Molecules in blastocyst implantation: Uterine and embryonic perspectives

VITAMINS AND HORMONES, VOL. 64

Molecules in Blastocyst Implantation: Uterine and Embryonic Perspectives HYUNJU~G LIM,. 1 HAEN,GSEOKSONG,* B. C. PARIA,*,T JEFF REESE,*'T SANJOY K. DAS,*'$ AND S. K .

DEY* 1

*Department of Molecular and Integrative Physiology, t Department of Pediatrics, and SDepartment of Obstetrics and Gynecology, Ralph L. Smith Research Center University of Kansas Medical Center, Kansas City, Kansas 66160-7338 I. Introduction II. Embryo-Uterine Interactions A. Mouse as a Model to Study Implantation B. Uterine Receptivity C. Blastocyst Activation III. Uterine Factors in Implantation A. Steroid Hormone Signaling B. Adhesion Molecules C. Histamine and Prostaglandin Signaling D. Growth Factor Signaling E. Cytokine Signaling Pathway F. Transcription Factors G. Cannabinoid Signaling Pathway IV. Embryonic Factors in Implantation A. Steroid Hormone Signaling B. Adhesion Molecules C. Growth Factor Signaling D. Cannabinoid Signaling V. Uterine Markers in Humans VI. Conclusion References

Synchronized development of the embryo to the active stage of the blastocyst, differentiation of the uterus to the receptive state, and a "cross talk" between the blastocyst and uterine luminal epithelium are essential to the process of implantation. In spite of considerable accumulation of information and the present state of the knowledge, our understanding of the definitive mechanisms t h a t regulate these events remains elusive. Although there are species variations in the process of implantation, m a n y basic similarities do exist among various species. This review focuses on specific aspects 1Correspondence and requests for reprints should be addressed to H. Lim or S. K. Dey. 43

Copyright © 2002 by Academic Press. All rightsofreproductionin any form reserved. 0083-6729/02 $35.00

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HYUNJUNGLIMETAL. of the implantation process in mice with the hope that many of the findings will be relevant to the process in humans. To establish signaling mechanisms of embryo-uterine interactions during implantation, studies on both embryonic and uterine consequences are required to generate more meaningful information. Due to ethical restriction and experimental limitation, it is difficult to generate such information in humans. This review has attempted to provide a comprehensive, but not complete, narration of a number of embryonic and uterine factors that are involved in the process of implantation in autocrine, paracrine, and/or juxtacrine manners in mice at the physiological, cellular, molecular, and genetic levels. © 2002 Academic Press.

I. INTRODUCTION

A considerable loss resulting from preimplantation embryonic death is common to many mammals. This can be regarded as a natural selection process that chooses superior embryos for implantation. In addition, defects in the events during and immediately after implantation often give rise to poor pregnancy rates in eutherian mammals (Cross et al., 1994). Therefore, a comprehensive understanding ofpreimplantation embryo development and implantation is a fundamental challenge in alleviating the problems of infertility, ensuring the birth of healthy offspring, and developing safe and useful contraceptive approaches to restrict world population. Successful implantation results from an intimate "two-way" interaction between the blastocyst and the uterus. After fertilization in the oviduct, the embryo undergoes a series of mitotic cell divisions ultimately forming the differentiated stage of the blastocyst. Blastocysts are comprised of two cell types, the inner cell mass (ICM) and the trophectoderm (Tr). The ICM forms the embryo proper, and the trophectoderm, the very first epithelial cell type in the developmental process, makes the first physical and physiological contact with the maternal endometrium. In mammals, the timing of embryonic development to the blastocyst stage and the nature of its initial interactions with the uterus for implantation vary from species to species. In mice, the fourth day of pregnancy after mating marks the day of implantation (Dey, 1996), whereas in humans, the window of implantation is between days 20 and 24 of the menstrual cycle (Anderson, 1990). This is the time when the steroid hormonal milieu becomes optimal for e m b r y o - u t e r i n e interactions. In the mouse, the first known molecular interaction between the blastocyst and the uterus occurs

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around 1600 h on day 4 of pregnancy (Das et al., 1994a). The first step in the implantation process, the apposition of the blastocyst to the uterine epithelium, is initiated by creation of an implantation chamber surrounding each blastocyst along the uterine lumen. This is preceded by generalized uterine edema and luminal closure, which result in close apposition of the blastocyst trophectoderm with the luminal epithelium. One of the earliest detectable signs of implantation is a localized increase in endometrial vascular permeability at the site of blastocyst apposition. This vascular response can be monitored by intravenously injecting a macromolecular blue dye (Psychoyos, 1973). With the initiation of the attachment reaction, the ICM is positioned toward the uterine lumen while the mural trophectoderm makes direct contact with the uterine epithelium (Fig. 1). Whereas this is the case in mice, human blastocysts orient themselves in the opposite direction in the uterus and the polar trophectoderm unites with the luminal epithelium. When this union is made within each implantation chamber, a plethora of molecular interactions commences in a coordinated fashion marking the attachment phase. The attachment reaction is followed

FIG. I. Attachment reaction of the blastocyst. Around midnight of day 4 of pregnancy, the attachment reaction occurs between the blastocyst and uterine luminal epithelium with respect to localized endometrial vascular permeability. Key: le, luminal epithelium; s, stroma; bl, blastocyst; ge, glandular epithelium.

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by trophoblast penetration through the underlying epithelial basement membrane and leads to an extensive stromal cell proliferation and differentiation to decidual cells (decidualization) (Enders, 1976; Parr et al., 1987). Molecular signals that render the uterus receptive for implantation and enable bidirectional communications between the uterus and the blastocyst are complex and yet to be clearly defined. This review presents accumulated and emerging data with regard to the molecules involved in these events from both the uterine and embryonic perspectives. Due to molecular and genetic advances during the last two decades, mice are being widely used as a model system to study the implantation process. Therefore, a large part of the work narrated here represents various descriptive and mechanistic approaches taken to elucidate the involvement of a number of molecules and their signaling pathways in implantation in mice. Since many of the findings in mice have been replicated in humans, those findings will also be summarized at the end of this review.

II. EMBRYO--UTERINE INTERACTIONS

A. MOUSE AS A MODEL TO STUDY IMPLANTATION For many decades, the mouse has served as an animal model for implantation. There are certain similarities between mouse and human preimplantation and implantation physiology. Both mouse and human preimplantation embryos can develop in vitro in a simple defined medium (Whitten and Biggers, 1968; Muggleton-Harris et al., 1990). Although the initial reaction to implantation in the mouse is eccentric, as opposed to interstitial in humans, in both species embryo implantation leads to stromal decidualization, and embryos embed in the antimesometrial stroma (Dey, 1996). Further, both the mouse and human have a hemochorial type of placentation. The implantation process is a two-way interaction between the embryo and uterus: uterine factors influence embryonic functions, and embryonic factors affect uterine events during implantation. Because it is not possible to use human embryos to study embryo-uterine interactions during implantation, the mouse fulfills this purpose and serves to provide a more mechanistic approach in defining the molecular basis of implantation. Physiological functions of specific factors can be examined more mechanistically by overexpression or targeted deletion of their genes in transgenic mice. Indeed, rapidly accumulating data using these

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approaches are helping us to generate novel concepts and ideas regarding implantation. B. UTERINE RECEPTWITY

In all eutherian mammals so far studied, the uterus must differentiate to a receptive state for successful implantation to occur. Uterine receptivity is defined as the window of time when the uterine environment is conducive to support blastocyst growth, attachment, and subsequent events of implantation (Psychoyos, 1973; Paria et al., 1993a; Dey, 1996). The major determinants of uterine receptivity are the ovarian steroids, progesterone (P4) and/or estrogens. While ovarian P4 and estrogen are essential for implantation in mice and rats, ovarian estrogen is not an essential component for implantation in pigs, guinea pigs, rabbits, and hamsters (Heap and Deanesly, 1967; Harper et al., 1969; Psychoyos, 1973; Kwun and Emmens, 1974; McCormack and Greenwald, 1974; Heap et al., 1981). However, the role of embryonic estrogen in implantation in these species cannot be ruled out. Indeed, estrogen-synthesizing capacity has been documented in rabbit and pig embryos (reviewed in Stromstedt et al., 1996). Whether preimplantation ovarian estrogen secretion is essential for implantation in humans is unknown. In mice, the coordinated actions of Pa and estrogen that regulate proliferation and/or differentiation of uterine cells establish the window of implantation (Huet-Hudson et al., 1989). For example, on the first day of pregnancy (vaginal plug), uterine epithelial cells undergo proliferation under the influence of the preovulatory estrogen surge. In contrast, rising levels of Pa secreted from freshly formed corpora lutea drive stromal cell proliferation from day 3 onward. The stromal cell proliferation is further potentiated by a small amount of ovarian estrogen secreted on the morning of day 4 of pregnancy. The coordinated effects of P4 and estrogen on this day result in the cessation of uterine epithelial cell proliferation with induction of differentiation (Huet-Hudson et al., 1989). In normal pregnancy, the presence of an active blastocyst in the uterus is the stimulus for the implantation reaction. Following the attachment reaction, stromal cells surrounding the implanting blastocyst begin to proliferate extensively and differentiate into decidual cells (decidualization) (Dey, 1996). In pseudopregnant mice, the steroid hormonal environment in the uterus is similarly maintained due to the presence of newly formed corpora lutea. Thus, the sensitivity of the pseudopregnant uterus for implantation during this period is quite similar to that in normal pregnancy, and blastocyst transfer into

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the uterine lumen during the receptive phase provokes normal implantation reactions and subsequent decidualization. Although the blastocyst is a normal inducer of these events, various nonspecific stimuli (intraluminal infusion of oil, air, and mechanical stimuli) can simulate certain aspects of the decidual cell reaction (deciduoma) in pseudopregnant or steroid hormonally prepared uteri (Dey, 1996). However, there is evidence that the initial uterine reactions induced by nonspecific stimuli are different from those induced by blastocysts (Lundkvist and Nilsson, 1982). Uterine sensitivity with respect to hormonal requirements and implantation has been classified into prereceptive, receptive, and nonreceptive (refractory) phases (Psychoyos, 1973; Dey, 1996). The various phases of uterine sensitivity to implantation have been defined by employing embryo transfer experiments in pseudopregnant mice (Fig. 2).

Pedimplant~ion Mouse Uterus

,|

i 5 1

2

3

4

5

Pre-recepttve - ~ l ~ R e c e p t i v e ~ l

--I

6

7

8

Refractory

Window cl~ed

FIG. 2. Window of receptivity in the mouse. Steroid hormonal status during the periimplantation period is shown. On day 1 of pregnancy, the uterus is under the influence of the preovulatory estrogen surge. From day 3 onward, increasing levels of progesterone are produced from the newly formed corpora lutea. On day 4 morning, a small amount of "preimplantation estrogen" is also secreted from the ovary. Using blastocyst transfer experiments into pseudopregnant recipients with similar hormonal status, it is possible to monitor the "window" of uterine receptivity for implantation.

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In the mouse, while the uterus is fully receptive on day 4, it is considered prereceptive on days 1-3 of pregnancy or pseudopregnancy, and blastocysts transferred into uteri on these days fail to implant. Experimental evidence demonstrates that the mouse uterus can only be rendered receptive for blastocyst implantation if exposed to a small amount of estrogen after 24-48 h of P4 priming (Huet and Dey, 1990). Collectively, the results showed that the uterus is most receptive for implantation on day 4 (Paria et al., 1993a; S. K. Dey et al., unpublished) and the efficiency of implantation decreases with time (S. K. Dey et al., unpublished). By day 6, the uterus becomes completely refractory to blastocyst implantation. Another critical factor determining the window of implantation is the blastocyst's state of activity, as described below. C. BLASTOCYSTACTIVATION

In mice, ovariectomy prior to the preimplantation estrogen secretion on the morning of day 4 of pregnancy induces delayed implantation (Yoshinaga and Adams, 1966; Paria et al., 1993a). This status can be maintained for many days if P4 treatment is continuously provided. Under this condition, although blastocysts show zona hatching, albeit at a slower pace, they undergo dormancy without initiating the attachment reaction. However, a single injection of estrogen initiates blastocyst activation with the initiation of implantation in the P4-primed uterus. Blastocyst dormancy, as opposed to blastocyst activation, is both molecularly and physiologically distinguishable (Fig. 3). Epidermal growth factor receptor (EGF-R), cyclooxygenase-2 (COX-2), and histamine type 2 receptor (n2), molecules implicated in blastocyst attachment reaction, are expressed in normal or active blastocysts, but are downregulated in dormant blastocysts (Paria et al., 1993b, 1998a; Raab et al., 1996; Lim et al., 1997; Zhao et al., 2000). On the contrary, a G protein-coupled cannabinoid receptor, CB1, which is activated by cannabinoid-like ligands, is downregnlated in active blastocysts, but remains upregnlated in dormant blastocysts (Paria et al., 2001 Fig. 3). A tight regulation of the ligand-receptor signaling with natural or endogenous cannabinoid-like molecules appears to be important for blastocyst functions and implantation (reviewed in Paria and Dey, 2000; see Sections III.G and IV.D). Collectively, these findings suggest that a complex array of molecular networking regulates blastocyst activation and dormancy. Although estrogen is essential for blastocyst activation and implantation in the P4-primed mouse uterus, the mechanisms by which estrogen initiates these responses remain elusive. We speculated that estrogen

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Normal Blastocyst

Dormant Blastocyst

~4-OH-E= (PGE=) (cAMP)

EGF-R cox-2 Hz-R CBI

EGF-R~ cox-2 Hz-R~ CBI~

FIG. 3. Molecular markers for the blastocyst's state of activity. Whereas normal and dormant blastocysts apparently show morphological differences, several molecular markers are regulated reversibly by the blastocyst's state of activity. For example, 4-OH-E2, PGE2, or cAMP can activate dormant blastocysts in vitro and can either up- or downregulate specific marker molecules as indicated. If normal blastocysts are induced to undergo dormancy by the employment of delayed implantation in the uterus prior to the attachment reaction, expression of these markers is shifted in the reverse direction. Key: EGF-R, epidermal growth factor receptor; COX-2, cyclooxygenase-2; H2-R, histamine type 2 receptor; CB1, brain-type cannabinoid receptor.

actions in uterine preparation and blastocyst activation for implantation are two distinct events. Indeed, our recent study using embryo transfer experiments in delayed-implanting recipient mice provides evidence that while the primary estrogen, estradiol-17fl (E2), initiates uterine events for implantation, its catechol metabolite, 4-hydroxy-E2 (4-OH-E2), participates in activating dormant blastocysts (Paria et al., 1998a; Fig. 4). Blastocyst activation by 4-OH-E2 involves COX-2-derived prostaglandins (PGs) and cAMP (Paria et al., 1998a). Furthermore, the use of the estrogen receptor antagonist ICI-182, 780 has shown that while E2 via its interaction with the nuclear estrogen receptors (ERs) participates in the preparation of the P4-primed uterus to the receptive state in an endocrine manner, 4-OH-E2 produced from E2 in the uterus mediates blastocyst activation in a paracrine manner that does not involve nuclear ERs (Fig. 4). This work established that both primary and catechol estrogens are required for embryo-uterine interactions for successful implantation and that implantation occurs only when uterine receptivity coincides with the blastocyst's state of activity. Potential molecular pathways that could be involved in blastocyst activation are discussed below in more detail.

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OH

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,~1~

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OH~~2~H'E2 OH ~ ' ~ f

2.M.E2 O~4Ho

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Target E2 4-OH-~

OH

Receptor

Signaling Pathway

ER Blastocyst Unknown

Transcription? cAMP production COX-2 pathway

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FIG. 4. Primary and catechol estrogens with distinct targets during implantation. Primary estrogen, estradiol-17fl(E2), can be converted to 2-OH-E2 by 2-hydroxylase (CYP1A1) or to 4-OH-E2 by 4-hydroxylase (CYP1B1). Paria et al. (1998a) have shown that E2 has a role in the preparation of the receptive uterus for implantation, while 4-OH-E2 activates blastocysts for implantation competency. The action of E2 apparently is mediated by classical nuclear ER affecting transcription of uterine genes, since this effect is neutralized by an ER antagonist. In contrast, the action of 4-OH-E2 in blastocyst activation is insensitive to the ER antagonist, and cAMP- and COX-2-derived PG signaling seems to be involved. This unique estrogenic action differs from classical genomic effects of estrogens in a variety of systems. However, a potential receptor for 4-OH-E2 in the blastocyst has not yet been discovered.

III. UTERINE FACTORS IN IMPLANTATION

A. STEROIDHORMONESIGNALING

As mentioned earlier, ovarian estrogen and P4 are critical to the process of implantation. Differential uterine expression of nuclear estrogen receptors (ER~ and ERp) and progesterone receptor (PR) during the periimplantation period in mice suggests that coordinated effects of estrogen and P4 in uterine events for implantation are mediated via these nuclear receptors (Tan et al., 1999). Furthermore, mouse models devoid of each receptor gene were informative as to how these receptors are involved in uterine biology. For example, E I ~ ( - / - ) mice exhibit infertility due to hyperstimulated ovaries and hypoplastic uteri (Lubahn et al., 1993). However, further experiments in E R a ( - / - ) mice showed that P4 alone is sufficient to support decidualization (deciduoma) in response to artificial stimuli (Paria et al., 1999a; Curtis et al., 1999).

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These results suggest that E R ~ ( - / - ) mice have defective implantation perhaps due to the failure of the attachment reaction, but not due to the failure of decidualization events (Das et al., 1997a). P R ( - / - ) mice also exhibit pleiotropic reproductive abnormalities including impaired ovulation, uterine hyperplasia, and defective decidualization (Lydon et al., 1995). A recent report of a selective ablation of the PR-A isoform also showed infertility with a milder phenotype, suggesting the PRA and PR-B serve as functionally distinct mediators of P4 action in vivo (Mulac-Jericevic et al., 2000). Experiments using both P R ( - / - ) and PR-A(-/-) mice further reinforced a requirement of P4 in decidualization (Lydon et al., 1995; Mulac-Jericevic et al., 2000). While both E R ( - / - ) and P R ( - / - ) mice show severe phenotypes of female reproductive failures, these mice have been used as model systems to study steroid hormonal regulation of several genes. Das et al. (1997a) reported a non-ER-mediated estrogen signaling pathway which is resistant to the ER antagonist ICI-182, 780 in E R a ( - / - ) uteri. Lactoferrin (LF) is a well-known estrogen-responsive gene and many natural or synthetic estrogens can induce this gene in wild-type uteri (Das et al., 1997a). In contrast, whereas E2 was not able to induce this gene in E R ~ ( - / - ) uteri, 4-OH-E2 was effective in this response. Since this induction by 4-OH-E2 was resistant to an ER antagonist (ICI-182, 780), it is also not considered to be mediated by ERfl. Das and coworkers further explored the possibility of an alternative estrogen signaling pathway independent of nuclear ERs in the mouse uterus (Das et al., 2000). Using a differential display technique, they found several genes that are similarly induced or downregulated by E2 and 4-OH-E2 both in wild-type and E P ~ ( - / - ) uteri independent of nuclear ERs. The genes that were rapidly upregulated include those for immunoglobulin heavy-chain-binding protein (Bip), calpactin I (CalP), calmodulin (CalM), and Sik-similar protein (Sik-SP), whereas the gene that encodes secreted frizzled related protein-2 (SFRP-2) was readily downregulated. An ER antagonist failed to neutralize these responses both in wild-type and E I ~ ( - / - ) mice, suggesting that they are ERindependent early responses. This provides compelling evidence that an alternative estrogen-signaling pathway is operative in the uterus. However, it is unclear whether this early estrogenic response is mediated by a putative cell-surface estrogen receptor or by other nuclear receptors, such as ERy or ERRs (Shigeta et al., 1997; Stefano et al., 2000). B. ADHESION MOLECULES

Many glycoproteins and carbohydrate ligands and their receptors have been reported to be expressed in the uterine luminal epithelium

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and the blastocyst (reviewed in Kimber and Spanswick, 2000). The list includes selectins, galectins, heparan sulfate proteoglycans (HSPGs), Muc-1, integrins, cadherins, and the trophinin complex. Among these, Muc-1 has a unique role as a masking molecule, that is, an antiadhesive molecule (Surveyor et al., 1995). Muc-1, as stretches of long carbohydrate moieties, is expressed in the mouse uterine epithelium prior to implantation. The physical hindrance rendered by these branches presumably plays a role in preventing the interaction between the embryos and the luminal epithelium prior to the attachment reaction. Timely downregulation of Muc-1 throughout the uterus before the attachment reaction is thought to allow the trophectoderm to make contact with the epithelium (Surveyor et al., 1995). The integrins serve as receptors for various extracellular matrix (ECM) ligands and modulate cell-cell adhesion and signal transduction cascades (Giancotti and Ruoslahti, 1999). They are composed of two subunits, u and fl, and each ufi combination has its own binding specificity and signaling properties. As membrane-associated receptors, integrins possess short cytoplasmic tails with no enzymatic activity. Thus, signaling by integrins is mediated by associating adaptor proteins that connect the integrin to the cytoskeleton, cytoplasmic kinases, and transmembrane growth factor receptors (Giancotti and Ruoslahti, 1999). Several members of the integrin family, including avP3, are known to interact with the RGD (Arg-Gly-Asp) peptide sequence present in many ECM proteins, such as fibronectin, laminin, and entactin. In mice, uvfl3 is expressed both in the uterine luminal epithelium and the blastocyst during implantation. It has been shown that an intrauterine injection of RGD peptide or neutralizing antibody against Uvfl3 reduces the number of implantation sites (Illera et al., 2000). However, the site of action by integrin signaling during implantation, whether it is the uterus or the embryo, has yet to be determined. Gene-targeting experiments ofintegrin subunits have not been very informative with respect to their roles in implantation due to other complex phenotypes or possible compensation by other subunits (Fassler and Meyer, 1995; Kreidberg et al., 1996; Gardner et al., 1996). The role of fl 1 integrin in blastocyst function is discussed later. Trophinin is a novel protein identified by cDNA library screening of a h u m a n trophoblastic cell line (Fukuda et al., 1995). This transmembrane protein was shown to mediate homophilic interactions between two different cell types, a h u m a n endometrial cell line and a trophoblastic cell line (Fukuda et al., 1995). Trophinin requires the presence of a cytoplasmic protein, tastin, to support adhesion between these two cell types. For the interaction between trophinin and tastin, the presence of bystin, another cytoplasmic protein, is required. This adhesion complex

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is expressed in both trophoblastic teratocarcinomas and endometrial adenocarcinomas and mediates adhesion between these two cell types. In humans and monkeys, trophinin is specifically expressed in cells involved in implantation. However, whereas trophinin expression coincides with the timing of implantation in the mouse, it is expressed in the luminal and glandular epithelia throughout the uterus regardless of the presence or absence ofblastocysts (Suzuki et al., 2000). C. HISTAMINE AND PROSTAGLANDIN SIGNALING

It has long been speculated that vasoactive agents such as histamine and prostaglandins (PGs) are involved in many aspects of reproduction including implantation and decidualization. Histamine is a ubiquitous cell-cell mediator. It is a product of mast cells and its release in the uterus by preimplantation ovarian estrogen secretion has been implicated in implantation and decidualization (Marcus et al., 1964). However, normal implantation in mast cell-deficient mice (Wordinger et al., 1986) and the virtual absence of mast cells from the mouse endometrium and deciduum argue against a role for mast cell-derived histamine in implantation. Thus, if histamine is involved in implantation, it should be provided by the blastocyst or uterine cell types other than resident mast cells. We have recently demonstrated that histidine decarboxylase (HDC), an enzyme involved in the biosynthesis of histamine from histidine, is expressed in the uterine epithelium prior to implantation in the mouse, but this gene is not expressed by the blastocyst (Paria et al., 1998b). In contrast, histamine receptor type 2 (H2), but not H1, is expressed only at the blastocyst stage. These observations as well as the inhibition of blastocyst zona hatching and implantation by H2 antagonists and an HDC inhibitor suggest that uterine histamine targets the blastocyst for implantation (Zhao et al., 2000). However, apparently normal implantation in mice deficient in HDC or H2 suggests a possible involvement of other vasoactive agents with overlapping functions in this process. Cyclooxygenase (COX) is the rate-limiting enzyme in PG biosynthesis and exists in two isoforms, COX-1 and COX-2 (Lim et al., 1997; reviewed in Lim and Dey, 2000). Whereas the expression of COX-1 is constitutive, that of COX-2 is inducible (Smith and DeWitt, 1996). COX genes exhibit specific spatiotemporal expression patterns in the uterus during the periimplantation period. For example, COX-1 is expressed in the epithelium throughout the uterus on day 4 of pregnancy prior to the time of implantation, whereas COX-2 is expressed in the luminal epithelium and stroma surrounding the blastocyst at the time of the attachment reaction. This observation suggested a role of COX-2 in implantation

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(Chakraborty et al., 1996). Indeed, gene-targeting experiments have demonstrated that COX-2-derived PGs are essential for implantation and decidualization (Dinchuk et al., 1995; Langenbach et al., 1995; Lim et al., 1997). Experiments with C O X - l ( - / - ) mice suggest that the loss of COX-1 is compensated by the expression of COX-2 for implantation (Reese et al., 1999). Among various PGs, the levels ofprostacyclin (PGI2) are highest at the implantation sites of wild-type mice and implantation defects are partially restored in COX-2(-/-) mice by administration of a more stable prostacyclin agonist, carbaprostacyclin (Lim et al., 1999a). The importance of COX-2 in implantation is also suggested in other species including sheep, mink, skunk, baboon, and humans (Charpigny et al., 1997; Song et al., 1998; Das et al., 1999a; Kim et al., 1999; Marions and Danielsson, 1999). PGs can act via dual receptor signaling systems. Receptors for PGE2, PGF2~, PGD2, PGI2, and thromboxanes have been identified as EP1EP4, FP, DP, IP, and TP, respectively, and they belong to the G-proteincoupled cell-surface receptors (reviewed in Negishi et al., 1995; Lim and Dey, 2000). Although EP receptors are expressed in the periimplantation mouse uterus (Yang et al., 1997), gene-targeting experiments have demonstrated that three of the four EP receptor subtypes (EP1-EP3) are not critical for implantation. EP4 deficiency results in embryonic lethality and therefore its role in implantation has not yet been determined (reviewed in Lim and Dey, 2000). Furthermore, mice deficient in FP or IP show normal implantation. Interestingly, PGs can also exert their effects by utilizing peroxisome proliferator-activated receptors (PPARs) that belong to a nuclear hormone receptor superfamily. Three members of the PPAR family are PPARa, PPARy, and PPARS. PPARs can respond to a wide variety of ligands including natural and synthetic eicosanoids, fatty acids, and hypolipidemic and hypoglycemic drugs (Desvergne and Wahli, 1999). To act as a transcriptional activator, PPARs must form a heterodimer with a member of the retinoid X receptor (RXR) subfamily (Kliewer et al., 1992; reviewed in Lim et al., 1999a). We have shown that COX-2-derived PGI2 participates in implantation via activation of PPAR~ (Lim et al., 1999a), as the implantation defects in COX-2(-/-) mice are reversible by a PGI2 agonist or a combination of PPAI~ and RXR agonists. These studies provided evidence that COX-2-PGI2-PPAR8 signaling is important for implantation. D. GROWTH FACTOR SIGNALING

The expression of various growth factors and their receptors in the uterus in a temporal- and cell-specific manner during the periimplantation period suggests that these factors are important for implantation

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(Cross et al., 1994). Although this review highlights primarily the importance of the epidermal growth factor (EGF) family of growth factors in implantation, the roles of other growth factors, such as transforming growth factor/~s (TGF-fls), fibroblast growth factors (FGFs), insulinlike growth factors (IGFs), platelet-derived growth factors (PDGFs), and vascular endothelial growth factors (VEGFs), should not be understated. The EGF family of growth factors includes EGF itself, TGF-a, heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, epiregulin, and neuregulins (Das et al., 1997b; Lim et al., 1998). HB-EGF is the first molecular marker to be found so far in the uterus exclusively at the sites of active blastocysts several hours before the attachment reaction (Das et al., 1994a). This induction is followed by the expression of betacellulin, epiregulin, neuregulin-1, and COX-2 around the time of the attachment reaction (Chakraborty et al., 1996; Das et al., 1997b; Reese et al., 1998; Lim et al., 1998). In contrast, amphiregulin (Ar) is expressed in the uterine epithelium on the morning of day 4 of pregnancy and is well characterized as a P4-responsive gene in the uterus (Das et al., 1995). Around the time of the attachment reaction, strong expression of Ar in the luminal epithelium is only found around the implanting blastocysts and this expression subsides by the morning of day 5. While these results suggested that Ar has a role in implantation, Ar-deficient mice or compound knockout mice for EGF/TGF-a/Ar do not exhibit implantation defects (Luetteke et al., 1993, 1999). Since HB-EGF, betacellulin, epiregulin, neuregulin, and Ar all show overlapping uterine expression patterns around the implanting blastocyst at the time of attachment reaction (reviewed in Das et al., 1997b), it is assumed that a compensatory mechanism rescues implantation in the absence of one or more members of the EGF family. The EGF-like growth factors interact with the receptor subtypes of the erbB gene family, which is comprised of four receptor tyrosine kinases: ErbB1 (EGF-R), ErbB2, ErbB3, and ErbB4. They share common structural features, but differ in their ligand specificity and kinase activity (Olayioye et al., 2000). The initial dimerization between coexpressed receptors upon ligand binding constitutes the classical mechanism of action of EGF-like ligands. Spatiotemporal expression patterns of EGF family members and ErbBs in the uterus during the periimplantation period suggest compartmentalized functions of EGF-like growth factors in implantation (Lim et al., 1998). E. CYTOKINE SIGNALINGPATHWAY

The expression of various cytokines and their receptors in the uterus and embryo during early pregnancy suggests their roles in various

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aspects of implantation (reviewed in Chard, 1995; Stewart and Cullinan, 1997; Sharkey, 1998; Carson et al., 2000). However, gene-targeting studies show that mice deficient in tumor necrosis factor-cz (TNF-~), interleukin-lp (IL-lp), IL-1 receptor antagonist (IL-lra), IL-1 receptor type 1, IL-6, and granulocyte/macrophage-colony stimulating factor (GM-CSF) apparently do not manifest overt reproductive defects (reviewed in Stewart and Cullinan, 1997). These observations suggest that either these molecules have minor roles in implantation or the loss of one cytokine is compensated by other cytokines with overlapping functions. In contrast, some cytokines are important for normal female fertility (Pollard et al., 1991; Stewart et al., 1992; Robb et al., 1998). For example, female op/op mice with a naturally occurring mutation of the CSF-1 gene have markedly impaired fertility (Pollard et al., 1991), leukemia inhibitory factor (LIF)(-/-) mice show complete failure of implantation, and blastocysts in L I F ( - / - ) mice undergo dormancy (Stewart et al., 1992; Escary et al., 1993). Gene-targeting studies using IL-11Ru(-/-) mice have also shown that IL-11 is crucial to decidualization, but not for the attachment reaction (Robb et al., 1998). Interestingly, both LIF and IL-11 are members of the interleukin-6 (IL-6) family, which includes IL-6 itself, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin (Kishimoto et al., 1994). LIF and IL-11 bind to the ligand-specific receptors LIFR and IL-11R, respectively, and share gpl30 as a signal transduction partner (Kishimoto et al., 1994), suggesting that gpl30 signaling is critically involved in implantation. Although the mechanism underlying implantation and decidualization failures in the absence of LIF remains to be elucidated, recent evidence shows that there is a loss or an aberrant expression of certain implantation-related genes in pregnant L I F ( - / - ) uteri (Song et al., 2000). For instance, there is loss of uterine expression of HB-EGF and epiregulin and aberrant expression of COX-2 at the sites of the blastocyst in L I F ( - / - ) mice during the anticipated time of implantation. LIF and its receptors, LIFR and gp130, exist both in soluble and membrane-bound forms, and soluble forms of these two receptors antagonize the actions of their ligands, implying the complexity of LIF signaling pathway (Rathjen et al., 1990; Narazaki et al., 1993; Layton et al., 1994). LIF was previously shown to be transiently expressed in uterine glands on day 4 of pregnancy in the mouse, suggesting its role in implantation (Bhatt et al., 1991). However, our recent studies show that LIF expression is biphasic in the mouse uterus on day 4 of pregnancy. Not only is LIF expressed in endometrial glands, but it is also expressed in stromal cells surrounding the blastocyst at the time of the attachment reaction (Song et al., 2000). This suggests that LIF has dual roles: first in the preparation of the uterus and later in

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the attachment reaction. Although previous studies demonstrated that LIF is critical to the implantation process, the molecular mechanism by which LIF executes its effects on implantation is not known, since the complicated ligand-receptor interaction and detailed expression patterns of its receptors during periimplantation period remain to be elucidated. L I F ( - / - ) mice are incapable of providing a suitable environment for blastocysts to implant, irrespective of the blastocyst genotypes, although L I F ( - / - ) blastocysts can implant after transfer to wild-type pseudopregnant recipients (Stewart et al., 1992; Escary et al., 1993). These reciprocal embryo transfer experiments suggest that maternal LIF is essential for blastocyst implantation. However, a role for this cytokine in embryonic functions cannot be ignored, since LIFR and gpl30 are expressed by the blastocyst stage, and administration of exogenous LIF improves embryo viability and hatching in several species (Fry, 1992; Dunglison et al., 1996; Nichols et al., 1996). Taken together, these data suggest that both the preimplantation embryo and the uterus are the sites of LIF action. However, embryos lacking either LIFR or gp 130 develop to the blastocyst stage and implant normally (Ware et al., 1995; Yoshida et al., 1996), raising questions about the critical role of LIF signaling in embryo development. LIF expression in the uterus is maximal around the time of implantation in most species examined, although the steroid hormonal requirements for the preparation of uterine receptivity and implantation differ depending on the species. While uterine LIF expression in several species appears to be regulated by P4 (reviewed in Vogiagis and Salamonsen, 1999), estrogen regulates LIF expression in the mouse uterus. This is evident by LIF expression on day 1 of pregnancy and during the estrus stage of the cycle when the uterus is under the influence of estrogen stimulation (Bhatt et al., 1991; Shen and Leder, 1992; Yang et al., 1996a). In addition, LIF is not expressed in the uterus during experimentally induced delayed implantation, but is rapidly induced by an injection of estrogen (Bhatt et al., 1991; Song et al., 2000). However, it has yet to be defined how estrogen induces LIF expression in the mouse uterus and the mechanism by which it is regulated by P4 in other species. F, TRANSCRIPTIONFACTORS

Hox genes code for developmentally regulated transcription factors and belong to a multigene family. They share a common highly conserved sequence element called the homeobox that encodes a 61-amino

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acid helix-turn-helix DNA-binding domain (Krumlauf, 1994). In humans and mice, Hox genes are organized in four clusters (A, B, C, and D) on four different chromosomes and follow a strict pattern of spatial and temporal collinearity during embryogenesis (Krumlauf, 1994). Several Hox genes at the 5' end of each cluster are classified as AbdBlike Hox genes, because they share homology with the Drosophila AbdB gene. AbdB-like Hox genes, similar to their Drosophila ortholog, are expressed in developing genitourinary systems in vertebrates (Benson et al., 1996). Hoxa-10 and Hoxa-ll, while highly expressed in developing genitourinary tracts, are also implicated in reproductive performance in adult mice (Hsieh-Li et al., 1995; Benson et al., 1996; Gendron et al., 1997). Hoxa-10(-/-) mice exhibit oviductal transformation of the proximal one-third of the uterus. Furthermore, adult female mice devoid of Hoxa-10 showed unexpected failure in blastocyst attachment and decidualization unrelated to this oviductal transformation (Benson et al., 1996). Follow-up investigation revealed that uterine stromal cells in Hoxa-10(-/-) mice show dramatically reduced proliferation in response to P4, leading to decidualization defects (Benson et al., 1996; Lim et al., 1999b). A similar but more severe phenotype was also observed in Hoxa-11(-/-) female mice (Gendron et al., 1997). For example, uteri from H o x a - l l ( - / - ) mice are severely hypoplastic and do not have uterine glands due to developmental defects. Two possibilities were suggested to explain the underlying mechanism of infertility in these mice (Lim et al., 1999b). First, since P4-responsive stromal cell proliferation in ovariectomized uteri is reduced in both m u t a n t mice (Lim et al., 1999b; H. Lim et al., unpublished), it is possible that these Hox genes are involved in cell cycle regulation of the uterine stroma by regulating cell-cycle molecules. Indeed, cyclin D3 is aberrantly expressed in Hoxa-10(-/-) uteri in response to a decidualization stimulus (Das et al., 1999b). Second, it has been shown that several P4-responsive genes are dysregulated in the stroma of Hoxa-10(-/-) uteri (Lim et al., 1999b). Therefore, as transcription factors, Hoxa-10 and Hoxa-ll may be involved in P4-responsiveness in the uterine stroma by regulating gene expression. The Hmx family of transcription factors is encoded by a distant homeobox gene family unrelated to other larger classes of homeobox genes, such as Hox genes (W. Wang et al., 1998). Whereas these genes exhibit overlapping expression during development and in the adult uterus, gene-targeting experiments revealed a unique role for Hmx3 in female reproduction (W. Wang et al., 1998). H m x 3 ( - / - ) female mice have normal fertilization and their embryos undergo normal preimplantation development to blastocysts. However, these embryos fail to implant

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in the uterus and subsequently die. Since Hmx3 is mainly expressed in the uterine myometrium during early pregnancy, it is suspected that the mechanism of infertility in these mice is different from that of Hoxa-10(-/-) or H o x a - l l ( - / - ) mice and is yet to be defined. G. CANNABINOIDSIGNALINGPATHWAY Psychoactive cannabinoids are active components of marijuana and work via G-protein-coupled cell surface receptors, CB1 and CB2 (Matsuda et al., 1990; Munro et al., 1993). The discovery ofcannabinoid receptors led to the identification of endogenous cannabinoid ligands, arachidonoylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) (Devane et al., 1992; Felder et al., 1993). The mouse uterus is a site of anandamide synthesis, and in fact, the uterine levels of anandamide are higher than in any other mammalian tissues so far examined (Schmid et al., 1997). Furthermore, levels of anandamide fluctuate during early pregnancy coincident with the window of uterine receptivity for implantation. For example, whereas anandamide levels are lower in the receptive uterus, the levels are higher in the nonreceptive uterus. Furthermore, its levels are lower at the implantation site than at the interimplantation sites (Schmid et al., 1997). Delayed-implanting uteri also exhibit high levels of anandamide, whereas termination of the delayed implantation by estrogen promptly downregulates its levels (Paria et al., 2001). Therefore, a strong correlation between the levels of anandamide and phases of uterine receptivity led to a hypothesis that an endocannabinoid ligand-receptor signaling is one important aspect of defining the window of uterine receptivity for implantation (Schmid et al., 1997). Recent experiments using L I F ( - / - ) pregnant mice support this speculation, since these mice with implantation failure have higher uterine levels of anandamide than do wild-type mice (Paria et al., 2001). The role of anandamide in uterine receptivity is related to the developmental potential of preimplantation embryos and is discussed later.

IV. EMBRYONIC FACTORS IN IMPLANTATION

A. STEROIDHORMONESIGNALING In mice, both P4 and estrogen are essential to implantation (Dey, 1996). The mouse uterus during early pregnancy expresses both ER and PR, and gene-targeting experiments have established requirements

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of these receptors in uterine preparation for implantation (Lubahn et al., 1993; Lydon et al., 1995; Tan et al., 1999). While the preimplantation estrogen secretion on the morning of day 4 of pregnancy is critical to implantation, it has a dual role as a primary estrogen and as a catechol estrogen with distinct targets (Paria et al., 1998a; see Section II.C). For example, whereas primary estrogen at appropriate doses acts on the uterine ER to prepare the uterus for implantation, catechol estrogen formed from the primary estrogen participates in blastocyst activation. However, it is unknown how catechol estrogen mediates activation of blastocysts (Paria et al., 1998a). Although nuclear ERa is present in both the active and dormant blastocyst (Hou et al., 1996), dormant blastocysts did not respond to estradiol-17fl (E2) and failed to attain implantation competency in vitro. In contrast, dormant blastocysts responded to a catecholestrogen, 4-OH-E2, and became implantation-competent in vitro. This response could not be neutralized by ICI-182, 780 (an ER antagonist), suggesting that nuclear ER signaling is not critical to blastocyst activation induced by 4-OH-E2 (Paria et al., 1998a). Primary estrogen undergoes hydroxylation to 4-OH-E2 by the P450linked enzyme CYP1B1 (Shen et al., 1994). This enzyme is present throughout the mouse uterus on day 4, but disappears from the implantation site on day 5 (Paria et al., 1998a). Activation of dormant blastocysts appears to involve an "early response" by 4-OH-E2, since dormant blastocysts transferred into delayed-implanting recipient uteri within 1 h of E2 injection of the recipients show implantation, whereas similar blastocysts transferred beyond this 1-h period fail to implant (Paria et al., 1998a). These results suggest that a rapid response occurs in utero that is critical to implantation. Furthermore, dormant blastocysts cultured in the presence of 4-OH-E2, but not E2, gain implantation competency, and upon transfer, implant in pseudopregnant recipients well beyond the 1-h "window" of E2 treatment. Similar results were obtained by culturing dormant blastocysts in the presence of PGE2 or a permeable analog of cAMP. This effect apparently involves the COX-2 signaling pathway (Paria et al., 1998a). For example, coincubation of dormant blastocysts with a selective COX-2 inhibitor and 4-OH-E2 efficiently blocks their activation and implantation upon transfer to suitable recipients, and this effect of the COX-2 inhibitor was partially reversed by addition of PGE2 in the culture media. The results strongly suggest that the action of 4-OH-E2 on dormant blastocysts is mediated via the COX-2 signaling pathway leading to an increase in intracellular cAMP levels. Further investigation will reveal the types of PGs and receptors involved in this event.

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B. ADHESION MOLECULES Several ECM proteins including fibronectin, laminin, entactin, and collagen type IV are known to stimulate blastocyst outgrowth in culture (Armant et al., 1986a, 1986b; Sutherland et al., 1988; Yelian et al., 1993). Since the outgrowth of trophoblast occurs in the presence of specific ECM proteins in serum-free media, it was speculated that blastocysts possess functional receptors of the integrin family. Among many s u b u n i t s , ot5~{1, Ot6Bfil , and avfi3 are expressed in the mouse embryo throughout periimplantation period, and severalothers exhibit stagespecific expression (Sutherland et al., 1993). Integrins are also expressed in the differentiating trophoblast cells at later stages (Sutherland et al., 1993), suggesting their roles in trophoblast differentiation and adhesion. A role for fibronectin via integrin binding in blastocyst outgrowth was further confirmed in vitro using antibodies against Uv, a5, ill, or f13, which inhibited trophoblast outgrowth inducible by fibronectin (Schultz and Armant, 1995). Furthermore, a gene-targeting experiment revealed that deletion of the fl 1 gene results in ICM defects and embryonic lethality (Stephens et al., 1995). For example, it was observed that while the m u t a n t embryos form morphologically normal blastocysts and initiate implantation, trophoblast invasion was defective (Stephens et al., 1995). E-cadherin, a calcium-dependent cell adhesion molecule, participates in the formation of the epithelial tight junctions in cooperation with zonula occludens-1 (ZO-1) and occludin (Itoh et al., 1993, 1997). E-cadherin is a critical factor for blastocyst formation, since its targeted deletion leads to defective embryonic development resulting from the failure to form the Tr (Larue et al., 1994; Riethmacher et al., 1995). E-cadherin is implicated in uterine-embryo interactions because of its homotypic adhesive nature (Paria et al., 1999b). Whereas the Tr highly expresses E-cadherin, the components of the tight junctional complex are also expressed in the uterine luminal epithelium at the time of the attachment reaction. The expression subsequently becomes evident in the subepithelial stroma surrounding the implanting blastocysts with apoptosis of the luminal epithelium (Paria et al., 1999b). Therefore, it is speculated that a molecular guidance resulting from temporal- and cell-specific expression of the tight-junction proteins in the uterus is important for blastocyst attachment and subsequent invasion.

C. GROWTH FACTOR SIGNALING

A number of growth factors and their receptors are expressed in preimplantation embryos of several species, suggesting their roles

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MOLECULES IN BLASTOCYSTIMPLANTATION TABLE I E G F FAMILY OF GROWTH FACTORS AND THEIR RECEPTORS: EXPRESSION IN THE BLASTOCYST AND THE UTERUS DURING IMPLANTATION a

Ligands

Blastocyst

Uterus

Reference

EGF

-

-

TGF-a

+

+

Johnson et al., 1994 Huet-Hudson et al., 1990 Rappolee et al., 1988 Tamada et al., 1991 Das et al., 1994a Tsark et al., 1997 Das et al., 1995 Das et al., 1997b Das et al., 1997b Reese et al., 1998 Johnson et al., 1994

HB-EGF Amphiregulin

Not known +

Epiregulin Betacellulin NDF Cripto

Not known Not known Not known +

Receptors ErbB1/EGF-R ErbB2

ErbB3 ErbB4

+ + + + + Not known

+

+, stroma

+

+, epithelium

Not known +

+, epithelium +, submyometrial stroma

Paria et al., 1993b Das et al., 1994b H. Lira and S. K. Dey, unpublished Lim et al., 1997 Lim et al., 1998 Lim et al., 1998

aDay 4, 2300 hr-day 5, 1000 hr. A majority of EGF-like ligands are expressed in the mouse uterus in overlapping patterns during the initial phase of the implantation process, while blastocysts perhaps express various combinations of receptor dimers. The periimplantation mouse uterus also expresses all of the receptors in various cell types. Therefore, ligands of uterine origin may work as paracrine and autocrine factors on the blastocyst and the uterus. Since some of the ligands are expressed in membraneanchored forms in the uterus, juxtacrine signaling between the blastocyst and the uterus is also possible during implantation.

in preimplantation mammalian development (Rappolee et al., 1988; Harvey et al., 1995). In this review, we focus on certain potential roles of signaling by the EGF family of ligands with respect to preimplantation embryo development and implantation (Table I). ErbB1 (EGF-R), ErbB2, and ErbB4, the receptor subtypes for the EGF family of growth factors, are expressed in the mouse blastocyst (Paria et al., 1993b, 1999c; Paria et al., 2001) and EGF or TGF-a have beneficial effects on embryonic development in vitro (Paria and Dey, 1990). Using genetic and biochemical approaches, roles of embryonic ErbB1 and/or ErbB4 in interacting with uterine HB-EGF in blastocyst implantation have recently been highlighted in mice (Raab et al., 1996; Paria et al., 1999c). HB-EGF is expressed as soluble and transmembrane forms in the uterine luminal epithelium at the site of the blastocyst, suggesting paracrine, and/or

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juxtacrine interactions with embryonic ErbBs, as well as autocrine, paracrine, and/or juxtacrine interactions with uterine ErbBs that are expressed in a spatiotemporal manner during the periimplantation period (Das et al., 1994a, 1994b; Lim et al., 1998). For example, the expression of both ErbB1 and ErbB4 is downregulated in dormant blastocysts during delayed implantation, but is readily upregulated with blastocyst activation and initiation of implantation (Paria et al., 1993b, 1999c). Furthermore, whereas a recombinant soluble HB-EGF can promote blastocyst growth and differentiation (Das et al., 1994a), freely dispersed cells in culture that express the transmembrane form of HBEGF can adhere to active, but not dormant, blastocysts in vitro (Raab et al., 1996), suggesting paracrine and juxtacrine functions of HB-EGF. In addition, by directing an HB-EGF-toxin conjugate toward wild-type and e r b B l ( - / - ) blastocysts, it was shown that HB-EGF could also interact with embryonic ErbB4 and heparan sulfate proteoglycan (HSPG) molecules (Paria et al., 1999c). Collectively, these results suggest that an interaction between uterine HB-EGF and blastocyst ErbBs is important for the attachment reaction. It is, however, to be noted that early events of implantation do not appear to be affected by blastocysts deficient in either ErbB1 or ErbB4 (Gassmann et al., 1995; Threadgill et al., 1995), although the implantation-initiating efficiency ofblastocysts deficient in more than one receptor type needs to be tested to delineate the functional redundancy among the receptor family. In conclusion, detailed expression and gene-targeting experiments with all of the ligands and receptors are required to define paracrine, autocrine, and/or juxtacrine roles of a specific ligand or its receptors in implantation. D.

CANNABINOIDSIGNALING

As mentioned earlier, fluctuation in uterine anandamide levels correlates with uterine receptivity for implantation. A strong implication for ligand-receptor signaling in preimplantation embryo development and implantation is evident from the observation that the mouse blastocysts highly express the cannabinoid receptor CB1, at a level even higher than the brain (Paria et al., 1995). CB1 is expressed from the two-cell stage at the time of zygotic gene expression through the blastocyst stage (Paria et al., 1995). Embryonic CB1 is functional, since two-cell embryos cultured in the presence of nanomolar concentrations of natural, synthetic, and endocannabinoids fail to develop to the blastocyst stage and this failure occurs between the eight-cell and blastocyst stages. This effect is completely reversed by a specific CB1 antagonist (Yang et al., 1996b). However, further lowering the levels of cannabinoids in culture stimulates blastocyst differentiation and trophoblast outgrowth

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(J. Wang et al., 1999). These results suggest that a narrow range of natural or endogenous cannabinoid concentrations regulates the embryonic developmental program. It is speculated that a tightly regulated level of uterine anandamide and embryonic cannabinoid receptors during early pregnancy is important for preimplantation embryonic development and implantation. This speculation is consistent with our recent observation of asynchronous preimplantation embryo development in C B I ( - / - ) and/or C B 2 ( - / - ) mice (Paria et al., 2001). Collectively, the expression of cannabinoid receptors in the preimplantation mouse embryo, the synthesis of anandamide (an endocannabinoid ligand) in the uterus, and the dose- and stage-specific effects of anandamide on embryo development and implantation suggest that ligand-receptor signaling with anandamide is important for these events.

V. UTERINE MARKERS IN HUMANS

The identification of factors important for h u m a n implantation is an extremely difficult task, since the availability of tissues of h u m a n implantation sites is rare and ethically restricted. Thus, most studies have used biopsy materials from h u m a n endometria during the menstrual cycle to identify molecular markers for uterine receptivity for implantation (Giudice, 1999). In humans, the window of uterine receptivity is considered to be between cycle days 20 and 24 (Anderson, 1990). Although a definitive answer as to which molecules are important for h u m a n implantation is hard to reach, it is likely that appropriate expression of some of these uterine markers may be implicated in uterine receptivity for implantation. Among many growth factors that have been studied in humans, HB-EGF appears to play a role in implantation and embryonic development. Its expression is maximal during the late secretory phase (cycle days 20-24) when the endometrium becomes receptive for implantation (Yoo et al., 1997; Leach et al., 1999). Furthermore, HB-EGF has been shown to be the most potent growth factor for enhancing the development of h u m a n in vitro fertilization-derived embryos to blastocysts (71%) and subsequent zona-hatching (82%) (Martin et al., 1998). Thus, cumulative evidence suggests that HB-EGF has a significant role in preimplantation embryo development and implantation as a paracrine and/or juxtacrine factor in various species. Among adhesion molecules, integrins have been studied extensively in h u m a n endometrium, and their cycle-dependent changes imply possible roles in uterine receptivity. Whereas many integrin heterodimers exhibit constitutive expression in the epithelium or stroma, ~ l f l l , 0t3•1,

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~6fli, avfl3, and Olvfi I a r e cycle-dependent (Tabibzadeh, 1992; Klentzeris et al., 1993; Lessey et al., 1992, 1996; Lessey, 1994). Among these, u1~1 exhibits implantation-related fluctuation in its expression. For example, alfll is restricted to early- and mid-secretory phases both in the epithelium and stroma and is restricted entirely to the stroma during the predecidual phase (Lessey et al., 1992). Whereas a lack of av~3 or a4fl 1 has been implicated in unexplained infertility (Lessey et al., 1995), precise definition of these markers in uterine receptivity or pathological conditions awaits further investigation. The trophinin complex is also implicated in h u m a n implantation, since this complex was detected in both trophoblast and decidual cells at the h u m a n fetal-maternal interface as early as the sixth week of pregnancy (Suzuki et al., 1999). Uterine LIF is one of the critical factors in implantation, as shown in L I F ( - / - ) mice (Stewart et al., 1992; Escary et al., 1993). In humans, LIF is expressed in endometrium and at higher levels in glandular epithelium of the secretory endometrium (Arici et al., 1995). Furthermore, LIF deficiency is associated with unexplained recurrent abortions and infertility in women (Hambartsoumian, 1998). Among other uterine markers, HOXA-10 and COX-2 have been studied in humans. HOXA-10 is a P4-regulated transcription factor and is implicated in P4-responsiveness in the mouse uterine stroma (Lira et al., 1999b). In humans, both HOXA- 10 and HOXA- 11 genes are markedly upregulated in the uterus during the mid-secretory phase in a steroid hormonedependent manner (Taylor et al., 1998), suggesting their roles in h u m a n implantation. COX-2 expression in h u m a n endometrium has also been reported (Critchley et al., 1999; Marions and Danielsson 1999). Whereas no steroidal regulation of COX-2 was shown in mice (Chakraborty et al., 1996), it seems to be under negative regulation by P4 in humans (Critchley et al., 1999; Marions and Danielsson, 1999).

VI. CONCLUSION Two major global concerns regarding women's health are pregnancy and infertility. A comprehensive understanding of preimplantation embryo development and uterine preparation for implantation is critical to addressing these concerns. Interactions between the heterogeneous cell types of the uterus and embryo during implantation are complex. Thus, establishing the signaling mechanism(s) during this critical time requires studies on both embryonic and uterine consequences to formulate a more meaningful picture. This cannot be addressed in humans due to ethical considerations and current restrictions on research with h u m a n embryos. Thus, the use of various animal models should be

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explored for studying embryo-uterine interactions that could be relevant to h u m a n implantation. Nonetheless, research to identify molecules associated with uterine receptivity ("window of implantation") during the menstrual cycles of women will continue to be pursued. Although a large number of factors have been identified to define embryo-uterine interactions during implantation in various species, the interactions among the various signaling pathways that direct this process are poorly understood. A difficult task lies ahead to dissect the intricate nature of the pathways, including whether they operate independently in a "cross talk" network or converge in a common pathway. Although gene-targeting and expression studies have helped us define some of the pathways in a limited manner, a comprehensive understanding of implantation is lacking. For example, many of the genes which appear to be important for uterine functions relevant to implantation cannot be studied mechanistically because mutation of these genes gives rise to embryonic lethality. Technology to achieve uterine- or embryo-specific conditional "knockout" is urgently needed to better understand the definitive roles of these gene products in uterine biology and implantation. In parallel, global gene expression profiling should be pursued to compare gene expression patterns between uterine receptive and nonreceptive phases, between implantation and interimplantation sites (Reese et al., in press), and between active and dormant blastocysts. This may help in identifying many more potential factors that regulate these events. Finally, although there is species-dependent variation in the process of implantation, certain basic similarities exist across species. They are (1) implantation of the embryo at the blastocyst stage, (2) a defined but differential "window" of uterine receptivity for implantation, (3) a "twoway" interaction between the blastocyst and the uterus during implantation, and (4) a localized increase in uterine vascular permeability at the site of the blastocyst during the attachment reaction. Thus, identification of signaling pathways for these events may give rise to a unifying scheme that could be relevant to h u m a n implantation. This review has focused on a selected number of factors that are involved in embryo-uterine interactions during implantation. A large number of other polypeptide growth factors, cytokines, lipid mediators, and vasoactive agents that could well be involved in implantation have not been addressed here due to space limitations. ACKNOWLEDGMENTS This work was supported by NIH grants HD12304, DA06668, and HD 29968 as part of the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation to S. K. Dey, by ES07814 to S. K. Das, by HD37394 to B. C. Paria, and by

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HD37677 to J. Reese. H. Lim was supported by a Mellon Foundation Junior Investigator Award made to the Center for Reproductive Sciences at the University of Kansas Medical Center. REFERENCES Anderson, T. I. (1990). Window of uterine receptivity. In ~Blastocyst Implantation" (K. Yoshinaga, Ed.), pp. 219-224. Adams Publishing, Boston. Arici, A., Engin, O., Attar, E., and Olive, D. L. (1995). Modulation of leukemia inhibitory factor gene expression and protein biosynthesis in human endometrium. J. Clin. Endocrinol. Metab. 80, 1908-1915. Armant, D. R., Kaplan, H. A., Mover, H., and Lennarz, W. J. (1986a). The effect of hexapeptides on attachment and outgrowth of mouse blastocysts cultured in vitro: Evidence for the involvementof the cell recognition tripeptide Arg-Gly-Asp. Proc. Natl. Acad. Sci. USA 83, 6751-6755. Armant, D. R., Kaplan, H. A., and Lennarz, W. J. (1986b). Fibronectin and laminin promote in vitro attachment and outgrowth of mouse blastocysts. Dev. Biol. 116, 519-523. Benson, G. V., Lim, H., Paria, B. C., Satokata, I., Dey, S. K., and Maas, R. L. (1996). Mechanisms of reduced fertility in Hoxa-10 mutant mice: Uterine homeosis and loss of maternal Hoxa-10 expression. Development 122, 2687-2696. Bhatt, H., Brunet, L. J., and Stewart, C. L. (1991). Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. Proc. Natl. Acad. Sci. USA 88, 11408-11412. Carson, D. D., Bagchi, I., Dey, S. K., Enders, A. C., Fazleabas, A. T., Lessey, B. A., and Yoshinaga, K. (2000). Embryo implantation. Dev. Biol. 223, 217-237. Chakraborty, I., Das, S. K., Wang, J., and Dey, S. K. (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. Charpigny, G., Reinaud, P., Tamby, J. P., Creminon, C., and Guillimot, M. (1997). Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine embryos during the implantation period. Biol. Reprod. 57, 1032-1040. Chard, T. (1995). Cytokines in implantation. Hum. Reprod. Update 1, 385-396. Critchley, H. O., Jones, R. L., Lea, R. G., Drudy, T. A., Kelly, R. W., Williams, A. R., and Baird, D. T. (1999). Role of inflammatory mediators in human endometrium during progesterone withdrawal and early pregnancy. J. Clin. Endocrinol. Metab. 84, 240248. Cross, J. C., Werb, Z., and Fisher, S. J. (1994). Implantation and the placenta: Key pieces of the development puzzle. Science 266, 1508-1518. Curtis, S. W., Clark, J., Myers, P., and Korach, K. S. (1999). Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor alpha knockout mouse uterus. Proc. Natl. Acad. Sci. USA 96, 3646-3651. Das, S. K., Wang, X. N., Paria, B. C., Damm, D., Abraham, J. A., Klagsbrun, M., Andrews, G. K., and Dey, S. K. (1994a). 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. Das, S. K., Tsukamura, H., Paria, B. C., Andrews, G. K., and Dey, S. K. (1994b). Differential expression of epidermal growth factor receptor (EGF-R) gene and

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