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Prostaglandin E2. Receptor Subtype EP2. Gene Expression in the Mouse Uterus Coincides with Differentiation of the Luminal Epithelium for Implantation*.
0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 11 Printed in U.S.A.

Prostaglandin E2 Receptor Subtype EP2 Gene Expression in the Mouse Uterus Coincides with Differentiation of the Luminal Epithelium for Implantation* H. LIM†

AND

S. K. DEY

Department of Molecular and Integrative Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7336 ABSTRACT Among the PGs, PGE2 is considered especially important for implantation and decidualization. Four major PGE2 receptor subtypes, EP1, EP2, EP3, and EP4, mediate various PGE2 effects via their coupling to distinct signaling pathways. Previously, we have shown that the EP1, EP3, and EP4 genes are expressed in the periimplantation mouse uterus in a spatio-temporal manner, suggesting compartmentalized actions of PGE2 during this period. In this study, we examined the expression of the EP2 gene in the mouse uterus during the periimplantation period (days 1– 8) and during experimentally induced progesterone (P4)-maintained delayed implantation and its resumption by 17b-estradiol (E2). We also examined its regulation in the uterus by ovarian steroid hormones. Our results establish that EP2 messenger RNA (mRNA) is expressed exclusively in the luminal epithelium primarily on day 4 (the day of implantation) and day 5 (early implantation) of pregnancy. In (P4)-maintained delayed implanting

mice, EP2 mRNA was present in the luminal epithelium, and the expression was further enhanced regardless of the location of the blastocysts after reinitiation of implantation. This observation suggests little or no embryonic influence in regulating EP2 expression and, instead, shows its regulation by P4 and E2. Indeed, treatment with E2 and/or P4 exhibited unique regulation of this gene. The treatment of adult ovariectomized mice with E2 down-regulated the basal levels of EP2 mRNA, whereas that with P4 up-regulated its levels in the luminal epithelium. The up-regulation of EP2 mRNA levels by P4 was further augmented by superimposition of the E2 treatment, suggesting a synergistic interaction between E2 and P4 in regulating this gene in the uterus. Collectively, the results suggest that EP2 could be a potential mediator of PGE2 actions in regulating luminal epithelial differentiation and serve as a marker for uterine receptivity for implantation. (Endocrinology 138: 4599 – 4606, 1997)

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diators of increased endometrial capillary permeability, epithelial cell differentiation, and stromal cell proliferation/ differentiation to decidualization during early events of implantation (5, 6). PGs are derived from arachidonic acid by the cyclooxygenases (COX) pathway. COX, which exists in two isoforms, COX-1 and COX-2, is the rate-limiting enzyme in the biosynthetic pathway that converts arachidonic acid into PGH2. PGH2 is then converted by specific PG synthases into diverse PG isoforms, including PGE2, PGF2a, PGD2, and PGI2 (4). These PGs exert diverse effects through their G protein-coupled cell surface receptors (7). Among the PGs, PGE2 mediates many biological functions in cardiovascular, pulmonary, renal, endocrine, gastrointestinal, neural, reproductive, and immune systems (8). PGE2 can bind to and activate a set of functionally distinct cell surface receptors, EP1, EP2, EP3, and EP4, which are classified on the basis of their responses to various agonists and antagonists to PGE2. They also exhibit different characteristics with respect to their structures, tissue distribution, and signal transduction mechanisms (7). Thus, EP1 is coupled to Ca21 mobilization, whereas both EP2 and EP4 subtypes are coupled to the stimulation of adenylyl cyclase via Gs. In contrast, EP3 is coupled to Gi, which inhibits adenylyl cyclase activity. Cell surface receptors for PGF2a, PGD2, PGI2, or thromboxane have also been identified as FP, DP, IP, and TP, respectively (9 –12). Previously, we demonstrated that EP1, EP3, EP4, and FP genes are expressed in the mouse uterus during the periimplantation period in a spatio-temporal manner (13). The expression of EP3 and FP in the circular muscle of the myo-

YNCHRONIZED development of the embryo to the blastocyst stage and preparation of the uterus for the receptive state are key to the process of successful implantation (1, 2). Coordinated effects of ovarian estrogen and progesterone (P4) in a temporal and cell type-specific manner make the uterus receptive to blastocyst implantation. In the mouse, uterine receptivity for implantation occurs on day 4 (day 1 5 vaginal plug) of pregnancy when the luminal epithelial cells cease to proliferate and become differentiated, as opposed to entrance of stromal cells into mitosis (3). In this species, the first conspicuous sign for the initiation of implantation (attachment reaction) is increased endometrial vascular permeability at the site of blastocyst apposition that occurs in the evening (2300 –2400 h) of day 4 (1). This is followed by extensive proliferation and differentiation of uterine stromal cells into decidual cells. The vasoactive, mitogenic, and/or differentiating properties of PGs (4) place these lipid molecules as potential meReceived June 6, 1997. Address all correspondence and requests for reprints to: Dr. S. K. Dey, Department of Molecular and Integrative Physiology, Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160-7338. E-mail: [email protected]. *This work was supported by NIH grants as part of the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (HD-29968) and HD-12304. 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. † Kansas Health Foundation predoctoral fellow.

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metrium on days 3–5 suggested that this muscle layer is the target for PG-mediated uterine contractions required for embryo transport and spacing. In contrast, the expression of EP3 in a subpopulation of stromal cells at the mesometrial pole and of EP4 in the epithelium and stroma on these days suggested that the activation of these receptor subtypes by PGE2 could be important for preparation of the uterus for implantation. Further, expression of EP1, EP3, and EP4 in site-specific decidual cells during the postimplantation period suggested PGE2’s role in various aspects of decidualization process. However, information regarding the expression of EP2, an important member of the PGE2 receptor subtypes, in the uterus is very limited, except for a report describing the expression of this messenger RNA (mRNA) in the mouse uterus during pseudopregnancy (14). However, data concerning whether the expression of this gene in the uterus is influenced by developing embryos, ovarian steroids, and/or decidualization are totally lacking. In the present investigation, we examined the temporal and cell-specific expression of the EP2 gene in the mouse uterus during the periimplantation period (days 1– 8 of pregnancy) as well as its regulation during experimentally induced delayed implantation and after termination of the delayed implantation. We also examined the regulation of this gene in the ovariectomized adult uterus by estrogen and P4. The results establish that EP2 is associated with the luminal epithelial cell differentiation that is necessary for blastocyst implantation and that this gene is regulated synergistically by estrogen and P4, but not by developing and/or implanting embryo. Materials and Methods Animals and tissue preparation CD-1 mice (Charles River Laboratories, Raleigh, NC) were housed in the animal care facility at the University of Kansas Medical Center in accordance with NIH standards for the care and use of experimental animals. Adult females were mated with fertile males of the same strain to induce pregnancy (day 1 5 vaginal plug). Mice on days 1– 8 were killed at 0900 h, and their uteri were collected for RNA preparation and in situ hybridization. Pregnancy on days 1– 4 was confirmed by recovering embryos from the reproductive tracts. On days 5 and 6, implantation sites were identified by monitoring the localized uterine vascular permeability at the sites of blastocysts after iv injection of Chicago blue B solution in saline. Implantation sites were demarcated by discrete blue bands along the uterus (2). On days 7 and 8, implantation sites were distinct, and their identification did not require any special manipulation. To examine the effects of neutralization of P4 or estrogen effects on EP2 expression during the preimplantation period, pregnant mice received sc either an injection of RU-486 (400 mg/mouse; Roussel-UCLAF, Romaineville, France), a P4 receptor (PR) antagonist, on days 3 and 4 at 0900 h or an injection of ICI 182780 (50 mg/mouse; ICI Pharmaceuticals, Macclesfield, UK), a specific estrogen receptor (ER) antagonist, on day 4 at 0900 h. The control mice received the vehicle (0.1 ml oil/mouse) on days 3 and 4. They were killed on day 5 at 0900 h after injections of a blue dye solution to examine implantation sites, and uteri were collected for in situ hybridization. To induce and maintain delayed implantation, mice were ovariectomized at 0800 – 0900 h on day 4 of pregnancy and received daily injections of P4 from days 5–7 (2 mg/mouse; Sigma Chemical Co., St. Louis, MO) (2, 15). To terminate delayed implantation and to induce blastocyst activation, the P4-primed delayed implanting mice were given an injection of 17b-estradiol (E2; 25 ng/mice; Sigma) on the third day of the delay (day 7). Mice were killed 24 h after treatment with the respective steroid hormones, and their uteri were collected for in situ hybridization. The first visually detectable implantation sites after blue dye injection become evident 18 –24 h after an E2 injection.

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To determine the effects of estrogen and P4, mice were ovariectomized regardless of the stage of estrous cycle and rested for 2 weeks. They were treated with P4 (2 mg/mouse) for 2 days with or without an injection of E2 (100 ng/mouse) on the second day of P4 treatment. To neutralize the effects of P4 or E2, mice were injected with RU-486 (400 mg/mouse) or ICI-182780 (50 mg/mouse), respectively. Control animals received vehicle (0.1 ml oil/mouse) only. Mice were killed 24 h after the last injection, and their uteri were collected for in situ hybridization. All steroids and antagonists were dissolved in sesame oil and injected sc (0.1 ml/mouse).

Hybridization probes A mouse-specific complementary DNA to EP2 was generously provided by Dr. Ichikawa (Kyoto University, Kyoto, Japan) (16). For Northern hybridization, antisense 32P-labeled complementary RNA (cRNA) probe was generated, whereas for in situ hybridization, sense or antisense 35S-labeled cRNA probe was generated using appropriate polymerases. A part of the ribosomal protein L7 (rpL7) complementary DNA was subcloned into pCR-Script vector containing promoter for T7 polymerase and used as a template for synthesis of 32P-labeled antisense rpL7 RNA probe (17). The probes had specific activities of about 2 3 109 dpm/mg.

Northern blot hybridization Total RNA was extracted from uteri by a modified guanidine thiocyanate procedure (18, 19). Polyadenylated [poly(A)1] RNA samples were isolated by oligo(deoxythymidine)-cellulose column chromatography (20). Poly(A)1 RNA samples (2.0 mg) were denatured, separated by formaldehyde-agarose gel electrophoresis, transferred, and crosslinked to nylon membranes by UV irradiation. Northern blots were prehybridized and hybridized as described previously (21). Briefly, hybridization was carried out for 20 h at 68 C in 3 3 SET (1 3 SET 5 150 mm NaCl, 5 mm EDTA, and 10 mm Tris-HCl, pH 8.0), 20 mm phosphate buffer (pH 7.2), 250 mg/ml transfer RNA, 10% dextran sulfate, and approximately 2 3 106 counts/min of 32P-labeled antisense RNA probe/ml hybridization buffer. After hybridization, the blots were washed once in 1 3 SSC (standard saline citrate)-0.1% SDS for 1 h at 68 C, followed by a second washing in 0.3 3 SSC-0.1% SDS for 1 h under the same conditions, and the hybrids were detected by autoradiography. Stripping of the hybridized probe before subsequent rehybridization was achieved as described previously (21). Each blot was first hybridized to the EP2 probe and then to the rpL7 probe (a housekeeping gene) to confirm integrity, equal loading, and blotting of RNA samples. Northern blot hybridization experiments were repeated three times using independent RNA samples.

In situ hybridization In situ hybridization was performed as described previously (17, 18). Frozen uterine sections (10 mm) were mounted onto poly-l-lysinecoated slides and stored at 270 C until used. When required, frozen sections were cut serially to detect the sites of blastocysts. After removal from 270 C, the slides with the uterine sections were placed on a slide warmer (37 C) for 1 min and then fixed in 4% paraformaldehyde in PBS for 15 min at 4 C. After prehybridization, uterine sections were hybridized to 35S-labeled antisense EP2 cRNA probe for 4 h at 45 C. As negative controls, uterine sections were hybridized with the 35S-labeled sense probe. After hybridization and washing, the slides were incubated with ribonuclease A (RNase A; 20 mg/ml) at 37 C for 20 min. RNase A-resistant hybrids were detected within 3–5 days of autoradiography using Kodak NTB-2 liquid emulsion. The slides were poststained with hematoxylin and eosin. In situ hybridization experiments were repeated at least twice, using independent samples.

Results Northern blot analysis of the EP2 mRNA in the periimplantation uterus

Steady state levels of the EP2 mRNA in the periimplantation uterus (days 1– 8) were analyzed by Northern blot

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hybridization using 32P-labeled mouse EP2 antisense cRNA probe (Fig. 1). As reported previously (16), two transcripts (2.2 and 2.8 kilobases) were detected in poly(A)1-enriched whole uterine RNA samples. EP2 mRNAs were primarily detected on days 4 and 5 of pregnancy, preceded or followed by very low levels of expression on other days of pregnancy. In situ hybridization analysis of the EP2 mRNA in the periimplantation uterus

In situ hybridization study revealed distinct temporal and cell-specific localization of the EP2 mRNA during the periimplantation period. On days 1 (Fig. 2, A and B) and 2 (data not shown), no specific signals for EP2 mRNA were detected in any uterine cell type. In contrast, weak signals were first detected exclusively in the luminal epithelium on day 3 (data not shown) followed by augmentation of these signals on days 4 and 5 of pregnancy (Fig. 2, C–F). Furthermore, the signals were present throughout the luminal epithelium regardless of the location of blastocysts. No accumulation, however, was noted in the glandular epithelium. Autoradiographic signals were also not observed in decidualizing stroma during the postimplantation period (days 6 – 8); only the remaining luminal epithelium exhibited signals for this mRNA during this period. The photomicrographs for day 6 are shown (Fig. 2, G and H). The heightened signals in the luminal epithelium on days 4 and 5 are consistent with the results of Northern blot hybridization (Fig. 1). No positive autoradiographic signals were detected in sections hybridized with the sense probe (data not shown). These results provided evidence that the increased luminal epithelial EP2 mRNA levels on days 4 and 5 are correlated with rising P4 levels and preimplantation estrogen secretion on the morning of day 4. This is further supported by our observation of inhibition of implantation and drastic downregulation of luminal epithelial accumulation of EP2 mRNA on day 5 after treatment with RU-486, a PR antagonist, on days 3 and 4 of pregnancy (Fig. 3, compare A and B vs. C and D). An interference with estrogen functions by ICI 182780, an ER antagonist, on day 4 also inhibited implantation on day 5 and down-regulated the luminal epithelial accumulation of this mRNA (Fig. 3, E and F). However, this down-regulation was modest than when P4 effects were neutralized.

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In situ hybridization of EP2 mRNA in the delayed implanting uterus before and after the initiation of implantation

To determine whether the short window of unique cell type-specific localization of EP2 mRNA is under embryonic regulation, in situ hybridization was performed on uterine sections obtained from P4-treated delayed implanting mice or after the initiation of blastocyst activation and implantation by an E2 injection (Fig. 4). Autoradiographic signals were detected along the entire uterine luminal epithelium of P4treated delayed implanting mice (Fig. 4, A and B). Signals were greatly augmented when the implantation process was resumed by an injection of E2 (Fig. 4, C and D). However, this up-regulation was again noted regardless of the location of the activated blastocysts. This suggested that luminal epithelial expression of EP2 mRNA is regulated primarily by steroid hormones, but not by resident blastocysts. Regulation of the EP2 mRNA by ovarian steroid hormones

To further confirm that the EP2 gene in the uterus is primarily regulated by ovarian steroid hormones, adult ovariectomized mice treated with E2 alone, or P4 for 2 days with or without E2 (Fig. 5, A–D) were used. Mice were killed 24 h after the last injection, and in situ hybridization was performed on uterine sections. Basal levels of EP2 mRNA accumulation were observed in the luminal epithelium of ovariectomized mice treated with oil (vehicle) only (Fig. 5A). Although a single injection of E2 down-regulated this basal level of expression, treating the mice with P4 for 2 days up-regulated the level of this mRNA in the luminal epithelium (Fig. 5, B and C). Furthermore, this P4-up-regulated EP2 mRNA expression was remarkably enhanced when E2 treatment was superimposed on P4 priming (Fig. 5D). P4 induction of EP2 mRNA was attenuated by prior treatment of mice with a PR antagonist, RU-486 (Fig. 5E). In contrast, although treatment with this P4 antagonist greatly reduced the levels of EP2 mRNA in P4- plus E2-treated uterus (Fig. 5F), treatment with a specific estrogen antagonist, ICI-182780, maintained levels comparable to those observed in P4-onlytreated uterus (Fig. 5G). These results suggest that P4 is the prime regulator of the EP2 gene in the luminal epithelium, although a cooperative effect of E2 is operative only when it is superimposed upon P4 priming. The results obtained from normal pregnancy and delayed implantation models support this unique pattern of steroidal regulation of EP2 in the uterus. Discussion

FIG. 1. Northern blot analysis of the EP2 mRNA in the periimplantation mouse uterus. The mRNA levels were detected in poly(A)1 samples obtained from whole uteri on days 1– 8 of pregnancy. The transcript sizes are indicated. Autoradiographic exposures were 7 h for EP2 mRNA and 1.5 h for rpL7.

The highlights of the present investigation are that the PGE2 receptor subtype EP2 is expressed exclusively in the mouse uterine epithelium in a temporal manner during the periimplantation period and that this unique expression pattern is obtained by a well orchestrated effect of P4 and E2. The establishment of the receptive uterus is achieved by the coordinated actions of P4 and E2 in a temporal and cell-specific manner. In the mouse, the uterus on days 1 and 2 of pregnancy is under the influence of the preovulatory estrogen surge that directs proliferation of the epithelium. In contrast,

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FIG. 2. In situ hybridization of EP2 mRNA in the periimplantation mouse uterus. Uterine sections on days 1– 4 or 5– 8 of pregnancy were mounted onto the same slides. Sections were hybridized with a 35S-labeled antisense cRNA probe. RNase A-resistant hybrids were detected by autoradiography after 3 days of exposure. Uterine EP2 mRNA distribution on days 1 (A and B), 4 (C and D), 5 (E and F), and 6 (G and H) of pregnancy is shown in brightfield (left column) and darkfield (right column) photomicrographs at 3100. le, Luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; pdz, primary decidual zone; bl, blastocyst; em, embryo; M, mesometrial side; AM, antimesometrial side.

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FIG. 3. In situ hybridization of EP2 mRNA in day 5 pregnant uteri after treatment with an antagonist to PR (RU-486) or ER (ICI 182780). Day 5 uterus treated with vehicle (A and B), RU-486 (C and D), and ICI 182780 (E and F) is shown at 340. Brightfield (left column) and darkfield (right column) photomicrographs are shown. le, Luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; bl, blastocyst.

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FIG. 4. In situ hybridization of the EP2 mRNA in the delayed implanting uterus before and after the initiation of implantation. EP2 mRNA distribution in uterine sections from P4-treated delayed implanting (A and B) or P4- plus E2-initiated implanting mice (C and D) is shown in brightfield (A and C) and darkfield (B and D) photomicrographs at 340. le, Luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; bl, blastocyst.

rising levels of P4 from newly formed corpora lutea result in switching of the proliferation from the epithelium to the stroma on day 3 of pregnancy, which is further stimulated by preimplantation estrogen secretion on day 4 (3). This superimposition of estrogen on P4 priming appears to direct epithelial cell differentiation, which is essential for blastocystuterine interactions during the initial events of implantation. The beginning of EP2 mRNA expression exquisitely in the luminal epithelium on day 3 and its remarkable increase on day 4 (the day of the attachment reaction) suggest that PGE2 action via activation of this receptor subtype is involved in the differentiation of the luminal epithelium required for implantation. This assumption is consistent with the fact that PGE2 acts as a differentiating factor for different cell types of epithelial origin (22, 23). Furthermore, persistent expression of this gene on day 5 could be implicated in the maintenance of luminal epithelial differentiation during the early events of the implantation process. In this regard, the disruption of implantation by RU-486 or ICI 182780 was associated with down-regulation of luminal epithelial accumulation of EP2, reinforcing the idea that EP2 could be associated with the luminal epithelial cell differentiation that is required for implantation. The elevated uterine levels of COX-1 expression

on day 4 and of COX-2 at the implantation sites on day 5 (24) suggests that PGE2 should be available to exert its action via EP2. Both PGE2 and cAMP are known to be involved in stromal cell proliferation and decidualization (5, 6, 25). As the deepitheliated uterus fails to undergo decidualization in response to experimental stimuli, a signal(s) emanating from the luminal epithelium has been considered important for the initiation of stromal cell decidualization (26). Thus, it is possible that elevated levels of cAMP resulting from the activation of epithelial EP2 by PGE2 may participate in transmission of the luminal epithelial cell signals to the stroma for decidualization. On the other hand, there is evidence that information emanating from the stroma also influences epithelial cell functions (27). Thus, a cross-talk between the luminal epithelium and stroma by the coordinated actions of epithelial EP2 and stromal EP4 (13), both of which are coupled to stimulation of adenylyl cyclase, could be important for the initiation of implantation and decidualization. The induction of implantation by intraluminal injection of (Bu)2cAMP in the P4-maintained delayed implanting mice in the absence of estrogen (28) or rapid increases in intracellular cAMP after

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FIG. 5. In situ hybridization of the EP2 mRNA in steroid-treated adult ovariectomized uterus in the absence or presence of specific antagonists. Ovariectomized mice were given a single injection of sesame oil (0.1 ml/mouse), E2 (100 ng/mouse), or P4 (2 mg/mouse) for 2 days with or without an E2 injection on the last day of treatment, and mice were killed 24 h after the last injection. An injection of PR antagonist (RU-486) or ER antagonist (ICI-182780) was given 30 min before each steroid injection. Darkfield photomicrographs of representative longitudinal uterine sections are shown at 3100. A, Oil (control); B, E2; C, P4; D, P4 plus E2; E, RU-486 plus P4; F, RU-486, P4, and E2; G, ICI-182780, P4, and E2. le, luminal epithelium; s, stroma; myo, myometrium.

induction of decidual cell reaction in the hormonally primed uterus (25) is supportive of this possibility. Our study using the experimentally induced delayed implantation model demonstrates that uterine expression of EP2 is not influenced by either dormant or activated blastocysts; rather, this expression is regulated by ovarian steroids

P4 and/or E2. The similar expression pattern of EP2 in the pseudopregnant mouse uterus (15) as that in the pregnant uterus on days 4 and 5 reinforces the fact that this gene in the mouse uterus is primarily regulated by steroid hormones. Our present investigation further demonstrates that the EP2 gene is regulated uniquely in the uterus by P4 and E2, in that

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P4 up-regulates its expression, and E2 further potentiates this P4 effects, although E2 alone down-regulates the expression. The effects of P4 and E2 could be either synergistic or antagonistic with respect to various uterine functions and gene expression in a temporal and cell-specific manner. For example, synergistic and antagonistic effects of P4 and E2 on epithelial and stromal cell proliferation and/or differentiation, and their antagonistic effects on epithelial Muc-1, lactoferrin, amphiregulin, and erbB2 genes are well documented (3, 17, 21, 29, 30). As both P4 and estrogen are essential for implantation in the mouse, synergistic as well as antagonistic effects of these steroids at the molecular and cellular levels appear to be essential for successful implantation. The upregulation of EP2 in the luminal epithelium by P4 and E2 is the first example of true synergism between these two steroids at the molecular level in preparing the uterus for implantation. Synergistic modulation of the EP2 gene by P4 and E2 is consistent with its expression pattern in normal pregnant uterus on days 4 and 5 when the uterus has been exposed to rising P4 levels and preimplantation estrogen secretion. The effects of P4 and/or E2 on uterine EP2 expression is mediated by classical nuclear PR and ER, as PR and ER antagonists abrogated the effects of P4 and/or E2. Although synergistic and antagonistic cross-talk between ER and PR in gene expression has been studied (31, 32), better understanding of this cross-talk requires further investigation. In this respect, the EP2 should serve as a candidate gene to study synergism between steroid hormone receptors. Our previous and present investigations point toward the importance of ligand receptor signaling with PGs in the process of implantation and decidualization, and these events are attributes of the compartmentalized generation of PGs and/or expression of their receptors in the uterus during the periimplantation period. Application of PGE2 receptor subtype-specific antagonists or targeting of these genes by homologous recombination will address their definitive roles in implantation and uterine biology. Acknowledgments We thank Dr. S. K. Das for his advice concerning the molecular biology experiments, and Wen-ge Ma for technical assistance.

References 1. Psychoyos A 1973 Endocrine control of egg implantation. In: Greep RO, Astwood EG, Geiger SR (eds) Handbook of Physiology. American Physiological Society, Washington DC, pp 187–215 2. Paria BC, Huet-Hudson YM, Dey SK 1993 Blastocyst’s state of activity determines the “window” of implantation in the mouse receptive uterus. Proc Natl Acad Sci USA 90:10159 –10162 3. 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 125:1638 –1690 4. Smith WL, DeWitt DL 1996 Prostaglandin endoperoxide H synthase-1 and -2. Adv Immunol 62:167–215 5. Tawfik OW, Sagrillo C, Johnson DC, Dey SK 1987 Decidualization in the rat: role of leukotrienes and prostaglandins. Prostaglandins Leukotr Med 29:221–227 6. Kennedy TG 1985 Evidences for the involvement of prostaglandins throughout the decidual cell reaction in the rat. Biol Reprod 33:140 –146 7. Negishi M, Sugimoto Y, Ichikawa A 1995 Molecular mechanisms of diverse actions of prostanoid receptors. Biochim Biophys Acta 1259:109 –120 8. Coleman RA, Smith WL, Narumiya S 1994 International union of pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev 46:205–229

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9. Sugimoto Y, Hasumoto K-Y, Namba T, Irie A, Katsuyama M, Negishi M, Kakizuka A, Narumiya S, Ichikawa A 1994 Cloning and expression of a cDNA for mouse prostaglandin F receptor. J Biol Chem 269:1356 –1360 10. Hirata M, Kakizuka A, Aizawa M, Ushikubi F, Narumiya S 1994 Molecular characterization of a mouse prostaglandin D receptor and functional expression of a cloned gene. Proc Natl Acad Sci USA 91:11192–11196 11. Namba T, Oida H, Sugimoto Y, Kakizuka A, Negishi M, Ichikawa A, Narumiya S 1994 cDNA cloning of a mouse prostacyclin receptor. J Biol Chem 269:9986 –9992 12. Namba T, Sugimoto Y, Hirata M, Hayashi Y, Honda A, Watabe A, Negishi M, Ichikawa A, Narumiya S 1992 Mouse thromboxane A2 receptor: cloning, expression and Northern blot analysis. Biochem Biophys Res Commun 184:1197–1203 13. 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 56:368 –379 14. Katsuyama M, Sugimoto Y, Morimoto K, Hasumoto K-Y, Fukumoto M, Negishi M, Ichikawa A 1997 Distinct cellular localization of the messenger ribonucleic acid for prostaglandin E receptor subtypes in the mouse uterus during pseudopregnancy. Endocrinology 138:344 –350 15. Yoshinaga K, Adams CE 1966 Delayed implantation in the spayed, progesterone treated adult mouse. J Reprod Fertil 12:593–595 16. Katsuyama M, Nishigaki N, Sugimoto Y, Morimoto K, Negichi M, Narumiya S, Ichikawa A 1995 The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and Northern blot analysis. FEBS Lett 372:151–156 17. Lim H, Dey SK, Das SK 1997 Differential expression of the erbB2 gene in the periimplantation mouse uterus: potential mediator of signaling by epidermal growth factor-like growth factors. Endocrinology 138:1328 –1337 18. Das SK, Wang X-N, Paria BC, Damm D, Abraham JA, Klagsbrun M, Andrews GK, Dey SK 1994 Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation. Development 120:1071–1083 19. 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 26:1617–1625 20. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor 21. Das SK, Chakraborty I, Paria BC, Wang X-N, Plowman GD, Dey SK 1995 Amphiregulin is an implantation-specific and progesterone-regulated gene in the mouse uterus. Mol Endocrinol 9:691–705 22. Evans CB, Pillai S, Goldyne ME 1993 Endogenous prostaglandin E2 modulates calcium-induced differentiation in human skin keratinocytes. Prostaglandins Leukotr Essent Fatty Acids 49:777–781 23. McArdle CA 1990 Chronic regulation of ovarian oxytocin and progesterone release by prostaglandins: opposite effects in bovine granulosa and early luteal cells. J Endocrinol 126:245–253 24. 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 16:107–122 25. Rankin JC, Ledford BE, Baggett B 1977 Early involvement of cyclic nucleotides in the artificially stimulated decidual cell reaction in the mouse uterus. Biol Reprod 17:549 –554 26. Lejeune B, Van Hoeck J, Leroy F 1981 Transmitter role of the luminal uterine epithelium in the induction of decidualization in rats. J Reprod Fertil 61:235–240 27. Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J, Korach KS, Taylor JA, Lubahn DB, Cunha GR 1997 Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. Proc Natl Acad Sci USA 94:6535– 6540 28. Holmes PV, Bergstrom S 1975 Induction of blastocyst implantation in mice by cyclic AMP. J Reprod Fertil 43:329 –332 29. Surveyor GA, Gendler SJ, Pemberton L, Das SK, Chakraborty I, Julian J, Pimental RA, Wegner CC, Dey SK, Carson DD 1995 Expression and steroid hormonal control of Muc-1 in the mouse uterus. Endocrinology 136:3639 –3647 30. McMaster MT, Teng CT, Dey SK, Andrews GK 1992 Lactoferrin in the mouse uterus: analyses of the preimplantation period and regulation by ovarian steroids. Mol Endocrinol 6:101–111 31. Bradshaw MS, Tsai SY, Leng X, Dobson ADW, Conneely OM, O’Malley BW, Tsai M-J 1991 Studies on the mechanism of functional cooperativity between progesterone and estrogen receptors. J Biol Chem 266:16684 –16690 32. Kraus WL, Weis KE, Katzenellenbogen BS 1995 Inhibitory cross-talk between steroid hormone receptors: differential targeting of estrogen receptor in the repression of its transcriptional activity by agonist- and antagonist-occupied progestin receptors. Mol Cell Biol 15:1847–1857