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J Biol Chem, Vol. 274, Issue 45, 32215-32224, November 5, 1999
From the Population Council and Rockefeller University,
New York, New York 10021
The steroid hormone estrogen profoundly
influences growth and differentiation programs in the reproductive
tract of cycling and pregnant mamals. It is thought that estrogen
exerts its cellular effects by regulating the expression of specific
target genes. We utilized a messenger RNA differential display method
to identify the genes whose expression is modulated by estrogen in the
preimplantation rat uterus. Here we report the cloning of a novel gene
(ERG1) that is tightly regulated by estrogen in two key reproductive tissues, the uterus and oviduct. Spatio-temporal analyses reveal that
ERG1 mRNA is expressed in a highly stage-specific manner in the uterus
and oviduct, and its expression is restricted to the surface epithelium
of both of these tissues. Nucleotide sequence analysis of the
full-length ERG1 cDNA indicates that it has an open reading frame of
1821 nuceotides encoding a putative protein of 607 amino acids with a
single transmembrane domain and a short cytoplasmic tail. The
extracellular part of the protein contains several distinct structural
motifs. These include a zona pellucida binding domain, which is present
in a number of proteins such as the zona pellucida sperm binding
proteins, and uromodulin, In addition, there is a repeat of a motif
called CUB domain, which exists in a number of genes involved in
development and differentiation such as bone morphogenetic protein 1 (BMP1). Although the precise function of ERG1 eludes us presently, its
unique pattern of expression in the uterus and oviduct and its
regulation by estrogen, a principal reproductive hormone, lead us to
speculate that this novel gene plays an important role in events during
the reproductive cycle and early pregnancy.
The steroid hormone estrogen modulates the structure and function
of the female reproductive tissues, such as the uterus and oviduct, by
eliciting an array of biochemical responses in these tissues. Estrogen
critically influences the transport of the fertilized egg through the
oviduct into the uterus (1, 2). Estrogen also promotes the growth,
differentiation, and remodeling of the uterus at various physiological
states, such as the reproductive cycle and pregnancy (3-6). The
cellular actions of this hormone are mediated through its nuclear
receptors, which function as ligand-inducible transcription factors
(7-10). Previous studies in rodents employing immature and
ovariectomized model systems have demonstrated that in the uterus,
estrogen modulates the expression of genes that are likely to be
involved in the regulation of cell growth and proliferation. These
include the genes encoding protooncogenes, such as c-fos and
c-myc, growth factors, such as epidermal growth factor and
insulin-like growth factor-1, and their receptors (11-21). The
identity of the majority of estrogen-regulated genes that mediate the
uterotropic hormonal responses to estrogen, however, remains largely
unknown. In order to understand the role of estrogen in complex
physiological processes, such as the changes in the endometrium during
the reproductive cycle, uterine receptivity, zygote transport,
implantation, stromal cell differentiation, and parturition, it is
essential to identify the spectrum of genes that are regulated by
estrogen in the reproductive tract.
It is known that in the rodents, the preovulatory ovarian estrogen is
important for uterine cellular proliferation and differentiation of
epithelium during early stages (days 1 and 2 post-fertilization) of
pregnancy (22-24). This transformation of the uterus in response to
estrogen is critical for subsequent uterine preparation for embryonic
implantation and successful establishment of pregnancy (22-24). To
identify genes that mediate estrogen action in the uterus during early
pregnancy, we employed the mRNA differential display
(DD)1 procedure devised by
Liang and Pardee (25). Using this approach, we compared mRNAs
obtained from uteri of non-pregnant (estrus stage) and pregnant (day 5)
rats and isolated several cDNA clones representing genes that are
either up- or down-regulated during the preimplantation stage of pregnancy.
In this study, we report the isolation of a novel gene that is
expressed in the uterus in a stage-specific manner during the ovarian
cycle and early pregnancy. The expression of this gene is induced in
the uteri of ovariectomized or immature rats upon administration of
estrogen and is abolished in the presence of antiestrogens. We,
therefore, termed this gene as estrogen-regulated gene 1 or ERG1. ERG1
is also highly expressed in the oviduct and this expression is under
estrogenic control. ERG1 mRNA is present predominantly in the
surface epithelium of the uterus and oviduct. The full-length cDNA
of ERG1 codes for a putative protein of 607 amino acids harboring a
single transmembrane domain. Nucleotide sequence analysis of the ERG1
cDNA clone revealed the presence of several interesting structural
features. The amino terminus of the predicted ERG1 protein harbors a
tandem repeat of a CUB domain, which exhibits striking homology to the
one that exists in the bone morphogenetic protein 1 (BMP1) (26, 27).
The carboxyl terminus of ERG1 consists mostly of a highly conserved ZP
domain that is also found in the zona pellucida sperm binding proteins and other proteins, such as uromodulin (28, 29).
Reagents--
Progesterone and 17 Animals--
All experiments involving animals were conducted
according to National Institutes of Health standards for the care and
use of experimental animals. Virgin female rats (Sprague-Dawley, from Charles River, Wilmington, MA; 60-75 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 14 days 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 under
"Results." The rats were killed 16 h after final injection.
For certain experiments immature rats (21 days old, ~40-45 g) were
injected with estrogen (40 µg/kg body weight) in sesame oil. Control
rats received an injection of sesame oil only.
DD--
Total RNAs were extracted from pregnant (day 5) and
nonpregnant (estrus) uteri using an RNAgents isolation system (Promega, Madison, WI). RNA samples were freed of DNA after treatment with DNase
I (Genehunter Corp., Brookline, MA) and subjected to differential display reactions as described previously (25, 30, 31) with certain
modifications. Briefly, 2 µg of DNA-free total RNA were reverse-transcribed with 200 units of Moloney murine leukemia virus
reverse transcriptase (Promega) in the presence of 1 mM T12MA, 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 dNTPs, 10 mCi of [35S]dATP
(Amersham Pharmacia Biotech), and two primers: 1 mM
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 unit of
AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). The cycling
parameters for PCR were: 94 °C for 30 s, 40 °C for 2 min,
72 °C for 30 s 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 before (25, 30, 31). 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 dNTP nor
radioisotope was used. The PCR products were cut from 2.5% low melting
point agarose gels, subcloned into TA vector (Invitrogen), and
subjected to nucleotide sequence analysis.
Northern Blot Analysis--
For Northern analysis 20 µg of
total RNA was separated by formaldehyde-agarose gel electrophoresis and
transferred to Duralon membrane (Stratagene). Blots were prehybridized
in 50 mM NaPO4, pH 6.5, 5× SSC, 5×
Denhardt's, 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 ERG1 cDNA fragment. The filters were washed
twice for 15 min in 1× SSC, 0.1% SDS at room temperature, then twice
for 20 min in 0.2× SSC, 0.1% SDS at 55 °C and the filters were
exposed to x-ray films for 24-72 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
glyceraldehyde-3-phosphate dehydrogenase (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
re-probed with a 32P-labeled GAPDH probe
(CLONTECH) as described above.
In Situ Hybridization--
Uterine or oviductal tissues from
pregnant animals were 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 (Sigma) coated slides.
In situ hybridization was performed with digoxigenin
(DIG)-labeled sense or antisense RNA probes complimentary to ERG1
cDNA. DIG-labeled RNA probes were synthesized from ERG1 cDNA
using T3 or T7 RNA polymerase and DIG-labeled nucleotides according to
manufacturer's specifications (Roche Molecular Biochemicals).
Prehybridization was carried out in a damp chamber at 55 °C for 60 min in hybridization buffer (50% formamide, 5× SSC, 2% blocking
reagent, 0.02% SDS, 0.1% N-laurylsarcosine). Hybridization
was carried out at 55 °C overnight in a damp humidified chamber. To
develop the substrate, sections were sequentially washed in 2× SSC,
1× SSC, and 0.1× 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
(nitro blue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate)
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 counterstained
in Nuclear Fast Red for 5 min. The slides were washed in water,
dehydrated, and coverslipped. Control incubations utilized a
DIG-labeled RNA sense strand and were performed under identical conditions.
Cloning of ERG1--
To isolate the uterine genes that are
regulated by steroid hormones in the preimplantation phase of
pregnancy, we employed the messenger RNA differential display method
(25). We compared RNA samples prepared from uteri of nonpregnant
(estrus) and pregnant (day 5 morning prior to implantation) rats.
Several differentially displayed bands representing cDNA clones
corresponding to genes whose expression in the uterus was up- or
down-regulated during the preimplantation phase were isolated. One of
these bands, which was present in the uteri of nonpregnant rats but
disappeared on day 5 of pregnancy (marked by an arrow in
Fig. 1, left lane), was selected for further characterization. The cDNA present in this
band was recovered from the gel and amplified by PCR (40 cycles). The
cDNA fragment was labeled with 32P and used to probe
Northern blots of mRNAs obtained from uteri of nonpregnant (estrus
stage) and pregnant (day 5) rats (Fig. 1, right
panel). A strong signal corresponding to 2.2 kilobase pairs
emerged in the RNA isolated from the nonpregnant uteri, while no
detectable signal was observed in the RNA from pregnant uteri.
Hybridization of the blot with a control probe (ferritin light chain)
indicated that the difference in intensities of the signals in the
lanes did not arise due to unequal loading of mRNAs in these lanes.
These results indicated that the gene we isolated is indeed
down-regulated in the uteri of pregnant rats. Using this cDNA
fragment as a probe, we isolated a full-length cDNA from a rat
uterus cDNA library. Nucleotide sequence analysis of the isolated
cDNA and comparison with the GenBankTM data base
indicated it to be a novel gene (GenBankTM accession no.
AF167170). Subsequent characterization of this gene in our laboratory
revealed that it is regulated by estrogen. Therefore, it has been
designated "estrogen-regulated gene 1" or ERG1.
The ERG1 cDNA harbors an open reading frame of 1821 base pairs in
length. The conceptual translation of the cDNA predicts a protein
of 607 amino acids (Fig. 2A).
A search of GenBankTM protein data bases showed that ERG1
exhibits homology to two kinds of protein families. In the
amino-terminal region, it possesses a repeat of a 100-amino acid motif
termed CUB domain that exhibits ~40% amino acid identity to a
similar sequence in bone morphogenetic type-1 protein, while its
carboxyl-terminal half contains a 250-amino acid ZP domain that
displays ~30% amino acid identity to the zona pellucida region of
uromodulin (Fig. 2B). ERG1 appears to be a membrane protein
with a single 16-amino acid transmembrane region and a short 19-amino
acid C-terminal area, which is presumably the intracellular part of the
protein.
ERG1 mRNA Expression Is Restricted to the Uterus and
Oviduct--
As a first step in understanding the biological function
of ERG1, we analyzed the expression pattern of this gene in a variety of tissues (Fig. 3A). Using
Northern blot analysis, we observed a band of about 2.2 kilobases
corresponding to the ERG1 mRNA in the uterus (estrus stage,
lane 10). We failed to detect any ERG1 mRNA
in several other tissue such as heart, brain, lung, liver, kidney,
skeletal muscle, and testes (Fig. 3A, lanes
1-9). We also studied its expression in other female
reproductive tissues such as the ovary and oviduct (Fig.
3B). While no ERG1 mRNA could be detected in the ovary,
it was expressed abundantly in the oviduct. By our estimation, the
level of ERG1 expression in the oviduct appeared to be at least 2-fold
higher than in the uterus. Taken together, these results indicate that
ERG1 is expressed predominantly in two reproductive tissues, uterus and
oviduct, at a high level.
ERG1 Expression Is Modulated in the Uterus and Oviduct during the
Reproductive Cycle--
To examine the pattern of ERG1 expression in
the uterus and oviduct during estrus cycle, we performed Northern blot
analysis of RNA isolated from uteri of nonpregnant rats at proestrus,
estrus, and diestrus stages. As shown in Fig.
4, when the blot was hybridized with a
32P-labeled ERG1 cDNA probe, a strong 2.2-kilobase
message was seen in the uterine RNA sample at the proestrus stage of
the cycle (lane 1). The signal corresponding to
ERG1 mRNA declined at estrus (lane 2) and
disappeared at the diestrus stage of the cycle (lane 3). The magnitude of the decline at the estrus phase was
estimated to be about 40% compared with proestrus stage of the cycle.
In the oviduct, the expression of ERG1 was high in both proestrus and
estrus but declined significantly at diestrus (Fig. 4, lanes 4-6). The expression of the ERG1 message in both uterus and
oviduct is therefore modulated in a stage-specific manner during the
reproductive cycle.
Localization of ERG1 mRNA in the Surface Epithelia of the
Uterus and Oviduct--
The site of expression of ERG1 mRNA in the
uterus and oviduct was investigated by employing in situ
hybridization (Fig. 5). Uterine and
oviductal sections from nonpregnant animals at proestrus stage were
hybridized with a 500-base pair digoxigenin-labeled antisense or sense
RNA probe containing sequences from ERG1 cDNA. While control
uterine sections hybridized with the sense RNA probe did not exhibit
any significant signal (Fig. 5A, panel
b), we observed a strong hybridization signal in the luminal
epithelial cells of these uterine sections upon hybridization with the
antisense probe (panels a and c). A
strong hybridization signal in the oviductal epithelial cells was also
observed upon hybridization with the antisense probe (Fig.
5B, panels a and c). These
results demonstrate that the surface epithelium is the primary site of
synthesis of ERG1 mRNA in rat uterus and oviduct.
Profile of ERG1 mRNA Expression in the Uterus and Oviduct
during Early Pregnancy--
We then investigated the pattern of ERG1
mRNA expression in rat uterus at various stages of early pregnancy.
We performed Northern blot analysis of RNA isolated from uteri of
pregnant animals at days 1-6 of gestation. As shown in Fig.
6A, a high level of ERG1
mRNA was observed in the uterus of day 1 pregnant animals
(lane 1). The level of this mRNA declined
dramatically on day 2 of pregnancy (lane 2). No
ERG1 expression was observed on days 3, 4, 5, and 6 of gestation
(lanes 3, 4, 5, and
6, respectively). In situ hybridization of
uterine sections at different days of gestation further confirmed the
down-regulation of ERG1 mRNA expression during early pregnancy
(Fig. 6B). The signal corresponding to ERG1 mRNA was
intense at the luminal epithelium of uterus on day 1 (panel
A), diminished on day 2 (panel B), and
disappeared on days 5 and 6 of pregnancy (panels
C and D, respectively). Collectively, these
results show that ERG1 is expressed in a highly stage-specific manner
in the luminal epithelial cells of the uterus during early pregnancy.
We also examined the expression of ERG1 in the oviduct during early
pregnancy by Northern blot analysis (Fig.
7A). In pregnant rats, ERG1
mRNA level was high in the oviduct on days 1 and 2 but declined
significantly on days 3 and 4. The magnitude of this decline on days
3-4 was estimated to be about 50% compared with either day 1 or 2 of
pregnancy (Fig. 7B). These results indicate that, as in the
uterus, the expression of ERG1 in the oviduct is also down-regulated
during early pregnancy.
Estrogen Up-regulates and Progesterone Down-regulates ERG1 Gene
Expression in the Uterus--
To determine whether ERG1 expression in
the uterus is regulated by steroid hormones, we administered estrogen
(E), progesterone (P), or a combination of both
to ovariectomized rats. Uteri were collected from animals 16 h
after the injection, and mRNAs were isolated from these tissues for
Northern blot analysis. The blot was probed with
32P-labeled ERG1 cDNA. As shown in Fig.
8A, the signal representing ERG1 mRNA was barely detectable in the uteri of overiectomized rats
(lane 1). While treatment with progesterone
(lane 3) failed to induce ERG1 mRNA, estrogen
alone (lane 2) markedly increased the level of
ERG1 mRNA in the uterus. The level of this mRNA declined (by
more than 60%) significantly when progesterone was administered together with estrogen to ovariectomized animals (Fig. 8, A
and B, lane 4). These results
demonstrate that estrogen alone can trigger the accumulation of ERG1
mRNAs in the uteri of ovariectomized animals. These results also
clearly show that progesterone antagonizes the action of estrogen on
induction of ERG1 expression in the uterus.
We next examined the time course of ERG1 gene induction by estrogen.
Ovariectomized rats were injected with estrogen for 4, 8, 16, and
24 h, and a Northern blot analysis was carried out using mRNAs
isolated from the uteri. As shown in Fig.
9A, the amount of ERG1
mRNA increased progressively with the duration of estrogen
treatment. A significant increase (10-fold) in the level of ERG1
mRNA was observed after 8 h of estrogen treatment. Estrogen
injections up to 16 or 24 h led to a further enhancement in the
level of ERG1 mRNA. We also performed in situ
hybridization analysis of uterine sections from ovariectomized animals
treated with estrogen (Fig. 9B). Consistent with the results
of Northern blot analysis, no ERG1 mRNA expression was observed in
the luminal epithelial cells of ovariectomized animals
(panel A). While ERG1 mRNA was barely visible
at the surface epithelial cells 4 h after estrogen treatment
(panel B), this mRNA became detectable 8 h after estrogen treatment (panel C). The level
of ERG1 mRNA increased further after 16 or 24 h of estrogen
treatment (panels D and E, respectively). Taken together, these results show that the level of
ERG1 mRNA at the surface epithelial cells of the uterus increases progressively with the duration of estrogen treatment.
An Antagonist of ER Blocks ERG1 mRNA Expression in the Uterus
and Oviduct--
We further investigated the estrogenic regulation of
ERG1 mRNA expression by employing an antiestrogen, ICI 182,780, which is known to severely impair the transcriptional activity of
estrogen receptors (32). Animals were ovariectomized and injected with vehicle or estrogen or a combination of estrogen and ICI 182,780. As
depicted in Fig. 10A, ERG1
expression was not observed in vehicle-treated ovariectomized animals
(lane 1). As expected, administration of estrogen
induced ERG1 in the uterus (lane 2). Treatment of
animals with estrogen in combination with ICI 182,780 (lane
3), however, caused a drastic decline in the level of ERG1
mRNA but did not affect the expression of GAPDH mRNA. These
results strongly suggest that ICI 182,780 switched off the ERG1 gene
expression in the uterus by inhibiting the estrogen receptors.
We also studied the effects of ICI 182,780 on ERG1 expression in the
uterus of cycling rats by in situ hybridization (Fig. 10B). Animals were injected at proestrus stage with either
vehicle (control) or ICI 182,780, and the uteri were isolated at estrus stage for in situ hybridization. As shown in Fig.
10B, a high level of ERG1 mRNA was observed in the
uterine epithelial cells of animals at estrus stage (panel
A). However, a single dose of ICI 182,780 dramatically
reduced the level of this mRNA in the uterine epithelial cells
(panel B). These studies further confirm the role
of estrogen in regulating ERG1 expression in the uterus.
We next examined the regulation of ERG1 mRNA by estrogen in the
oviduct of cycling animals. Animals were injected at proestrus stage
with either vehicle (control) or ICI 182,780. Uteri were isolated and a
Northern blot analysis was performed. As shown in Fig.
11, a single dose of ICI 182,780 reduced the level of ERG1 mRNA in the oviduct. By our estimate, the
level of ERG1 mRNA was reduced to about 50% of that of the control
within 16 h of treatment. These results indicate that, as in the
uterus, estrogen regulates ERG1 expression in the oviduct.
Progesterone Suppresses ERG1 Expression in the Uterus during Early
Pregnancy--
Since our results indicated that progesterone
antagonizes estrogen-induced ERG1 expression in the uterus (Fig. 8), we
considered the possibility that the down-regulation of ERG1 expression
following day 1 of pregnancy could be caused by the surge of
progesterone at the onset of pregnancy. The level of progesterone
increases markedly on day 2 and remains high until the end of
gestation. To test this hypothesis, animals were injected on day 3 of
pregnancy either with a vehicle or a dose of RU486, which is known to
block the action of progesterone receptors in the uterus (33, 34). As
shown in Fig. 12A, ERG1 was
not detected in the uterus of day 4 pregnant animals (lane
1). A single dose of RU486, however, induced a remarkable
expression of ERG1 mRNA in the uterus within 24 h of treatment
(lane 2). The RU486-induced expression of ERG1 mRNA in the uterus was also examined by in situ
hybridization. Consistent with the results of the Northern blot
analysis, RU486 treatment led to an increase in the accumulation of
ERG1 mRNA in the epithelial cells relative to the signal seen in
the uteri of vehicle-treated pregnant (day 4) animals (Fig.
12B, compare panels B and
C). Collectively, these results confirm the role of
progesterone in down-regulating estrogen-induced ERG1 gene expression
in the uterus of pregnant rats.
Estrogen Induces ERG1 Gene Expression in the Uterus of Immature
Rats--
Estrogen-induced proliferation of immature rodent epithelium
is a long standing and widely used model of uterine actions of estrogen
(35-37). We, therefore, examined whether ERG1 is expressed in the
uterus of immature rats in response to estrogen. Immature rats were
treated with estrogen for 4, 8, and 24 h; mRNA was isolated and subjected to Northern blot analysis using ERG1 cDNA probe. As
shown in Fig. 13A, ERG1
mRNA was not detected in the uteri of immature rats
(lane 1). However, treatment with estrogen alone for 4 or 8 h dramatically increased the level of this mRNA
(lanes 2 and 3). The level of ERG1
mRNA increased further after 24 h of estrogen treatment.
We then performed in situ hybridization to localize
estrogen-induced expression of ERG1 mRNA in the uterine
compartments of immature rats. As shown in Fig. 13B
(panel A), no signal corresponding to ERG1
mRNA was observed in the uterus of immature rats. A specific signal
corresponding to ERG1 mRNA, however, appeared in the uterus after
24 h of estrogen treatment (panel B), which
was restricted to the luminal epithelial cells. These results indicate
that, as in the adult rats, estrogen is the primary inducer of ERG1 mRNA expression at the surface epithelium of immature rats.
In the present study, we have isolated a full-length cDNA of
ERG1 from rat uterus. In the mRNA differential display, ERG1 cDNA appeared to represent a gene that is down-regulated during early pregnancy (Fig. 1). Indeed, spatio-temporal analyses of ERG1
expression in the endometrium confirm that its expression in the
surface epithelium is strongly repressed as gestation progresses from
day 2 to day 5. The level of ERG1 mRNA is high on day 1 of pregnancy, declines on day 2, and is barely detectable on days 3, 4, 5, or 6 of gestation. ERG1 is also expressed in the uterus in a
stage-specific manner during the ovarian cycle. While maximal expression of ERG1 occurred at the proestrus stage of the ovarian cycle
coincident with the estrogen-induced uterine cell proliferation, we
observed minimal, if any, ERG1 expression at the diestrus stage of the
cycle. The tight spatio-temporal regulation of ERG1 expression during
the reproductive cycle and early pregnancy suggest that it might have a
critical regulatory role(s) in these processes.
Recently Kasik reported the isolation of a cDNA that is highly
expressed in mouse uterus during late pregnancy (38). This cDNA
exhibits 82% homology with the rat ERG1 clone isolated by us. The
investigator performed Northern blot analysis using mRNAs isolated
from nonpregnant and pregnant uteri and observed that expression of
this cDNA is absent in nonpregnant uterus but is restricted to
pregnant uterus a few days before parturition (38). If this cDNA
represents the mouse homologue of ERG1, its reported lack of expression
in the non-pregnant uterus is surprising, especially in light of the
high expression of rat ERG1 in proestrus and estrus uteri. However, it
cannot be ruled out that the altered expression pattern of the mouse
gene arises from species difference. It is also possible that, although
it exhibits strong sequence similarity, the mouse gene is distinct from ERG1.
Our study demonstrates that expression of ERG1 in the uterus is under
estrogenic control. Consistent with its up-regulation by estrogen, the
profile of ERG1 expression in the uterus during the estrus cycle
overlaps closely with that of plasma estrogen. In the nonpregnant
animals, the circulating level of estrogen is high at proestrus and,
consistent with this, we detect abundant ERG1 mRNA at this stage of
the ovarian cycle. In the diestrus phase, concomitant with a fall in
estrogen concentration, there is a sharp drop in the level of ERG1
mRNA (Fig. 4). We observe a high level of ERG1 mRNA in the
uterus also on day 1 of pregnancy, presumably in response to the
preovulatory ovarian estrogen, which is known to be crucial for
proliferation of uterine epithelium on days 1 and 2 of pregnancy
(22-24).
Our studies also indicate that the ERG1 mRNA level in the uterus is
regulated by progesterone. While estrogen induces ERG1 expression,
progesterone inhibits estrogen-mediated ERG1 expression in the uterus.
The estrogen concentration remains low from pregnancy day 2 onward,
except for a transitory rise on day 4 immediately prior to implantation
(24). The progesterone level, on the other hand, starts to increase by
day 2 and remains high until the end of gestation (39). It is likely
that the estrogen-induced ERG1 expression in the uterus on day 2 and
subsequent days of pregnancy is antagonized by the rising progesterone
level. This concept is supported by the observation that treatment of
animals with RU486, which antagonizes the action of progesterone,
dramatically enhances ERG1 expression on day 4 of gestation (Fig. 12).
ERG1 expression in the uterus is therefore regulated by a complex
interplay of estrogen and progesterone.
Besides uterus, ERG1 is also highly expressed in the oviduct. In
mammals, oviduct is the site at which fertilization of the egg and
early development of the zygote occurs. The zygote travels through the
oviduct and enters the uterus to arrive at the site of implantation.
Previous studies indicated that the correct programming of egg or
zygote transport through the oviduct is regulated by estrogen and
progesterone (1, 2). It was shown in a number of animal models that
estrogen accelerates egg/zygote transport and progesterone blocks this
estrogen-mediated transport. Allen showed that estrogen stimulates
differentiation of the oviductal epithelium in the rhesus monkey (40,
41). Since the receptors for steroid hormones are present in oviductal
tissue, it is likely that the actions of estrogen and progesterone on
gamete transport and differentiation of the epithelium are mediated by
receptor-activated mechanisms. Despite the well known regulatory role
of estrogen in the oviduct, to the best of our knowledge, there is only
one published report of an estrogen-regulated gene in the oviduct (42).
Therefore, our finding that estrogen regulates ERG1 expression in the
epithelial cells of the oviduct establishes a novel marker of estrogen
action in this tissue. The expression level of ERG1 in the oviduct,
however, is significantly higher than that in the uterus. We therefore
cannot rule out the possibility that another factor(s) in addition to
estrogen modulates ERG1 expression in the oviduct.
The biological implication of ERG1 expression in the uterus and oviduct
is unclear. The ERG1 gene product is predicted to exhibit several
distinct structural motifs. In the carboxyl-terminal region, a stretch
of 260 amino acids harbors a ZP domain. The ZP domain was first
identified in ZP1, ZP2, and ZP3, major glycoproteins of the
extracellular matrix surrounding oocytes (28). These proteins are
involved in sperm-egg recognition. The ZP domain was later found in
several other receptor-like proteins, such as uromodulin, and the major
zymogen granule membrane glycoprotein (GP-2), defining a new family of
distantly related proteins involved in binding functions (28, 29).
Uromodulin, a major glycoprotein secreted by the mammalian kidney,
functions as an immune suppressor and prevents urinary tract infections
by binding mannose-sensitive fimbriated microorganisms. The homologous
region common to all ZP domain proteins contains eight strictly
conserved cysteines, which are likely to form disulfide bridges. In
addition to the conserved cysteines, these proteins also harbor few
aromatic or hydrophobic amino acids that are highly conserved among the
family members. Interestingly, in each of these proteins the common
domain occurs next to the putative membrane-spanning regions suggesting a possible conserved biological role of the domain. Consistent with the
ascribed features of the ZP family proteins, ERG1 possesses the
characteristics of an integral membrane protein since it harbors a
single transmembrane signal near the carboxyl terminus. Future studies
will determine whether ERG1 exists in a soluble or secreted form and
displays specific binding functions.
The nucleotide sequence of ERG1 cDNA also reveals the presence of
two CUB domains at the amino-terminal end of the predicted protein. The
CUB domain is a 110-amino acid module that was first identified in the
complement subcomponents Cls and Clr (Cls/Clr) (26). Later on, a
similar domain was found in bone morphogenetic protein 1 (BMP1) and an
embryonic sea urchin protein Uegf. Recently, several other proteins
containing CUB domain have been reported (26, 27). These include the
Drosophila dorso-ventral patterning gene product tolloid,
the sea urchin blastulla protein Bp10, and a family of sperm adhesins
(27). In all the proteins identified so far, four cysteine residues are
conserved in all CUB modules, which display a typical antiparallel
We thank Michele Macaraig for excellent
technical assistance. We also thank Evan Read for the artwork and Jean
Schweis for carefully reading the manuscript.
*
This work was supported in part by National Institutes of
Health Grant R01 HD-34527 (to I. C. B.) and National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation Grant U01 HD-34760 (to I. C. B.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF167170.
§
To whom correspondence should be addressed: Population Council,
1230 York Ave., New York, NY 10021. Tel.: 212-327-8758; Fax: 212-327-7678; E-mail: indrani@popcbr.rockefeller.edu.
The abbreviations used are:
DD, differential
display;
ZP, zona pellucida;
PCR, polymerase chain reaction;
DIG, digoxigenin;
bp, base pair(s);
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
Cloning and Uterus/Oviduct-specific Expression of a Novel
Estrogen-regulated Gene (ERG1)*
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-estradiol were purchased
from Sigma. Mifepristone (RU 38486) and ICI 182,780 were kind gifts of
Roussel-Uclaf, France and Zeneca Pharmaceuticals, Cheshire, United Kingdom.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 1.
Profiles of differentially expressed
mRNAs in uterine tissues of nonpregnant and pregnant rats.
Left panel, total RNA (2 µg) isolated from
uteri of nonpregnant (estrus) and pregnant (day 5) rats was subjected
to differential display. A band representing mRNA that is
down-regulated during pregnancy is marked by an arrowhead.
Right panel, Northern blot analysis of uterine
mRNAs of nonpregnant and pregnant rats. Poly(A+) RNA (1 µg/lane) was analyzed by RNA gel blot analysis. The upper
panel shows the pattern of signals obtained after
hybridization with a 32P-labeled cDNA fragment that was
isolated from the DD gel shown in the left panel
(indicated by arrowhead). The position of this mRNA is
indicated by ERG1 mRNA. The lower panel shows
the same blot after hybridization with a control
32P-labeled ferritin light chain probe.
View larger version (44K):
[in a new window]
Fig. 2.
Structure and deduced amino acid sequence of
ERG1. A, the complete nucleotide sequence of rat ERG1
cDNA has been submitted to the GenBankTM (accession
no. AF167170). The longest open reading frame of ERG1 yields a deduced
amino acid sequence of 607 residues. The positions of the CUB and ZP
domains are indicated in a linear structure. The position of the signal
peptide is shown at the amino-terminal end and a putative transmembrane
domain, indicated by TM, is depicted at the
carboxyl-terminal region. B, description of CUB and ZP
domains of ERG1, bone morphogenetic protein 1 (BMP1), or
uromodulin (UROM) is indicated.
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Fig. 3.
Northern blot analysis of mRNA obtained
from various rat tissues. A, poly(A+) RNA
(2 µg/lane) from heart, brain, spleen, lung, liver, skeletal muscle,
kidney, testis, ovary, and uterus. B, total RNA (20 µg/lane) from ovary (lane 1), uterus
(lane 2), and oviduct (lane
3) were analyzed by Northern blot analysis. Hybridization
was performed with 32P-labeled ERG1 and GAPDH cDNA
probes.
View larger version (55K):
[in a new window]
Fig. 4.
Profile of expression of ERG1 mRNA in rat
uterus and oviduct at different stages of the reproductive cycle.
Total RNA (20 µg/lane) from uteri of animals at proestrus
(PE), estrus (E), and diestrus (DE)
was analyzed by Northern blotting. Hybridization was performed with a
32P-labeled ERG1 and GAPDH cDNA probes.
View larger version (58K):
[in a new window]
Fig. 5.
Localization of ERG1 mRNA in the rat
uteri and oviduct by in situ hybridization.
A, uterine sections from nonpregnant (estrus) rats were
subjected to in situ hybridization. The hybridization was
performed employing 500-bp-long digoxigenin-labeled antisense
(panels a and c) or sense
(panel b) cRNA probes specific for ERG1 gene as
described under "Experimental Procedures." g and
le indicate the glandular and luminal epithelium,
respectively. Magnification: a and b, ×40;
c, ×400. B, sections of oviduct from nonpregnant
(estrus) rats were hybridized with antisense (panels A and
C) and sense (panel B) ERG1 probes.
View larger version (57K):
[in a new window]
Fig. 6.
Profile of expression of ERG1 mRNA in rat
uterus during early pregnancy. A, total RNA (20 µg)
was isolated from uteri of animals at 1, 2, 3, 4, 5, and 6 days of
pregnancy and subjected to Northern blot analysis using ERG1
(upper panel) or GAPDH (lower
panel) cDNA probes. The results are representative of
two independent experiments. B, uterine sections at
different days of early pregnancy were subjected to in situ
hybridization. A-D represent sections of uteri on days 1, 2, 5, and 6 of gestation, respectively. The hybridization was performed
employing a 500-bp-long digoxigenin-labeled cRNA probe specific for
ERG1 cDNA as described under "Experimental Procedures."
l and le indicate the lumen and luminal
epithelium, respectively.
View larger version (21K):
[in a new window]
Fig. 7.
Profile of expression of ERG1 mRNA in rat
oviduct during early pregnancy. A, total RNA (20 µg)
was isolated from oviducts of animals at 1, 2, 3, and 4 days of
pregnancy and subjected to Northern blot analysis using ERG1
(upper panel) or GAPDH (lower
panel) cDNA probes. B, the intensities of the
signals corresponding to the ERG1 transcript was 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.
View larger version (20K):
[in a new window]
Fig. 8.
Effects of progesterone and estrogen on ERG1
gene expression in uteri of ovariectomized animals. A,
total RNA (20 µg/lane) was subjected to Northern blot analysis and
hybridized with a 32P-labeled ERG1 (upper
panel) or GAPDH (lower panel) probes.
Lane 1 represents RNA from uteri of animals
injected with vehicle only after ovariectomy; lane
2, RNA from uteri of animals injected with estrogen (2 µg/kg body weight); lane 3, RNA from uteri of
animals injected with progesterone (40 mg/kg body weight);
lane 4, RNA from uteri of animals injected with
estrogen (2 µg/kg body weight) plus progesterone (40 mg/kg body
weight). B, the intensities of the signals corresponding to
the ERG1 transcript were quantitated by densitometric scanning of the
autoradiogram and normalized with respect to the GAPDH signals of the
Northern blot shown in panel A.
View larger version (35K):
[in a new window]
Fig. 9.
Time course of induction of ERG1 mRNA by
estrogen in the uteri of ovariectomized animals. A,
upper panel, poly(A+) RNA (2 µg/lane) were analyzed by Northern blotting and hybridized with a
32P-labeled ERG1 cDNA probe. Following ovariectomy
animals were treated with estrogen for 4, 8, 16, and 24 h
(lanes 1-5, respectively). Lower
panel, the relative levels of ERG1 mRNA induced by
different doses of estrogen in ovariectomized animals. Quantitation was
performed by densitometric scanning of the bands in the Northern blot
shown in the upper panel followed by
normalization with respect to GAPDH signals (data not shown) in the
same blot. B, in situ hybridization of uterine
sections from ovariectomized animals (panel A)
and after treatment with estrogen for 4 h (panel
B), 8 h (panel C), 16 h
(panel D), and 24 h (panel
E). The hybridization was performed employing a 500-bp-long
digoxigenin-labeled cRNA probe specific for ERG1 cDNA.
Magnification, ×100.
View larger version (31K):
[in a new window]
Fig. 10.
ERG1 mRNA in the uterus of nonpregnant
rats declines after treatment with antiestrogen ICI 182,780. A, total RNA (20 µg/lane) was subjected to Northern blot
analysis and hybridized with a 32P-labeled ERG1
(upper panel) or GAPDH (lower
panel) probes. Lane 1, RNA from uteri
of animals injected with vehicle only after ovariectomy;
lane 2, RNA from uteri of animals injected with
estrogen; lane 3, RNA from uteri of animals
injected with estrogen (2 µg/kg body weight) and ICI 182,780 (1 mg/kg
body weight). B, in situ hybridization of uterine
sections from nonpregnant animals at the estrus stage (panel
A) and following treatment with a single dose of
antiestrogen ICI 182,780 (1 mg/kg body weight) (panel
B).
View larger version (27K):
[in a new window]
Fig. 11.
ERG1 mRNA in the oviduct of nonpregnant
rats declines after treatment with antiestrogen ICI 182,780. Total
RNA (20 µg/lane) was subjected to Northern blot analysis and
hybridized with a 32P-labeled ERG1 (upper
panel) or S16 RNA (lower panel) probe.
Lanes 1 and 2 represent oviductal RNA
at the estrus stage and upon treatment with a single dose of
antiestrogen ICI 182,780 (1 mg/kg body weight), respectively.
View larger version (28K):
[in a new window]
Fig. 12.
Effects of the antiprogestin RU486 on ERG1
mRNA synthesis in the uterus. A, total RNA (20 µg/lane) was analyzed by Northern blotting. Lane
1, RNA from the uteri of animals injected with vehicle on
day 3 of pregnancy and killed on day 4; lane 2,
RNA from the uteri of animals given a single dose of RU486 (8 mg/kg
body weight) on day 3 and killed on day 4 of pregnancy. B,
in situ hybridization of uterine sections from day 1 pregnant animals (panel A). Panels
B and C represent uterine sections of animals
treated with vehicle or RU486 (8 mg/kg body weight) on day 3 of
pregnancy and killed on day 4, respectively.
View larger version (39K):
[in a new window]
Fig. 13.
ERG1 mRNA is induced by estrogen in the
uteri of immature animals. A, upper
panel, total RNA (20 µg/lane) was isolated from immature
rats after treatment with estrogen for 4, 8, and 24 h,
respectively. RNA samples were analyzed by Northern blotting and
hybridized with a 32P-labeled ERG1 cDNA probe.
Lower panel, the relative levels of ERG1 mRNA
induced by different doses of estrogen in immature animals.
Quantitation was performed by densitometric scanning of the bands in
the Northern blot shown in the upper panel
followed by normalization with respect to GAPDH signals (data not
shown) in the same blot. B, in situ hybridization
of uterine sections from immature rats before (panel
A) and after treatment with estrogen (panel
B). The hybridization was performed employing a 500-bp-long
digoxigenin-labeled cRNA probe specific for ERG1 cDNA.
le represents luminal epithelium.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet structure. Although a common structural framework exists for
all CUBs, there is no clear indication for functional relations.
Nevertheless, there are several indications of an involvement of all
CUB domain proteins in the developmental and differentiation processes.
It is intriguing that ERG1 shows maximal similarity to BMP1, a factor
that induces mesenchymal cells to differentiate into cartilage-forming
cells and further into bone cells. Interestingly, ERG1 is induced in the uterine epithelium of immature rodents during estrogen-induced cell
proliferation and differentiation. Future studies will determine whether ERG1 expression in response to estrogen plays a role in the
proliferation and differentiation of epithelial cells during the estrus
cycle and early pregnancy.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by National Institutes of Health Grants RO1-DK-50257 and
U54-HD-13541.
ABBREVIATIONS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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