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Endocrinology Vol. 139, No. 12 5235-5246
Copyright © 1998 by The Endocrine Society

ARTICLES

Coordination of Differential Effects of Primary Estrogen and Catecholestrogen on Two Distinct Targets Mediates Embryo Implantation in the Mouse1

Bibhash C. Paria, Hyunjung Lim2, Xiao-Ning Wang3, Joachim Liehr, Sanjoy K. Das and Sudhansu K. Dey

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the mouse, estrogen is essential for blastocyst implantation in the progesterone (P4)-primed uterus. The mechanism(s) by which estrogen initiates this response still remains elusive. The present investigation, using delayed implantation in the mouse, examined the differential role of estradiol-17ß (E2) and its catechol metabolite 4-hydroxy-E2 (4-OH-E2) in uterine and blastocyst activation for implantation. The conditions of delayed implantation were induced by ovariectomizing mice on day 4 (day 1 = vaginal plug) of pregnancy or pseudopregnancy and maintaining them with P4 from days 5–7. The binding of EGF to blastocysts was used as a marker for blastocyst activation. Our results show that whereas E2 fails to activate dormant blastocysts (with respect to EGF binding in vitro), 4-OH-E2, cAMP, or prostaglandin E2, is effective in this response. Further, whereas 4-OH-E2 induced-activation is not blocked by an antiestrogen, an inhibitor of PG synthesis, adenylyl cyclase or protein kinase A effectively blocks this activation. These results suggest that 4-OH-E2 effects on blastocysts are mediated by PGs, which, in turn, stimulate cAMP production and thus activation of protein kinase A. Two-fluoro-E2 is a poor substrate and an inhibitor of catecholestrogen synthesis, but it is estrogenic, with respect to uterine growth and gene expression. Using blastocyst transfer experiments, we observed that dormant blastocysts incubated with 4-OH-E2 in vitro, but not with E2, are capable of implanting in P4-treated delayed implanting mice receiving two-fluoro-E2. The results suggest that whereas E2 is necessary for preparation of the uterus, uterine-derived catecholestrogen is important for blastocyst activation for implantation. Indeed, the receptive uterus has the capacity to synthesize 4-OH-E2. Collectively, we demonstrate that the primary ovarian estrogen E2, via its interaction with nuclear estrogen receptors, participates in the preparation of the P4-primed uterus to the receptive state in an endocrine manner, whereas its metabolite 4-OH-E2, produced from E2 in the uterus, mediates blastocyst activation for implantation in a paracrine manner. Our results also establish that these target-specific effects of primary estrogen and catecholestrogen are both essential for implantation and that successful implantation occurs only when the activated stage of the blastocyst coincides with the receptive state of the uterus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SYNCHRONIZED development of the preimplantation embryo to the blastocyst stage and differentiation of the uterus to the receptive state are essential to the process of implantation (1, 2). In rodents, the earliest conspicuous sign for the initiation of the implantation process is an increased endometrial vascular permeability at the sites of blastocyst apposition (1). This coincides with the initial attachment reaction between the uterine luminal epithelium and blastocyst trophectoderm (3). In the mouse, this event occurs in the evening (2200–2300 h) of day 4 of pregnancy (4) and is followed by apoptosis of the luminal epithelium and stromal decidualization at the sites of blastocyst (5).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
E2, estrone (E1), estriol (E3), P4, dibutyryl cAMP (dbcAMP), epinephrine, dopamine, indomethacin, and acetylsalicylic acid (aspirin), a preferential COX-1 inhibitor (26, 27), were purchased from Sigma Chemical Co. (St. Louis, MO). DuP697 [5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl) thiophene], a COX-2 selective inhibitor (28), was a generous gift from Dr. Trzaskos, DuPont-Merck Pharmaceutical Co. (Wilmington, DE). PGE2 was obtained from Cayman Chemical Co. (Ann Arbor, MI), and 2-OH-E2 and 4-OH-E2 were purchased from Steraloids (Wilton, NH). ICI-182,780, an ER antagonist (29), was a gift of ICI Pharmaceuticals, Macclesfield, UK. RMI-12,350A, an inhibitor of adenylyl cyclase (30), was provided by Merrill National Laboratory (Cincinnati, OH); and H8, an inhibitor of cAMP-dependent protein kinase A (31), was purchased from Seikagaku America (Rockville, MD). 2-Fl-E2 was synthesized as described previously (32). Primers for RT-PCR were obtained from our Institutional Biotechnology Center.

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. 3Go, 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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  		Figure 3. Experimental protocols for blastocyst transfer experiments. Scheme A was used to generate results that are described in Table 2Go, and scheme B was used to generate results that are described in Tables 3Go and 4Go. Ovx, Ovariectomy.

 
EGF binding to blastocysts
Autoradiographic binding of 125I-EGF to blastocysts was performed as described previously (34). In brief, blastocysts were incubated for 30 min at 37 C in an atmosphere of 5% CO2 in air in 25-µl microdrops of Whitten’s medium containing 1 nM 125I-EGF (specific activity, 150 µCi/µg) in the absence or presence of 500-fold molar excess of unlabeled EGF. After termination of the incubation, blastocysts were washed in medium and fixed in 4% paraformaldehyde in PBS for 15 min at 4 C. Fixed blastocysts were placed onto poly-L-lysine coated slides by cytocentrifugation. Slides were dehydrated through ascending grades of alcohol, coated with Kodak NTB-2 emulsion (Rochester, NY), exposed for 7 days, and poststained with hematoxylin. The autoradiographic signals (silver grains), under a darkfield, were quantitated using OPTIMA II program with an image analysis system. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls test.

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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dormant blastocysts are activated by catecholestrogen, 4-OH-E2
The levels of EGF receptor (EGF-R) are down-regulated in dormant blastocysts but are rapidly up-regulated with blastocyst activation after termination of the delay by E2 in utero, suggesting possible interaction with uterine EGF-like ligands during implantation (36, 42, 43, 44). Furthermore, failure of E2 to up-regulate EGF-R levels in dormant blastocysts in vitro suggested that other inducers, generated from or by E2 in the uterus, were responsible for up-regulation of blastocyst EGF-R (36). Thus, EGF binding was used as a marker of blastocyst activation. We demonstrate that catecholestrogen 4-OH-E2, but none of the primary estrogens (E2, E1, or E3), consistently up-regulated the levels of EGF binding to dormant blastocysts in vitro. This effect of 4-OH-E2 was specific and was not caused by the catechol structure of the A-ring in 4-OH-E2, because epinephrine or dopamine was ineffective in this response (Fig. 1Go, A and B).



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  		Figure 1. EGF binding to blastocysts. A, Autoradiographic localization of 125I-EGF binding to dormant blastocysts after various treatments in culture. Pregnant mice, ovariectomized on the morning of day 4, were treated with P4 (2 mg/mouse) from days 5–7 to induce delayed implantation. On day 7, dormant blastocysts were recovered and subjected to various agents in culture. Representative photomicrographs, showing autoradiographic signals under brightfield, are shown at 200x. a, Day-4 normal blastocyst; b, day-4 normal blastocyst, nonspecific binding; c–e, dormant blastocysts, cultured for 24 h in medium alone, E2, and 4-OH-E2, respectively; f, 4-OH-E2, nonspecific binding; g, PGE2; h, PGE2, nonspecific binding. Nonspecific binding was determined in the presence of 500-fold molar excess of unlabeled EGF. The concentrations of various agents are indicated in B. ICM, Inner cell mass; Tr, trophectoderm. B, Quantitation of 125I-EGF binding to dormant blastocysts exposed to various agents. Autoradiographic grains were counted, under darkfield, using the OPTIMA II program with an image analysis system. Nonspecific binding was subtracted from the total binding to obtain specific binding. Each experiment used 15–20 blastocysts and was repeated 4–10 times with similar results. Five blastocysts in each experiments were used for quantitation. The concentrations of agents used were: E1, E2, E3, 4-OH-E2, or ICI-182,780 (ICI), each at 10-9 M; indomethacin (INDO, 10-5 M); PGE2 (1.4 x 10-5 M); dbcAMP (2 x 10-8 M); RMI 12,350A (2.6 x 10-8 M); H8 (3 x 10-8 M); and epinephrine or dopamine (3 x 10-8 M). Four-OH-E2, but not E2, at 10-10 M or 10-12 M concentration, also induced EGF binding (data not shown). Arrows indicate absence of any specific binding (see A for representative autoradiographic grain distribution patterns in blastocysts). Results are mean ± SEM. Bars marked with an asterisk are not significantly different from each other (P > 0.05).

 
Although ER{alpha} (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. 1Go, 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. 1Go, 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. 2Go). 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. 2AGo). 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. 2BGo). Nonetheless, 2-Fl-E2 (50 or 75 ng/mouse) was unable to induce implantation in P4-treated delayed-implanting mice (Table 1Go); whereas E2, at these doses (data not shown) or even at 10 ng/mouse, induced a normal number of implantations (Table 1Go). 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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  		Figure 2. Uterine estrogenic responses of 2-Fl-E2. A, Autoradiographic localization of nuclear 3H-thymidine incorporation in ovariectomized mouse uterus after steroid treatment. Ovariectomized mice were given an injection of (a) vehicle (oil, 0.1 ml/mouse), (b) E2 (75 ng/mouse), (c) 2-Fl-E2 (75 ng/mouse), (d) E2 plus P4 (2 mg/mouse), or (e) 2-Fl-E2 plus P4. Each mouse was injected ip with 25 µCi of 3H-thymidine, 18 h after the last injection. Mice were killed 2 h later, and their uteri were fixed in 4% paraformaldehyde and processed for paraffin sectioning and autoradiography. le, Luminal epithelium; ge, glandular epithelium; s, stroma; m, myometrium. B, Northern blot hybridization of uterine Muc-1 and LF mRNAs in steroid-treated adult ovariectomized mice. Ovariectomized mice were given a single injection of oil (0.1 ml/mouse), E2 (75 ng/mouse), or 2-Fl-E2 (75 ng/mouse). They were killed 12 h later, and uterine RNA was isolated. Total RNA (6 µg) samples were separated by formaldehyde-agarose gel electrophoresis, transferred, UV cross-linked to nylon membranes, and hybridized sequentially to 32P-labeled Muc-1, LF, and rpL7 probes, as indicated. These experiments were repeated twice with similar results.

 

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  		Table 1. Effects of 2-Fl-E2 on implantation in P4-treated delayed implanting mice

 
Whereas normal day-4 blastocysts implanted when transferred into uteri of P4-treated delayed pseudopregnant recipients receiving an injection of 2-Fl-E2, dormant blastocysts failed to do so (Table 2Go, see Fig. 3AGo). Dormant blastocysts, cultured with 4-OH-E2, implanted in all mice examined (Table 3Go). 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 3Go, see Fig. 3BGo). 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 4Go, see Fig. 3BGo). 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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  		Table 2. Implantation of normal day 4 or dormant day 7 blastocysts after transfer into uteri of delayed pseudopregnant recipients receiving an injection of 2-Fl-E2

 

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  		Table 3. Implantation of dormant blastocysts cultured with 4-OH-E2 and/or ICI-182,780 after transfer into the uteri of delayed pseudopregnant recipients injected with 2-Fl-E2

 

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  		Table 4. Implantation of dormant blastocysts cultured with estrogens and PGE2 after transfer into the uteri of delayed pseudopregnant recipients injected with 2-Fl-E2

 
Apparently, 4-OH-E2 effects are mediated via generation of PGs (Fig. 1Go). 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 4Go (see Fig. 3BGo), 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 4Go), 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. 4Go) are consistent with this suggestion.



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  		Figure 4. Immunolocalization of COX-2 in dormant and activated blastocysts. Dormant blastocysts recovered from P4-primed delayed implanting mice on day 7 were cultured for 24 h in Whitten’s medium, in the presence of (a) vehicle; (b) E2; (c) 4-OH-E2. Blastocysts were processed for immunostaining. Red deposits indicate the sites of immunoreactive COX-2 at 200x. Blastocysts were counterstained with hematoxylin. ICM, inner cell mass; Tr, trophectoderm.

 
As stated earlier, the conversion of 2-Fl-E2 to catechol metabolites in vivo is poor, although renal microsomes can catalyze 2-Fl-E2 into 2-Fl-4-OH-E2 in vitro (47). However, this metabolite is not detected in the rat injected with 2-Fl-E2 (48). It is still a possibility that 2-Fl-E2 in the uterus is converted to 2-Fl-4-OH-E2 and could activate dormant blastocysts. To address this possibility, P4-treated delayed implanting mice were injected with 2-Fl-4-OH-E2 (75 ng/mouse) to induce implantation. None of the 5 mice had implantation, and 27 dormant blastocysts were recovered, suggesting rapid clearance of this catechol metabolite as methyl ether by catechol-O-methyltransferase (47).

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. 5Go, 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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  		Figure 5. Estradiol-4-hydroxylase activity in uterine microsomes of P4-treated day-7 delayed-implanting mice. Luminal epithelium, with the underlying stroma, was isolated by gently squeezing the uterine horns from the ovarian to the cervical end. E-2/4-H activity was measured in microsomes by a direct product isolation assay. The assay was run with [4-14C]E2, in the presence of NADPH. Basal activities were measured, in the absence of exogenous NADPH. Effects of 2-Fl-E2 were measured by incubating microsomes for 2 min before the enzyme reaction was initiated. LES, Microsomes of luminal epithelium plus underlying stroma; LES+2-Fl-E2, LES incubated with 2-Fl-E2; Ut-LES, whole uterus stripped of LES. The results are mean ± SEM. Statistical analysis was performed by ANOVA and Newman-Keuls tests (*, P < 0.05).

 
CYP1B1 mRNA is expressed in the mouse uterus
The presence of NADPH-dependent E-4-H in the uterus prompted us to examine whether CYP1B1, the enzyme involved in NADPH-dependent 4-hydroxylation of estrogens, is expressed in the mouse uterus. Northern blot hybridization detected a transcript of approximately 5.1 kb CYP1B1 mRNA in uterine poly(A)+ RNA samples obtained from days 1–8 of pregnancy. The steady-state levels of this mRNA were low on days 1 and 2 but showed gradual increases from day 3 of pregnancy onward (Fig. 6AGo). 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. 6BGo). 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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  		Figure 6. CYP1B1 mRNA in the mouse uterus. A, Northern blot hybridization of CYP1B1 mRNA in the pregnant (days 1–8) mouse uterus, as indicated. Poly (A)+ RNA (2 µg) samples were separated by formaldehyde-agarose gel electrophoresis, transferred, UV cross-linked to nylon membranes, and hybridized sequentially to 32P-labeled CYP1B1 and rpL7 probes, as indicated. B, Hybridization of CYP1B1 mRNA in the mouse uterus. Brightfield (left column) and darkfield (right column) photomicrographs are shown at 40x. a and b, Day 1; c and d, day 4; e and f, day 5; myo, myometrium; s, stroma; le, luminal epithelium; ge, glandular epithelium; bl, blastocyst. These experiments were repeated twice with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study establishes a novel concept, that estrogen effects on implantation are diversified in a bimodal and target-specific manner. The highlight of this concept is that whereas E2 prepares the P4-primed uterus to the receptive state via its interaction with the classical ER, its catechol metabolite 4-OH-E2, produced locally in the uterus, makes the blastocyst implantation-competent via generation of PGs. The superimposition of these two events is essential for implantation. This may explain why preimplantation ovarian estrogen secretion on the morning of day 4 is essential for implantation and why its elimination results in blastocyst dormancy and delayed implantation. Thus, estrogen acts as an endocrine mediator in the uterus, whereas its catechol metabolite serves as a paracrine factor.

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{alpha} 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{alpha} or ERß. It is possible that ER{alpha}, and perhaps ERß, in the blastocyst is unresponsive to E2. This is consistent with normal development and implantation of ER{alpha}-negative mouse embryos resulting from heterozygous crossings (50). However, alternatively spliced forms of ER{alpha} 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{alpha} 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{alpha}-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.


   Acknowledgments
 
We thank Jue Wang for her excellent technical assistance in hybridization experiments.


   Footnotes
 
1 This work was supported, in part, by NIH Grant HD-12304 and as part of the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation [NIH Grants HD-29968 (to S.K.D.), HD-35114 (to B.C.P.), and ES-07814 (to S.K.D.)]. A center grant in Reproductive Biology (HD-33994) and a center grant in Mental Retardation and Developmental Disabilities (HD-02528) provided access to various core facilities. Back

2 A Kansas Health Foundation predoctoral fellow. Back

3 Present address: Ligand Pharmaceuticals, Inc., La Jolla, California 92037. Back

Received June 23, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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