Potential Regulation of Membrane Trafficking by Estrogen Receptor
via Induction of Rab11 in Uterine Glands during Implantation
Dahu Chen,
Padmaja Ganapathy,
Li-Ji Zhu,
Xueping Xu,
Quanxi Li,
Indrani C. Bagchi and
Milan K. Bagchi
Population Council and The Rockefeller University New York, New
York 10021
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ABSTRACT
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The steroid hormone estrogen profoundly influences
the early events in the uterus leading to embryo implantation. It is
thought that estrogen triggers the expression of a unique set of genes
in the preimplantation endometrium that in turn control implantation.
To identify these estrogen-induced genes, we used a delayed
implantation model system in which embryo attachment to endometrium is
dependent on estrogen administration. Using a mRNA differential display
(DD) method, we isolated a number of cDNAs representing mRNAs whose
expression is either turned on or turned off in response to an
implantation-inducing dose of estrogen. We identified one of these
cDNAs as that encoding rab11, a p21ras-like GTP-binding protein (G
protein), which functions in the targeting of transport vesicles to the
plasma membrane. In normal pregnant rats, rab11 mRNA was expressed at
low levels on days 12 of pregnancy, but its expression was markedly
enhanced (
6- to 8-fold) between days 35 immediately before
implantation. In situ hybridization and immunocytochemistry
revealed that rab11 expression in the uterus was predominantly in the
glandular epithelium. In ovariectomized rats, the expression of rab11
mRNA was induced in the endometrium in response to estrogen. To
determine whether this effect of estrogen was mediated through its
nuclear receptors, we examined rab11 expression in a transformed
endometrial cell line, Ishikawa. In transient transfection experiments,
we observed that overexpression of estrogen receptor (ER)
or ß
induced endogenous rab11 mRNA in a hormone-dependent manner. ER bound
to an antagonist, ICI 182,780, failed to activate this gene expression.
These findings, together with the observation that ER
but not ERß
is detected in the glands of the preimplantation uterus, indicate that
rab11 is one of the proteins that are specifically induced by
estrogen-complexed ER
in rat endometrium at the onset of
implantation. Our results imply that estrogen, which induces the
synthesis of many growth factors and their receptors and other
secretory proteins that are thought to be critical for implantation,
may also facilitate their transport to the membrane and/or secretion by
stimulating the expression of rab11, a component of the
membrane-trafficking pathway. This study therefore provides novel
insights into the diverse cellular mechanisms by which estrogen, acting
via its nuclear receptors, may influence blastocyst implantation.
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INTRODUCTION
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Initiation of implantation leading to the establishment of
pregnancy results from the culmination of a series of complex
interactions between the developing embryo and the uterus (1, 2, 3, 4).
Although the details of implantation vary in different species, the
basic features of the blastocyst attachment and penetration of the
uterine surface epithelium are common to many mammals. In humans and
rodents, implantation occurs 46 days after fertilization when the
blastocyst reaches the uterus. It is generally believed that the
endometrium and the fertilized ovum must undergo synchronized
developmental changes for implantation to succeed. Studies by Psychoyos
(1) demonstrated that rat uterus can accept the blastocyst to implant
only for a brief period of time known as the receptive phase. The
specific modifications leading to acquisition of the receptive state of
the uterus are regulated by a timely interplay of the maternal steroid
hormones, estrogen and progesterone (5, 6).
Estrogen profoundly influences uterine functions at various phases of
the menstrual cycle and pregnancy. In the rat, the preovulatory ovarian
estrogen is important for uterine cellular proliferation and
differentiation during early stages (days 1 and 2 postfertilization) of
pregnancy (7, 8). This transformation of the uterus in response to
estrogen is important for subsequent uterine preparation for embryonic
implantation and successful establishment of pregnancy (7, 8). After
fertilization the level of estrogen declines and remains low throughout
gestation, except a transitory rise in estrogen level that occurs on
day 4 of pregnancy (9). Although estrogen is essential to create the
receptive state of the uterus that allows implantation on day 5, the
molecular basis of this hormonal effect remains unclear.
The cellular actions of estrogen are mediated through estrogen
receptors, ER
and ERß, which function as ligand-inducible
transcription factors (10, 11, 12, 13). The uterotropic hormonal
responses to estrogen are believed to be mediated through the
expression of specific estrogen-regulated genes in the uterus (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24).
Studies in rodents employing immature and ovariectomized model systems
have demonstrated that in the uterus estrogen modulates the expression
of several genes that are likely to be involved in the regulation of
cell growth and division. These include the genes encoding
protooncogenes, such as c-fos and c-myc, growth
factors, such as epidermal growth factor (EGF) and insulin-like growth
factor-I, and their receptors (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). However, the
relationship of these gene activation events to the complex
estrogen-regulated physiological processes, such as uterine
receptivity, secretory protein production, and embryonic implantation,
have not been clearly established.
To investigate the molecular basis of the estrogen regulation of
implantation, we sought to identify the genes whose expression in the
preimplantation endometrium is induced by estrogen. For this purpose,
we employed a delayed implantation rat model that allows one to
determine the independent functional roles of estrogen and progesterone
on implantation (1, 2). In these rats, which have undergone ovariectomy
on day 4 of gestation, implantation is blocked in the absence of
ovarian estrogen. Continued administration of progesterone allows the
blastocysts to remain viable, but the attachment of the embryo to the
uterine epithelium will not occur in the absence of estrogen. Such a
delayed state can be maintained for up to 710 days after
fertilization. Administration of estrogen to the ovariectomized
pregnant rats triggers implantation within 1224 h, emphasizing the
critical role of estrogen in this process (25, 26).
We used the mRNA differential display (DD) technique to isolate a
number of cDNAs representing mRNAs whose expression are either
turned on or turned off in response to an implantation-inducing dose of
estrogen in rats undergoing delayed implantation. Our studies revealed
that rab11, which is present at a low level in the progesterone-treated
delayed rats, is markedly induced when estrogen is administered to
these rats to overcome the delay. rab11 belongs to the rab family of
low-molecular mass G proteins that share significant homologies with
p21 ras (27, 28, 29). The members of the rab family are thought to be
required at distinct steps in transport along the secretory pathway,
especially for the targeting and fusion of transport vesicles with the
plasma membrane. The timing of its enhanced expression in the
endometrial glands prompts us to propose that rab11 may function by
regulating secretory activities that are critical for blastocyst
implantation.
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RESULTS
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Isolation of rab11 cDNA by mRNA DD Analysis
To isolate the uterine genes that are regulated by estrogen during
delayed implantation, we employed the mRNA DD method (30, 31, 32). We
compared RNA samples prepared from uteri of delayed rats treated with
either progesterone alone or progesterone plus an
implantation-initiating dose of estrogen. Our studies revealed several
cDNA clones representing genes whose expression in the uteri of rats
undergoing delayed implantation is up- or down-regulated within 24
h of estrogen treatment (D. Chen, P. Ganapathy, and I. Bagchi,
unpublished observation). One of the differentially displayed bands,
which was absent in the uteri of delayed rats but appeared after
estrogen treatment (marked by * in Fig. 1
, panel A, right lane), was
selected for further characterization. The band of interest was
recovered from the gel and amplified by PCR (40 cycles). Using this
cDNA fragment as a probe, we isolated a full-length cDNA from the rat
uterus cDNA library. Nucleotide sequence analysis of the isolated cDNA
and comparison with the Genbank data base revealed its identity as
rab11, a member of the rab family of GTPases (33).

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Figure 1. Profiles of Differentially Expressed mRNAs in
Uterine Tissues of Delayed Implanting Rats
A, Delayed implantation was performed according to the protocol
described in Materials and Methods. Total RNA (2 µg)
isolated from uteri of ovariectomized pregnant rats treated with either
progesterone or progesterone and estrogen was subjected to DD. A band
representing mRNA that is up-regulated with estrogen treatment is
marked by * in the right lane. B, RNA was analyzed by
Northern blotting followed by hybridization with a
32P-labeled rab11 or GAPDH cDNA probe. Lane 1: RNA from
delayed rats treated with progesterone alone; lane 2: RNA from delayed
rat after progesterone and estrogen treatment. The experiment was
repeated twice for reproducibility. The results of a representative
experiment are shown.
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The differential expression of rab11 mRNA during estrogen-induced
delayed implantation was confirmed by Northern blotting. A rab11 cDNA
fragment was labeled with 32P and used to probe blots of
mRNAs obtained from uteri of delayed rats on day 8 of pregnancy
before estrogen treatment (Fig. 1
, panel B, lane 1) and after
administration of estrogen (lane 2). Two weak signals corresponding to
2.3-kb and 1-kb transcripts emerged in lane 1, while signals of
markedly enhanced intensity were observed in lane 2. Hybridization of
the blot with a control probe [glyceraldehyde 3-phosphate
dehydrogenase (GAPDH)] indicated that the difference in intensities of
the signals in lanes 1 and 2 did not arise due to unequal loading of
mRNAs in these lanes. These results indicated that the rab11 mRNA is
indeed up-regulated in delayed rats upon administration of an
implantation-initiating dose of estrogen. It has been reported
previously that rab11 is encoded by a single gene from which two
transcripts, 2.3 kb and 1 kb, are generated by alternative use of two
polyadenylation sites (34). Both of these transcripts give rise to the
same protein. The sizes of the induced mRNAs in our Northern blot in
Fig. 1B
are in excellent agreement with those reported previously for
the rab11 transcripts (34).
Rab11 Transcripts Are Induced in Pregnant Rat Uterus within the
Window of Implantation
We next examined the profiles of expression of the 2.3-kb
and 1-kb rab11 transcripts in normal pregnant rat uterus during days
16 of gestation by employing Northern blot analysis. As shown in Fig. 2
, the overall profiles of expression of
the two transcripts were similar. Both transcripts were detected on
days 12 of pregnancy. However, the shorter transcript was
consistently expressed at a higher level than the longer one. The
levels of both transcripts increased significantly on day 3, attained a
maximum on days 45, and then declined sharply by day 6 of pregnancy.
These results indicated that a transient surge of rab11 mRNA expression
occurs in the uterus in a highly stage-specific manner, that is between
days 35 of gestation, and this temporally coincides with the
preparatory events leading to implantation. The relative levels of
expression of the 2.3-kb and 1-kb transcripts were estimated by
densitometric scanning, followed by normalization with respect to the
control GAPDH mRNA signal. By our estimate, the level of either
transcript on day 45 was about 6- to 8-fold higher than that on day 1
of gestation (Fig. 2B
).

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Figure 2. Profile of Expression of rab11 mRNA in Rat Uterus
at Different Stages of Early Pregnancy
A, Total RNA (20 µg/lane) from uteri of animals at days 16 of
pregnancy was analyzed by Northern blotting. Hybridization was
performed with a 32P-labeled rab11 cDNA probe. The
positions of the 1-kb and 2.3-kb rab11 transcripts are indicated. The
lower panel shows the same blot after hybridization with
a control 32P-labeled GAPDH probe. B, The intensities of
the signals corresponding to the 2.3-kb (upper panel) and 1-kb (lower panel)
rab11 transcripts were quantitated by densitometric scanning of the
autoradiogram and normalized with respect to the GAPDH signals of the
Northern blot shown in panel A. The results are representative of two
independent experiments.
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rab11 mRNA and Protein Are Localized in the Endometrial Glands
The site of expression of rab11 mRNA in the pregnant rat uterus
was investigated by employing in situ hybridization (Fig. 3A
). Uterine sections from day 4 pregnant
animals were hybridized with a 400-bp digoxigenin-labeled (DIG)
antisense or sense RNA probe containing sequences from rab11 cDNA.
While control uterine sections (pregnant, day 4) hybridized with the
sense RNA probe did not exhibit any significant signal (panel B), we
observed a strong hybridization signal in the glandular epithelial
cells of these uterine sections upon hybridization with the antisense
probe (panel A). A weaker but specific hybridization signal was also
found in the luminal epithelium. These results demonstrated that the
glandular epithelium is the primary site of synthesis of rab11 mRNA in
the uteri of pregnant rats.

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Figure 3. Localization of rab11 mRNA in the Rat Uteri by
in Situ Hybridization
A, Uterine sections from pregnant (day 4) rats were subjected to
in situ hybridization. The hybridization was performed
employing a 400-bp long DIG-labeled antisense (panel A) or sense (panel
B) cRNA probe specific for rab11 gene as described in Materials
and Methods. g and l, Glandular and luminal epithelium,
respectively. Magnification, 43x. B, Uterine sections from pregnant
rats at days 16 of gestation (panels D1D6) were subjected to
in situ hybridization as described in panel A.
Magnification, 215x.
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We performed in situ hybridization of uterine sections at
different days of gestation to further confirm the stage-specific
expression of rab11. The level of rab11 mRNA in glandular epithelial
cells markedly increased on day 3, reached a peak on day 4, and then
diminished on day 6 of pregnancy (Fig. 3B
, panels D1D6). These
results confirmed our Northern blot results (Fig. 2
) that rab11
expression is induced in the endometrial glands in an implantation
stage-specific manner.
We also investigated the site of accumulation of rab11 protein by
immunocytochemical staining of sections of uteri isolated from day 4
pregnant animals using a polyclonal antibody specific for this protein.
As shown in Fig. 4
(left
panel), rab11-specific immunostaining was present predominantly in
the glandular epithelial cells. Weaker but specific immunostaining was
observed in the luminal epithelium. No significant staining was
observed in the stroma or myometrium. Control sections of the same
uterine tissue, when incubated with preimmune serum, showed no
immunoreactivity, indicating the specificity of the immunostaining
(Fig. 4
, right panel). These results, consistent with the
in situ hybridization analysis, showed that rab11 protein is
expressed in the glandular epithelial cells around the time of
implantation.

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Figure 4. Localization of rab11 in Rat Uterus by
Immunocytochemistry
Immunocytochemistry was performed employing polyclonal rabbit
anti-rab11 using sections from day 4 pregnant (panel A) rat uterus as
described in Materials and Methods. Left
panel, Red deposits indicate site of specific
immunoreactivity. Right panel, Control sections of day 4
pregnant uterus incubated with preimmune serum. G and L, Glandular
epithelium and luminal epithelium, respectively. Magnification, A and
B, 150x.
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Estrogen Induces rab11 mRNA Expression in the Uterus
Since expression of rab11 is stimulated during estrogen-induced
delayed implantation, we considered the possibility that rab11 is under
estrogenic regulation. To determine whether rab11 expression in the
uterus is indeed regulated by estrogen, we administered steroid
hormones to ovariectomized rats. A week after ovariectomy, the animals
were treated with either estrogen or progesterone or a combination of
both. Uteri were collected from animals 16 h after the last
injection, and mRNAs were isolated from these tissues for Northern blot
analysis. As shown in Fig. 5A
, rab11 mRNA
was barely detectable in uteri of ovariectomized rats (lane ovex).
Progesterone treatment for 4 days (lane P4) did not significantly
increase rab11 mRNA. In contrast, both rab11 transcripts were markedly
induced after treatment with estrogen for 4 consecutive days (lane E4).
The net estrogen-dependent stimulation in rab11 mRNA after 4 days of
treatment was quantitated to be about 20-fold relative to the level in
the ovariectomized animals (Fig. 5B
). The slight increase in lane E+3P
is most likely due to the brief estrogen treatment before progesterone
administration. Simultaneous administration of estrogen and
progesterone [lane E+3(P+E)] elicited the same response as estrogen
alone, indicating that progesterone does not significantly affect rab11
expression. In a time-course experiment, we observed that the levels of
both rab11 transcripts in ovariectomized rat uterus rose significantly
(
5-fold) within 24 h of injection with a single dose of
estrogen (data not shown). Collectively, these results established that
rab11 expression in the uterus is under estrogenic regulation.

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Figure 5. Estrogen Regulation of rab11 Gene Expression
A, Total RNA (20 µg per lane) was subjected to Northern blot analysis
and hybridized with 32P-labeled rab11 or GAPDH (not shown)
probe. Lane "ovex" represents RNA from uteri of animals injected
with vehicle only after ovariectomy; lane 4P, RNA from uteri of animals
injected with progesterone for 4 consecutive days; lane 4E, RNA from
uteri of animals injected with estrogen for 4 consecutive days; lanes E+3P
and E+3(P+E), RNA from uteri of animals injected with estradiol on day
1 followed by progesterone, and estrogen plus progesterone,
respectively, for 3 consecutive days. B, The bars represent the levels
of rab11 mRNAs quantitated by densitometric scanning of the signals in
the autoradiogram of the Northern blot (shown in panel A). The rab11
signals were normalized with respect to GAPDH mRNA signals in the same
blot. The normalized value of 4E sample was set to 100%. The results
are representative of two independent experiments. Panel C, lane 1, RNA
from the uteri of animals injected with vehicle on days 13 of
pregnancy and killed on day 4; lane 2, RNA from the uteri of animals
given ICI 182,780 during days 13 and killed on day 4 of pregnancy.
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An Antagonist of ER Blocks rab11 mRNA Expression during
Implantation
We further investigated the estrogenic
regulation of rab11 mRNA expression by employing an antiestrogen, ICI
182,780, which is known to severely impair the transcriptional activity
of ER. Animals were injected in the preimplantation phase of pregnancy
either with a vehicle or ICI 182,780, which blocks implantation in rats
(35). As depicted in Fig. 5C
, treatment with this antiestrogen drug
caused a drastic (
85%) decline in the level of rab11 mRNA but did
not affect the expression of GAPDH mRNA. These results strongly
suggested that ICI 182,780 switched off the rab11 gene expression
during implantation by inhibiting the ERs.
ER
Level Increases in the Glandular Epithelium during rab11
Expression
If rab11 mRNA synthesis during implantation is mediated by ERs,
one would expect to find these receptor proteins in the glandular cells
at the time of this gene induction. To monitor the expression of ERs in
glandular epithelium during early pregnancy, we performed
immunohistochemical staining of sections of uteri isolated from
pregnant animals (days 15) using polyclonal antibodies specific for
each ER isoform. When we used an antibody against ER
, the uterine
sections of animals on day 1 of pregnancy exhibited very little
immunostaining in the glandular epithelium, while those from day 4 or
day 5 pregnant animals showed significant ER
-specific staining (Fig. 6
, panels D1, D4, and D5, respectively).
The ER
immunostaining declined to undetectable levels on day 6 of
gestation (panel D6). We noted considerable ER
stromal
immunostaining on days 16 (panels D1D6). Sections of uteri (days
16) incubated with control serum did not exhibit any specific
immunostaining (data not shown). We failed to detect any expression of
ERß in the endometrial sections using an ERß-specific antibody,
although this antibody efficiently immunoreacted with ERß present in
other tissues such as prostate (data not shown). These results are
consistent with recent reports indicating that ER
is the predominant
isoform of ER in the endometrium (36). Sufficient quantities of ER
are therefore present in the glandular epithelial cells while rab11
mRNA is induced between days 35 of gestation. This observation is
consistent with a direct regulatory role for ER
in rab11 gene
expression during implantation.

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Figure 6. Immunocytochemical Localization of ER in Rat Uteri
at Different Days of Pregnancy
Immunocytochemistry of uterine sections from day 1 (D1), day 4 (D4),
day 5 (D5), and day 6 (D6) pregnant rats was performed employing an
ER antibody. g, Glands. Magnification, 100x.
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Overexpression of ER
or ERß in Ishikawa Cells Induces rab11
mRNA
To analyze the role of ER in rab11 mRNA induction, we examined the
regulation of the rab11 gene by each ER isoform in human endometrial
Ishikawa cells using transient transfection. The rab11 expression was
monitored by RT-PCR using total RNA isolated from these cells. Ishikawa
cells contain only low levels of endogenous ER and displayed a low,
basal level of rab11 expression irrespective of hormone addition (Fig. 7
, lanes 1 and 2). This basal promoter
activity did not change significantly when expression vectors harboring
either ER
or ERß was transfected into these cells in the absence
of estrogen (lanes 3 and 7). However, when either ER expression vector
was transfected in the presence of 100 nM estrogen, a 6- to
8-fold induction in rab11 mRNA was observed (lanes 4 and 8). This
ER-mediated induction of rab11 transcripts was completely dependent on
estrogen, since ER
complexed with ICI 182,780 failed to promote
rab11 expression (lane 5 and 6). These results clearly indicated that
the rab11 gene induction by estrogen is mediated by the ER, and both
receptor isoforms have the ability to perform this regulatory
function.

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Figure 7. Induction of rab11 mRNA in Response to Estrogen in
Ishikawa Cells
Ishikawa cells were cultured and transiently transfected using
the lipofectamine procedure as described in Materials and
Methods. Experiments were performed with medium containing
charcoal-stripped serum. Semiconfluent cells received either empty
vector (lanes 1 and 2) or expression vectors containing ER (lanes
36) or ERß (lanes 7 and 8). After 24 h, the cells were
incubated in fresh medium with ligands (10-7 M
estrogen or estrogen along with 10-5 M ICI
182,780) or solvent. Twenty four hours after treatment with ligands,
cells were harvested and mRNA was isolated and subjected to RT-PCR
analysis employing rab11 or GAPDH-specific primers. PCR reactions using
both rab11 and GAPDH primers were performed through multiple rounds
ranging from 1540 cycles to ensure that the amplifications were in
the linear range. The data shown above represent 25 cycles of PCR
amplification. The authenticity of the PCR products was confirmed
by Southern blotting employing 32P-labeled rab11 and
GAPDH cDNA probes. Lanes 1, 3, and 7 represent cells treated with
vehicle; lanes 2, 4, and 8 represent cells treated with estrogen;
lane 6 represents cells treated with ICI 182,780; and lane 5 represents
cells treated with estrogen in combination with ICI 182,780. The
relative levels of rab11 mRNA were as follows: lane 1 (13 ±
1.75), lane 2 (12 ± 2), lane 3 (13 ± 1.25), lane 4
(100 ± 8), lane 5 (22 ± 2.5), lane 6 (11 ± 2), lane 7
(15 ± 1.5), and lane 8 (90 ± 5).
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DISCUSSION
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Blastocyst implantation in rodents is controlled by the
synergistic action of both ovarian steroids, estrogen and progesterone.
In these species, implantation fails to occur unless estrogen becomes
available. An increase in the estrogen level immediately before
implantation appears to be essential for blastocyst attachment (1, 2).
The precise mechanisms by which estrogen acts within the uterus to
influence implantation remain unclear. It is believed that one of the
major actions of estrogen in inducing implantation is related to its
effect in promoting the synthesis of new proteins that are secreted
into the uterine lumen at the time of implantation to critically
regulate this process. Indeed, it has been known for a long time that
the uterine epithelium secretes a variety of proteins in response to
estrogen, and some of these might regulate implantation. The
estrogen-regulated secretory proteins include growth factors such as
EGF, insulin-like growth factor-I, leukemia inhibitory factor (LIF),
and heparin-binding EGF-like growth factor (37, 38, 39, 40), and other proteins
such as lactoferrin, keratan sulfate proteoglycan, and complement C3
(41, 42, 43, 44).
The control of estrogen-dependent secretions of important regulatory
factors during implantation may occur at multiple levels. At one level,
estrogen may induce the synthesis of a number of growth factors and
other proteins in the glands that are positive modulators of the
implantation process. At another level, estrogen may facilitate
secretion of these induced factors by regulating critical components of
the secretory pathway. It has been reported that in mice undergoing
delayed implantation, no LIF is synthesized in the uteri containing
viable but unattached blastocysts. A single injection of estrogen,
which interrupts the delay, also induces secretion of LIF by the
endometrial glands within 1824 h (39, 45). It is conceivable that
estrogen plays a dual regulatory role by controlling both synthesis and
secretion of LIF during implantation. Our finding in this paper
supports such a scenario, as estrogen significantly enhances the
expression of rab11, which is known to function in membrane trafficking
and cellular secretory pathway.
Rab11 is a recently discovered member of a large superfamily of small
ras-related GTP-binding proteins that has been grouped into several
main branches: ras, rho, and rab, according to their sequence
homologies and functional features (27, 28, 29). The members of the rab
subgroup function as key signal components that regulate many aspects
of vesicular transport along both the endocytotic and exocytotic
pathways (27, 28, 29). Different rab proteins exhibit cell type-specific
variations in expression, as well as differential localization to
distinct vesicular compartments and organelles that perform multiple
sorting functions. The cDNA encoding rab11 has been isolated and is
found to be highly conserved among different species (27).
Transcription of the mammalian rab11 gene yields two mRNAs, 1 and 2.3
kb, which share identical coding sequences but differ in the length and
sequence of their 3'-untranslated regions (34). Sequence analysis of
the cDNAs identified two different poly A signals at positions 927 and
2302 of the larger transcript (34). It has been suggested that the two
mRNA species originate from a single gene by the use of different
polyadenylation sites (34). Both rab11 transcripts yield the same
protein product.
Although ubiquitously expressed, rab11 is more abundant in tissues with
a high level of secretion (33). This observation led to the suggestion
that rab11 might function in the exocytic pathway. All cells secrete
some proteins in a constitutive fashion. Urbe et al. (49)
found rab11 associated with the trans-Golgi network-derived
vesicles involved in both the constitutive and the regulated secretory
pathway in the neuroendocrine cell line PC12. Goldenring et
al. (50) localized rab11 in the apical tubulovesicles of the
acid-secreting parietal cells in the gastrointestinal tract. In
addition, rab11 was found associated with the apical vesicular
populations in a variety of polarized epithelial cells (51). Rab11
interconverts between GDP and GTP-bound conformations, as part of a
catalytic cycle of vesicle delivery and rab protein recycling.
Rab11 expression, however, is not restricted to the secretory cells.
This protein has been localized in the pericentriolar recycling
endosomes of certain nonpolarized cells in which it is thought to
function in the recycling pathway of plasma membrane receptors (52).
Studies using rab11 mutants that are preferentially in the GTP- or
GDP-bound state showed that GTP activation is necessary for rab11 to
function in protein trafficking through the sorting
endosomes to either the recycling compartment or the plasma membrane
(52, 53). The precise role of rab11 in the membrane trafficking in
endometrial glands is unknown. However, it is reasonable to assume that
rab11 is in some way involved in targeting the transport vesicles
destined to fuse with the plasma membrane. In this way, it may
facilitate secretion of estrogen-induced secretory proteins.
Additionally, rab11 may also be involved in recycling of
estrogen-induced growth factor receptors.
How does estrogen modulate rab11 expression? Estrogen mediates its
gene-regulatory activity through intracellular ER isoforms, ER
and
ERß, which regulate transcription of target cellular genes (13). Our
studies show clearly that both isoforms of ER are able to induce rab11
expression in Ishikawa endometrial cells. In the uterine glands,
however, the expression of ER
is far greater than that of ERß,
which is virtually undetectable. It is therefore reasonable to infer
that ER
, rather than ERß, mediates estrogen regulation of rab11
expression in the glandular epithelium. Consistent with this scenario,
the profile of rab11 expression in the uterine glands overlaps closely
that of ER
(Fig. 6
).
Previous studies indicated that a transient surge of estrogen on day 4
of gestation in the rat is essential for implantation on the following
day (1, 2). The relationship between this preimplantation estrogen
surge and rab11 expression remains unclear. Our studies indicate that
rab11 expression, which starts to rise on day 3 and attains a peak on
day 45, slightly precedes the preimplantation estrogen surge.
Nevertheless, it is clear from our antiestrogen studies (Fig. 5C
) that
the expression of rab11 between days 35 of gestation is primarily
under estrogenic regulation. Furthermore, there is an excellent
correlation between the expression profiles of rab11 and ER
in the
glands (Figs. 3B
and 6
). However, we cannot rule out the possibility
that other factor(s) in addition to ER
modulates rab11 expression in
the preimplantation uterus. This view is supported by a low but
detectable level of uterine rab11 expression in ICI 182,780-treated or
delayed implanting rats (see Figs. 1B
and 5C
).
The possibility that ER directly regulates rab11 gene expression in
glandular epithelium has interesting implications for cell
type-specific actions of ER within the endometrium. It was previously
thought that estrogen acting through ER stimulates uterine epithelial
cell growth, proliferation, and differentiation. Recent studies by
Cooke et al. (54) however, question the role of epithelial
ER
in the normal uterine response to estrogen. Using tissue
recombinants prepared with uterine tissue from adult ER
knockout
(ERKO) and normal neonatal mice, these workers observed that epithelial
ER
is neither necessary nor sufficient to mediate the mitogenic
response to estrogen in the epithelium. Interestingly, estrogen-induced
epithelial proliferation appears to be mediated through stromal ER
in a paracrine manner. In contrast, the same group ofworkers
recently reported that both epithelial and stromal ER
are essential
for secretory protein production in uterine epithelial cells (55).
These findings are consistent with our hypothesis that glandular ER
facilitates uterine secretions by stimulating the cellular levels of
specific components of the secretory pathway such as rab11. We have
noted strong stromal ER
expression during the entire preimplantation
period (Fig. 6
). However, it is not clear whether these stromal ER
s
play a role in estrogen-mediated rab11 induction in the tissue. Further
studies in primary cell culture systems and tissue recombinants using
rab11 as a specific marker of estrogen action in the epithelium will
help us to understand the intra- and intercellular signaling mechanisms
that modulate the gene-regulatory activity of ER in specific uterine
cell types.
 |
MATERIALS AND METHODS
|
---|
Reagents
Progesterone and 17ß-estradiol were purchased from Sigma Chemical Co. (St. Louis, MO). ICI 182,780 was a gift from
Zeneca Pharmaceuticals (Cheshire, U.K.). Rab11 antibody
was purchased from Zymed Laboratories, Inc. (Burlingame,
CA). ER
and -ß antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Animals
All experiments involving animals were conducted according to
NIH standards for the care and use of experimental animals. Virgin
female rats (Sprague-Dawley, from Charles River Laboratories, Inc., Wilmington, MA; 6075 days of age), in proestrus, were
mated with adult males. The different stages of the cycle in the
nonpregnant rats were ascertained by examining vaginal smears. The
presence of a vaginal plug after mating was designated as day 1 of
pregnancy. The animals were killed at various stages of gestation and
the uteri collected. In some experiments, animals were ovariectomized
and 2 weeks later were injected subcutaneously with either estradiol (2
µg/kg body weight), progesterone (40 mg/kg body weight), or a
combination of both hormones or vehicle (sesame oil) as described in
Results. The rats were killed 16 h after final
injection.
To induce and maintain delayed implantation, rats were ovariectomized
on day 4 of pregnancy and injected daily with progesterone (10 mg) from
days 58. To terminate delayed implantation and induce blastocyst
activation, the progesterone-primed delayed implanting rats were given
an injection of estrogen (0.25 µg) on the third day of the delay (day
8). Rats were killed at 24 h after the estrogen injection.
DD
Total RNAs were extracted from delayed rats before and after
estrogen treatment using an RNAgents isolation system (Promega Corp., Madison, WI). RNA samples were freed of DNA after
treatment with DNAse I (Genehunter Corp., Brookline, MA) and subjected
to DD reactions as described previously (30, 31, 32) with certain
modifications. Briefly, 2 µg of DNA-free total RNA were
reverse-transcribed with 200 U of MMLV reverse transcriptase
(Promega Corp.) in the presence of 1 mM
T12MA, or T12MC or T12MG primer
(Genehunter Corp.), where M is a mixture containing dG, dA, and
dC. The reaction was performed at 37 C for 1 h. One tenth of this
reaction was then used in a PCR amplification reaction containing 2
mM each of deoxynucleoside triphosphates, 10
mCi of [35S] dATP (Amersham, Arlington
Heights, IL), two primers: 1 mM of a T12
oligonucleotide and 0.2 mM of one of the five arbitrary
decamers, AP-1 (5'-AGCCAGCGAA-3'); AP-2 (5'-GACCGCTTGT-3'); AP-3
(5'-AGGTGACCGT-3'); AP-4 (5'-GGTACTCCAC-3'); AP-5 (5'-GTTGCGATCC-3').
These reactions also contained 1 U of AmpliTaq DNA polymerase
(Perkin Elmer Corp., Norwalk, CT). The cycling parameters
for PCR were 94 C for 30 sec, 40 C for 2 min, and 72 C for 30 sec for
40 cycles. After PCR amplification, samples were analyzed on a 6%
polyacrylamide sequencing gel, dried without fixation, and exposed to
Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) for
72 h. Bands exhibiting differential expression were cut out from
the gel, and DNA was eluted by boiling as described previously
(30, 31, 32). Eluted DNA samples were then reamplified by PCR using the
corresponding pair of primers under the same conditions as described
above, except that neither 25 mM deoxynucleoside
triphosphate nor radioisotope was used. The PCR products were
cut from 2.5% low-melt agarose gels, subcloned into Pinpoint Vector
(Promega Corp.), and subjected to nucleotide sequence
analysis.
Northern Blot Analysis
For Northern analysis 20 µg of total RNA were separated by
formaldehyde agarose gel electrophoresis and transferred to Duralon
membrane (Stratagene, La Jolla, CA). After transfer, the
membranes were baked at 80 C for 2 h. Blots were prehybridized in
50 mM NaPO4, pH 6.5/5x SSC/5x
Denhardts/50% formamide/0.1% SDS and 100 µg/ml salmon sperm DNA
for 4 h at 42 C. Hybridization was carried out overnight in the
same buffer containing 106 cpm/ml of a
32P-labeled rab11 cDNA fragment. The filters were washed
twice for 15 min in 1x SSC/0.1% SDS at room temperature, and then
twice for 20 min in 0.2x SSC/0.1% SDS at 55 C, and the filters were
exposed to x-ray films for 2472 h. The intensities of signals on the
autoradiogram were estimated by densitometric scanning. To correct for
RNA loading, the obtained signals were normalized with respect to the
GAPDH signal in the same blot. For this the filters were stripped of
the radioactive probe by washing for 10 min in 0.5% SDS at 95 C. The
blots were then reprobed with a 32P-labeled GAPDH probe
(CLONTECH Laboratories, Inc., Palo Alto, CA) as described
above.
In Situ Hybridization
Uterine tissue from pregnant animals was collected and frozen.
Tissues were fixed in 4% paraformaldehyde at 4 C. Cryostat sections
were cut at 8 µm and attached to 3-aminopropyl triethyl silane-coated
slides (Sigma Chemical Co.). In situ
hybridization was performed with DIG-labeled sense or antisense RNA
probes complimentary to nucleotides 341730 of rat rab11 gene.
DIG-labeled RNA probes were synthesized from rab11 cDNA using T3 or T7
RNA polymerase and DIG-labeled nucleotides according to manufacturers
specifications (Boehringer Mannheim, Indianapolis, IN).
Prehybridization was carried out in a damp chamber at 37 C for 60 min
in hybridization buffer (50% formamide, 5x SSC, 2% blocking reagent,
0.02% SDS, 0.1% N-laurylsarcosine). Hybridization was
carried out at 42 C overnight in a damp humidified chamber. To develop
the substrate, sections were sequentially washed in 2x SSC, 1x SSC,
and 0.1x SSC for 15 min in each buffer at 37 C. Sections were then
incubated with anti-DIG alkaline phosphatase-conjugated antibody.
Excess antibody was washed away, and the color substrate (nitroblue
tetrazolium salt and 5-bromo-4-chloro-3-indoylphosphate) was
added. Slides were allowed to develop in the dark, and the color was
visualized under light microscopy until maximum levels of staining were
achieved. The reaction was stopped and the slides were counterstained
in Nuclear Fast Red for 5 min. The slides were washed in water,
dehydrated, and coverslipped. Control incubations used a DIG-labeled
RNA sense strand and were performed under identical conditions.
Immunohistochemistry
Polyclonal antibodies against rab11 (Zymed Laboratories, Inc., Burlingame, CA) and ER
and ERß (Santa Cruz Biotechnology, Inc.) were diluted 1:1000 for
immunohistochemistry. Frozen uteri were sectioned at 7 µm, mounted on
slides, and then fixed in 5% formaldehyde in PBS. Sections were washed
in PBS for 20 min and then incubated in a blocking solution containing
10% normal goat serum for 10 min before incubation in primary antibody
overnight at 4 C. Immunostaining was performed using a
Streptavidin-Biotin kit for rabbit primary antibody (Zymed Laboratories, Inc.). Red deposits indicate the sites of
immunostaining.
Transient Transfection Experiments
Ishikawa endometrial adenocarcinoma cells were maintained in
DMEM (Gibco BRL, Grand Island, NY) supplemented with 5%
FBS (HyClone Laboratories, Inc., Logan, UT). Cells (5
x 105) were plated on 10-cm tissue culture dishes in
phenol red-free medium containing 5% charcoal-stripped serum. After
2448 h, cells were transiently transfected with plasmid DNAs using
Lipofectamine (Life Technologies, Grand Island, NY)
according to the manufacturers guidelines. Typically, cells received
1 µg of ER
or ERß plasmids. After 24 h the cells were
washed with PBS and incubated in fresh phenol red-free medium with
either 10-7 M estrogen or 10-7
M estrogen and 10-5 M ICI, or
solvent. Cells were harvested after 24 h and RNA was isolated for
RT-PCR analysis. The experiment was repeated at least three times.
RT-PCR
Total RNA (5 µg) was subjected to RT reaction using a
Stratascript RT-PCR kit. Briefly, the RNA samples were mixed with oligo
(dT) primer, incubated at 65 C for 5 min, and annealed at room
temperature. First-strand cDNA was synthesized using MMLV reverse
transcriptase at 37 C, and the reaction was stopped by heating the
tubes at 95 C for 5 min. PCR reaction was then performed in 100 µl
total volume using 35 ng of primers, 200 µM each of dATP,
dGTP, dCTP, and dTTP, 1.5 mM Mg++, and 0.5 µl
of Taq DNA polymerase (Perkin-Elmer Corp.). The
nucleotide sequences of the oligonucleotide primers were
ATAGTAACATTGTTATCATG and ACCACAAGAATTACAAATAT. The conditions for PCR
were 94 C (30 sec) for 1 cycle followed by 94 C (30 sec), 65 C (30
sec), and 68 C (2 min) for 25 cycles. PCR products were electrophoresed
on agarose gels and processed for Southern blot analysis.
Southern Blot Analysis
PCR products (2 µl each) were run on 1% agarose gel. After
electrophoresis, the gel was transferred to Duralon membrane
(Stratagene). The membrane was prehybridized in 6x SSC,
5x Denhardts, 0.5% SDS, and 100 µg/ml salmon sperm DNA for 2
h at 68 C. Hybridization was performed in the same buffer containing
106 cpm/ml of 32P-labeled cDNA fragment of
rab11 overnight at 68 C. The membrane was washed with 2x SSC and 0.1%
SDS for 15 min at room temperature, in 0.1x SSC containing 0.5% SDS
at 68 C for 45 min and exposed to x-ray film for 12 h.
 |
ACKNOWLEDGMENTS
|
---|
We acknowledge the excellent technical assistance of Michelle
Macaraig. We also acknowledge Donald McDonnell for ER
and ERß
expression vectors and Bruce Lessey for Ishikawa cells. We thank
Evan Read for the artwork and Jean Schweis for carefully
reading the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Milan K. Bagchi, Ph.D., The Population Council, 1230 York Avenue, New York, New York 10021. E-mail:
milan{at}popcbr.rockefeller.edu
This work was supported by NIH Grants R01 HD-34527 and National
Cooperative Program on Markers of Uterine Receptivity for Blastocyst
Implantation HD-34760 to I.C.B. M.K.B. is supported by NIH Grants
R01DK-5025702 and HD-1354118.
Received for publication February 3, 1999.
Revision received March 1, 1999.
Accepted for publication March 5, 1999.
 |
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