminal epithelium and to inhibit
blastocyst implantation (26). Thus, EP2 expressed in the
luminal epithelium might be involved in transmission of the signal from
the blastocyst to the underlying stromal cells.
The expression of EP4, which is also positively coupled to
adenylate cyclase, sharply increased on day 3 of pseudopregnancy. This
increase may be reflecting the change in stromal cells; EP4
mRNA was expressed in the stroma on day 5, but not on day 0. As the
serum progesterone level was reported to rise and reach its maximum on
day 5 of pseudopregnancy in the rat (27), it is possible that the
EP4 expression in stromal cells is induced by progesterone,
as reported previously (22). PGE2 was reported to cause an
increase in alkaline phosphatase activity in the stromal cells during
decidualization via elevation of the cAMP level (28). This effect may
be mediated by EP4. Thus, EP4 may mediate
signals for decidualization of the stromal cells.
The mRNA encoding EP3, which is coupled to the inhibition
of adenylate cyclase, was specifically localized to the myometrium.
Previous pharmacological studies demonstrated that PGE2
causes uterine contraction through elevation of the intracellular
Ca2+ concentration (8). Mouse EP3 expressed in
Chinese hamster ovary cells is coupled not only to the inhibition of
adenylate cyclase, but also to Ca2+ mobilization via
Gi (29). Therefore, PGE2-induced
uterine contraction may be mediated by an EP3-mediated
Ca2+ signal in the myometrium.
As in the cases of EP2 and EP4, the level of
EP3 expression also changed during pseudopregnancy;
EP3 expression was low on day 0, but increased to reach a
maximal level on day 5. The temporal changes in EP3
expression coincided well with the change in myometrial activity in
pregnant and pseudopregnant rats; the myometrial activity was reported
to increase after mating and reached a maximal level on day 5 (30).
Therefore, the level of EP3 expression might affect
myometrial activity. The cell populations expressing EP3
also changed in a temporally specific manner; EP3
transcript was localized to the longitudinal smooth muscle on day 0,
but expression was confined to the circular smooth muscle on day 5. To
date, there have been no reports concerning changes in
PGE2-induced myometrial contractility in these two distinct
types of smooth muscle during pregnancy or pseudopregnancy. However,
EP3 expressed in the longitudinal smooth muscle on day 0
might be involved in longitudinal contraction observed during the
periovulatory period (31). EP3-mediated longitudinal
contraction might contribute to sperm transport (32). On the other
hand, EP3 highly expressed in the circular smooth muscle on
day 5 might be involved in circular contraction necessary for retention
and spacing of embryos (30). At present, there is no information
regarding the mechanism underlying such time-dependent changes in the
expression of EP3 mRNA in two distinct types of smooth
muscle cells. More detailed analyses are, therefore, required.
In summary, the results presented in this paper indicated distinct
cellular localizations of PGE receptor subtypes in the mouse uterus
during pseudopregnancy. Furthermore, each of the receptors showed
time-dependent changes in the level of expression in individual cells,
suggestive of hormonal regulation. Thus, it is suggested that
PGE2 modulates uterine functions through at least three
receptors; EP2 is involved in blastocyst implantation
signaling, EP4 is involved in decidual transformation of
the stromal cells, and EP3 is involved in regulation of
myometrial contractility.
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Acknowledgments
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We thank Dr. K. Takabatake of the Department of Gynecology and
Obstetrics, Kyoto University Faculty of Medicine, for helpful
discussion. We also thank Mr. K. Tsuboi and Mr. E. Funakoshi of the
Department of Physiological Chemistry, Kyoto University Faculty of
Pharmaceutical Sciences, for their technical assistance.
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Footnotes
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1 This work was supported in part by Grants-in-Aid for Scientific
Research 07672353, 07278220, 07772167, and 07557156 from the Ministry
of Education, Science, and Culture of Japan and by grants from the
Sankyoh Life Science Research Foundation and the Katoh Memorial
Foundation. 
2 Recipient of research fellowships from the Japan Society for the
Promotion of Science for Young Scientists.
Received June 11, 1996.
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Top
Abstract
Introduction
