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Department of Molecular and Integrative Physiology (B.C.P., H.L., X.-N.W., Sa.K.D., Su.K.D.), University of Kansas Medical Center, Ralph L. Smith Research Center, Kansas City, Kansas 66160-7338; and the Department of Pharmacology and Toxicology (J.L.), University of Texas Medical Branch, Galveston, Texas 77555-1031
Address all correspondence and requests for reprints to: B. C. Paria or Sudhansu K. Dey, Department of Molecular and Integrative Physiology, MRRC 37/3017, University of Kansas Medical Center, Kansas City, Kansas 66160-7338. E-mail: deylab{at}kumc.edu
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Introduction |
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Estrogen is essential for implantation in the progesterone (P4)-primed mouse uterus. Ovariectomy on day 4 morning, before preimplantation ovarian estrogen secretion, results in blastocyst dormancy and implantation failure. This condition, known as delayed implantation, can be maintained by continued P4 treatment, but it is terminated by an injection of estrogen with blastocyst activation and implantation (2, 6). The mechanism(s) by which estrogen mediates these events still remains undefined. Uterine sensitivity to implantation is comprised of prereceptive, receptive, and nonreceptive (refractory) phases (2). Uterine receptivity occurs only for a limited period during pregnancy or pseudopregnancy. In pregnant or pseudopregnant mice, the prereceptive uterus on day 3 becomes receptive on day 4 (the day of implantation), whereas by day 5 (as examined by blastocyst transfer), the uterus becomes refractory and fails to respond to blastocysts. These uterine phases can also be produced in delayed implanting pregnant or pseudopregnant mice by appropriate P4 and estrogen treatments. The uterus becomes neutral (similar to prereceptive phase) when exposed to P4 alone and fails to respond to the presence of blastocysts for implantation. The neutral uterus can be rendered receptive for implantation for a limited period if exposed to estrogen after 24 h of P4 priming (reviewed in Ref. 2). The mechanisms by which estrogen transforms the P4-primed uterus to the receptive state, activates blastocysts, and initiates implantation are not known. Implantation in the receptive uterus has previously been assumed to occur irrespective of the blastocyst’s state of activity. Using blastocyst transfers in delayed implanting mice, we have recently shown that the blastocyst’s state of activity is also an essential determinant in defining the so-called window of implantation in the receptive uterus (2). The results demonstrated that dormant blastocysts transferred into uteri of P4-treated delayed pseudopregnant recipients implant successfully only if they are transferred within 1 h of estradiol-17ß (E2) treatment of the recipients. In contrast, day 4 normal or E2-treated in utero-activated blastocysts successfully implant when transferred into uteri of P4-treated delayed recipients even at 16 h of E2 treatment. Dormant blastocysts cultured in vitro are known to acquire metabolic activation, leading to the tenet that the uterus, during the delayed implantation, elaborates an inhibitor(s) that renders the blastocysts dormant (7, 8). However, our studies have established that dormant blastocysts cultured in vitro fail to implant upon transfer into P4-treated delayed uterus beyond the critical period of 1 h of E2 treatment (2). These results led to the following conclusions. First, the window of implantation is tightly regulated and is achieved when the activated state of the blastocyst coincides with the receptive state of the uterus. Second, E2 induces very rapidly, but transiently, a factor(s) in the P4-primed uterus that activates the dormant blastocysts in utero for implantation in the receptive uterus. Finally, although dormant blastocysts acquire metabolic activation in vitro, they do not become implantation-competent. However, the mechanism by which embryos achieve activation in the P4-treated uterus in vivo by estrogen remains unanswered.
The focus of this investigation was to identify the factor(s) and the mechanism(s) by which blastocysts are activated for implantation. Our previous work suggests that estrogen action in implantation may involve catecholestrogens (9). Catecholestrogens are active metabolites formed by aromatic hydroxylation of phenolic estrogens at either the C-2 or C-4 position and catalyzed by NADPH-dependent cyctochrome P-450 enzyme system, estrogen-2/4-hydroxylase (10). In human extrahepatic tissues, including the uterus, cytochrome P-4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1) catalyze estrogen hydroxylation at C-2 and C-4 positions, respectively (11, 12). Catecholestrogens can also be formed by the peroxidase system (13). They can function via classical nuclear estrogen receptor (ER) and/or membrane receptors, and they can also influence metabolism of catecholamines by inhibiting catechol-O-methyltransferase (14, 15). Catecholestrogens are not likely to function as circulating hormones, because of their rapid metabolism and clearance. However, they can be formed in the target tissues to mediate their functions in an autocrine and/or paracrine manner (15). Indeed, catecholestrogens are synthesized in various neoplastic and normal tissues, including the embryo and uterus (9, 16, 17). Further, catecholestrogens, but not E2, can stimulate PG synthesis in blastocysts and endometrial cells in vitro (18). Because PGs are considered important for embryonic and uterine functions during implantation (reviewed in Refs. 19, 20), we speculated that a catecholestrogen produced in the P4-primed mouse uterus after E2 treatment initiates blastocyst activation via local production of PGs. Cyclooxygenase (COX) is the rate-limiting enzyme that catalyzes the conversion of arachidonate to PGH2, the precursor for various PGs. COX exists in two isoforms (reviewed in Ref. 21), and they are expressed in the mouse uterus and blastocyst during implantation (19, 22).
Two-fluoroestradiol-17ß (2-Fl-E2) is a potent estrogen, but it inhibits estrogen-2/4-hydroxylase activity and is a poor substrate for catecholestrogen formation (9, 23, 24, 25). Using 2-Fl-E2 and the delayed implanting mouse model, we demonstrate herein that E2, interacting via the nuclear ERs, is required for the preparation of the receptive uterus; whereas a catecholestrogen 4-OH-E2, formed from E2 in the uterus, activates the embryo for implantation via generation of PGs.
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Materials and Methods |
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Animals
Adult CD-1 female mice (7–8 weeks old; Charles River, Raleigh,
NC) 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 or
pseudopregnancy. To induce conditions of delayed implantation, mice
were ovariectomized on the morning (0800–0900 h) of day 4 of pregnancy
or pseudopregnancy and maintained with daily injections of
P4 (2 mg/mouse) from days 5–7 (2). To initiate
implantation, P4-primed delayed-implanting pregnant mice
were injected either with E2 or 2-Fl-E2. In
embryo transfer experiments, pregnant mice served as blastocyst donors,
whereas pseudopregnant mice served as recipients. For experiments
requiring the depletion of endogenous ovarian steroids, females were
ovariectomized without regard to their estrous cycle, and they rested
for 2 weeks before receiving any treatment.
Embryo culture and transfer
Two-cell embryos were recovered by flushing oviducts on day 2 of
pregnancy, whereas blastocysts were collected by flushing uteri with
Whitten’s medium (33). Embryos were pooled from several mice for
various experiments. Normal blastocysts were recovered on day 4 (1400
h) of pregnancy. Dormant blastocysts were recovered from ovariectomized
P4-treated delayed mice on day 7 (0900 h). Two-cell embryos
were cultured in Whitten’s medium for development to blastocysts for
72 h (34). Dormant blastocysts were cultured for 24 h in
Whitten’s medium, in the presence or absence of various agents. After
termination of the cultures, blastocysts were washed several times in
the same medium. They were used for either autoradiographic binding of
125I-EGF (34) or were transferred to P4-treated
delayed pseudopregnant recipients (2), injected iv with
2-Fl-E2, 15 min before transfers. Normal blastocysts
recovered at 1400 h on day 4 were also transferred into delayed
pseudopregnant mice after an injection of 2-Fl-E2
(see Fig. 3
, A and B). Blastocysts developed from 2-cell embryos were
transferred to uteri of P4-treated pseudopregnant
recipients receiving E2 (25 ng/mouse) or
2-Fl-E2 (75 ng/mouse). Implantation sites (increased
uterine vascular permeability at the sites of blastocyst apposition)
were determined by iv injections (0.1 ml/mouse) of a Chicago Blue B dye
solution (1% in saline) 24 h after transfer of blastocysts (2).
They were killed 5 min later to examine implantation sites as
demarcated by discrete blue bands in the uterus. If implantation sites
were absent, uterine horns were flushed with saline to recover
unimplanted blastocysts. Mice without implantation sites or blastocysts
were excluded from the experiments.
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Assay of estradiol-2/4-hydroxylase (E-2/4-H)
To determine the E-2/4-H activity, luminal epithelia (with the
underlying stroma) were isolated from the delayed
P4-treated pseudopregnant mice by gently squeezing the
uterine horns from the ovarian to the cervical end with a pair of small
curved forceps. Microsomes were assayed for E-2/4-H by the product
isolation method using 14C-estradiol as described by us
previously (9). The separation of catecholestrogens was performed using
an LC300 liquid chromatograph equipped with a radioactive flow
detector. An IBM computer, connected with the detector, integrated the
peak areas of the separated products recorded in the
14C-channel corresponding to the authentic
2-OH-E2 and 4-OH-E2 in the electrochemical
channel. The integrated radioactive peaks were used for calculation of
E-2/4-H activity. The products were also authenticated by using a
neutral alumina column to adsorb catecholestrogens and separate them
from the parent estrogen. To examine the effects of 2-Fl-E2
on E-2/4-H activity, microsomes were preincubated with
2-Fl-E2 for 2 min before initiating the enzyme
reactions.
Nuclear [3H]thymidine incorporation in the uterus
To determine uterine cell-specific growth, nuclear
[3H]thymidine incorporation (DNA synthesis) was performed
as described previously (35). After 2 weeks of ovariectomy, the mice
were injected sc with oil (vehicle), 2-Fl-E2 (75 ng/mouse),
or E2 (75 ng/mouse), with or without P4
priming, for 2 days. At 18 h after the last injection, they
received an ip injection of [methyl-3H]thymidine (25
µCi/0.1 ml saline, specific activity, 40 mCi/mmol; RPI Corp., Mount
Prospect, IL) and were killed 2 h later. Uteri were fixed in 4%
paraformaldehyde and processed for paraffine embedding, sectioning, and
autoradiography. Slides were exposed for 3 weeks, developed, and
poststained with hematoxylin.
Cloning of the mouse CYP1B1 partial complementary DNA
(cDNA)
RT-PCR was used to generate the mouse-specific CYP1B1 partial
cDNA clone. RT-PCR conditions were essentially the same as described
previously by us (36). Oligonucleotide primers were synthesized, based
on the cDNA sequence of the mouse CYP1B1 cDNA (12). The primers were
(5'-TGCTCATCCTCTTTACCAGATACCC-3') (sense) and
(5'-TTGCCTACTGAGAATATCATCACAA-3') (antisense). The sense strand primer
corresponds to nucleotides 1381–1404, and the antisense strand
primer encompasses nucleotides 1732–1756 of the mouse CYP1B1
cDNA (12). Day-4 pregnant mouse uterine total RNA (1 µg) was
reverse transcribed using the antisense primer, as described (36). RT
products (3 µl) were amplified by PCR for 45 cycles using the cycle
parameters: 94 C, 1 min and 30 sec; 55 C, 2 min; 72 C, 2 min and 30
sec. The predicted RT-PCR product (376 base pairs) was analyzed by gel
electrophoresis, and this product was cloned into pCR-Script SK(+)
cloning vector (Stratagene, La Jolla, CA). Several
colonies were analyzed by restriction digestion; and finally,
nucleotide sequence of one clone was determined on both strands by the
dideoxynucleotide chain termination method (37) and the Sequenase
version 2.0 kit (United States Biochemical, Cleveland, OH).
Northern blot hybridization
To compare the estrogenic effects of 2-Fl-E2 with
those of E2, the expression of two estrogen-responsive
genes, lactoferrin (LF) and Muc-1, in the mouse uterus (38, 39) was
detected by Northern blot hybridization, as described previously (4).
Ovariectomized mice were given an sc injection of sesame oil (vehicle,
0.1 ml/mouse), E2 (75 ng/mouse), or 2-Fl-E2 (75
ng/mouse). Mice were killed at 24 h after steroid injections, and
uteri were processed for RNA extraction and hybridization. To determine
the levels of CYP1B1 messenger RNA (mRNA), poly(A)+ RNA was
extracted from uteri on days 1–8 of pregnancy. Total RNA (6.0 µg) or
poly(A)+ RNA (2 µg) was denatured, separated by
formaldehyde agarose gel electrophoresis, transferred to nylon
membranes, and cross-linked by UV irradiation. Blots were hybridized
with 32P-labeled LF, Muc-1, or CYP1B1 complementary RNA
(cRNA) probes (4). Hybrids were detected by autoradiography. The same
RNA blots were rehybridized to rpL7 (ribosomal protein L7) cRNA probe,
a house-keeping gene, to confirm RNA integrity and equal loading.
In situ hybridization
In situ hybridization was performed as described
previously (4). Uteri were cut into 4- to 6-mm pieces and flash frozen
in freon. Frozen sections (10 µm) from days 1, 4, or 5 of pregnancy
were mounted onto poly-L-lysine-coated slides and fixed in
cold 4% paraformaldehyde solution in PBS, acetylated, and hybridized
at 45 C for 4 h in 50% formamide hybridization buffer containing
the 35S-labeled antisense CYP1B1 cRNA probe. After
hybridization and washing, the sections were incubated with
ribonuclease (RNase) A (20 µg/ml) at 37 C for 15 min. RNase
A-resistant hybrids were detected by autoradiography using Kodak NTB-2
liquid emulsion. Parallel sections, hybridized with the sense probe,
served as negative controls. Slides were poststained with hematoxylin
and eosin.
Immunocytochemistry
Antipeptide antibodies to COX-2 were developed in rabbits using
the synthetic peptide encompassing the unique sequence in the
C-terminal region of mouse COX-2 protein. The peptide, ASASHSRLDDINPT,
corresponding to amino acids 563–577 of the mature COX-2 protein, was
coupled to thyroglobulin and used to raise antiserum to COX-2 (19, 20).
The antibody was affinity-purified over Affi-gel 15 column coupled with
the antigenic peptide. In brief, delayed blastocysts, cultured in the
presence of either the vehicle, E2, or 4-OH-E2
for 24 h, were cytospun onto poly-L-lysine-coated slides,
air-dried, and fixed in Bouin’s solution for 10 min. The embryos were
washed three times (10 min each) in PBS and processed for
immunocytochemistry using a Zymed-Histostain-SP Kit for rabbit primary
antibody (Zymed Laboratories, San Francisco, CA) (19, 20). After
immunostaining, sections were lightly counterstained with hematoxylin.
Red deposits indicated the sites of immunoreactive proteins. Control
slides were incubated in preneutralized antibodies with a 100-fold
molar excess of the antigenic peptide, and these blastocysts did not
show any positive immunostaining. The specificity of this antibody has
already been established (20).
Analysis of ERß mRNA in the blastocyst
RT-PCR was employed to detect ERß mRNA in the blastocyst (36).
The primers for ERß transcript were 5'-CAGAACCTCAAAAGAGTCC-3'(sense)
and 5'-GACCATTCCTACTTCATAACAC-3'(antisense) (40). The primers for mouse
rpL7 clone (a house-keeping gene) were 5'-TCAATGGAGTAAGCCCAAAG-3'
(sense) and 5'-CAAGAGACCGAGCAATCAAG-3' (antisense) (41). RNA was
isolated from day-4 pregnant uterus or ovary, or 70–80 day-4
blastocysts (36). Total uterine or ovarian RNA (1 µg) or 1/4
of embryonic RNA was treated with RNase-free deoxyribonuclease and was
reverse transcribed using specific antisense primers. RT products (3
µl) were subjected to PCR using the sense and antisense primers, as
described (36). PCR products were run on agarose gels and subjected to
Southern blotting using (32P)end-labeled internal primers,
5'-GATCACTAGAGCACACCTTAC-3' (ER-ß) and 5'-GATTGCCTTGACAGATAATTC-3'
(rpL7). Experimental and negative controls (without primers) were run
simultaneously.
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Results |
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, A and B).
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(45) and ERß (data not shown) are expressed in the
blastocyst, the effects of 4-OH-E2 were not influenced by a
specific ER antagonist ICI 182,780, suggesting participation of a
pathway distinct from the classical nuclear ERs. In contrast,
participation of PGs in mediating 4-OH-E2 effects was
evident, because indomethacin, an inhibitor of PG synthesis, blocked
4-OH-E2 effects on EGF binding. This block was reversed by
coaddition of PGE2, and PGE2 alone also
increased EGF binding to dormant blastocysts (Fig. 1
, A and B). Because
PGE2 can stimulate cAMP production, we speculated that
PGE2 effects are mediated via generation of cAMP, leading
to the activation of protein kinase A (PKA) pathway. Indeed, addition
of dbcAMP, a cell-permeable analog of cAMP, in culture medium,
up-regulated the levels of EGF binding to dormant blastocysts; and
the up-regulation by 4-OH-E2 was blocked by a PKA inhibitor
H8 (31). Inability of 4-OH-E2 to up-regulate EGF binding to
dormant blastocysts in the presence of an adenylyl cyclase inhibitor,
RMI 12,330A (30), is consistent with a role for cAMP in embryonic
activation (Fig. 1, A and B). Collectively, these results suggest that
the catecholestrogen 4-OH-E2, but not E2,
activates dormant blastocysts. This effect is mediated by generation of
PGs that activate adenylyl cyclase and, thus, the PKA system. The
implantation competence of dormant blastocysts activated in
vitro was examined by blastocyst transfer experiments.
Blastocyst implantation fails in the absence of uterine
catecholestrogen formation
To examine the site-specific roles of primary estrogen and
catecholestrogen in uterine preparation and blastocyst activation for
implantation, 2-Fl-E2 was used. As stated earlier,
2-Fl-E2 is a poor substrate and a potent inhibitor of
estrogen-2/4-hydroxylation (reviewed in Ref. 9). Although
2-Fl-E2 is a known potent estrogen (24, 46), we further
compared the estrogenic effects of 2-Fl-E2 and
E2. We observed that 2-Fl-E2 is equipotent to
E2 at a dose of 75 ng/mouse, with respect to cell-specific
uterine growth and gene expression (Fig. 2). Similar to E2 (35),
2-Fl-E2 stimulated nuclear 3H-thymidine uptake
in the luminal and glandular epithelia, and in the stroma when combined
with P4 in ovariectomized adult mice (Fig. 2A). Further,
the effects of E2 and 2-Fl-E2 in up-regulating
the levels of estrogen-responsive genes Muc-1 and LF in uteri of
ovariectomized mice were comparable (Fig. 2B). Nonetheless,
2-Fl-E2 (50 or 75 ng/mouse) was unable to induce
implantation in P4-treated delayed-implanting mice (Table 1); whereas E2, at these
doses (data not shown) or even at 10 ng/mouse, induced a normal number
of implantations (Table 1). These results suggest that
2-Fl-E2 prepared the uterus to the receptive state but
failed to activate the dormant blastocysts for implantation, possibly
because of its failure to form catecholestrogens. This issue was
further addressed by blastocyst transfer experiments.
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, see Fig. 3A). Dormant blastocysts, cultured with
4-OH-E2, implanted in all mice examined (Table 3). The effects of 4-OH-E2
were dose-dependent; and the ER antagonist ICI 182,780, at equimolar,
or even at 5-fold excess molar concentration, did not interfere with
4-OH-E2 effects on implantation (Table 3, see Fig. 3B). In
contrast, dormant blastocysts that had been cultured for 24 h, in
the presence or absence of E2, failed to implant in such
mice (Table 4, see Fig. 3B). Further, in
a separate set of experiments, only 2 of 65 blastocysts, developed
in vitro from the 2-cell stage, implanted when transferred
into uteri of P4-treated delayed mice receiving
2-Fl-E2 (n = 7), whereas 53 of 81 blastocysts,
similarly developed in vitro, successfully implanted upon
transfer within 1 h of E2 treatment of the recipients
(n = 4). It is to be noted that the observed implantation rates
are within the expected range, considering the various steps involved
in embryo transfers, especially handling of zona-free blastocysts,
which are fragile and sticky in nature. These results establish that
blastocysts become implantation-competent in the presence of
catecholestrogen and successfully implant when transferred into
receptive uterus.
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). To examine whether the effects of
4-OH-E2 were mediated by specific isoforms of COX, dormant
blastocysts were cultured, in the presence of 4-OH-E2 with
DuP697, a selective COX-2 inhibitor (28), or with acetylsalicylic acid
(aspirin), a preferential COX-1 inhibitor (26). After termination of
the cultures, blastocysts were transferred into uteri of
P4-treated recipients after an injection of
2-Fl-E2. As shown in Table 4 (see Fig. 3B), dormant
blastocysts, cocultured with 4-OH-E2 and COX-2 inhibitor,
failed to implant; whereas the COX-1 inhibitor was ineffective. These
results suggest that 4-OH-E2 activation of blastocysts is
mediated by COX-2. Activation of dormant blastocysts by
PGE2 and reversal of DuP697 effects by this PG (Table 4),
as well as up-regulation of COX-2 in the mural trophectoderm of dormant
blastocysts exposed to 4-OH-E2, not E2,
in vitro (Fig. 4) are
consistent with this suggestion.
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The mouse uterus synthesizes catecholestrogen 4-OH-E2
We surmised that, if catecholestrogen
4-OH-E2 is physiologically important for blastocyst
activation, it should be produced locally in the uterus and/or
blastocyst. Because E2 was ineffective in activating
dormant blastocysts in vitro, we postulated that blastocysts
lack the enzyme (E-2/4-H) for catecholestrogen formation. Thus, our
attention was focused on uterine E-2/4-H (9). As shown in Fig. 5, we observed that mostly
4-OH-E2 (
30.0 pmol/mg protein·30 min), but very little
or no 2-OH-E2, was formed from E2 by extracts
of the luminal epithelium and its underlying stroma isolated from the
P4-treated uteri. The formation of 4-OH-E2 from
E2 was inhibited by 2-Fl-E2 (8 pmol/mg
protein·30 min). This explains why E2 is ineffective in
inducing implantation in P4-treated delayed-implanting
mice, in the presence of 2-Fl-E2 (9).
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). We thought
that if CYP1B1 expression is important for generating
4-OH-E2 for blastocyst activation, it would be through cell
type-specific expression in the uterus. Thus, we examined the
distribution of CYP1B1 mRNA in the pregnant uterus by in
situ hybridization. On day 1, very low levels of accumulation were
noted. However, distinct accumulation of this mRNA occurred primarily
in stromal cells closely apposed to the luminal epithelium throughout
the pregnant uterus on day 4 morning, although low levels of signals
were also present in epithelial cells. In contrast, on day 5 (after the
initiation of implantation), accumulation of CYP1B1 mRNA declined at
the sites of implantation but was retained at the interimplantation
sites (Fig. 6B). The presence of E-4-hydroxylase activity in the
P4-treated uterus and the expression of CYP1B1 mRNA in the
day-4 uterus strongly suggest that 4-OH-E2 locally produced
in the uterus makes the blastocyst implantation-competent. The
significance of CYP1B1 expression at the interimplantation sites is
presently not clear.
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Discussion |
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Distinguishing the effects of estrogen on the uterus and embryo during implantation has been a major challenge since the inception of the concept that the process of implantation is the result of an intimate interaction between the blastocyst and uterus. Because our present and previous data show that the mouse uterus and blastocyst express classical nuclear ERs (reviewed in Refs. 45, 49), and because E2 can induce implantation in P4-treated delayed-implanting mice (2, 6), it is conceivable that estrogens should influence the functions of these two targets directly via interactions with the nuclear ERs. However, the failure of 2-Fl-E2 to induce implantation, or its capacity to block E2-induced implantation in the P4-treated delayed-implanting mice, suggests that a catecholestrogen is involved in implantation (9). The speculation that blastocysts are the target for catecholestrogens was strengthened by our observation that dormant blastocysts implant in the P4-primed uterus only if transferred within 1 h of E2 administration, as opposed to day-4 blastocysts, which implant even several h after an E2 injection (2). We reasoned that a catecholestrogen was formed from E2 very rapidly, but transiently, in the uterus and made the dormant blastocysts implantation-competent. The successful implantation of normal blastocyts, but not dormant blastocysts or blastocysts developed in vitro from 2-cell embryos, in P4-primed uterus after an injection of 2-Fl-E2, establishes that the uterine receptivity was achieved under this condition; but formation of catecholestrogen was absent to activate the blastocysts. This is consistent with the observation of implantation of blastocysts grown in vitro from the 2-cell stage, after transfer, within 1 h of E2 treatment of the recipients. Although either 2-OH-E2 (data not shown) or 4-OH-E2 is able to activate dormant blastocysts in culture, with respect to EGF binding, we considered 4-OH-E2 as a physiologically relevant catecholestrogen, because the day-4 pregnant (9) and P4-primed mouse uterus is capable of producing mainly this catecholestrogen. CYP1B1, which has been shown to catalyze estradiol 4-hydroxylation in humans (11, 12), is expressed in the mouse uterine stromal cells. Our observations of the presence of E-4-hydroxylase activity in extracts of luminal epithelial-stromal cell complex, and accumulation of CYP1B1 mRNA in stromal cells intimately apposed to the luminal epithelial cells before the attachment reaction on day 4, provide evidence that 4-OH-E2 is available to influence blastocyst functions locally. In addition, the peroxidase activity of COXs that are present in the periimplantation mouse uterus may also serve as an alternate source of catecholestrogens (13, 19, 20).
Our finding that a specific ER antagonist ICI-182,780 interferes with
normal implantation or E2-induced implantation in
P4-treated delayed-implanting mice (data not shown) further
convinced us that uterine preparation by estrogen is mediated by
nuclear ER. In contrast, the ability of 4-OH-E2 to make
dormant blastocysts implantation-competent in culture, and the failure
of this ER antagonist to prevent this effect, establish that embryonic
activation is not mediated by the classical ERs that are expressed in
the blastocyst. Because E2 and antiestrogens can bind to
the ligand-binding domains of both the ER and recently identified
ERß (40), and because E2 was ineffective in blastocyst
activation, we believe that 4-OH-E2 effects are not
mediated via the ligand-binding domains of ER or ERß. It is
possible that ER, and perhaps ERß, in the blastocyst is
unresponsive to E2. This is consistent with normal
development and implantation of ER-negative mouse embryos resulting
from heterozygous crossings (50). However, alternatively spliced forms
of ER or ERß (reviewed in Ref. 29) may exist in the blastocyst,
for mediating the 4-OH-E2 effects. There is also evidence
for the existence of estrogen-responsive genes with no
recognizable estrogen response element (reviewed in Ref. 29). Further,
a heterodimeric complex formed by ER and ERß, or combinatorial
complexes generated by many coactivator proteins or basal transcription
factors, may regulate gene expression differentially, in response to
specific estrogenic ligands (reviewed in Ref. 30). Thus, it is possible
that 4-OH-E2 effects are mediated via the formation of such
complexes. Alternatively, the effects are mediated via binding of
4-OH-E2 to membrane receptors (14). The participation of
nuclear orphan receptors and/or membrane receptors for steroids in
target tissues are, in fact, becoming increasingly evident (51, 52). In
this respect, we have recently demonstrated that 4-OH-E2,
but not E2, induces estrogen-responsive gene, LF, in the
uteri of ER-deficient mice, and this response is not negated by
antiestrogen (29). In addition, 4-OH-E2 may activate
blastocysts via generation of oxyradicals transiently.
Catecholestrogens are known to generate free radicals (53, 54, 55), and it
has recently been shown that a programed oxyradical burst occurs in
mouse blastocysts during their escape from the zona-pellucidae before
implantation (56).
Our results demonstrate that 4-OH-E2 activates dormant blastocysts via generation of PGs. Further, differential responses of blastocysts to inhibitors of COX-1 and COX-2 suggest that 4-OH-E2 activates blastocyts primarily via stimulation of the COX-2 pathway. Because the attachment reaction during implantation involves an interaction between the luminal epithelium and mural trophectoderm epithelium of the implantation-competent blastocyst, the accumulation of COX-2 immunoreactive protein in the mural trophectoderm of the dormant blastocysts, after activation by 4-OH-E2, is intriguing; and it suggests that these blastocysts acquired implantation competence via this pathway. Because COX-2 is inducible by a variety of stimuli (reviewed in Refs. 20, 21), it is not surprising that 4-OH-E2 induces this isoform in dormant blastocysts. Although binding of catecholestrogens to cell membranes has been reported (14), whether 4-OH-E2 influenced the dormant blastocysts by binding to its cell surface receptor is not known. We have recently shown that COX-2 is expressed in the uterus solely at the sites of blastocyst implantation and that this expression requires the presence of active blastocysts (19, 20). Using COX-1 and COX-2 null mice, we have further demonstrated that uterine COX-2, not COX-1, is essential for implantation (20). Collectively, the results suggest that blastocyst activation by 4-OH-E2 is associated with the induction of uterine COX-2 at the sites of blastocyst apposition. If blastocyst COX-2 is also important for its activation, it could be argued as to why COX-2 null embryos derived by heterozygous crossings implant and develop to term. It is possible that null embryos are rescued by PGs generated by wild-type or heterozygous blastocysts located in close proximity of the null embryos. Cooperative interactions among preimplantation embryos for their development have been documented in vitro (34). In addition to its accumulation in the cytoplasm, COX-2 is also localized in the perinuclear envelope in uterine cells at the sites of blastocyst implantation and is considered to influence nuclear events in an intracrine fashion (20). In contrast, COX-2 was exclusively localized in the cytoplasm of activated blastocysts, as described here, and is likely to generate PGs that exit cells and act as autocrine and/or paracrine functions. Inability to restore implantation defects in COX-2-deficient mice by exogenous administration of PGE2 (20), but resumption of activation of dormant blastocysts by PGE2, in the presence of a selective COX-2 inhibitor, are consistent with this consideration. The other possibility is that PG deficiency in COX-2 null embryos derived by heterozygous-crossings is compensated by COX-1. Indeed, using COX-1 and COX-2 null mice, it has recently been reported that compensatory PG synthesis occurs in absence of alternate COX isozymes (57). Collectively, the results suggest that both the embryonic and uterine COX-2 could be critical for embryo-uterine interactions during implantation. Using a physiologically relevant model of delayed implantation, we have been able to distinguish, for the first time, the embryonic and uterine events, with respect to estrogen actions in implantation. Under normal pregnancy conditions, separating these events (with respect to estrogen actions) has not been possible, because the system is never completely depleted of estrogen and/or P4, although their levels fluctuate with the stages of pregnancy.
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2 A Kansas Health Foundation predoctoral fellow.
3 Present address: Ligand Pharmaceuticals, Inc., La
Jolla, California 92037.
Received June 23, 1998.
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