Dysregulation of EGF Family of Growth Factors and COX-2 in the Uterus during the Preattachment and Attachment Reactions of the Blastocyst with the Luminal Epithelium Correlates with Implantation Failure in LIF- Deficient Mice
Haengseok Song,
Hyunjung Lim,
Sanjoy K. Das,
Bibhash C. Paria and
Sudhansu K. Dey
Departments of Molecular and Integrative Physiology (H.S., H.L.,
S.K.Dey) Obstetrics and Gynecology (S.K.Das), and Pediatrics
(B.C.P.) Ralph L. Smith Research Center University of Kansas
Medical Center Kansas City, Kansas 66160-7338
 |
ABSTRACT
|
---|
Various mediators, including cytokines,
growth factors, homeotic gene products, and prostaglandins (PGs),
participate in the implantation process in an autocrine, paracrine, or
juxtacrine manner. However, interactions among these factors that
result in successful implantation are not clearly understood. Leukemia
inhibitory factor (LIF), a pleiotropic cytokine, was shown to be
expressed in uterine glands on day 4 morning before implantation and is
critical to this process in mice. However, the mechanism by which LIF
executes its effects in implantation remains unknown. Moreover,
interactions of LIF with other implantation-specific molecules have not
yet been defined. Using normal and delayed implantation models, we
herein show that LIF is not only expressed in progesterone
(P4)-primed uterine glands before implantation
in response to nidatory estrogen, it is also induced in stromal cells
surrounding the active blastocyst at the time of the attachment
reaction. This suggests that LIF has biphasic effects: first in the
preparation of the receptive uterus and subsequently in the attachment
reaction. The mechanism by which LIF participates in these events was
addressed using LIF-deficient mice. We observed that while uterine
cell-specific proliferation, steroid hormone responsiveness, and
expression patterns of several genes are normal, specific members of
the EGF family of growth factors, such as amphiregulin (Ar),
heparin-binding EGF-like growth factor (HB-EGF), and
epiregulin, are not expressed in
LIF(-/-) uteri before and during the
anticipated time of implantation, although EGF receptor family members
(erbBs) are expressed correctly. Furthermore,
cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG
synthesis and essential for implantation, is aberrantly expressed in
the uterus surrounding the blastocyst in
LIF(-/-) mice. These results suggest that
dysregulation of specific EGF-like growth factors and COX-2 in the
uterus contributes, at least partially, to implantation failure in
LIF(-/-) mice. Since estrogen is essential
for uterine receptivity, LIF induction, and blastocyst activation, it
is possible that the nidatory estrogen effects in the
P4-primed uterus for implantation are mediated
via LIF signaling. However, we observed that LIF can only partially
resume implantation in P4-primed, delayed
implanting mice in the absence of estrogen, suggesting LIF
induction is one of many functions that are executed by estrogen for
implantation.
 |
INTRODUCTION
|
---|
A cross-talk between the active blastocyst and receptive uterus is
essential to implantation (1, 2, 3). Coordinated effects of ovarian
progesterone (P4) and estrogen play critical
roles in establishing uterine receptivity for implantation. The
initiation of implantation is first marked by an increased endometrial
vascular permeability at the sites of blastocyst apposition, and this
coincides with the attachment reaction (2, 4). The uterine preparation
for implantation is sequentially programmed into three stages termed
prereceptive, receptive, and nonreceptive phases (1, 5). In the mouse,
the uterus becomes prereceptive on day 3 of pregnancy or
pseudopregnancy by rising ovarian P4 levels. On
day 4, the P4-primed uterus becomes receptive
when complemented with preimplantation ovarian estrogen secretion.
Blastocysts implant only in the receptive uterus, which spontaneously
enters into the nonreceptive state (2). Similar uterine phases can be
produced by inducing delayed implantation by ovariectomy on the morning
of day 4 of pregnancy or pseudopregnancy before the preimplantation
estrogen secretion. Under this condition, blastocysts in the pregnant
uterus or transferred blastocysts into the pseudopregnant uterus
undergo dormancy and failed to initiate the attachment reaction. This
condition can be maintained by continued P4
treatment when the uterus remains in the neutral (prereceptive phase)
state, but is terminated by an estrogen injection with blastocyst
activation and attainment of uterine receptivity. Again, the limited
period of uterine receptivity is followed by the nonreceptive phase in
the absence of implantation-competent blastocysts (1, 2, 6). The
mechanisms by which estrogen renders the uterus receptive, activates
dormant blastocysts, and initiates implantation are not clearly
understood.
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine that
regulates various cellular functions including proliferation and/or
differentiation (7, 8, 9, 10). LIF is a member of the interleukin-6 (IL-6)
family, which includes IL-6 itself, oncostatin M, ciliary
neurotrophic factor, IL-11, and cardiotrophin-1. LIF functions
through heterodimerization of LIF receptor (LIFR) and gp130. The IL-6
family shares gp130 as a common receptor for signal transduction,
resulting in considerable functional overlap among the cytokines (11).
LIF signaling pathway is suggested to involve activation of
mitogen-activated protein (MAP) and/or Janus kinases, triggering
phosphorylation of signal transducer and activator of
transcription (STAT) factors for translocation in the nucleus
and gene activation. However, the mechanism(s) by which LIF transmits
cellular signaling in vivo is not clearly understood.
The epidermal growth factor (EGF) family includes EGF, transforming
growth factor-
(TGF-
), heparin binding-EGF (HB-EGF), amphiregulin
(Ar), betacellulin, epiregulin, and heregulins/neu differentiation
factors (NDFs) (12, 13, 14, 15, 16, 17, 18). They are synthesized as transmembrane proteins
that are proteolytically processed to release the mature forms (19).
These ligands interact with the receptor subtypes of the
erbB gene family, which is comprised of four receptor
tyrosine kinases: ErbB1 (EGF-R), ErbB2, ErbB3, and ErbB4. They share a
common structural feature, but differ in their ligand specificity and
kinase activity (20, 21, 22). In general, the EGF-like ligands can interact
with ErbB family members via homodimerization or heterodimerization
(23, 24, 25). Thus, cross-talk between the receptor subtypes with various
ligands can serve as a potential signaling mechanism (26, 27). The
expression patterns of the EGF family of growth factors and their
receptors in the periimplantation mouse uterus have been examined (4, 25, 28, 29, 30). Overall, the results show that the expression of
Ar, HB-EGF, and epiregulin in the
periimplantation uterus is unique. Ar is expressed in a
P4-dependent manner on day 4 (the day of
implantation) (28), while HB-EGF is expressed solely in the
luminal epithelium at the sites of blastocyst apposition several hours
before the attachment reaction, and this expression requires the
presence of active blastocysts (4). In contrast, epiregulin
is expressed in the luminal epithelium and underlying stroma
surrounding the blastocysts during the attachment reaction (29). This
expression pattern of Ar, HB-EGF, and
epiregulin suggests their roles in uterine preparation and
subsequent attachment reaction. Further, HB-EGF influences blastocyst
activities for implantation in paracrine and juxtacrine manners
(31).
Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in PG biosynthesis,
is expressed in luminal epithelial and stromal cells surrounding the
active blastocyst during the attachment reaction (32). We have recently
established that COX-2-derived prostacyclin
(PGI2) is essential for implantation and
decidualization (33, 34). The molecular mechanisms by which EGF-like
growth factor and COX-2 genes are induced locally by
blastocysts are poorly understood. Cytokines are implicated to
participate in various events of implantation in an autocrine/paracrine
manner (reviewed in Ref. 35). Among the cytokines, LIF is a potential
candidate since it was reported to be transiently expressed in mouse
uterine glands on day 4 of pregnancy or pseudopregnancy, and this
expression precedes blastocyst implantation (36, 37). In addition,
LIF-deficient mice exhibit implantation and decidualization failures
(38). Reciprocal embryo transfer experiments have shown that
LIF(-/-) blastocysts can implant after transfer
into wild-type pseudopregnant uteri, although wild-type blastocysts
fail to implant in LIF(-/-) uteri. This
suggests that maternal LIF is essential for implantation. Uterine LIF
appears to be also important for implantation in other species
including humans (39, 40, 41, 42). Indeed, LIF deficiency is noted in infertile
women and in those with unexplained recurrent abortions (43, 44).
Blastocysts express both LIFR and gp130, and LIF influences their
development (45, 46, 47, 48, 49). These results suggest that both the embryo and
uterus are potential targets for LIF signaling.
The molecular mechanism by which LIF regulates the implantation process
and its interactions with other implantation-specific molecules still
remains unexplained. This study is the first attempt to define the
mechanism of LIF action in implantation. Using multiple approaches and
LIF(-/-) mice, we herein demonstrate that the
expression of several members of the EGF family is down-regulated in
LIF-deficient uteri during implantation, although their receptors
(erbBs) are correctly expressed. Furthermore,
COX-2, which is expressed in an implantation-specific manner
and essential for implantation (32, 33), is aberrantly expressed in the
LIF(-/-) uteri during implantation. The
deficiency of EGF-like growth factors and abnormal expression of
COX-2 may, in part, be responsible for implantation failure
in LIF(-/-) mice.
 |
RESULTS
|
---|
LIF Is Spatiotemporally Expressed in the Uterus during the
Periimplantation Period
LIF was reported to be expressed transiently in endometrial glands
on day 4 of pregnancy before implantation, and LIF-deficient mice show
implantation failures (36, 38). However, the mechanism by which LIF
executes its effects on implantation is not yet known. To better
understand its role in implantation, we examined the spatiotemporal
expression of LIF throughout the periimplantation period by
Northern blot and in situ hybridization. As reported
previously (36, 37), Northern blot hybridization detected a single
transcript (4.2 kb), and the levels of this mRNA were higher on days 1
and 4 of pregnancy (data not shown). However, analysis of in
situ hybridization experiments showed unique expression pattern of
LIF mRNA. Distinct LIF mRNA accumulation was
noted in the luminal epithelium on day 1 and in the glandular
epithelium on day 4 of pregnancy (Fig. 1
, a and d). However, contrary to the previous report of transient
expression before implantation (36), LIF mRNA was clearly
detected in the glandular epithelium and also in subluminal stromal
cells surrounding the implanting blastocyst on day 5, but not
thereafter (Fig. 1
, e and f). To determine whether the presence of an
active blastocyst is necessary for the induction of LIF,
delayed implantation model was employed. As expected, LIF
was not induced in the P4-primed uterus during
delayed implantation (Fig. 2a
). However,
LIF was rapidly expressed in the glandular epithelium
within 12 h of an estrogen injection that terminated the delayed
implantation with blastocyst activation (Fig. 2b
). Furthermore, as
observed during normal implantation on day 5, LIF mRNA
accumulation occurred in stromal cells surrounding the blastocyst at
24 h of an estrogen injection (Fig. 2
, c and d). These results add
new information that LIF is not only expressed in the uterus before the
initiation of implantation, but the expression persists in the glands
and appears in the stromal cells surrounding the implanting blastocyst.
This expression pattern suggests that LIF could be closely
associated with other implantation-specific genes, such as EGF-like
growth factors and COX-2, that are also induced by active
blastocysts before or around the time of the attachment reaction.

View larger version (199K):
[in this window]
[in a new window]
|
Figure 1. Spatiotemporal Expression of LIF
during the Periimplantation Period
In situ hybridization of LIF mRNA in
uterine sections on days 1 (a), 2 (b), 3 (c), 4 (d), 5 (e), and 6 (f)
are shown under darkfield at 40x. le, Luminal epithelium; s, stroma;
myo, myometrium; am, antimesometrial side; m, mesometrial side.
Arrowheads and arrows indicate the
location of glands and blastocysts, respectively. These experiments
were repeated using three mice for each day of pregnancy with similar
results.
|
|

View larger version (71K):
[in this window]
[in a new window]
|
Figure 2. In Situ Hybridization of
LIF mRNA in Uteri of P4-Primed, Delayed
Implanting Mice before and after E2 Treatment
Darkfield photomicrographs of representative longitudinal uterine
sections are shown at 40x (ac) or 100x (d). a, Uterine section from
a delayed implanting mouse treated with P4 from days 57
and killed 24 h after the last injection. b and c, Sections of
uteri from P4-treated, delayed implanting mice killed 12
and 24 h after an E2 injection, respectively. d,
Higher magnification of c. Arrowheads and
arrows indicate the location of glands and blastocysts,
respectively. le, Luminal epithelium; s, stroma; myo, myometrium. These
experiments were repeated with three mice in each group with similar
results.
|
|
Uterine Cell Proliferation in LIF(-/-)
Mice Is Normal
The uterus is comprised of heterogeneous cell types that respond
differentially to estrogen and P4. In the mouse,
the establishment of a receptive uterus for supporting implantation is
regulated by coordinated effects of P4 and
estrogen (50). Preovulatory estrogen directs proliferation of
epithelial cells on days 1 and 2 of pregnancy. In contrast, rising
levels of P4 from newly formed corpora lutea from
day 3 onward direct stromal cell proliferation, which is further
potentiated by ovarian estrogen secretion on the morning of day 4. At
this time, the luminal epithelium ceases proliferating and becomes
differentiated for its interactions with the blastocyst for the
attachment reaction.
We suspected that the failure of implantation in
LIF(-/-) mice could arise from uterine
delinquency to attain receptivity resulting from abnormal proliferation
and/or differentiation. Thus, we compared uterine cell proliferation
profiles between the wild-type and LIF(-/-)
mice on day 4 of pregnancy by proliferating cell nuclear antigen (PCNA)
immunostaining (data not shown) and nuclear
[3H]thymidine incorporation (Fig. 3A
). As previously observed (50),
proliferation was restricted to stromal cells, and no difference in
proliferation pattern was noted between the wild-type and
LIF(-/-) mice. These data suggest that uterine
LIF expression on days 1 or 4 is not essential for uterine cell
proliferation and differentiation in response to steroid hormones
during the preimplantation period.

View larger version (83K):
[in this window]
[in a new window]
|
Figure 3. Uterine Responsiveness to Ovarian Steroids and
Expression of Angiogenic Factors in Wild-Type and
LIF(-/-) Mice
A, Nuclear [3H]thymidine (3H-thy)
incorporation in day 4 pregnant uteri of wild-type and
LIF(-/-) mice. B, In
situ hybridization of VEGF and
Flk-1 mRNAs in day 4 pregnant uteri of wild-type and
LIF(-/-) mice. C, In
situ hybridization of P4-responsive
(Ar and Hoxa-10) or estrogen-responsive
(PR and LF) genes in ovariectomized
wild-type and LIF(-/-) mice after
E2 or P4 treatment. Mice were injected with
E2 (100 ng/mouse) or P4 (2 mg/mouse) 2 weeks
after ovariectomy. E2-treated mice were killed at 24
h, while those treated with P4 were killed at 8 h.
Patterns of nuclear [3H]thymidine incorporation and gene
expression are shown under darkfield at 40x. ge, Glandular epithelium;
le, luminal epithelium; s, stroma; myo, myometrium. These experiments
were repeated in three mice with similar results.
|
|
Angiogenic Factors Are Normally Expressed in
LIF(-/-) Uteri
In the adult, angiogenesis occurs primarily in the ovary and
uterus during cycles and pregnancy. Increased uterine vascular
permeability and angiogenesis are critical to the process of
implantation and decidualization (51). Vascular endothelial growth
factor (VEGF) is an inducer of vascular permeability and a potent
angiogenic factor. VEGF mediates these effects by activating its
receptors, Flt-1 and Flk-1 (reviewed in Ref. 52). While activation of
Flk-1 is involved in vascular permeability changes, both Flt-1 and
Flk-1 are involved in angiogenesis. We surmised that implantation
failure in LIF(-/-) mice could be due to
defective uterine vascular permeability and/or angiogenesis. However,
uterine expression patterns of VEGF and Flk-1
were normal in LIF(-/-) mice on day 4 of
pregnancy (Fig. 3B
), suggesting that the failure of uterine response to
implantation was not the result of abnormal uterine vascular
permeability or angiogenesis before the attachment
reaction.
Steroid-Responsive Genes Are Normally Expressed in
LIF(-/-) Mice
We speculated that the implantation defect in
LIF(-/-) mice could result from altered
responsiveness of the uterus to steroids. Thus, we compared the
expression of several steroid-responsive genes in uteri of
ovariectomized LIF(-/-) mice with that of
wild-type mice. Lactoferrin (LF) and progesterone
receptor (PR) are estrogen-responsive genes, while Ar
and Hoxa-10 are P4-responsive genes
(28, 53, 54). The expression pattern of LF to estrogen and
of Ar to P4 in the uterine epithelium
of both wild-type and LIF(-/-) mice was
similar, although the level of expression of Ar in the
ovariectomized uteri was little lower in response to
P4 than that of the day 4 pregnant wild-type
uterus (see Fig. 3C
vs. Fig. 5
). This reduced response to
P4 could be due to lower uterine PR levels in
ovariectomized mice devoid of estrogen which is required for PR
induction. However, PR expression pattern in both the
epithelium and stroma in response to estrogen was also normal in
LIF(-/-) uteri, and so is the stromal cell
expression of Hoxa-10 to P4 (Fig. 3C
).
These results show that ovariectomized LIF(-/-)
uteri respond to steroid hormones normally with respect to specific
marker genes. Although these genes are normally expressed in the
ovariectomized LIF(-/-) uteri by estrogen and
P4, we sought to know whether these and other
implantation-related genes are correctly expressed in the
LIF(-/-) uteri during the preimplantation
period. The results are described below.

View larger version (138K):
[in this window]
[in a new window]
|
Figure 5. Altered Uterine Expression of EGF-Like Growth
Factors around the Time of Implantation in Wild-Type and
LIF(-/-) Mice
Photomicrographs of representative uterine sections showing in
situ hybridization of Ar, HB-EGF,
and epiregulin mRNAs on day 4 or 5 of pregnancy in
wild-type and LIF(-/-) mice are shown
at 40x (ad) or 100x (ep). Note the absence of hybridization
signals for Ar, HB-EGF, and
epiregulin in uteri of LIF(-/-) mice.
Arrows indicate the location of blastocysts. le, Luminal
epithelium; ge, glandular epithelium; s, stroma; myo, myometrium. These
experiments were repeated using three mice in each group with similar
results.
|
|
Expression of Estrogen Receptor-
(ER-
), PR, and
Hoxa-10 Is Normal in LIF(-/-)
Uterus
ER-
and PR are expressed in the mouse uterus in a cell-specific
manner during the periimplantation period (55), and these receptors are
essential for normal fertility in mice, since female mice deficient for
either ER-
or PR are infertile (56, 57). To examine whether
these two nuclear receptors are correctly expressed in the uterus of
LIF(-/-) mice, we compared their localization
patterns in LIF(-/-) uteri with those of
wild-type uteri on day 4 of pregnancy. No significant differences were
noted between the two groups (Fig. 4
).
Hoxa-10 is a P4-responsive gene and
expressed in stromal cells on day 4 of pregnancy. The deletion of this
gene by null mutation results in female infertility in mice resulting
from defective decidualization (58). Uterine expression of
Hoxa-10 was normal in LIF(-/-) mice
on day 4 of pregnancy (Fig. 4
). These results suggest that the primary
steroid responsive machinery is apparently normal and that any
aberration in uterine gene expression in LIF-deficient mice should not
result from the delinquency of the uterus to respond to steroid
hormones. With this data in hand, we examined the expression of EGF
family of growth factors and receptors in uteri of
LIF(-/-) mice.

View larger version (144K):
[in this window]
[in a new window]
|
Figure 4. Expression of Steroid Receptors and
Hoxa-10 in Pregnant Wild-Type and
LIF(-/-) Mice
Immunolocalization of ER- and PR and in situ
hybridization of Hoxa-10 mRNA in day 4 pregnant
wild-type and LIF(-/-) uteri are shown
under brightfield (200x) and darkfield (40x), respectively.
Red deposits indicate positive nuclear immunostaining
for ER- or PR. le, Luminal epithelium; ge, glandular epithelium; s,
stroma; myo, myometrium. These experiments were repeated in four mice
with similar results.
|
|
Uterine Expression of EGF Family of Growth Factors in the Uterus Is
Down-Regulated in LIF (-/-) Mice
We have previously suggested that EGF-like growth factors act as
local mediators for uterine preparation and embryo-uterine interactions
during implantation (4, 25, 28, 29, 30). Thus, Ar, which is
expressed in the entire epithelium on day 4, is considered to be
important for uterine preparation, while HB-EGF, because of its unique
spatiotemporal localization and effects on blastocyst functions, is
considered to be involved in embryo-uterine interactions during the
preattachment and attachment periods (4, 31, 59). In contrast,
epiregulin, because of its expression during the attachment reaction,
is also likely to be involved in the attachment reaction
(29). Thus, we investigated the expression of Ar,
HB-EGF, and epiregulin in
LIF(-/-) mice. Ar was expressed in
the luminal and glandular epithelia of wild-type uteri, but its
expression was not detectable in LIF(-/-) uteri
on the morning of day 4 of pregnancy (Fig. 5
, a and b vs. c and d). It
should, however, be recalled that Ar was induced in
ovariectomized LIF(-/-) uteri after
P4 treatment, albeit at lower levels. It is
possible that plasma levels of P4 are different
in LIF(-/-) mice than those in wild-type mice,
resulting in the down-regulation of P4-responsive
Ar expression. However, the plasma levels of
P4 were comparable between the two groups (data
not shown), suggesting that the repression of Ar expression
on day 4 of pregnancy is not the result of altered
P4 levels, but may result from lack or presence
of other factors in LIF(-/-) pregnant mice.
HB-EGF is the first known molecular marker that is induced in the
uterine luminal epithelium exclusively at the site of blastocyst
apposition several hours before the attachment reaction (4) and is
known to interact with EGF receptors expressed in the blastocyst in
paracrine and juxtacrine manners (4, 31, 59). This gene was not induced
in the luminal epithelium at the site of blastocyst apposition in
LIF(-/-) mice at the anticipated time (1800 h)
before the attachment reaction on day 4 (compare Fig. 5
, e and f
vs. g and h) or even on the morning (0900 h) of day 5 of
pregnancy (compare Fig. 5
, i and j vs. k and l). However,
HB-EGF was expressed in the luminal epithelium on day 1 of
pregnancy or in the brain of LIF(-/-)
mice in a similar manner as observed in wild-type mice (data not
shown). Epiregulin, which is expressed exclusively in the
luminal epithelium and underlying stroma adjacent to the implanting
blastocyst at the time of the attachment reaction (22002300 h) on day
4 and thereafter on day 5 (27), was also not induced on day 5 of
pregnancy in LIF(-/-) mice (compare Fig. 5
, m
and n vs. o and p). In contrast, TGF-
(another
member of the EGF family), which is expressed in the uterus during the
entire periimplantation period, showed similar pattern of expression in
both the wild-type and LIF(-/-) mice on day 4
(data not shown). Collectively, the results suggest that implantation
failure in LIF(-/-) mice could result from
sequential deficits of specific EGF-like growth factors in the uterus
around the time of implantation. This finding led us to examine whether
EGF receptor family members (erbBs) are correctly expressed
in LIF(-/-) uteri during early pregnancy.
Uterine erbBs Are Correctly Expressed and Responsive to
EGF-Like Ligands in LIF(-/-) Mice
We examined the expression patterns of erbBs and
phosphorylation status of ErbB1 in response to ligands in
LIF(-/-) mice on day 4 of pregnancy. To our
surprise, the expression profiles of erbBs in
LIF(-/-) uteri were similar to those in
wild-type uteri (Fig. 6A
). As noted in
wild-type mice, erbB1 mRNA was normally expressed in major
uterine cell types of LIF(-/-) mice. Similarly,
erbB2 and erbB3 mRNAs, which are expressed
predominantly in the epithelium (25, 30), were correctly expressed in
LIF(-/-) uteri (Fig. 6A
). Normally,
erbB4 mRNA is primarily expressed in the submyometrial
stroma and myometrial connective tissues with low levels of expression
in epithelial cells and other areas of the stroma (30). Again, no
aberrant expression of erbB4 was noted in LIF-deficient
uteri (Fig. 6A
).

View larger version (82K):
[in this window]
[in a new window]
|
Figure 6. Expression and Phosphorylation of erbBs in
LIF(-/-) Mice
A, In situ hybridization of the
erbBs (erbB1, erbB2,
erbB3, and erbB4) in uteri of day 4
pregnant wild-type and LIF(-/-) mice.
Photomicrographs of representative uterine sections are shown under
darkfield at 40x. le, Luminal epithelium; ge, glandular epithelium; s,
stroma; myo, myometrium. These experiments were repeated two times
using independent samples with similar results. B, Phosphorylation of
uterine ErbB1 by EGF-like ligands. Phosphorylation of ErbB1 was
determined using day 4 uterine membranes of wild-type or
LIF(-/-) mice after preincubation with
100 ng/ml of EGF, HB-EGF, or betacellulin, or without these ligands
(control). The labeling reaction was initiated by the addition of
[ 32P] ATP. After labeling, immunoprecipitation was
performed with antibodies to ErbB1. Immunoprecipitates were separated
by SDS-PAGE, transferred to nitrocellulose membrane, and detected by
autoradiography. These experiments were repeated two times with similar
results.
|
|
This finding led us to determine whether these receptors undergo
phosphorylation in response to ligands in
LIF(-/-) uteri (Fig. 6B
). As a prototypic
response, day 4 pregnant uterine membranes were used to determine
phosphorylation of ErbB1 in response to EGF, HB-EGF, and betacellulin.
All of these ligands induced phosphorylation of ErbB1 in uterine
membranes of either wild-type or LIF(-/-) mice,
suggesting that ErbB1 and perhaps other members are biologically
active.
COX-2, but not COX-1, Is Aberrantly
Expressed in Uteri of LIF (-/-) Mice
PGs are considered important for mediating increased localized
endometrial vascular permeability during implantation and
decidualization (reviewed in Ref. 32). COXs, which exist in two
isoforms, COX-1 and COX-2, are the rate-limiting enzymes in PG
biosynthesis. We have shown that COX-2(-/-),
but not COX-1(-/-), female mice show multiple
reproductive defects including ovulation, fertilization,
implantation, and decidualization (33). COX-1 and
COX-2 genes are differentially expressed in the
periimplantation mouse uterus. COX-1 is expressed in the
luminal epithelium irrespective of the location of blastocysts on the
morning of day 4 of pregnancy, but this expression disappears during
the attachment reaction. In contrast, COX-2 is exclusively
induced in the uterine luminal epithelium and subluminal stromal cells
surrounding the blastocyst during the attachment reaction but is not
induced in the presence of dormant blastocysts during delayed
implantation (32). We examined COX-1 and COX-2
expression in LIF(-/-) mice on days 4 and 5 of
pregnancy, respectively, to assess whether implantation failures in
LIF(-/-) mice could result from abnormal
expression of COX isoforms. While COX-1
expression on day 4 was normal in LIF(-/-) mice
(compare Fig. 7
, a vs. b),
COX-2 expression on day 5 was aberrant, i.e. the
expression in the luminal epithelium was present, but the expression in
underlying stromal cells surrounding a blastocyst was absent in
LIF(-/-) mice (compare Fig. 7
, c and e
vs. d and f). These results suggest that aberrant expression
of COX-2 contributes to the failure of implantation and
decidualization in LIF-deficient mice.

View larger version (151K):
[in this window]
[in a new window]
|
Figure 7. Uterine Expression of COX-1 and
COX-2 in Wild-Type and
LIF(-/-) Mice
In situ hybridization of COX-1 and
COX-2 on days 4 and 5 of pregnancy, respectively, is
shown in wild-type and LIF(-/-) uterine
sections. Photomicrographs of representative uterine sections are shown
at 40x (a and b) or 100x (cf). Arrows indicate the
location of blastocysts. Note the absence of hybridization signals for
COX-2 in stroma adjacent to the blastocyst in
LIF(-/-) mice. le, Luminal epithelium;
s, stroma; myo, myometrium. These experiments were repeated two times
with similar results.
|
|
LIF Partially Replaces Preimplantation Ovarian Estrogen
Required for Implantation
Our results show that LIF is not induced in
P4-treated, delayed implantation mouse uterus,
but is readily induced by an injection of E2.
Thus, it can be assumed that implantation-inducing effects of estrogen
are mediated via uterine induction of LIF. However, our results using a
delayed implantation model show that LIF can partially replace estrogen
required for implantation. While LIF given systemically by constant
infusion (5 ng/h or 10 ng/h, sc) by miniosmotic pumps for a period of
48 h or 72 h (n = 8) failed to induce implantation in
P4-treated, delayed-implanting wild-type mice in
the absence of estrogen, a bolus ip injection of LIF (2 µg/mouse)
showed few implantations (2.7 ± 0.7) in only 3 of 12 similarly
treated mice. Moreover, two of these three mice each had implantation
sites in only one uterine horn. A lower dose of LIF (1 µ/mouse)
showed no effects on implantation (n = 5). Unimplanted embryos
with morphological appearance of dormant blastocysts were recovered
from uterine horns that did not show any sign of implantation. In
contrast, a sc injection of E2 (3 ng or 10
ng/mouse) induced expected number of implantation sites (3 ng: 6.0
± 0.8; 10 ng: 7.4 ± 0.7) in all of the
P4-treated, delayed-implantation mice (n = 5
in each group). These results suggest that although LIF can partially
replace estrogen required for implantation, uterine LIF induction is
not the only function of estrogen in implantation.
 |
DISCUSSION
|
---|
It has recently been recognized that there are close interactions
between steroid hormones and local factors during implantation. These
local factors are generated by the uterus or embryo independently or
cooperatively under the guidance of steroid hormones. Recent progress
in molecular biology and genetics has significantly contributed to our
understanding on the roles of locally derived peptide growth factors
and cytokines in embryo-uterine interactions during implantation and
decidualization (4, 25, 28, 29, 30, 31, 38, 59, 60, 61, 62). Expression and gene
targeting studies have established LIF as one of the uterine cytokines
that is critical to the implantation process (36, 38). However, the
molecular mechanism by which LIF executes its effects on implantation
is not yet known. Our present results show that before and during
implantation LIF is expressed in a biphasic manner; first in uterine
glands on day 4 morning and then again in stromal cells surrounding the
implanting blastocyst on day 5. This suggests that LIF participates in
both uterine preparation for implantation and attachment reaction.
However, normal cell type-specific proliferation/differentiation and
expression of angiogenic factors and several other implantation-related
genes in LIF(-/-) uteri on the morning of day 4
of pregnancy suggest that the first phase of LIF expression
in uterine glands for preparation of the uterine receptivity is not as
critical as the second phase of stromal LIF expression
during implantation. In contrast, aberrant expression of Ar,
HB-EGF, epiregulin, and COX-2 during
the preattachment and attachment phases in LIF(-/-) uteri suggests
that the second phase of stromal LIF expression is more critical for
implantation. This is supported by our observation of stromal cell
induction of LIF surrounding activated blastocysts within 24 h of
an estrogen injection in P4-primed,
delayed-implanting mice.
The expression of Ar in the epithelium on day 4 followed by
HB-EGF expression in the luminal epithelium surrounding the
blastocyst during the preattachment and attachment periods, and of
epiregulin in the luminal epithelium and underlying stromal
cells at the sites of blastocysts during the attachment period,
suggests their distinct and overlapping functions in implantation. The
down-regulation of these genes in LIF(-/-)
uteri during these periods raises interesting questions. Is this
down-regulation due to direct effects of LIF deficiency or the
consequence of implantation failure from LIF deficiency or
down-regulation of one member in the absence of LIF leads to repression
of other members? Answers to these questions will require experiments
to rescue implantation in LIF-deficient mice by sequential replacement
of these growth factors singly or in combination.
PGs have long been considered important for implantation.
Recently, expression and gene deletion studies have established that
COX-2-derived PGI2 is essential for implantation
and decidualization (33, 34). COX-2 induction in the luminal
epithelium and underlying stroma at the site of the attachment reaction
requires the presence of an active blastocyst, since this expression is
absent at the sites of dormant blastocysts during delayed implantation,
but reappears with blastocyst activation after termination of the delay
by estrogen (32). Implantation failure in
LIF(-/-) mice reassembles delayed implantation
with morphologically appearing dormant blastocysts (38). Our present
observation of COX-2 expression exclusively in the luminal
epithelium, but not in the underlying stroma, at the sites of
blastocysts in LIF(-/-) mice on day 5 suggests
that the conditions of delayed implantation in the absence of LIF are
different from those produced experimentally in wild-type mice by
ovariectomy and P4 treatment. This is an
intriguing observation and suggests that uterine and blastocyst
quiescence produced by estrogen deficiency is different from that
executed by LIF deficiency.
PGs can mediate their effects via G protein-coupled cell
surface receptors or peroxisome proliferator-activated receptors
(PPARs), members of the steroid hormone nuclear receptor superfamily
(63, 64). Gene targeting experiments have shown that three of the four
PGE2 cell surface receptor subtypes
(EP1, EP2, and
EP3) are not critical for implantation (65, 66), while EP4 deficiency results in embryonic
lethality and thus its role in implantation remains unknown (67).
Nonetheless, EP4 is normally expressed in
LIF(-/-) uteri on day 4 of pregnancy (data not
shown). Furthermore, mice deficient in PGI2 cell
surface receptor (IP) or PGF2
receptor (FP) do
not exhibit overt implantation defects (68, 69). In contrast, we have
recently shown that COX-2-derived PGI2 can induce
implantation via activation of PPAR
in
COX-2(-/-) mice (34), suggesting that
PGI2 mediates its effects on implantation via
activation of a nuclear receptor. Normally, PPAR
is
expressed in stromal cells and becomes more localized surrounding the
blastocyst with the initiation of the attachment reaction (34).
However, the expression of PPAR
remains diffuse in the
stroma surrounding a blastocyst in the LIF(-/-)
uterus on day 5 of pregnancy (data not shown). Collectively, these
results suggest that COX-2-coupled nuclear PG signaling is defective in
the LIF-deficient uteri during early pregnancy. Again, the cause and
effect relationship between aberrant COX-2 expression and
LIF deficiency is not clear. Future experiments to rescue implantation
in LIF(-/-) mice with
PGI2 or PPAR
agonists will be required to
address this question.
Gene deletion experiments have demonstrated that LIF, COX-2, and
Hoxa-10 are each essential for implantation and/or
decidualization. The hierarchical arrangement of these molecules in
directing uterine functions during implantation is not clearly
understood. However, it is interesting to note that while
COX-2 expression is aberrant in
LIF(-/-) uteri, uterine LIF
expression is normal in COX-2(-/-) mice (33).
This suggests that LIF functions upstream of COX-2 in implantation.
Interestingly, LIF expression is normal in
Hoxa-10(-/-) uteri (58) and Hoxa-10
expression is normal in LIF(-/-) uteri during
the preimplantation period (Fig. 4
), insinuating that the regulation of
these genes is independent of each other. However, COX-2
expression is down-regulated in Hoxa-10(-/-)
uteri. Collectively, these results raise the following issues. First,
it is possible that the information sequentially originating from the
LIF, EGF-like growth factor, and Hoxa-10 signaling pathways finally
converges to the COX-2 pathway for implantation. Second, each pathway
may stand on its own right and has distinctive functions in
implantation. Thus, disruption of one pathway will lead to implantation
failure, and apparent dysregulation of other pathways could be the
consequence of implantation failure. Finally, two of the three pathways
may be interconnected, and interruption of either pathway will lead to
implantation failure. These complex relationships may hinder rescue of
implantation defects by supplementation of specific factors.
LIF is synthesized in soluble and matrix-associated forms and
was shown to be transiently expressed in the glandular epithelium on
day 4 of pregnancy in mice (36, 37). As we show herein, LIF
is also expressed in the stroma at the sites of blastocysts during the
attachment reaction. The targets for LIF action during the
preattachment and attachment periods are not fully understood. However,
the results of reciprocal embryo transfer experiments and failure to
induce deciduoma in LIF(-/-) mice suggest that
LIF targets its own creator for implantation, but the exact site of
action is not known. Spatiotemporal expression of LIFR and
gp130 in the uterus during implantation is required to
address this issue. Previous observations (36, 37) and our present
finding with delayed implantation show that estrogen is an absolute
requirement for LIF induction in the mouse uterus. Is it
then possible that LIF replaces estrogen as a down stream effector for
implantation? Our present results show that LIF can only partially
replace nidatory estrogen for implantation, suggesting that
LIF induction is one of many functions estrogen exerts
during implantation. However, it is possible that systemic delivery of
LIF did not reach the target in appropriate time and concentration for
exerting its full effects. In conclusion, the present investigation
provides evidence for relative importance of biphasic expression of
uterine LIF in implantation and its interactions with other
implantation-specific genes.
 |
MATERIALS AND METHODS
|
---|
Mice
Adult CD-1 wild-type (Charles River Laboratories, Inc., Wilmington, MA) and LIF(+/-)
mice on a mixed background were housed in the Animal Care Facility at
the University of Kansas Medical Center according to NIH and
institutional guidelines for laboratory animals.
LIF(+/-) mice were kindly provided by Story
Landis (NINDS/NIH). These mice were originally generated by Philipe
Brulet (Pasteur Institute, Paris, France). The disruption of the
LIF gene was achieved in (129/Sv) ES cells by homologous
recombination as described (70). PCR analysis of tail genomic DNA was
used for genotyping of mutant mice. CD-1 and
LIF(-/-) mice were mated with fertile or
vasectomized males of the same strain to induce pregnancy or
pseudopregnancy, respectively. The morning of finding a vaginal plug
was designated day 1 of pregnancy. Implantation sites on day 5 were
visualized by iv injection (0.1 ml/mouse) of Chicago Blue dye solution
in 0.1% saline. To induce delayed implantation, mice were
ovariectomized on the morning (0900 h) of day 4 of pregnancy and
maintained with daily injections of progesterone
(P4, 2 mg/mouse) from days 57. To initiate
implantation, P4-primed, delayed-implanting
pregnant mice were injected with 17ß-estradiol
(E2, 25 ng/mouse) (71). All steroids were
dissolved in sesame oil and injected subcutaneously (0.1 ml/mouse).
Uterine tissues were processed for various analyses.
Systemic Administration of LIF in Delayed-Implanting Wild-Type
Mice
Miniosmotic pumps (Alzet Corp., Palo Alto, CA) containing LIF
(Life Technologies, Inc., Gaithersburg, MD) at a constant
infusion rate of 5 ng/h or 10 ng/h were placed under the back skin on
the third day of P4-treated delayed implantation
(day 7) as described above and continued for 48 h or 72 h.
Another batch of P4-treated, delayed-implanting
mice were given a single injection of E2 (3 ng or
10 ng/mouse) or LIF (1 µg or 2 µg/mouse) on the second day (day 6)
of the delay and killed 48 h later. P4 (2
mg/mouse) treatment was continued throughout the experimental period.
At the termination of the experiments, implantation sites were examined
by the blue dye method (71). If implantation sites (blue bands)
were absent, uterine horns were flushed with saline to recover
unimplanted blastocysts. Mice without implantation sites or blastocysts
were excluded from the experiments.
Uterine Responsiveness to E2 and
P4 in LIF(-/-)
Mice
To determine whether the LIF(-/-) uteri respond to
E2 and/or P4, wild-type and
LIF(-/-) mice were ovariectomized irrespective of the
stage of the estrous cycle and rested for 2 weeks. They were given an
injection of E2 (100 ng/mouse) or
P4 (2 mg/mouse). The control mice received oil
(0.1 ml/mouse) alone. They were killed at different times, and uteri
were collected for in situ hybridization to study specific
gene expression.
To examine uterine cell-specific proliferation on day 4 of pregnancy
under the influence of P4 and estrogen, day 4
pregnant wild-type or LIF(-/-) mice received an injection
(ip) of [methyl-3H]thymidine (25 µCi/0.1 ml,
NEN Life Science Products, Boston, MA) at 0900 h on
day 4 and were killed 2 h later. Nuclear uptake of
[3H]thymidine was detected in uterine sections
by autoradiography (33).
Antibodies
The affinity-purified rabbit polyclonal antibody, C1355 for rat
ER-
, was kindly provided by Margaret Shupnik (University of Virginia
Medical Center, Charlottesville, VA). This antibody was raised against
a peptide for the last 14 amino acids in the C-terminal end of the rat
ER-
(72). Mouse monoclonal antihuman PR was purchased from
Zymed Laboratories, Inc. (South San Francisco, CA). This
antibody was developed against a peptide in the N-terminal proline-rich
region of human PR. Both ER-
and PR antibodies cross-react with
corresponding mouse proteins. Mouse monoclonal antibody for PCNA was
purchased from Sigma (St. Louis, MO). These antibodies
were used for immunohistochemistry.
Immunohistochemistry
Uteri were collected from wild-type and LIF(-/-)
mice on day 4 of pregnancy, fixed in 10% neutral formalin, and
embedded in paraffin. After dehydration, nuclear antigens were
retrieved by microwave processing with 10 mM
Na-citrate (pH 6.0) for 8 min followed by washings (5 min each x
2) in PBS. Sections were processed for immunohistochemical staining for
ER-
, PR, and PCNA using Histostain-SP Kits (Zymed Laboratories, Inc., South San Francisco, CA) appropriate for the
respective antibodies (55). After immunostaining, sections were lightly
counterstained with Fast Green. Red deposits indicated the sites of
immunoreactive proteins.
Hybridization Probes
Sense or antisense 35S-labeled cRNA probes
were generated using appropriate polymerases from cDNAs to
LIF, Ar, HB-EGF,
epiregulin, erbBs (erbB1,
erbB2, erbB3, erbB4),
Hoxa-10, COX-1, COX-2,
Flk-1, VEGF, LF, PR,
PGE2 receptor subtype
(EP4), and PPAR
for in
situ hybridization as described previously (4, 25, 29, 30, 33, 34). For Northern hybridization, antisense
32P-labeled cRNA probe for LIF was
generated. The probes had specific activities of
2 x
109 dpm/µg.
In Situ Hybridization
In situ hybridization was performed as described
previously (4, 33). Small pieces of tissues were flash-frozen in liquid
Histo-Freeze (Fisher Scientific, Pittsburgh, PA). Frozen
sections (11 µm) were mounted onto
poly-L-lysine-coated slides, fixed in cold 4%
paraformaldehyde solution in PBS, acetylated, and hybridized at 45 C
for 4 h in hybridization buffer containing the
35S-labeled antisense cRNA probes. After
hybridization, the sections were incubated with ribonuclease A (RNase
A, 20 µg/ml) at 37 C for 20 min. RNase A-resistant hybrids were
detected by autoradiography using NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Sections hybridized with sense probes
served as negative controls.
Northern Blot Hybridization
Total RNA were extracted from whole uteri pooled from 710 mice
on the indicated days of pregnancy by a modified guanidine thiocyanate
procedure. Polyadenylated [poly (A+)] RNAs were
isolated from total RNAs by oligo(dT)-cellulose column chromatography
as previously described (55). Poly (A+) RNA (2.0
µg) was denatured, separated by formaldehyde-agarose gel
electrophoresis, and transferred to nylon membranes. RNA was
cross-linked to the membranes by UV irradiation (Spectrolinker,
XL-1500; Spectronics Corp., Westbury, NY), and the blots were
prehybridized, hybridized, and washed as previously described by us
(4). The hybrids were detected by autoradiography.
Autophosphorylation of Uterine ErbB1 in
LIF(-/-) Mice
The autophosphorylation of ErbB1 was studied in day 4 pregnant
uterine membranes of wild-type or LIF(-/-) mice
by the protocol as previously described (25, 30). Membranes (150 µg
protein) were suspended in 50 ml phosphorylation reaction buffer
containing 0.1 mM Na-vanadate and preincubated
with or without a specific ligand (100 ng/ml) for 10 min at 4 C. The
labeling reaction was performed for 2 min at 4 C after addition of 5
µCi[
32P]ATP (1 mM).
The reaction was terminated by the addition of 15 µl of an ice-cold
mixture of 1 mM ATP and 0.1% BSA followed by an
equal volume (wt/vol) of 10% trichloroacetic acid. After incubation on
ice for 1 h, the mixture was centrifuged. The precipitate was
washed with a mixture of diethylether and ethanol (1:1) and suspended
in 50 µl of 50 mM Tris buffer (pH 7.5). An
equal volume of protein A/Sepharose-antibody conjugate (3 mg: 0.5 mg)
was added to this mixture and incubated for 90 min at 4 C. The protein
A/Sepharose-antibody conjugates were washed sequentially with buffer A
(50 mM HEPES, 0.1% Triton X-100, 0.1% SDS, 5
mM EGTA, pH 8.0), buffer B (50
mM HEPES, 0.1% Triton X-100, 0.1% SDS, 150
mM NaCl, pH 8.0), and buffer C (10
mM Tris-HCl, pH 8.0). The pellets were boiled in
1x SDS sample buffer for 3 min and centrifuged. The supernatants were
subjected to 7.5% SDS-PAGE. The gel was transferred to nitrocellulose
membrane, and the products were visualized by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Y. Zhu for her help in genotyping of LIF knock-out
mice.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Sudhansu K. Dey, Department of Molecular and Integrative Physiology, Kansas University Medical Center, 3901 Rainbow Boulevard, MRRC 37/3017, Kansas City, Kansas 66160-7338. E-mail: sdey{at}kumc.edu
This work was supported by NIH Grants HD-12304 and HD-29968, as part of
the National Cooperative Program on Markers of Uterine Receptivity for
Blastocyst Implantation (S. K. Dey), ES 07814 (S. K. Das),
and HD-37394 (B. C. Paria). H. Lim was a Kansas Health Foundation
predoctoral fellow. Center grant in Reproductive Biology (HD-33994) and
Mental Retardation (HD-02528) provided access to various core
facilities. S. K. Dey is a MERIT Awardee of the NICHD/NIH.
Received for publication February 29, 2000.
Revision received April 14, 2000.
Accepted for publication April 18, 2000.
 |
REFERENCES
|
---|
-
Psychoyos A 1973 Endocrine control of egg implantation.
In: Greep RO, Astwood EG, Geiger SR (eds) Handbook of Physiology.
American Physiological Society, Washington DC, pp 187215
-
Paria BC, Huet-Hudson YM, Dey SK 1993 Blastocysts state of
activity determines the "window of implantation in the receptive
mouse uterus. Proc Natl Acad Sci USA 90:1015910162[Abstract]
-
Dey SK 1996 Implantation. In: Adashi EY, Rock JA, Rosenwaks Z
(eds) Reproductive Endocrinology, Surgery and Technology.
Lippincott-Raven Publishers, Philadelphia, pp 421434
-
Das SK, Wang X-N, Paria BC, Damm D, Abraham JA, Klagsbrun M,
Andrews GK, Dey SK 1994 Heparin-binding EGF-like growth factor gene is
induced in the mouse uterus temporally by the blastocyst solely at the
site of its apposition: a possible ligand for interaction with
blastocyst EGF-receptor in implantation. Development 120:10711083[Abstract/Free Full Text]
-
Yoshinaga K 1980 Inhibition of implantation by advancement of
uterine sensitivity and refractoriness. In: Leroy F, Finn CA, Psychoyos
A, Hubinot PO (eds) Blastocyst-Endometrium Relationships: Progress in
Reproductive Biology. Karger, Switzerland, vol 7:189199
-
Yoshinaga K, Adams CE 1966 Delayed implantation in spayed,
progesterone-treated adult mouse. J Reprod Fertil 12:593595[CrossRef][Medline]
-
Hilton DJ 1992 LIF: lots of interesting functions. Trends
Biochem Sci 17:7276[CrossRef][Medline]
-
Metcalf D 1992 Leukemia inhibitory factor a puzzling
polyfunctional regulator. Growth Factors 7:169173[Medline]
-
Shellard J, Perreau J, Brulet P 1996 Role of leukemia
inhibitory factor during mammalian development. Eur Cytokine Network 7:699712[Medline]
-
Dani C, Chambers I, Johnstone S, Robertson M, Ebrahimi B,
Saito M, Taga T, Li M, Burdon T, Nichols J, Smith A 1998 Paracrine
induction of stem cell renewal by LIF-deficient cells: a new ES cell
regulatory pathway. Dev Biol 203:149162[CrossRef][Medline]
-
Kishimoto T, Taga T, Akira S 1994 Cytokine signal
transduction. Cell 76:253262[Medline]
-
Cohen S 1962 Isolation of a mouse submaxillary gland protein
accelerating incisor eruption and eyelid opening in the mouse newborn.
J Biol Chem 237:15551562[Free Full Text]
-
Derynck R, Roberts AB, Winkler ME, Chen YE, Goeddel DV 1984 Human transforming growth factor-
: precursor structure and
expression in E. coli. Cell 38:287297[Medline]
-
Shoyab M, McDonald VL, Bradley JG, Todaro GJ 1988 Amphiregulin: a bifunctional growth-modulating glycoprotein
produced by the phorbol 12-myristate 13-acetate-treated human breast
adenocarcinoma cell line MCF-7. Proc Natl Acad Sci USA 85:65286532[Abstract]
-
Higashiyama S, Abraham JA, Miller J, Fiddes JC, Klagsbrun M 1991 A heparin-binding growth factor secreted by macrophage-like cells
that is related to EGF. Science 251:936939[Medline]
-
Holmes WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, Park JW,
Yansura D, Abadi N, Raab H, Lewis GD, Shepard HM, Kuang W-J, Wood WI,
Goeddel DV, Vandlen RL 1992 Identification of heregulin, a specific
activator of p185erbB2. Science 256:12051210[Medline]
-
Shing Y, Christofori G, Hanahan D, Ono Y, Sasada R, Igarashi
K, Folkman J 1993 Betacellulin: a mitogen from pancreatic ß cell
tumors. Science 259:16041607[Medline]
-
Toyoda H, Komurasaki T, Uchida D, Takayama Y, Isobe T, Okuyama
T, Hanada K 1995 Epiregulin: a novel EGF with mitogenic activity for
rat primary hepatocytes. J Biol Chem 270:74597500
-
Massague J, Pandiella A 1993 Membrane-anchored growth factors.
Annu Rev Biochem 62:515541[CrossRef][Medline]
-
Prigent SA, Lemoine NR 1992 The type I (EGFR-related) family
of growth factor receptors and their ligands. Prog Growth Factor Res 4:124[Medline]
-
Peles E, Yarden Y 1993 Neu and its ligands: from an oncogene
to neural factors. Bioessays 15:815824[Medline]
-
Heldin CH 1995 Dimerization of cell surface receptors in
signal transduction. Cell 80:213223[Medline]
-
Riese IIDJ, Bermingham Y, van Raaij TM, Buckley S, Plowman GD,
Stern DF 1996 Betacellulin activates the epidermal growth factor
receptor and erbB-4 and induces cellular response patterns distinct
from those stimulated by epidermal growth factor or
neuregulin-ß. Oncogene 12:345353[Medline]
-
Elenius K, Paul S, Allison G, Sun J, Klagsbrun M 1997 Activation of HER4 by heparin-binding EGF-like growth factor stimulates
chemotaxis but not proliferation. EMBO J 16:12681278[Abstract/Free Full Text]
-
Lim H, Dey SK, Das SK 1997a Differential expression of the
erbB2 gene in the periimplantation mouse uterus: potential
mediator of signaling by epidermal growth factor-like growth factors.
Endocrinology 138:13281337
-
Hynes NE, Stern DF 1994 The biology of erbB2/neu/HER2 and its
role in cancer. Biochim Biophys Acta 1198:165184[CrossRef][Medline]
-
Earp HS, Dawson TL, Li X, Yu H 1995 Heterodimerization and
functional interaction between EGF receptor family members: a new
signaling paradigm with implications for breast cancer research. Breast
Cancer Res Treat 35:115132[Medline]
-
Das SK, Chakraborty I, Paria BC, Wang X-N, Plowman G, Dey SK 1995 Amphiregulin is an implantation-specific and
progesterone-regulated gene in the mouse uterus. Mol Endocrinol 9:691705[Abstract]
-
Das SK, Das N, Wang J, Lim H, Schryver B, Plowman GD, Dey SK 1997 Expression of betacellulin and epiregulin genes in the mouse
uterus temporally by the blastocyst solely at the site of its
apposition is coincident with the "window" of implantation. Dev
Biol 190:178190[CrossRef][Medline]
-
Lim H, Das SK, Dey SK 1998 erbB genes in the mouse
uterus: cell-specific signaling by epidermal growth factor (EGF) family
of growth factors during implantation. Dev Biol 204:97110[CrossRef][Medline]
-
Raab G, Kover K, Paria BC, Dey SK, Ezzell RM, Klagsbrun M 1996 Mouse preimplantation blastocysts adhere to cells expressing the
transmembrane form of heparin-binding EGF-like growth factor.
Development 122:637645[Abstract/Free Full Text]
-
Chakraborty I, Das SK, Dey SK 1996 Developmental expression of
the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the
peri-implantation mouse uterus and their differential regulation by the
blastocyst and ovarian steroids. J Mol Endocrinol 16:107122[Abstract]
-
Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos
JM, Dey SK 1997b Multiple female reproductive failures in
cyclooxygenase 2-deficient mice. Cell 91:197208
-
Lim H, Gupta RA, Ma W, Paria BC, Moller DE, Morrow JD, DuBois
RN, Trzaskos JM, Dey SK 1999a Cyclo-oxygenase-2-derived prostacyclin
mediates embryo implantation in the mouse via PPAR
. Genes Dev 13:15611574
-
Sharkey A 1998 Cytokines and implantation. Rev Reprod 3:5261[Abstract/Free Full Text]
-
Bhatt H, Brunet LJ, Stewart CL 1991 Uterine expression of
leukemia inhibitory factor coincides with the onset of blastocyst
implantation. Proc Natl Acad Sci USA 88:1140811412[Abstract]
-
Shen MM, Leder P 1992 Leukemia inhibitory factor is expressed
by the preimplantation uterus and selectively blocks primitive ectoderm
formation in vitro. Proc Natl Acad Sci USA 89:82408244[Abstract]
-
Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F,
Abbondanzo SJ 1992 Blastocyst implantation depends on maternal
expression of leukaemia inhibitory factor. Nature 359:7679[CrossRef][Medline]
-
Yang Z-M, Le S-P, Chen D-B, Harper MJK 1994 Temporal and
spatial expression of leukemia inhibitory factor in rabbit uterus
during early pregnancy. Mol Reprod Dev 38:148152[Medline]
-
Song JH, Houde A, Murphy BD 1998 Cloning of leukemia
inhibitory factor (LIF) and its expression in the uterus during
embryonic diapause and implantation in the mink (Mustela
vison). Mol Reprod Dev 51:1321[CrossRef][Medline]
-
Hirzel DJ, Wang J, Das SK, Dey SK, Mead RA 1999 Changes in
uterine expression of leukemia inhibitory factor during pregnancy in
the western spotted skunk. Biol Reprod 60:484492[Abstract/Free Full Text]
-
Vogiagis D, Salamonsen LA 1999 The role of leukaemia
inhibitory factor in the establishment of pregnancy. J Endocrinol 160:181190[Abstract/Free Full Text]
-
Laird SM, Tuckerman EM, Dalton CF, Dunphy BC, Li TC,
Zhang x 1997 The production of leukaemia inhibitory factor by
human endometrium: presence in uterine flushings and production by
cells in culture. Hum Reprod 12:569574[Medline]
-
Piccinni M-P, Beloni L, Livi C, Maggi E, Scarselli G,
Romagnani S 1998 Defective production of both leukemia inhibitory
factor and type 2 T-helper cytokines by decidual T cells in unexplained
recurrent abortions. Nat Med 4:10201024[CrossRef][Medline]
-
Fry RC, Batt PA, Fairclough RJ, Parr RA 1992 Human leukemia
inhibitory factor improves the viability of cultured ovine embryos.
Biol Reprod 46:470474[Abstract]
-
Lavranos TC, Rathjen PD, Seamark RF 1995 Trophic effects of
myeloid leukaemia inhibitory factor (LIF) on mouse embryos. J Reprod
Fertil 105:331338[Abstract]
-
Dunglison GF, Barlow DH, Sargent IL 1996 Leukaemia inhibitory
factor significantly enhances the blastocyst formation rates of human
embryos cultured in serum-free medium. Hum Reprod 11:191196[Abstract]
-
Nichols J, Davidson D, Taga T,Yoshida K, Chambers I, Smith A 1996 Complementary tissue-specific expression of LIF and LIF-receptor
mRNAs in early mouse embryogenesis. Mech Dev 57:123131[CrossRef][Medline]
-
Chen HF, Shew JY, Ho HN, Hsu WL, Yang YS 1999 Expression of
leukemia inhibitory factor and its receptor in preimplantation embryos.
Fertil Steril 72:713719[CrossRef][Medline]
-
Huet-Hudson YM, Andrews GK, Dey SK 1989 Cell-type
specific localization of c-myc protein in the mouse uterus: modulation
by steroid hormones and analysis of the preimplantation period.
Endocrinology 125:16831690[Abstract]
-
Chakraborty I, Das SK, Dey SK 1995 Differential expression of
vascular endothelial growth factor and its receptor mRNAs in the mouse
uterus around the time of implantation. J Endocrinol 147:339352[Abstract]
-
Shibuya M, Ito N, Welsh-Claesson L 1999 Structure and function
of vascular endothelial growth factor receptor-1 and -2. Curr Top
Microbiol Immunol 237:5983[Medline]
-
McMaster MT, Teng CT, Dey SK, Andrews GK 1991 Lactoferrin in
the mouse uterus: analyses of the preimplantation period and regulation
by ovarian steroids. Mol Endocrinol 5:101111
-
Lim H, Ma L, Ma W, Maas RL, Dey SK 1999b Hoxa-10
regulates uterine stromal cell responsiveness to progesterone during
implantation and decidualization in the mouse. Mol Endocrinol 13:10051017
-
Tan J, Paria BC, Dey SK, Das SK 1999 Differential
uterine expression of estrogen and progesterone receptors correlates
with uterine preparation for implantation and decidualization in the
mouse. Endocrinology 140:53105321[Abstract/Free Full Text]
-
Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies
O 1993 Alteration of reproductive function but not prenatal sexual
development after insertional disruption of the mouse estrogen receptor
gene. Proc Natl Acad Sci USA 90:1116211166[Abstract]
-
Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery
Jr CA, Shyamala G, Conneely OM, OMalley BW 1995 Mice lacking
progesterone receptor exhibit pleiotropic reproductive abnormalities.
Genes Dev 9:22662278[Abstract]
-
Benson GV, Lim H, Paria BC, Satokata I, Dey SK, Mass RL 1996 Mechanisms of reduced fertility in Hoxa-10 mutant
mice:uterine homeostasis and loss of maternal Hoxa-10
expression. Development 122:26872696[Abstract/Free Full Text]
-
Paria BC, Elenius K, Klagsbrun M, Dey SK 1999 Heparin-binding
EGF-like growth factor interacts with mouse blastocysts independently
of ErbB1: a possible role for heparan sulfate proteoglycans and ErbB4
in blastocyst implantation. Development 126:19972005[Abstract/Free Full Text]
-
Cross JC, Werb Z, Fisher SJ 1994 Implantation and the
placenta: key pieces of the development puzzle. Science 266:15081518[Medline]
-
Rice A, Chard T 1998 Cytokines in implantation. Cytokine
Growth Factor Rev 9:287296[CrossRef][Medline]
-
Robb L, Li R, Hartley L, Nandurkar HH, Koentgen F, Begley CG 1998 Infertility in female mice lacking the receptor for interleukin 11
is due to a defective uterine response to implantation. Nat Med 4:303308[Medline]
-
Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan
receptors. Cell 83:841850[Medline]
-
Negishi M, Sugimoto Y, Ichikawa A 1995 Molecular mechanism of
diverse actions of prostanoid receptors. Biochim Biophys Acta 1259:109120[Medline]
-
Ushikubi F, Segi E, Sugimoto Y, Murata T, Matsuoka T,
Kobayashi T, Hizaki H, Tuboi K, Katsuyama M, Ichikawa A, Tanaka T,
Yoshida N, Narumiya S 1998 Impaired febrile response in mice lacking
the prostaglandin E receptor subtype EP3. Nature 395:281284[CrossRef][Medline]
-
Kennedy CRJ, Zhang Y, Brandon S, Guan Y, Coffee K, Funk CD,
Magnuson MA, Oates JA, Breyer MD, Breyer RM 1999 Salt-sensitive
hypertension and reduced fertility in mice lacking the prostaglandin
EP2 receptor. Nat Med 5:217220[CrossRef][Medline]
-
Nguyen M, Camenisch T, Snouwaert JN, Hicks E, Coffman TM,
Anderson PAW, Malouf NN, Koller BH 1997 The prostaglandin receptor
EP4 triggers remodelling of the cardiovascular
system at birth. Nature 390:7881[CrossRef][Medline]
-
Murata T, Ushikubi F, Matsuoka T, Hirata M, Yamasaki A,
Sugimoto Y, Ichikawa A, Aze Y, Tanaka T, Yosida N, Ueno A, Oh-ishi S,
Narumiya S 1997 Altered pain perception and inflammatory response in
mice lacking prostacyclin receptor. Nature 388:678682[CrossRef][Medline]
-
Sugimoto Y, Yamasaki A, Segi E, Tsuboi K, Aze Y, Nishimura T,
Oida H, Yoshida N, Tanaka T, Katsuyama M, Hasumoto K, Murata T, Hirata
M, Ushikubi F, Negishi M, Ichikawa A, Narumiya S 1997 Failure of
parturition in mice lacking the prostaglandin F receptor. Science 277:681683[Abstract/Free Full Text]
-
Escary J-L, Perreau J, Dumenil D, Ezine S, Brulet P 1993 Leukaemia inhibitory factor is necessary for maintenance of
haematopoietic stem cells and thymocyte stimulation. Nature 363:361364[CrossRef][Medline]
-
Paria BC, Lim H, Wang X-N, Liehr J, Das SK, Dey SK 1998 Coordination of differential effects of primary estrogen and
catecholestrogen on two distinct targets mediates embryo implantation
in the mouse. Endocrinology 139:52355246[Abstract/Free Full Text]
-
Friend KE, Resnick EM, Ang LW, Shupnik MA 1997 Specific modulation of estrogen receptor mRNA isoforms in rat pituitary
throughout the estrous cycle and in response to steroid hormones. Mol
Cell Endocrinol 131:147155[CrossRef][Medline]