Hoxa-10 Regulates Uterine Stromal Cell Responsiveness to Progesterone during Implantation and Decidualization in the Mouse
Hyunjung Lim,
Liang Ma,
Wen-ge Ma,
Richard L. Maas and
Sudhansu K. Dey
Department of Molecular and Integrative Physiology (H.L.,
W-G.M., S.K.D.) Ralph L. Smith Research Center University of
Kansas Medical Center Kansas City, Kansas 66160-7338
Division of Genetics (L.M., R.L.M.) Brigham and Womens
Hospital and Harvard Medical School Boston, Massachusetts 02115
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ABSTRACT
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Hoxa-10 is an
AbdominalB-like homeobox gene that is expressed in the
developing genitourinary tract during embryogenesis and in the adult
uterus during early pregnancy. Null mutation of Hoxa-10 in
the mouse causes both male and female infertility. Defective
implantation and decidualization resulting from the loss of maternal
Hoxa-10 function in uterine stromal cells is the cause of
female infertility. However, the mechanisms by which
Hoxa-10 regulates these uterine events are unknown.
We have identified two potential mechanisms for these uterine defects
in Hoxa-10(-/-) mice. First, two
PGE2 receptor subtypes,
EP3 and
EP4, are aberrantly expressed in the
uterine stroma in Hoxa-10(-/-) mice, while expression of
several other genes in the stroma (TIMP-2,
MMP-2, ER, and PR) and epithelium
(LIF, HB-EGF, Ar, and
COX-1) are unaffected before implantation. Further,
EP3 and EP4
are inappropriately regulated by progesterone
(P4) in the absence of Hoxa-10, while
PR, Hoxa-11 and c-myc, three other
P4-responsive genes respond normally. These
results suggest that Hoxa-10 specifically mediates
P4 regulation of
EP3 and EP4
in the uterine stroma. Second, since Hox genes are
implicated in local cell proliferation, we also examined
steroid-responsive uterine cell proliferation in
Hoxa-10(-/-) mice. Stromal cell proliferation in mutant
mice in response to P4 and 17ß-estradiol
(E2) was significantly reduced, while
epithelial cell proliferation was normal in response to
E2. These results suggest that stromal cell
responsiveness to P4 with respect to cell
proliferation is impaired in Hoxa-10(-/-) mice, and that
Hoxa-10 is involved in mediating stromal cell proliferation.
Collectively, these results suggest that Hoxa-10 mutation
causes specific stromal cell defects that can lead to implantation and
decidualization defects apparently without perturbing epithelial cell
functions.
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INTRODUCTION
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Hox genes are developmentally regulated transcription
factors belonging to a multigene family. They share in common a highly
conserved sequence element called the homeobox that encodes a 61-amino
acid helix-turn-helix DNA-binding domain. While one Hox
cluster (the HOM-C) is present in Drosophila, four mammalian
Hox clusters (A, B, C, and D) exist on different chromosomes
and have been generated by gene duplication (1). These genes follow a
strict pattern of spatial and temporal colinearity during
embryogenesis. For example, while genes at the 3'-end of each cluster
are activated during early embryogenesis in the anterior region of the
developing embryo, genes located toward the 5'-end are restricted to
posterior regions of the embryo and are expressed during later stages
of embryogenesis (1, 2). AbdominalB (AbdB) is the
most 5'-gene within the Drosophila homeotic complex. In
mammals, several AbdB-like genes exist at the 5'-ends of the
Hox a, c, and d clusters corresponding to paralogous groups
913 (3). The AbdB genes constitute a distinct subfamily of
homeobox genes that exhibit posterior domains of expression including
the genital imaginal disc in Drosophila and the developing
genitourinary system in vertebrates (4, 5).
Hoxa-10 is one AbdominalB-like homoeobox gene
that is located in the Hoxa cluster and expressed in the
developing genitourinary tract during mouse embryogenesis (3). Its
distinct role in development has been defined by gene targeting
experiments (6, 7). Hoxa-10-deficient mice exhibit male and
female infertility along with a homeotic transformation of the lumbar
vertebrae. Although the proximal uterus of Hoxa-10(-/-)
mice shows partial homeosis into an oviduct-like structure, this is not
the major cause of infertility in these mice. Hoxa-10 is
strongly expressed in the stroma and decidua of the pregnant mouse
uterus (6), and decidualization in Hoxa-10(-/-) mice is
severely compromised during blastocyst implantation (8), thus
reflecting a maternal requirement for Hoxa-10 in the
periimplantation uterus. However, the mechanism by which Hoxa-10
regulates uterine stromal cell proliferation and differentiation during
decidualization remains unknown. Recent investigations have revealed
that Hoxa-10 (9, 10) and other AbdB-like
Hoxa genes (9) are regulated by progesterone
(P4) in the mouse and human endometrium. Hoxa-10
is induced in the mouse uterine stroma within 4 h of a
P4 injection in a protein synthesis-independent fashion,
and the up-regulation of Hoxa-10 by P4 is
inhibited by the progesterone receptor (PR) antagonist RU-486,
suggesting a requirement for PR for this induction (9). These studies
imply that Hoxa-10 is a primary steroid
hormone-responsive gene and that it is involved in implantation as a
direct mediator of P4 actions. The adjacent AbdB
gene, Hoxa-11, is also regulated by ovarian steroids in the
uterine stroma (9), and Hoxa-11 mutant mice also exhibit
female infertility originating from uterine defects similar to those in
Hoxa-10(-/-) mice (11, 12).
A precise coordination between the establishment of uterine receptivity
and blastocyst activation is essential to the process of implantation
(13, 14). Ovarian P4 and estrogen play key roles in
implantation and subsequent decidualization. While preovulatory
estrogen secretion induces epithelial cell proliferation on day 1 of
pregnancy, superimposition of estrogen on P4 priming on day
4 directs stromal cell proliferation and epithelial cell
differentiation necessary for implantation in the mouse (15). This
profile of uterine cell proliferation can be mimicked in the
ovariectomized mouse uterus by ovarian steroids. For example, a single
injection of 17ß-estradiol (E2) induces epithelial cell
proliferation, while P4 induces stromal cell proliferation
by 24 h, which is further potentiated by E2 (15).
However, the mechanism by which these steroid hormones regulate uterine
cell-specific proliferation and differentiation is unclear. The initial
attachment reaction of the blastocyst trophectoderm with the luminal
epithelium, which coincides with increased stromal vascular
permeability and occurs around 22002300 h on day 4 of pregnancy, is
followed by stromal cell proliferation and differentiation
(decidualization) at the sites of blastocyst apposition (14).
P4 is an absolute requirement for decidualization since
PR-deficient mice fail to exhibit decidualization, while estrogen
receptor-
(ER-
) deficient mice are capable of responding to a
deciduogenic stimulus only in the presence of P4 (16 16A ).
Because of their vasoactive and mitogenic nature, PGs are implicated in
implantation and decidualization (reviewed in Ref. 17). PGs are
generated via cyclooxygenase (COX), which exists in two isoforms,
COX-1 and COX-2 (18). COX-2, but not COX-1, is essential for
implantation and decidualization (17). Among PGs, prostacyclin
(PGI2) and PGE2 are believed to be important
mediators of implantation (reviewed in Ref. 17). PGE2 binds
and activates a set of functionally distinct cell surface receptors,
EP1, EP2, EP3, and EP4,
which are classified on the basis of their pharmacological responses to
various agonists and antagonists of PGE2. They also exhibit
different characteristics with respect to their structures, tissue
distribution, and signal transduction mechanisms (19). While
PGI2 can bind to one G protein-coupled receptor known as IP
(19), PGI2 also functions as a ligand for peroxisome
proliferator-activated receptors (PPAR
and PPAR
), members of a
nuclear hormone receptor superfamily (20, 21). Previous investigation
on periimplantation defects in Hoxa-10(-/-) mice
demonstrated poor vascular response and defective decidualization (8).
These results suggested that altered PG signaling could be one cause of
uterine failure in Hoxa-10(-/-) mice. Here we provide
evidence that uterine stromal responsiveness to P4 with
respect to both PG signaling and cell proliferation is defective in
Hoxa-10(-/-) mice.
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RESULTS
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PGE2 Receptor Subtypes in the Uteri of
Hoxa-10(-/-) Mice Are Aberrantly Expressed
We examined the expression of several implantation-related genes,
such as LIF (22), HB-EGF (23), amphiregulin
(Ar) (24), and COX-1 (25) in the
Hoxa-10(-/-) mouse uterus on day 4 of pregnancy (Ref. 8
and data not shown). All of these genes are normally expressed in the
uterine epithelium of Hoxa-10(-/-) mice. We have recently
shown that PGI2 is the primary initiator of implantation
and decidualization (17), and our recent investigation using
COX-2 mice shows that PGE2 functions as an
ancillary factor with PGI2 for implantation (H. Lim, R. A.
Gupta, B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois,
J. M. Trzaskos, and S. K. Dey, in preparation). Since
IP is not detectable in the mouse uterus during implantation
(H. Lim, R. A. Gupta, B. C. Paria, D. E. Moller, J. D. Morrow, R. N.
DuBois, J. M. Trzaskos, and S. K. Dey, in preparation), we
examined expression of PGE2 receptor subtypes in
Hoxa-10(-/-) mice. Among the PGE2 receptor
subtypes, EP2, EP3, and EP4 exhibit
spatiotemporal expression in the periimplantation uterus, suggesting a
role of PGE2 in implantation (26, 27).
EP2 is solely expressed in the luminal
epithelium primarily on days 4 and 5 of pregnancy (27). In contrast,
EP3 is expressed in the mesometrial stroma and
throughout the myometrium, while EP4 is
expressed in the epithelium and stroma during the periimplantation
period (26). In day 4 pregnant Hoxa-10(-/-) uteri,
EP2 expression in the epithelium was normal
(Fig. 1
, a and b), but the exclusive
mesometrial stromal localization of EP3 was lost
with both mesometrial and lateral antimesometrial stromal expression at
low levels (Fig. 1
, cf). Myometrial expression of this gene was also
reduced in Hoxa-10(-/-) mice. With respect to
EP4, the stromal expression was considerably
down-regulated in Hoxa-10(-/-) uteri with little change in
epithelial expression (Fig. 1
, gj).

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Figure 1. Expression of PGE2 Receptor Subtypes in
Hoxa-10(-/-) Mice
In situ hybridization of EP2,
EP3, and EP4
mRNAs in day 4 pregnant uteri of wild-type and
Hoxa-10(-/-) mice is shown under darkfield at 20x
(ad, g, and h) or at 40x (e, f, i, and j).
EP2 (a and b);
EP3 (cf); EP4
(gj). Longitudinal sections (ad, g, h); transverse sections (e,f,
i, and j). Mesometrial pole of the uterus is directed toward the top of
each picture. le, Luminal epithelium; s, stroma; myo, myometrium; cm,
circular muscle; lm, longitudinal muscle. Similar results were observed
in six mice.
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These results could suggest that the aberrant
EP3 and EP4 expression in
the stroma reflects a global problem resulting from developmental
alteration in uterine cell types. To address this, we examined the
expression of several other genes, including tissue inhibitor of
metalloproteinase-2 (TIMP-2), matrix metalloproteinase-2
(MMP-2), ER-
, and PR that are
expressed in the stroma (16A, 28, 29). These genes were correctly
expressed in the uteri of Hoxa-10(-/-) mice on day 4 of
pregnancy (data not shown). Thus, the aberrant expression of
EP3 and EP4 in the
uterine stroma of Hoxa-10(-/-) mice appears to result
specifically from stromal Hoxa-10 deficiency.
EP3 and
EP4 Are Not Correctly Regulated by
P4 in Ovariectomized Hoxa-10(-/-)
Uteri
Since both EP3 and
EP4 are regulated by P4 in the mouse
uterus (26), we examined their regulation in Hoxa-10(-/-)
uteri under steroid hormonal stimulation. Levels of ovarian
P4 and estrogen and uterine responsiveness to these
steroids are important for preparing the uterus and embryo for
implantation (14). EP3 and
EP4 expression in the uterus is up-regulated by
P4 24 h after steroid injection of ovariectomized mice
(26), and the uterine distribution of these genes after P4
injection resembles that on day 4 of pregnancy. In
Hoxa-10(-/-) uteri, expression of
EP3 in the stroma and myometrium by
P4 was greatly reduced compared with that in wild-type mice
(Fig. 2
, ad). In contrast,
EP4 expression persisted at basal levels in
ovariectomized wild-type uteri in the absence of steroids. However,
P4 treatment up-regulated EP4
expression both in the stroma and epithelium in wild-type mice (Fig. 2
, e and g). In Hoxa-10(-/-) uteri,
EP4 expression was generally lower but
especially in the stroma after P4 treatment (Fig. 2
, f and
h). No differences were noted in EP2 expression
under similar conditions (data not shown). These results suggest that
Hoxa-10 mediates the effects of P4 in regulating the
correct expression of EP3 and
EP4 in the uterine stroma. Moreover, the
abnormal regulation of stromal EP3 and
EP4 in Hoxa-10(-/-) uteri is
apparently due neither to reduced levels of ovarian steroids nor
aberrant expression of their nuclear receptors, since exogenous
P4 injection in the ovariectomized
Hoxa-10(-/-) mice did not correct this aberration (Fig. 2
), and PR and ER-
are normally expressed in
these mice (data not shown).

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Figure 2. Regulation of EP3 and
EP4 mRNAs by Ovarian Steroid P4
in the Uteri of Ovariectomized Wild-Type and
Hoxa-10(-/-) Mice
Wild-type and Hoxa-10(-/-) mice were injected with oil
(vehicle, 100 µl) or P4 (2 mg/mouse) 2 weeks after
ovariectomy. They were killed 24 h later, and uteri were collected
for in situ hybridization. Darkfield photomicrographs
are shown at 40x. EP3 (ad, transverse
sections); EP4 (eh, longitudinal
sections). Oil (a, b, e, and f); P4 (c, d, g, and h). le,
Luminal epithelium; s, stroma; myo, myometrium; cm, circular muscle.
Similar results were observed in three (EP3)
or five (EP4) mice.
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Altered P4 Responsiveness in the Stroma of
Hoxa-10(-/-) Mice Is Restricted to Specific Genes
The above results suggest that intrinsic defects exist in
Hoxa-10(-/-) uterine stroma in the regulation of uterine
EP3 and EP4 expression.
Since Hoxa-10 itself is regulated by P4 in the
mouse and human endometrium (9, 10), it is possible that Hoxa-10
regulates the P4-induced expression of these genes.
Alternatively, Hoxa-10(-/-) uteri could express a general
defect in P4 responsiveness. Therefore, we analyzed the
expression of three other uterine genes that are differentially
regulated by P4 and/or E2 in the uterus.
PR is induced in the ovariectomized mouse uterus in a
cell-specific manner within 6 h of P4 and/or
E2 administration (Ref. 29 and our unpublished
results). Hoxa-11, another AbdB-like
homeodomain gene, is also up-regulated by P4 and/or
E2 by 6 h (9). c-myc is induced by
P4 and E2 by 6 h in the uterus (30).
Expression of PR (2 h, 6 h, and 24 h),
Hoxa-11 (6 h) and c-myc (6 h) was examined by
ribonuclease (RNase) protection assay and/or by in situ
hybridization in uteri of ovariectomized wild-type and
Hoxa-10(-/-) mice treated with P4 with or
without E2. PR was induced at similar levels in
the ovariectomized wild-type and Hoxa-10(-/-) uteri
treated with P4 and E2, reaching a peak at
6 h (Fig. 3A
). Further, in
situ hybridization revealed similar localization and levels of
Hoxa-11 in P4-treated wild-type and
Hoxa-10(-/-) uteri (Fig. 3B
). Lastly, the up-regulation of
c-myc by P4 and E2 at 6 h also
remained indistinguishable between wild-type and
Hoxa-10(-/-) mice (Fig. 3C
). These results further confirm
that the aberrant regulation of EP3 and
EP4 in Hoxa-10(-/-) uteri is a
specific consequence of stromal deficiency of Hoxa-10 and that Hoxa-10
mediates the steroid hormonal regulation of these genes in the uterine
stroma.

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Figure 3. Uterine Responsiveness to P4 in
Hoxa-10(-/-) Mice
A, Quantitation of the steroidal regulation of PR in the
uteri of ovariectomized wild-type and Hoxa-10(-/-)
mice by RNase protection assay. Mice were injected with oil or
P4 (2 mg/mouse) plus E2 (100 ng/mouse) 2 weeks
after ovariectomy. They were killed at indicated times, and uteri were
collected for RNA extraction. Three sets of RNase protection assays
were analyzed, and band intensities were quantitated by phosphorimager
and normalized for loading differences with rpL19.
Relative values after normalization are shown (mean ±
SEM). B, Regulation of Hoxa-11 by
P4 in the uteri of ovariectomized wild-type and
Hoxa-10(-/-) mice. Ovariectomized mice were injected
with oil or P4 (2 mg/mouse). They were killed 6 h
later, and uteri were collected for in situ
hybridization. Darkfield photomicrographs are shown at 40x. le,
Luminal epithelium; s, stroma; myo, myometrium. Similar results were
observed in three mice in each group. C, Quantitation of the steroidal
regulation of c-myc in the uteri of ovariectomized
wild-type and Hoxa-10(-/-) mice by RNase protection
assay. Mice were injected with oil or P4 (2 mg/mouse) plus
E2 (100 ng/mouse) 2 weeks after ovariectomy. They were
killed 6 h later, and uteri were collected for RNA extraction.
Three sets of RNase protection assays were analyzed, and band
intensities were quantitated by phosphorimager and normalized for
loading differences with rpL19. Relative values after
normalization are shown (mean ± SEM).
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Stromal Cell Proliferation in Response to
P4 and E2 is Reduced in
Uteri of Ovariectomized Hoxa-10(-/-) Mice
Epithelial cell proliferation in the ovariectomized mouse
uterus is induced by E2, while that of stromal cells is
induced by P4 and further potentiated by E2;
these profiles closely mimic the patterns observed during early
pregnancy (15). We compared cell proliferation profiles in response to
P4 and/or E2 between ovariectomized wild-type
and Hoxa-10(-/-) uteri. While an E2 injection
induced epithelial cell proliferation at equal levels in both wild-type
and Hoxa-10(-/-) uteri, stromal cell proliferation in
response to P4 and E2 was
7-fold lower in
Hoxa-10(-/-) mice (Fig. 4
and Table 1
). These data indicate that
Hoxa-10 is required for basal and for P4-induced stromal
cell proliferation.

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Figure 4. Uterine Cell Proliferation in Wild-Type and
Hoxa-10(-/-) Mice after Steroid Treatments
Mice were injected with oil, E2 (100 ng/mouse), or
P4 (2 mg/mouse) plus E2 2 weeks after
ovariectomy. Twenty-two hours after steroid injection, they received an
intraperitoneal injection of
[methyl-3H]thymidine (25 µCi/0.1 ml
saline) and were killed 2 h later. Nuclear uptake of
[3H]thymidine was detected in uterine sections by
autoradiography after 7 days of exposure. le, Luminal epithelium; s,
stroma; myo, myometrium. Similar results were observed in six mice (see
Table 1 ).
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COX-2 Expression Is Aberrant in
Hoxa-10(-/-) Uteri at the Initial Stage of
Decidualization
Since PGs are implicated in cell proliferation (31, 32), the
involvement of Hoxa-10 in cell proliferation could be via a
PG-signaling pathway. PGs produced via uterine COX-2 are
critical for implantation and decidualization (17) and are also
implicated in the proliferation and transformation of several cell
types (reviewed in Refs. 33, 34). Thus, we examined COX-2
induction in Hoxa-10(-/-) mice using an experimentally
induced decidualization model. A biphasic induction of COX-2
was noted in the day 4 pseudopregnant mouse uterus after intraluminal
oil infusion as a deciduogenic stimulus. COX-2 expression
was induced in the epithelium of the oil-infused horn at 2 h, but
disappeared by 8 h (17). A second phase of COX-2 expression was
focally induced in the subepithelial stroma at 24 h (Fig. 5B
), resembling COX-2 induction at the
time of blastocyst attachment reaction (25). In
Hoxa-10(-/-) mice, epithelial COX-2 induction at 2 h
was normal, but stromal COX-2 induction at 24 h was considerably
reduced or absent in Hoxa-10(-/-) mice (Fig. 5
, A and B).
A significant reduction of stromal COX-2 was also confirmed
by RNase protection assay (Fig. 5C
). This provides evidence that loss
of COX-2 in the absence of Hoxa-10 is related to defective
decidualization in these mice. Alternatively, this could be a
consequence of decidualization failure in Hoxa-10(-/-)
mice.

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Figure 5. Induction of COX-2 mRNA in Day 4
Pseudopregnant Wild-Type And Hoxa-10(-/-) Uteri with
or without Intraluminal Oil Infusion
A, Northern blot hybridization of COX-2 mRNA at 2 h
after oil infusion. Pseudopregnant wild-type or
Hoxa-10(-/-) mice received an intraluminal oil
infusion on the morning of day 4. Two hours later, infused and
noninfused uteri were collected separately and pooled from eight to
nine mice. They were subjected to RNA extraction and Northern blot
hybridization. Lane 1, Wild-type noninfused horns; lane 2, wild-type
infused horns; lane 3, Hoxa-10(-/-) noninfused horn;
lane 4, Hoxa-10(-/-) infused horns.
rpL7 serves as a loading control. B, In
situ hybridization of COX-2 mRNA in the stroma
at 24 h after intraluminal oil infusion. Pseudopregnant wild-type
or Hoxa-10(-/-) mice received an intraluminal oil
infusion on the morning of day 4. Uteri were collected 24 h later
for in situ hybridization. le, Luminal epithelium; s,
stroma; myo, myometrium. Reduced stromal induction of
COX-2 was noted in 10 of 12 mice. C, RNase protection
assay of stromal COX-2 induction in wild-type and
Hoxa-10(-/-) mice. Pseudopregnant wild-type or
Hoxa-10(-/-) mice received an intraluminal oil
infusion on the morning of day 4. Uteri were collected 24 h later
for RNase protection assays. Three sets of assays were analyzed, and
band intensities were quantitated by phosphorimager and normalized for
loading differences with rpL19. Relative values after
normalization are shown (mean ± SEM).
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Supplementation of PGs Cannot Restore Decidualization in
Hoxa-10(-/-) Mice
Because stromal COX-2 was poorly induced in the
Hoxa-10(-/-) uteri, we administered PGE2
and/or carbaprostacyclin (cPGI, a stable agonist of PGI2)
to Hoxa-10(-/-) mice in an attempt to correct the
decidualization defects that were observed in the experimentally
induced decidualization model. PGE2 and/or cPGI was
administered from days 47 of pseudopregnancy in
Hoxa-10(-/-) mice after an intraluminal oil infusion on
day 4. Supplementation of PGE2 and/or cPGI was ineffective
in improving the decidual response in Hoxa-10(-/-) mice.
While four of five wild-type mice showed
13 fold uterine weight
increases in response to intraluminal oil infusion, none of the five
Hoxa-10(-/-) mice showed any response. Further,
cPGI (n = 4), PGE2 (n = 5), or cPGI plus
PGE2 (n = 5) treatment did not improve the decidual
response in these mutants. The results are consistent with the idea
that the defective decidualization in Hoxa-10(-/-) mice
reflects altered downstream signaling of PGs due to aberrant expression
of their receptors.
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DISCUSSION
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Across metazoa, functions of Hox homeoproteins are conserved as
factors that specify embryonic segmental structures (1). Genetic
studies have provided insight into the mechanisms by which these
transcription factors affect such precisely regulated cellular events.
However, apparent differences exist in the way Drosophila
Hox genes exert their actions during development from those
of mammalian Hox genes (35). One recent view on mammalian
Hox functions envisages their roles as local regulators of cell
proliferation (35), although their definitive functions in different
systems remain unknown. Defective implantation and decidualization
resulting in female infertility in the absence of Hoxa-10
(8) demonstrate an unexpected function for a Hox gene.
Appropriate levels of P4 and responsiveness of the uterus
to this steroid are absolute requirements for decidualization (16 16A, 17). The failure of P4 to restore decidualization in
Hoxa-10(-/-) mice (8) and the regulation of uterine
Hoxa-10 by P4 (9) suggest that this gene is
involved in mediating some important P4 actions in the
uterus, and the identification of functional PR response elements
(PREs) in the Hoxa-10 and Hoxa-11 intergenic
region supports this notion (L. Ma and R. L. Maas, unpublished). Since
Hoxa-10 is expressed in stromal cells in the
periimplantation uterus and after steroid hormonal stimulation (6, 9),
aberrant expression of EP3 and
EP4 under these conditions in
Hoxa-10(-/-) mice suggests that this homeobox protein acts
as a direct or indirect mediator of P4 function in
regulating these genes during implantation and decidualization. This is
consistent with the observation that P4 induction of
Hoxa-10 temporally precedes that of
EP3/EP4. The correct
expression of other P4-regulated uterine genes expressed in
the epithelium (EP2 and Ar) or in the
stroma (PR, Hoxa-11, and c-myc) in
Hoxa-10(-/-) uteri further supports this hypothesis. The
normal expression of ER and PR indicates that
aberrant expression of EP3 and
EP4 in the stroma is not the result of altered
expression of these nuclear steroid receptors.
Reduced stromal cell proliferation in response to P4 and
E2 in Hoxa-10(-/-) mice could potentially
represent a consequence of altered PG signaling and may contribute to
the defective decidual response in these mice. Alternatively, defective
stromal cell proliferation in these mice could constitute a distinct
phenomenon unrelated to PG signaling, since mammalian Hox
genes are implicated in cell proliferation events (35). Cyclin D3, one
of the G1 phase cyclins, is expressed in stromal cells at
the onset of decidualization and is likely to activate cell cycle
progression during this time (36). We have recently observed that
expression of cyclin D3 fails to be up-regulated in
Hoxa-10(-/-) uteri after application of a deciduogenic
stimulus (36). This is also consistent with impaired stromal cell
proliferation and points toward an underlying basis for the defective
decidual response in Hoxa-10(-/-) mice.
Uterine COX-2 is induced by activated blastocysts at the
time of the attachment reaction and produces PGs that are essential for
implantation and decidualization (17, 25). Uterine and/or embryonic
COX-2 also appears to be important for implantation in
various species including sheep, mink, skunk, and baboon (37, 38, 39, 40).
COX-2 exhibits a unique biphasic cell-specific induction at
2 h and 24 h during the initial stages of decidualization
(Ref. 17 and Fig. 5
), and the stromal cell expression at 24 h
resembles the expression during blastocyst implantation (17, 25). The
loss of stromal COX-2 expression in
Hoxa-10(-/-) mice is intriguing, since these
COX-2 expressing cells exhibit the first decidual cell
reaction (17). Since uterine COX-2 is not directly regulated
by P4 and/or E2 (25), loss of stromal
COX-2 in these mice could be an indirect consequence of
defective decidualization. Alternatively, the loss of stromal
COX-2 in Hoxa-10(-/-) mice could indicate that
Hoxa-10 regulates COX-2 in this cell type. The second
speculation is supported by the observation that Hoxa-10 gene is
correctly expressed in the COX-2(-/-) uteri (17), implying
that Hoxa-10 is functionally upstream of COX-2 expression in
the subepithelial stroma.
The inability of exogenously administered PGs to improve decidual
responsiveness in Hoxa-10 mutants could be due to
inappropriate selection of time and doses, rapid metabolism, and/or
suboptimal delivery of these agents to the target cells. On the other
hand, the defective decidualization in Hoxa-10(-/-) mice
could result from altered PGE2 signal transduction due to
aberrant EP receptor expression. This phenotype of Hoxa-10deficiency is clearly distinct from that of
COX-2(-/-) mice (17). COX-2(-/-) mice, which
exhibit normal uterine cell proliferation and expression of
EP3 and EP4, respond to
exogenous cPGI and PGE2 with improved implantation and
decidualization (Ref. 17 and H. Lim, R. A. Gupta, B. C. Paria, D. E.
Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos, and S. K. Dey, in
preparation). Therefore, aberrant expression of
EP3 and EP4 in
Hoxa-10(-/-) uteri suggests that signaling via these two
receptors is important for decidualization. Although
EP3-deficient female mice exhibit apparently
normal reproductive performance (41),
EP4-deficient mice exhibit neonatal lethality
and are therefore uninformative for this function (42). Thus,
EP4 could represent a potential candidate for
PGE2 signaling in decidualization. Although the
PGI2 cell surface receptor IP and nuclear
receptor PPAR
are not detected in the mouse uterus at the
time of implantation, another PGI2 nuclear receptor
PPAR
is expressed in stromal cells around the blastocyst
with the initiation of the attachment reaction and subsequent
decidualization. We have also found that COX-2 and
PPAR
are coordinately expressed in stromal cells after
application of a deciduogenic stimulus (H. Lim, R. A. Gupta,
B. C. Paria, D. E. Moller, J. D. Morrow, R. N. DuBois, J. M. Trzaskos,
and S. K. Dey, in preparation). These observations suggest that
PGI2 signaling in implantation and decidualization is
mediated via PPAR
, but not by IP or PPAR
. We have preliminary
evidence that PPAR
, like COX-2, fails to be
up-regulated in stromal cells after application of a deciduogenic
stimulus in Hoxa-10(-/-) mice (data not shown). Thus,
aberrant expression of EP3,
EP4, PPAR
, and COX-2 in
Hoxa-10(-/-) uteri suggests that PG signaling plays an
important role in implantation and decidualization and that Hoxa-10 is
involved in regulating these signaling systems. Finally, implantation
and decidualization defects in Hoxa-10(-/-) mice are also
correlated with defective stromal cell proliferation in response to
steroid hormones. Based on our observation of reduced expression of
cyclin D3 with the onset of decidualization in
Hoxa-10(-/-) mice (36), this result is consistent with a
defect in progression through the G1 phase of the cell
cycle. Although PGs are involved in cell proliferation, whether
defective cell proliferation in Hoxa-10(-/-) mice results
from defective PG signaling will require further investigation.
The process of implantation involves regulated mitogenesis and vascular
permeability changes in the uterus, and ovarian steroids play pivotal
roles in these uterine events (14). P4 mediates a variety
of female reproductive functions as demonstrated in
PR-deficient mice (16). Our results show that
Hoxa-10(-/-) mice provide a good model to define the role
of P4 actions in the uterine stroma during implantation.
Interestingly, although a smaller percentage of
Hoxa-10(-/-) mice achieve successful pregnancy, about 40%
of Hoxa-10(-/-) mice succeed in initiating the
implantation reaction (8), suggesting that stromal defects in
Hoxa-10(-/-) mice do not completely negate the functions
of luminal epithelial cells for initial blastocyst contact. It is
possible that Hoxa-10 induces genes that are important for stromal cell
proliferation and differentiation in a P4-dominant
environment. Our results potentially identify the PG-signaling system
as acting functionally downstream of uterine Hoxa-10 in
implantation. Whether EP3,
EP4, PPAR
, or COX-2 are
directly or indirectly regulated by Hoxa-10 requires further
investigation. Hoxa-10 is also implicated in the proliferation and
differentiation of the myeloid lineage during hematopoiesis (43, 44),
implying that this protein may also be capable of functioning similarly
in both contexts. Since Hox functions during embryogenesis have been
considered as regulators of local cell proliferation (35), the uterine
cell proliferative defect in Hoxa-10(-/-) mice could
provide a potentially powerful system with which to study the role of
Hox genes in cell proliferation and cell cycle control.
Collectively, our present findings suggest a novel role of Hoxa-10
in mediating certain actions of P4 in the uterine stroma
with respect to implantation and decidualization.
 |
MATERIALS AND METHODS
|
---|
Gene-Targeted Mice
The disruption of the Hoxa-10 gene was performed by
insertion of a neomycin resistance cassette into an XhoI
site within the homeobox by homologous recombination in 129/SvJ ES
cells and generation of chimeric mice (6). PCR analysis of tail genomic
DNA determined the genotypes. All of the mice used were housed in the
animal care facility at the University of Kansas Medical Center
according to NIH and institutional guidelines on the care and use of
laboratory animals. Females were mated with fertile or vasectomized
males of the same strain to induce pregnancy or pseudopregnancy (day
1 = vaginal plug), respectively.
Hybridization Probes
For Northern hybridization and RNase protection assay,
32P-labeled antisense cRNA probes were generated, while for
in situ hybridization, sense and antisense
35S-labeled cRNA probes were generated using the
appropriate polymerases. Mouse-specific cDNAs to c-myc,
LIF, Ar, Hoxa-10, Hoxa-11,
COX-1, COX-2, PGE2 receptor subtypes
(EP2, EP3,
EP4), estrogen receptor-
(ER-
),
progesterone receptor (PR), tissue inhibitor of
metalloproteinase-2 (TIMP-2), matrix metalloproteinase-2
(MMP-2), rpL7, and rpL19 were used as
templates for generating probes for Northern blot hybridization,
in situ hybridization, or RNase protection assay (9, 17, 24, 25, 26, 27, 28, 29, 30).
In Situ Hybridization
In situ hybridization was performed as described
previously (23). Briefly, uteri were cut into 46 mm pieces and flash
frozen in Histo-Freeze (Fisher Scientific, Pittsburgh,
PA). Frozen sections (11 µm) were mounted onto
poly-L-lysine coated slides and fixed in cold 4%
paraformaldehyde in PBS. The sections were prehybridized and hybridized
at 45 C for 4 h in 50% formamide hybridization buffer containing
the 35S-labeled antisense cRNA probe (specific
activities
2 x 109 dpm/µg). After hybridization
and washing, the sections were incubated with RNase A (20 µg/ml) at
37 C for 20 min. RNase A-resistant hybrids were detected by
autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak Co., Rochester, NY). Sections hybridized with the corresponding
sense probe served as negative controls. Slides were poststained with
hematoxylin and eosin.
Northern Blot Hybridization
Total RNA was extracted from whole uteri pooled from 1015 mice
at indicated times by a modified guanidine thiocyanate procedure (23, 45). Total RNA (6 µg) was denatured, separated by
formaldehyde-agarose gel electrophoresis, transferred to nylon
membranes, and cross-linked by UV irradiation (Spectrolinker, XL-1500,
Spectronics Corp., Westbury, NY). The blots were prehybridized,
hybridized with 32P-labeled antisense cRNA probe (specific
activities
2 x 109 dpm/µg), and washed as
described previously (23). After hybridization, the blots were washed
under stringent conditions, and the hybrids were detected by
autoradiography. The blots were stripped and rehybridized with
rpL7 probe as described previously (27).
RNase Protection Assay
32P-labeled cRNA probes were prepared as described
above. Analyses were performed as described previously (9, 46). For
each lane, 20 µg total RNA were hybridized for 16 h at 45 C
simultaneously with 3 x 105 cpm of RNA probes for
each of c-myc, PR, or COX-2 and with
2 x 104 cpm of rpL19 RNA probe and then
digested with 20 µg/ml RNase A and 1.5 µg/ml RNase T1. Protected
fragments were electrophoresed in 6% denaturing polyacrylamide gel and
analyzed by autoradiography. Band intensities were quantitated by
phosphorimager and normalized for loading differences with
rpL19.
Expression of Uterine Genes on Day 4 of Pregnancy in
Hoxa-10(-/-) Mice
Sections of day 4 (0900 h) pregnant uteri from wild-type or
Hoxa-10(-/-) mice were processed for in situ
hybridization for LIF, Ar, COX-1,
EP2, EP3,
EP4, TIM-2, MMP-2,
ER-
, and PR mRNAs.
Uterine Responsiveness to E2 and
P4 in Hoxa-10(-/-) Mice
To determine whether Hoxa-10(-/-) mice respond
appropriately to P4, wild-type and
Hoxa-10(-/-) mice were ovariectomized and treated with
sesame oil (vehicle) or P4 (2 mg/mouse) with or without
E2 (100 ng/mouse) after 2 weeks of rest. Sesame oil and
steroid hormones were purchased from Sigma Chemical Co.
(St. Louis, MO). Uteri were collected 2, 6, or 24 h after the last
injection. Induction of PR (2, 6, and 24 h),
Hoxa-11 (6 h), c-myc (6 h)
EP3 (24 h), and EP4 (24
h) genes was assayed by RNase protection and/or in situ
hybridization.
To examine uterine cell-specific proliferation in response to
E2 and/or P4, ovariectomized wild-type or
Hoxa-10(-/-) mice were given an injection of
E2 or P4 plus E2. After 22 h,
they received an injection of
[methyl-3H]thymidine (25 µCi/0.1 ml saline
ip, specific activity, 40 mCi/mmol; RPI Corp., Mount Prospect, IL) and
were killed 2 h later. Uteri were flash frozen and fixed in 4%
paraformaldehyde after sectioning. Nuclear uptake of
[3H]thymidine was detected in uterine sections by
autoradiography (15) after 710 days of exposure. The autoradiographic
signals (silver grains) were quantitated under a darkfield using the
OPTIMA II program with an image analysis system (47).
Induction of COX-2 in the Hoxa-10(-/-) Uteri after
Application of a Deciduogenic Stimulus
To examine COX-2 induction in the Hoxa-10(-/-)
uteri in response to a deciduogenic stimulus, wild-type and
Hoxa-10(-/-) mice received intraluminal oil (25 µl) on
day 4 of pseudopregnancy. Uteri were collected at 2 or 24 h after
the oil infusion for Northern blot hybridization, in situ
hybridization, or RNase protection assay.
Decidual Response in Hoxa-10(-/-) Mice after
Supplementation of PGs
To induce decidualization, sesame oil (25 µl) was infused
intraluminally in one uterine horn on day 4 of pseudopregnancy; the
contralateral horn served as control. Mice were killed on day 8, and
uterine weights of the infused and noninfused (control) horns were
recorded to assess the extent of decidualization. PGE2
and/or cPGI (Cayman Chemical Co., Ann Arbor, MI) were prepared in 10%
EtOH-90% saline solution (20 µg/injection) and supplemented
intravenously at 1700 h on day 4 of pseudopregnancy followed by an
intraperitoneal injection on days 57.
 |
FOOTNOTES
|
---|
Address requests for reprints to: S. K. Dey, Ph.D., Department of Molecular and Integrative Physiology, MRRC 37/3017, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7338. E-mail: sdey{at}kumc.edu
This work was supported by NICHD/NIH grants as part of the National
Cooperative Program on Markers of Uterine Receptivity for Blastocyst
Implantation [(HD-29968) and HD-12304 to S.K.D.], and by NICHD Grant
HD-35580 (to R.L.M.). H. L. was supported by a Kansas Health Foundation
predoctoral fellowship, and L. M. is supported by an NIH National
Research Service Award (1F32 HD-0826401). Center grants in
Reproductive Biology (HD-33994) and Mental Retardation (HD-02528) at
the University of Kansas Medical Center provided access to various core
facilities.
Received for publication January 6, 1999.
Revision received February 23, 1999.
Accepted for publication February 25, 1999.
 |
REFERENCES
|
---|
-
Krumlauf R 1994 Hox genes in vertebrate development. Cell 78:191201[Medline]
-
McGinnis W, Krumlauf R 1992 Homeobox genes and axial
patterning. Cell 68:283302[Medline]
-
Benson GV, Nguyen TE, Maas RL 1995 The expression pattern of
the murine Hoxa-10 gene and the sequence recognition and its
homeodomain reveal specific properties of Abdominal B-like
genes. Mol Cell Biol 15:15911601[Abstract]
-
Dolle P, Izpisúa-Belmonte JC, Tickle C, DuBoule D 1991 HOX4 genes and the morphogenesis of mammalian genitalia. Genes Dev 5:17671776[Abstract]
-
Izpisúa-Belmonte JC, Falkenstein H, Dolle P, Renucci A,
DuBoule D 1991 Murine genes related to the Drosophila AbdB
homeotic genes are sequentially expressed during development of the
posterior part of the body. EMBO J 10:22792289[Abstract]
-
Satokata I, Benson G, Maas R 1995 Sexually dimorphic
sterility phenotypes in Hoxa10-deficient mice. Nature 374:460463[CrossRef][Medline]
-
Rijli FM, Matyas R, Pellegrini M, Dietrich A, Gruss P, Dolle
P, Chambon P 1995 Cryptorchidism and homeotic transformation of spinal
nerves and vertebrae in Hoxa-10 mutant mice. Proc Natl Acad
Sci USA 92:1858189[Abstract]
-
Benson GV, Lim H, Paria BC, Satokata I, Dey SK, Maas RL 1996 Mechanisms of reduced fertility in Hoxa-10 mutant mice:
uterine homeosis and loss of maternal Hoxa-10 expression.
Development 122:26872696[Abstract/Free Full Text]
-
Ma L, Benson GV, Lim H, Dey SK, Maas RL 1998 Abdominal
B (AbdB) Hoxa genes: regulation in adult
uterus by estrogen and progesterone and repression in Mullerian duct by
the synthetic estrogen diethylstilbestrol (DES). Dev Biol 197:141154[CrossRef][Medline]
-
Taylor HS, Arici A Olive D, Igarashi P 1998 HOXA10 is
expressed in response to sex steroids at the time of implantation in
the human endometrium. J Clin Invest 101:13791384[Abstract/Free Full Text]
-
Hsieh-Li HM, Witte DP, Weinstein M, Branford W, Li H, Small K,
Potter SS 1995 Hoxa 11 structure, extensive antisense
transcription, and function in male and female fertility. Development 121:13731385[Abstract/Free Full Text]
-
Gendron RL, Paradis H, Hsieh-Li HM, Lee DW, Potter SS, Markoff 1997 Abnormal uterine stromal and glandular function associated with
maternal reproductive defects in Hoxa-11 null mice. Biol
Reprod 56:10971105[Abstract]
-
Paria BC, Huet-Hudson YM, Dey SK 1993 Blastocysts state of
activity determines the "window" of implantation in the mouse
receptive uterus. Proc Natl Acad Sci USA 90:015910162
-
Dey SK 1996 Implantation. In: Adashi EY, Rock JA, Rosenwaks Z
(eds) Reproductive Endocrinology, Surgery, and Technology.
Lippincott-Raven Publishers, Philadelphia, pp 421434
-
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 periimplantation
period. Endocrinology 125:16831690[Abstract]
-
Lydon JP, DeMayo FJ, Funk CR, Mani SK, Hughes AR, Montgomery
CA, Shyamala G, Conneely OM, OMalley BW 1995 Mice lacking
progesterone receptor exhibit pleiotropic reproductive abnormalities.
Genes Dev 9:22662278[Abstract]
-
Paria BC, Tan J, Lubahn DB, Dey SK, Das SK 1999 Uterine
decidual response occurs in estrogen receptor-
deficient mice.
Endocrinology 140:27042710[Abstract/Free Full Text]
-
Lim H, Paria BC, Das SK, Dinchuk JE Langenbach R, Trzaskos JM,
Dey SK 1997 Multiple female reproductive failures in cyclooxygenase-2
deficient mice. Cell 91:197208[Medline]
-
Smith WL, DeWitt DL 1996 Prostaglandin endoperoxide H
synthase-1 and -2. Adv Immunol 62:167215[Medline]
-
Negishi M, Sugimoto Y, Ichikawa A 1995 Molecular mechanisms of
diverse actions of prostanoid receptors. Biochim Biophys Acta 1259:109120[Medline]
-
Forman BM, Tontonoz P, Chen J, Brun RP, Spiegelman BM, Evans
RM 1995 15-deoxy-
12,14-prostaglandin J2 is a
ligand for the adipocyte determination factor PPAR
. Cell 83:803812[Medline]
-
Forman BM, Chen J, Evans RM 1997 Hypolipidemic drugs,
polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome
proliferator-activated receptors
and
. Proc Natl Acad Sci USA 94:43124317[Abstract/Free Full Text]
-
Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F,
Abbondanzo SJ 1992 Blastocyst implantation depends on maternal
expression of leukemia inhibitory factor. Nature 359:7679[CrossRef][Medline]
-
Das SK, Wang WN, 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]
-
Das SK, Chakraborty I, Paria BC, Wang XN, Plowman G, Dey SK 1995 Amphiregulin is an implantation-specific and
progesterone-regulated gene in the mouse uterus. Mol Endocrinol 9:691705[Abstract]
-
Chakraborty I, Das SK, Wang J, 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]
-
Yang ZM, Das SK, Wang J, Sugimoto Y, Ichikawa A, Dey SK 1997 Potential sites of prostaglandin actions in the periimplantation mouse
uterus: differential expression and regulation of prostaglandin
receptor genes. Biol Reprod 56:368379[Abstract]
-
Lim H, Dey SK 1997 Prostaglandin E2 receptor
subtype EP2 gene expression in the mouse uterus coincides
with differentiation of the luminal epithelium for implantation.
Endocrinology 138:45994606[Abstract/Free Full Text]
-
Das SK, Yano S, Wang J, Edwards DR, Nagase H, Dey SK 1997 Expression of matrix metalloproteinases and tissue inhibitors of
metalloproteinases in the mouse uterus during the peri-implantation
period. Dev Dyn 21:4454
-
Das SK, Tan J, Johnson DC, Dey SK 1998 Differential
spatiotemporal regulation of lactoferrin and progesterone receptor
genes in the mouse uterus by primary estrogen, catechol estrogen, and
xenoestrogen. Endocrinology 139:29052915[Abstract/Free Full Text]
-
Das SK, Paria BC, Andrews GK, Dey SK 1993 Effects of
9-ene-tetrahydrocannabinol on expression of ß-type transforming
growth factors, insulin-like growth factor-1 and c-myc genes
in the mouse uterus. J Steroid Biochem Mol Biol 45:459465[CrossRef][Medline]
-
Pentland AP, Needleman P 1986 Modulation of keratinocyte
proliferation in vitro by endogenous prostaglandin
synthesis. J Clin Invest 77:246251[Medline]
-
Hashimoto N, Watanabe T, Ikeda Y, Yamada H, Taniguchi S,
Mitsui H, Kurokawa K 1997 Prostaglandins induce proliferation of rat
hepatocytes through a prostaglandin E2 receptor
EP3 subtype. Am J Physiol 272:G597604
-
Fischer SM 1997 Prostaglandins and cancer. Front Biosci 2:482500
-
DuBois RN, Abramson SB, Crofford L, Gupta RA, Simon LS, Van de
Putte LB, Lipsky PE 1998 Cyclooxygenase in biology and disease. FASEB J 12:10631073[Abstract/Free Full Text]
-
Duboule D 1995 Vertebrate Hox genes and
proliferation: an alternative pathway to homeosis? Curr Opin Genet Dev 5:525528[Medline]
-
Das SK, Lim H, Paria BC, Dey SK 1999 Cyclin D3 in the mouse
uterus is associated with decidualization process during early
pregnancy. J Mol Endocrinol 22:91101[Abstract/Free Full Text]
-
Charpigny G, Reinaud P, Tamby JP, Creminon C, Guillomot M 1997 Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine
embryos during the implantation period. Biol Reprod 57:10321040[Abstract]
-
Song JH, Sirois J, Houde A, Murphy BD 1998 Cloning,
developmental expression and immunohistochemistry of cyclooxygenase 2
(COX-2) in the endometrium during embryo implantation and gestation in
the mink. Endocrinology 139:36293636[Abstract/Free Full Text]
-
Das SK, Wang J, Dey SK, Mead RA 1999 Spatiotemporal expression
of cyclooxygenase-1 and cyclooxygenase-2 during delayed implantation
and periimplantation period in the Western spotted skunk. Biol Reprod 60:893899[Abstract/Free Full Text]
-
Kim JJ, Wang J, Bambra C, Das SK, Dey SK, Fazleabas AT 1999 Expression of cyclooxygenase-1 and -2 in the baboon endometrium during
the menstrual cycle and pregnancy. Endocrinology 140:26722678[Abstract/Free Full Text]
-
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]
-
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]
-
Thorsteinsdottir U, Sauvageau G, Hough MR, Dragowska W,
Lansdorf PM, Lawrence HJ, Largman C, Humphries RK 1997 Overexpression
of HOXA10 in murine hematopoietic cells perturbs both myeloid and
lymphoid differentiation and leads to acute myeloid leukemia. Mol Cell
Biol 17:495505[Abstract]
-
Tenen DG, Hromas R, Licht JD, Zhang DE 1997 Transcription
factors, normal myeloid development, and leukemia. Blood 90:489519[Free Full Text]
-
Han JH, Stratowa C, Rutter WJ 1987 Isolation of full-length
putative rat lysophopholipase cDNA using improved methods for mRNA
isolation and cDNA cloning. Biochemistry 26:16171625[Medline]
-
Krieg PA, Melton DA 1987 In vitro RNA synthesis
with SP6 RNA polymerase. Methods Enzymol 155:313324
-
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]