Embryo Implantation

Developmental Biology 223, 217–237 (2000) doi:10.1006/dbio.2000.9767, available online at http://www.idealibrary.com on

REVIEW Embryo Implantation Daniel D. Carson,* ,1 Indrani Bagchi,† Sudhandsu K. Dey,‡ Allen C. Enders,§ Asgerally T. Fazleabas, ¶ Bruce A. Lessey,㥋 and Koji Yoshinaga** *Department of Biological Sciences, University of Delaware, Newark, Delaware 19716; †Population Council, Rockefeller University, New York, New York 10021; ‡Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7338; §Department of Cell Biology and Human Anatomy, University of California at Davis, Davis, California 95616-8643; ¶Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois 60612-7313; 㛳Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of North Carolina, Chapel Hill, North Carolina 27599; and **Reproductive Sciences Branch, Center for Population Research, NICHD, National Institutes of Health, Bethesda, Maryland 20892-7510

INTRODUCTION A lesson learned from modern developmental biology is the striking degree of conservation of strategies and molecules used to program developmental events in species as diverse as insects, birds, amphibians, and mammals. Nonetheless, mammals retain distinctions with regard to the strategies used to protect and nourish their offspring during development, namely the processes of implantation and placentation. While involving relatively few cell types, placentation is a complex process. Furthermore, genes associated with this process display remarkably high spontaneous mutational rates, suggesting a strong adaptive/ selection pressure on this tissue (Roberts et al., 1999). In the case of implantation, a highly coordinated process is set into motion whereby specialized cells of the embryo, the trophectoderm and trophoblast, establish contact with a specialized tissue of the mother, the uterus. The exquisite coordination involves the regulated production of growth factors, cytokines, and hormones by embryonic as well as maternal tissues of both uterine and extrauterine origins. In concert, complementary receptors for these factors must be expressed by the appropriate tissues to propagate implantation signals. In addition, cell surface components must become functionally available to support attachment of trophectoderm/trophoblast and uterine cells. To add to the challenge, it has been shown that, in most mammals, there is only a restricted time during the uterine cycle during which implantation can occur (Psychoyos, 1986). Failure to Dedicated to the memory of Loren Hoffman. 1 To whom correspondence should be addressed. E-mail: [email protected]. 0012-1606/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

initiate the critical early events of implantation during this “window of receptivity” results in early pregnancy failure. In addition to processes occurring in the embryo during the pre- and peri-implantation period, uterine events also may be considered in a developmental context. The uterus undergoes dynamic changes during the cycle and displays many features typical of developmental processes, including differential and ordered activation or repression of gene expression and programmed changes in posttranscriptional and posttranslational modifications of mRNA and proteins. While the progression of these events is largely driven by endocrine actions, they display the same sequential nature as classical developmental processes. In the absence of an embryo, the uterus will progress through a predictable series of stages ultimately terminating in tissue regression and apoptosis. In the presence of an embryo, the endometrium not only is maintained, but also progresses through an additional program of events, i.e., the decidual cell response, leading to prolonged maintenance and additional programs of gene expression that otherwise are not observed (Parr and Parr, 1989). Thus, multipotentiality is displayed by uterine tissue. From the above, it is apparent that, even though embryonic development may proceed normally, there remain many opportunities for implantation failure. In this regard, while marked improvements in in vitro fertilization and embryo culture techniques have been made over the past 20 years, pregnancy success rates following these procedures have improved only marginally (ASRM Report, 1999). This has led to the proposition that additional uterine factors, critical for the implantation process, must be limiting. Identification of such parameters could lead to tests used in conjunction with available serum and histological assays to

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determine if the physiological state of the uterus is appropriate for embryo transfer. In addition, it is possible that genetic or epigenetic differences between individuals may impact the ability of the uterus to develop to a functionally receptive state. Such individuals would not be good candidates for embryo transfer, under any conditions. Other, more subtle, problems may occur as well. For instance, factors that significantly accelerate or delay the transition to the receptive state would disrupt the normal coordination between embryonic and uterine development even though all molecular players seem otherwise normal. In the discussion below, we will present the current understanding of the events of implantation in various model systems and humans, primarily focusing on uterine developments. In addition, information on a number of genes and gene products that appear to play a role in the implantation process in multiple species systems also will be presented.

TABLE 1 Markers of Functional Stages of Mouse Blastocysts a Marker/functional state

Stage I

Stage II b

Stage III

Zona EGF-R HSPG (perlecan) Attachment competence

⫹ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺

⫺ ⫹ ⫹ ⫹

a Three functional stages are proposed: (I) unhatched, attachment incompetent, typical of blastocysts encased in zonae pellicidae. Such blastocysts fail to attach even if zonae are removed by artificial means (Sherman, 1978); (II) hatched, incompetent, typical of blastocysts in implantation delay (Mead, 1993; Paria et al., 1993); (III) hatched, attachment competent. Progression from Stage I to III is very rapid in vivo. The existence of stage II can be demonstrated during implantation delay. b This normally transient stage can be stabilized in delayed implantation models.

BLASTOCYST/TROPHOBLAST EVENTS The preimplantation stage embryo normally develops from the one-cell zygote to the blastocyst stage within the zona pellucida; however, development in vitro proceeds at similar rates in embryos which have had their zonae removed by various means (Sherman, 1978). These observations suggest that the zona is not critical for developmental progression, e.g., by including (or excluding) growth factors. Nonetheless, the nonadhesive nature of the zona is likely to facilitate transport of the developing embryo through the oviduct to the uterus. The development of the trophectoderm and, subsequently, trophoblast creates the embryonic tissue responsible for establishing embryonic contact with the mother. Trophoblast, in particular, are quite effective in producing various hormones and cytokines that display profound effects on maternal physiology (Petraglia et al., 1998; Roberts et al., 1999). In addition, trophoblast cells express a number of extracellular matrix receptors and matrix-degrading activities that support interaction with and invasion through the endometrium (Cross et al., 1994; Alexander et al., 1996). In situ studies generally indicate that the matrix receptors and degrading activities are expressed in an ordered fashion that correlates well with the composition of the associated maternal matrix (Damsky et al., 1994; Alexander et al., 1996); however, in vitro studies suggest that embryos display matrix receptors in response to the matrix, rather than as a result of a rigid program (Schultz and Armant, 1995). As discussed in more detail below, studies of delayed implanting mouse blastocysts have revealed that expression of certain components of the trophectodermal cell surface, i.e., EGF 2 receptor and perlecan, are very carefully coordinated with the acquisition of attachment compe2 Abbreviations used: Ar, amphiregulin; BTC, ␤-cellulin; COX-1, COX-2, cycloxygenase-1 and -2; CSF-1, colony-stimulating factor; Er, epiregulin; E 2, estrogen; EGF, epidermal growth factor; HB-EGF,

tence (Paria et al., 1993; Smith et al., 1997; Tables 1 and 2). These studies also indicate that, while normally quite brief, even hatched blastocysts can display two functional states with regard to attachment competence. Despite the many studies demonstrating profound changes in growth factor/ growth factor receptor or adhesion protein expression during peri-implantation stage development in mice, there are no clear examples of genetic mutations manifest at the level of the embryo that result in defects in embryo attachment to the uterine epithelium. Thus, either genes essential to the embryonic events are novel or there is considerable redundancy of function at this step. The potential roles of particular embryonically expressed genes and gene products are discussed in conjunction with their uterine complements in the discussion below.

UTERINE EVENTS In the mouse, the first discernible sign of implantation is an increased uterine stromal vascular permeability at the site of blastocyst apposition (Psychoyos, 1986). This can be visualized as distinct blue bands along the uterus after an intravenous injection of a macromolecular blue dye solution. This increased vascular permeability coincides with the attachment reaction between the blastocyst and the uterine luminal epithelium. The attachment reaction oc-

heparin-binding, epidermal growth factor-like growth factor; HS, heparan sulfate; LIF, leukemia inhibitory factor; LNF-I, lacto-Nfucopentaose-I; NDF, heregulin/neu differentiating factor; P 4, progesterone; PGE 2, PGF 2␣, PGJ 2, and PGD 2, prostaglandins E 2, F 2␣, J 2, and D 2; PGI 2, prostacyclin; PPAR, peroxisome proliferatoractivated receptors; RXR, retinoid X receptor; TGF-␣, transforming growth factor-␣; TIMP, tissue inhibitor of metalloproteinase.

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TABLE 2 Markers of Pre- and Peri-implantation Uterine Development in the Mouse a

Note. M, 0800 – 0900 h; A, 1600 –1800 h; N, 2300 –2400 h (attachment reaction) period. a Day 1 represents the day on which the vaginal plug is observed. Bars indicate periods of expression in pregnant animals. Due to its critical nature in the timing of implantation, day 4 has been subdivided into three sections (morning, M, at 0800 – 0900 h; afternoon, A, at 1600 –1800 h; and night, N, at 2300 –2400 h). Abbreviations used: Ar, amphiregulin; BTC, betacellulin; COX-1, COX-2, cyclooxygenases -1 and -2; EGF, epidermal growth factor; EPI, epiregulin; HB-EGF, heparin-binding, EGF-like growth factor; LIF, leukemia inhibitory factor; MUC1, Muc1 mucin; NDF, neu differentiating factor; TGF-␣, transforming growth factor alpha.

curs in the mouse around midnight on day 4 of pregnancy (day 1 ⫽ vaginal plug) (Das et al., 1995). This event is preceded by generalized uterine edema and luminal closure that lead to close apposition of the blastocyst trophectoderm with the luminal epithelium. After the attachment reaction, epithelial cells undergo apoptosis, while stromal cells enter into extensive proliferation and differentiation into decidual cells (decidualization) at the site of blastocyst apposition (Parr and Parr, 1989). Although incompletely defined, it is clear that a number of molecular signals render the uterus receptive and direct the reciprocal interactions between the blastocyst and the uterus to initiate the process of implantation (see below). The temporal and celltype-specific expression of these factors and their receptors in the embryo and uterus during the peri-implantation period suggests their involvement in different aspects of embryo development and embryo– uterine interactions during implantation (Pollard, 1990). In the rat (Canivenc et al., 1956), mouse (Yoshinaga and Adams, 1966), and Mongolian gerbil (Norris and Adams, 1971), both progesterone and estrogen are required for induction of implantation. In the hamster (Prasad et al., 1960) and guinea pig (Deanesly, 1960), on the other hand, only progesterone is required. In the rabbit, both progesterone and estrogen are required when ovaries are removed in early pregnancy (Pincus and Werthessen, 1938; Chambon,

1949). In the rhesus monkey, some investigators report that progesterone only can induce implantation (Meyer et al., 1969), but others claim that removal of estrogen can prevent implantation despite progesterone supplement (Ravindranath and Moudgal, 1988). Although species differences in steroid requirements for implantation may be explained, at least in part, by the differences in gene expression in response to steroids, comparative analysis of the expression of molecules that play essential roles in implantation is needed for clarification of the species variation in steroid requirements for implantation. For example, it is worth elucidating how the uterus becomes receptive and then refractory when progesterone alone continuously acts on the hamster endometrium. The ability of blastocysts to remain dormant for a prolonged period of time creates a unique physiological condition of delayed implantation (Mead, 1993). Rats and mice ovulate within 24 h of parturition. When they are mated at this ovulation and nurse the suckling young, the embryos conceived at the postpartum ovulation fail to implant after shedding of the zona pellucida (Lataste, 1891). Implantation is delayed because the suckling stimulus stimulates high prolactin secretion that, in turn, suppresses gonadotropin secretion (Fox and Smith. 1984). The ovaries respond to a high level of prolactin and low gonadotropin to secrete a large amount of progesterone and little estrogen (Yoshinaga

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et al., 1971). This imbalance of progesterone/estrogen renders the endometrium nonreceptive to blastocysts despite a high level of progesterone secretion because implantation is not delayed if suckling stimulus is low. When suckling stimulus subsides, the pituitary gland secretes less prolactin and gonadotropin secretion starts to increase. In response to this increase the ovaries secrete estrogen. The synergistic actions of progesterone and estrogen render the uterus receptive for blastocyst implantation (Yoshinaga, 1977). This physiological phenomenon of estrogen/ progesterone synergism is probably a suitable subject for molecular analysis of coactivators and corepressors of steroid receptors. A delay in implantation is also observed in some wild animals. In the tammar wallaby, a suckling stimulus maintains the blastocyst in a dormant stage; however, cessation of lactation longer than 72 h alters the endocrine system of the mother to reactivate the dormant embryo (Renfree, 1993). In the spotted skunk, embryonic diapause usually takes place for approximately 200 days. It has been shown that the ovary produces an unidentified substance that is essential to stimulate the dormant embryo (Mead, 1989). Little attention is being paid to identify this substance. Investigation into the mechanisms involved in implantation in wild animals will broaden our knowledge of the schemes created to protect and rear the embryo while adapting to the environment in which these species survive. Ovarian steroid hormones are essential for preparation of the receptive uterus. The major uterine cell types respond differentially to P 4 and/or E 2. In the adult mouse, E 2 directs proliferation of uterine epithelial cells, while this process in the stroma requires both P 4 and E 2 (Pollard, 1990). Similar uterine effects occur in the mouse during early pregnancy in response to ovarian P 4 and E 2 (HuetHudson et al., 1989). On days 1 and 2, preovulatory ovarian E 2 stimulates epithelial cell proliferation. On day 3, P 4 from newly developed corpora lutea induces stromal cell proliferation which is further potentiated by preimplantation ovarian E 2 secretion on day 4. In contrast, epithelial cells stop proliferating and become differentiated on day 4. The uterus becomes receptive to blastocysts on this day and a “cross-talk” between the blastocyst and the uterus ensures initiation of implantation. In the rodent, uterine sensitivity with respect to implantation has been classified into prereceptive, receptive, and nonreceptive (refractory) phases. In the mouse, the prereceptive uterus on day 3 of pregnancy or pseudopregnancy becomes receptive on day 4 (the day of implantation) (Noyes et al., 1963). The receptive uterus subsequently enters into the nonreceptive phase and fails to respond to the presence of blastocysts. Similar uterine events can also be induced in delayed implanting of pregnant or pseudopregnant mice. Ovariectomy before the preimplantation E 2 secretion on the morning of day 4 results in blastocyst dormancy and inhibition of implantation, a condition termed delayed implantation. This condition can be maintained by daily P 4 treatment which maintains the

uterus at neutral phase (analogous to prereceptive phase) (Paria et al., 1993a). The neutral uterus enters into the receptive phase if exposed to E 2 after priming with P 4. Treatment with E 2 also activates dormant blastocysts in utero, resulting in the initiation of implantation (Paria et al., 1993a; Yoshinaga and Adams, 1966). In the absence of blastocysts, the receptive uterus proceeds to the nonreceptive phase. Thus, the receptive state is defined as the “window” when the uterus is conducive to blastocysts for implantation and lasts for a limited period. The mechanisms by which E 2 activates dormant blastocysts, stimulates uterine receptivity, and initiates implantation are not clearly understood. Blastocyst’s state of activity determines the window of implantation in the receptive uterus. Implantation in the receptive uterus has long been considered to occur irrespective of the blastocyst’s state of activity. Blastocyst transfer experiments in delayed implanting of mice have shown that the blastocyst’s state of activity is also an important determinant in defining the window of implantation in the receptive uterus (Paria et al., 1993a). The results of this investigation demonstrated that dormant blastocysts transferred into uteri of P 4-treated delayed pseudopregnant recipients implant successfully only if they are transferred within 1 h of E 2 treatment of the recipients. In contrast, day 4 normal or E 2-treated in utero-activated blastocysts successfully implant even when transferred at 16 h after E 2 treatment of P 4-treated delayed recipients. Dormant blastocysts cultured in vitro acquire “metabolic” activation with respect to increased oxygen consumption, leading to the suggestion that the delayed-implanting uterus produces an inhibitor(s) that renders the blastocysts dormant (McLaren, 1973; Weitlauf, 1974). However, a recent investigation showed that dormant blastocysts cultured in vitro fail to implant upon transfer into P 4-treated delayed uterus beyond the critical period of 1 h of E 2 treatment (Paria et al., 1993a). These results suggested the following conclusions. First, the window of implantation is tightly regulated and opens when the activated state of the blastocyst coincides with the receptive state of the uterus. Second, E 2 induces very rapidly, but transiently, a factor(s) in the P 4-primed uterus that activates dormant blastocysts in utero for implantation in the receptive uterus. Finally, although dormant blastocysts acquire metabolic activation in vitro (Weitlauf, 1974), they do not become implantation competent. Coordination of differential effects of E 2 and catecholestrogen on two distinct targets mediates implantation in mice. Although E 2 is essential for blastocyst implantation in the P 4-primed uterus, the mechanism(s) by which E 2 initiates this response still remains elusive. It has recently been demonstrated that the primary ovarian E 2 via its interaction with nuclear E 2 receptors participates in the preparation of the P 4-primed uterus for the receptive state in an endocrine manner, while its metabolite catecholestrogen produced from primary E 2 in the uterus mediates blastocyst activation for implantation in a paracrine manner. These results established both that these target-specific

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FIG. 1. Implantation strategies. (A) Murid rodents. Penetration of the uterine epithelium (shaded cells) is accomplished by polytene trophoblast cells, aided by apoptosis of uterine epithelial cells followed by penetration of the residual luminal epithelial basal lamina by processes of underlying decidual cells. (B) Guinea pig. The syncytial trophoblast of the implantation cone penetrates through the zona pellucida, then between uterine epithelial cells, after which the rest of the blastocyst, including the inner cell mass, is drawn into the uterine stroma. (C) Rabbit. Syncytial trophoblast knobs fuse with one or two uterine epithelial cells creating pegs in the epithelium prior to penetration into the stroma. (D) Primates. Syncytial trophoblast formed near the inner cell mass intrudes between uterine epithelial cells before penetrating the basal lamina. Abbreviations: D, decidual cells; En, embryonic endoderm; Ep, uterine epithelium; ICM, inner cell mass; S, stroma; T, trophoblast; ZP, zona pellucida.

effects of primary E 2 and catecholestrogen are essential for implantation and that implantation occurs only when the activated stage of the blastocyst coincides with the receptive state of the uterus (Paria et al., 1998b).

ANIMAL MODELS OF IMPLANTATION Implantation begins when the blastocyst both assumes a fixed position in the uterus and establishes a more intimate relationship with the endometrium. In species in which the eventual placenta is hemochorial, a series of events must occur during implantation, i.e., apposition of the blastocyst to the uterine luminal epithelium, adhesion of trophoblast to this epithelium, penetration of the epithelium followed by penetration of the epithelial basal lamina, and stromal invasion including penetration of the superficial endometrial vessels (Schlafke and Enders, 1975). Despite the common steps and the eventual relationship of trophoblast bathed with maternal blood, the way in which these stages are accomplished turns out to be different in different species. These differences provide opportunities to examine distinct features of cell interactions (Enders et al., 1995).

Murid rodents. Closure of the slot-like uterine lumen in rodents brings the blastocyst into close apposition to the luminal epithelium after loss of the zona pellucida (see Parr and Parr, 1989). The blastocyst elicits a decidual response from the endometrium during the apposition stage of implantation. Subsequently, the epithelium within the primary decidual area undergoes apoptosis and is phagocytized by the polytene trophoblast cells of the wall of the blastocyst, facilitating epithelial penetration (Fig. 1A). Decidual cells subsequently penetrate the residual uterine luminal basal lamina and reorganize the surrounding stroma. The forming decidua creates a chamber for the blastocyst, orienting it so that the ectoplacental cone region of the blastocyst is directed toward its eventual mesometrial position. Modification of the primary decidua, including its degeneration, brings blood in contact with the trophoblast to form the yolk sac placenta and allows space for enlargement of the conceptus. The removal of the luminal epithelium immediately above the implanting blastocyst and even toward the mesometrial end is again accomplished prior to trophoblast penetration into these areas (Welsh and Enders, 1991). It is not even certain that adhesion between trophoblast and the apical surfaces of luminal epithelial

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cells need be any greater than that involved in normal phagocytosis of sloughed uterine cells. The facts that certain rodents (mice and rats) can have delayed implantation, i.e., the uterine environment controls blastocyst development, and that the blastocyst induces the decidual response prior to epithelial penetration make these species particularly useful for studying signaling between endometrium and blastocyst (Das et al., 1994b; Chakraborty et al., 1996; Lim et al., 1999a; Paria et al., 1999b). The relative ease of finding implantation sites using markers of local edema also enhances the usefulness of these species. Guinea pig. The guinea pig, a caviomorph rodent, has an implantation pattern quite different from that of the murid rodents. While the blastocyst is in the uterine lumen, it develops an implantation cone at the opposite pole from the inner cell mass. This implantation cone is composed of syncytial trophoblast and sends processes through the zona pellucida to adhere to the uterine luminal epithelium (Enders and Schlafke, 1969). The processes not only penetrate between uterine epithelial cells, but also do not pause at the uterine basal lamina. Thus, the entire blastocyst intrudes into the endometrial stroma beneath the luminal epithelium, losing the zona pellucida in the process (Fig. 1B). Only after the blastocyst is within the endometrium does the endometrium undergo a decidual response similar to that of murid rodents. This mechanism of direct invasion into the endometrium should make the guinea pig a useful model for epithelial penetration. However, the relatively long estrous cycle, the fact that only three to five blastocysts are formed, and the great difficulty in detecting the initial implantation sites make the guinea pig a more difficult model to use. Rabbit. The rabbit is an extreme example of blastocyst adhesion to the apices of epithelial cells in that a large number of trophoblastic knobs, comprising syncytial trophoblast, first adhere to the apical ends of the epithelial cells, then fuse with them (Enders and Schlafke, 1971; Hoffman and Winfrey, 1989). Although such fusion with the apical ends of cells is not common in other species, it does indicate that apical–apical cell adhesion can be an important initiator of implantation (Fig. 1C). The predictable ovulation 10 h after mating and the fact that trophoblast vesicles can be used to attach to uterine epithelium in vitro make this an interesting model for the study of apical cell adhesion (Hoffman et al., 1998). Although trophoblast– uterine cell fusion may occur in other animals, the rabbit constitutes the only well-documented example of fusion at the beginning of the process of epithelial penetration. Primates. Primate blastocysts implant in a simplex uterus that has a slot-like lumen. Although the initial stages of implantation in the human have never been seen, the orientation of the blastocyst with the inner cell mass adjacent to the endometrium suggests that in this species, like other primates studied, the orientation of the blastocyst is brought about by trophoblast near the inner cell mass adhering to the epithelium (Fig. 1D). In primate

species in which the early stages have been examined (macaque, marmoset, baboon), syncytial trophoblast first forms near the inner cell mass, and in the macaque and marmoset it can be shown to be the type of trophoblast that penetrates the uterine epithelium by intruding between uterine epithelial cells (Enders et al., 1983; Smith et al., 1987). The stage in which the blastocyst adheres but does not penetrate the epithelium has yet to be described by electron microscopy in primates; however, blastocysts flushed from the baboon uterus at about the time of implantation show areas of syncytial trophoblast with cytoplasmic protrusions on their apices (Enders et al., 1997). Although primate endometrium can be readily studied in both normal and artificial cycles, it is not practical to obtain large numbers of early implantation stages in these species since their fertility is relatively low. Furthermore, at the present time good methods of ascertaining early stages of pregnancy with certainty prior to autopsy are not available. By about day 12 of pregnancy (3 days after implantation) the implantation site in the cynomolgus macaque can be located by ultrasound (Tarantal and Hendrickx, 1988). At this stage the syncytial trophoblast not only has penetrated the epithelium, but also has penetrated into maternal blood vessels, and the lacunar stage of implantation has begun. This species thus forms a good model for late implantation, and since cytotrophoblast begins to invade the endometrial blood vessels at this stage it is also useful to study arterial invasion and modification by cytotrophoblast (Enders and Blankenship, 1999). However, the lacunar stage in this species is not identical to that in the human in that the human shows more stromal invasion by individual cytotrophoblast cells and less vessel invasion at this early stage (Enders, 1997). To study trophoblast adhesion to and penetration of the uterine epithelium, the marmoset should prove particularly useful (Enders and Lopata, 1999). In this species the blastocyst remains superficial for several days, with masses of syncytial trophoblast repeatedly invading the epithelium as the implantation site spreads peripherally. Furthermore marmosets have one to three blastocysts per pregnancy, the presence of early pregnancy can be determined with at least 50% accuracy by P 4 levels, and the epithelial invasion stage takes place over several days. Marmoset blastocysts do well in vitro, and a successful in vitro implantation model has been established (Lopata et al., 1995). Thus it would be possible to compare in vitro and in vivo results and to manipulate conditions for implantation. Such a model is particularly important since attempts to use human blastocysts in vitro have not yet provided models comparable to the in vivo situation. Domestic animals. The methods of implantation in domestic animals vary widely. In the pig, implantation remains superficial, i.e., there is no penetration of the uterine epithelium by trophoblast (Wooding and Flint, 1994). Although the trophoblast and luminal epithelium become closely interdigitated and the blood vessels that

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develop in the blastocyst and those of the uterus indent the respective epithelia, at no time is the integrity of the uterine lumen breached. In the sheep, goat, and cow, much of the placenta remains superficial, but there is an interesting formation of trophoblast binucleate cells which fuse with uterine luminal epithelial cells, forming transient heterokaryons in the case of the cow and longer lasting plaques in the sheep and goat (Wooding, 1992). This is another example of apical cell adhesion and cell fusion and has been studied extensively (Wooding et al., 1994). The gonadotropin-producing trophoblast cells of the horse (endometrial cup cells) are even more intimately associated with the endometrium than the binucleate cells of ungulates since these cells migrate through the uterine luminal epithelial cells and into the endometrial stroma (Allen et al., 1973; Enders and Liu, 1991). The domestic dog and cat have a central implantation with many isolated islands or plaques of syncytial trophoblast that invade the epithelium by penetrating between epithelial cells (Leiser and Koob, 1993). Eventually all of the luminal epithelium in the area where the placental band will form is eliminated, and after the buildup of more syncytial and cytotrophoblast the syncytium penetrates the epithelial basal lamina and surrounds the basal lamina of the maternal vessels, which subsequently elongate greatly to form the maternal portion of the placental labyrinth. These species or another carnivore, the ferret, therefore could be used to study the way in which syncytial trophoblast adheres to and penetrates luminal epithelium since it has a built-in control of the noninvasive cytotrophoblast between the numerous areas of invasive syncytial trophoblast plaques.

APPOSITION/ATTACHMENT MODULATORS Common features of implantation. Depending on the species, trophoblast penetration of the endometrium may continue deep into the stroma/decidua (mice, rats, humans) or remain superficial and not even pass through the lumenal epithelium (domestic species). Nonetheless, these implantation strategies all share the interaction of the trophectoderm with the apical surface of the uterine epithelium. During the conversion to the receptive state, the uterine epithelium undergoes not only a dramatic functional, but also a morphological transition. As discussed in more detail below, the apical glycocalyx decreases in amount and negative charge character (Schlafke and Enders, 1975). In addition, normally abundant apical microvilli gradually retract, creating a flattened surface in many areas (Schlafke and Enders, 1975; Murphy, 1993). This process may be related to the observed disruption of the terminal actin web (Luxford and Murphy, 1989, 1992). Thie and co-workers (1996, 1997, 1998) have performed a series of studies using human trophoblast– uterine epithelial adhesion models examining the relationship between

attachment and integrity of the cytoskeleton and associated components. These studies have included examination of the physical forces associated with attachment. The results indicate that features of the epithelium associated with the receptive phase, i.e., remodeling of tight junctions, adherens junctions, and the actin cytoskeleton, are correlated with strong binding between the model cell lines. It should be noted that the RL95-2 cell line used for many of these studies expresses very little MUC1 (Hey and Aplin, 1996). It would be of value to generate MUC1-expressing lines of RL95-2 to determine if the lack of mucin, rather than the other changes noted, accounted for the increased adhesion between these cell lines. In many species, the receptive phase is strongly associated with the generation of large apical protrusions believed to be pinopodes (Given and Enders, 1989; Nikas, 1999). The functionality of the pinopode-like structures is not clear. It is possible that they reflect an important retrieval system for components in uterine lumenal fluid; however, decreased expression of apically disposed catabolic enzymes occurs during the receptive phase, suggesting that resorption may not be a critical function at this time (ClassenLinke et al., 1987). The pinopode-bearing cells might represent primary sites for embryo interaction since these structures physically tower above the rest of the uterine surface. No molecular characterization of the pinopodes has been performed. Thus, it is unclear if these structures lack antiadhesive components, e.g., mucins, or bear adhesionpromoting molecules, e.g., integrins and proteoglycans, properties that might be expected if these pinopodes function as embryo attachment sites. Uterine epithelial cells are highly polarized under most conditions in vivo or in vitro (reviewed Wegner and Carson, 1994). It appears that, in addition to the changes described above, alterations in the polarized characteristics of these cells change during the conversion to the receptive state. Among these include loss or redistribution of various markers of apical and basolateral membrane domains (reviewed in Wegner and Carson, 1994; Denker, 1994). In a number of species, basal processes emerge and penetrate the basal lamina (Denker, 1994, and references within). Denker (1994) also has suggested that these changes reflect aspects of epithelial–mesenchymal transition and may facilitate interactions between the apical poles of the uterine epithelia and trophoblast. Indeed, trophoblast also undergo phenotypic changes during implantation giving rise to both sessile and migratory cell populations. These changes in trophoblast are characterized by multiple changes in integrin and metalloprotease expression reflecting the composition of the uterine extracellular matrix and the invasive behavior of these populations (Damsky et al., 1994; Alexander et al., 1996). Nonetheless, while there are a number of impressive changes in the behavior of both uterine epithelium and trophoblast during the implantation process, there is little evidence to indicate a true epithelial–mesenchymal conversion for either cell type.

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Mucins. Apposition and attachment of the embryo to the endometrium initially involves interactions between the external surface of the trophectoderm and the apical surface of the lumenal epithelium of the uterus. Much interest is placed on understanding what binding factors or receptors support the interactions between these tissues. Nonetheless, an equally important principle is that both the blastocyst and the apical surface of the uterine epithelium are nonadhesive prior to the time of attachment. Therefore, both tissues must be converted to an adhesioncompetent state to support these interactions. In the case of the embryo, the nonadhesive state may be considered passive in the sense that, for most of the preimplantation period, the embryo is encased in the nonadhesive zona pellucida; however, the zona is not the only factor in this regard since zona removal is not sufficient to convert preimplantation embryos to an adhesive state (see Wegner and Carson, 1994). As discussed above, the studies with delayed implantation models demonstrate that the act of hatching from the zona is distinct from and precedes the expression of key attachment-promoting proteins. It is clear that as early as the morula stage embryos express cell– cell adhesion molecules, e.g., E-cadherin, critical for preimplantation development (Riethmacher et al., 1995). Nonetheless, these components do not appear to be sufficient to support attachment to uterine cells in vivo (Noyes et al., 1963) or extracellular matrix components in vitro (Carson et al., 1990). Attachment-competent blastocysts display the ability to bind to a diverse array of cell types and extracellular matrix components (Carson et al., 1990). Nonetheless, studies by Noyes et al. (1963) have demonstrated that attachmentcompetent rodent blastocysts transferred into uteri prior to the receptive phase fail to implant. There are likely to be multiple reasons for this, including inadequate expression of attachment-promoting molecules. One key factor also relates to the generalized barrier function that the uterine mucosa must provide. The uterus and other parts of the female reproductive tract must provide a barrier to microbial infection, particularly during the mating process. As is the case in other mucosae, high-molecular-weight mucin glycoproteins are abundantly expressed at the apical surface of uterine epithelia under most conditions and provide a physical barrier to enzymatic attack and infection (Hilkens et al., 1992). The best studied mucin in reproductive tract tissues is MUC1 (mouse nomenclature, Muc-1). MUC1 expression is restricted to uterine epithelia and is strongly influenced by steroid hormones in many species including mice (Surveyor et al., 1995), pigs (Bowen et al., 1996), rabbits (Hoffman et al., 1998), and baboons (Hild-Petito et al., 1996). In mice, Muc-1 expression is strongly stimulated by E 2 and repressed by P 4 (Surveyor et al., 1995). Moreover, anti-estrogens and anti-progestins appropriately antagonize these steroid hormone actions, suggesting that the activities are mediated by nuclear steroid hormone receptors. Nonetheless, analysis of the mouse Muc-1 promoter does not indicate direct regulation by steroid hormone receptors,

suggesting that other factors mediate the actions on Muc-1 expression by uterine epithelia (Zhou et al., 1998). Uterine expression of a variety of growth factors and cytokines is modulated by steroid hormones and may account for the alterations in Muc-1 expression; however, little information is available on what cytokines or growth factors directly regulate MUC1 gene expression in any system. MUC1 has been shown to effectively inhibit cell– cell and cell– extracellular matrix adhesion (Ligtenberg et al., 1992; Wesseling et al., 1995). This property requires a critical number of tandem repeats in the extracellular domain and appears to reflect steric hindrance to access to cell surface adhesion-promoting receptors (Wesseling et al., 1996). Similar results have been obtained with another transmembrane mucin, MUC4/sialomucin complex (Komatsu et al., 1997). Although not carefully studied in other systems, MUC4/sialomucin complex is readily detected in rat uterine epithelia in a pattern similar to that observed for Muc-1 in mice and is greatly reduced during the receptive phase (McNeer et al., 1998). A number of other mucin genes have been identified; however, limited information is available on their expression in female reproductive tract tissues. In the context of blastocyst attachment, mucins, in general, and MUC1, in particular, greatly impair access to the surface of uterine epithelia (DeSouza et al., 1999). General removal or reduction of mucins increases apical access to both enzymatic attack and embryo attachment. Remarkably, while Muc-1 accounts for only about 10% of the total cell surface mucin complement in mouse uterine epithelia (Pimental et al., 1996), several lines of evidence demonstrate that high level expression of MUC1 alone is sufficient to block embryo attachment (DeSouza et al., 1999). In keeping with these observations, MUC1 expression is severely reduced in uterine lumenal epithelia during the receptive phase, enhancing access for blastocyst attachment (Surveyor et al., 1995). In mice, treatment with the anti-progestin RU486 both restores Muc-1 expression during the receptive phase and inhibits implantation (Surveyor et al., 1995). In contrast, in rabbits and humans MUC1 expression actually increases during the receptive phase (Hey et al., 1994; Hoffman et al., 1998). Careful examination of implantation sites in rabbits reveals that MUC1 is locally reduced at the site of blastocyst attachment, suggesting that embryonic influences trigger reduction of MUC1 expression in this species (Hoffman et al., 1994). Candidate influences would include any factors secreted or expressed at the external surface of the trophectoderm that might trigger MUC1 down-regulation or activate release from the cell surface, e.g., activation of cell surface proteases. In this regard, it has recently been shown that the ADAM, MDC9, is found in uterine epithelia in several species, although MUC1-releasing activity has not been demonstrated (Olson et al., 1998). It is not clear if local loss of MUC1 occurs at human implantation sites and it has been suggested that MUC1 may actually promote embryo attachment in humans (Hey et al., 1998). It will be important to determine if human blastocysts or products of

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human blastocysts, e.g., chorionic gonadotropin, can stimulate removal of MUC1 to clarify this issue. For ethical reasons, it is not possible to use human blastocysts to determine if they can bind to MUC1 directly. Heparan sulfate (HS) proteoglycans and HS-binding proteins. Heparan sulfate proteoglycans are proteins bearing one or more high-molecular-weight, linear, highly negatively charged glycosaminoglycan chains of the HS variety. HS polysaccharides consist of glucosamine units alternating with either glucuronic or iduronic acid and variously sulfated on glucosamine and uronic acids. The ordered nature of the HS chain modification process leads to sequences within the polysaccharide, some of which contribute to a high degree of specific binding to various extracellular matrix proteins and growth factors/cytokines (Taipale and Keski-Oja, 1997). HS proteoglycans participate in cell adhesion processes in a variety of systems in which they appear to promote early stages of these processes (Lyon and Gallagher, 1998). HS chains are detected at the external aspect of peri-implantation stage mouse blastocysts and HS synthesis markedly increases in mouse embryos at the blastocyst stage (Farach et al., 1987). A number of functional assays indicate that HS participates in early stages of embryo attachment (Farach et al., 1987, 1988). Similar studies have been performed using human trophoblastic and uterine epithelial cell lines, suggesting a similar role for HS in promotion of adhesion between these cell types (Rohde and Carson, 1993). A number of core proteins that carry HS chains have been identified (David and Bernfield, 1998), but few have been studied in the context of embryo implantation. Syndecan is expressed by peri-implantation stage mouse embryos; however, it appears to be primarily expressed at the interior aspect of the blastocyst (Sutherland et al., 1991). Thus, syndecan is not normally located at a site to promote blastocyst attachment to uterine epithelia. Perlecan is a large HS proteoglycan typically found in basal lamina of many tissues and strongly expressed at the human fetal– maternal interface throughout pregnancy (Rohde et al., 1998). While perlecan can be detected in mouse embryos at the two- to four-cell stage (Dziadek et al., 1985), its expression sharply increases in blastocysts during the transition to the attachment-competent state and correlates well with acquisition of attachment competence in vivo, in vitro, and during activation from implantation delay in vivo (Smith et al., 1997). These observations are consistent with a role for perlecan in interactions between blastocysts and uterine epithelia. Other HS proteoglycans are likely to be involved as well since recent studies with perlecan null mice do not indicate implantation defects (Costell et al., 1999; Hassell et al., 1999). Knockouts have been generated in certain other HS proteoglycan genes without reported implantation defects (Xu et al., 1998; Cano-Gauci et al., 1999). Thus, there may be redundancy in function at this critical step requiring the generation of multiple null proteoglycan mutants for such studies. In addition, ectopic activation of HS proteoglycan genes not normally expressed during the peri-

implantation period or redistribution to the external blastocyst surface, e.g., syndecans, to support implantation function may occur in null contexts, requiring detailed proteoglycan analyses of such mutants. While there are a number of HS-binding proteins that could mediate interactions between the blastocyst and the epithelium, only a small subset of HS-binding proteins has been detected at embryo attachment sites. Perhaps the most intriguing example is that of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF expression by mouse uterine tissue is highly regulated by steroid hormones and is essentially lost during the pre-and peri-implantation period; however, expression is dramatically increased and restricted to implantation sites (Das et al., 1994b). Interestingly, it is the transmembrane, rather than the secreted, form of HB-EGF that appears to be induced at these sites, suggesting that this protein could mediate embryo attachment and juxtacrine signaling between uterine epithelia and trophectoderm. Additional studies have demonstrated that HB-EGF binding to blastocysts requires the presence not only of the EGF receptor, but also of HS proteoglycan (Raab et al., 1996; Paria et al., 1999b). Consistent with this are observations using a delayed implanting mouse model. HB-EGF expression by uterine epithelia is not induced at implantation sites in this model. Conversely, both EGF receptor and HS proteoglycan (perlecan) expression is repressed during delayed implantation and is restored rapidly upon activation from implantation delay (Paria et al., 1993; Smith et al., 1997). HB-EGF is expressed by uterine epithelia of other species, although the association of its pattern of expression with implantation is not as striking as in the mouse (Kennedy et al., 1994; Birdsall et al., 1996; Yoo et al., 1997). Other HS-binding proteins have been detected in uterine epithelia of various species during the receptive phase and include amphiregulin (Das et al., 1995) and heparin/heparan sulfateinteracting protein/L29 (Rohde et al., 1996). In both cases, expression is not restricted to implantation sites, but rather is observed throughout the uterine epithelia. Finally, ␤ 3and ␤ 1-containing integrin complexes have been reported to bind perlecan, although this appears to be mediated by interactions with the protein core (Hayashi et al., 1992; Brown et al., 1997). As noted above, expression of ␣ v integrins is greatly enhanced in human uterine epithelia during the receptive phase. Carbohydrate ligands and their receptors. Kimber and colleagues have provided a provocative series of studies implicating a class of oligosaccharides as mediators of embryo attachment. The lacto-N-fucopentaose-I (LNF-I) motif is detected on a subset of cells in mouse uterine epithelia during the receptive phase and soluble LNF-I inhibits mouse blastocyst attachment to uterine epithelia in vitro (Kimber et al., 1988; Lindenberg et al., 1988). In addition, fluoresceinated LNF-I specifically binds to attachment-competent mouse embryos (Lindenberg et al., 1990). The nature of the uterine carriers of LNF-I has not been determined, but could include both proteins and

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lipids. Furthermore, the identity of the LNF-I binding sites on embryos has not been determined. Such binding sites presumably would fall into the category of mammalian lectins. Expression of galactose-binding lectins (galectins) has been examined during early mouse embryogenesis, but these do not appear to be associated with the sites of embryo– uterine attachment (see Colnot et al., 1996). Furthermore, various mouse mutants that are null for galectin genes with no implantation phenotype have been created (see Colnot et al., 1996). Thus, it appears unlikely that galectins play a critical role in the implantation process. Similarly, despite multiple efforts by a variety of labs in the field, no evidence has been obtained for the presence of selectins at implantation sites. Again, double and triple nulls have been created for these genes with no apparent implantation phenotype (Jung and Ley, 1999). Still, as noted above, aspects of implantation strategies may differ among species. MUC1 persists in human uteri during the receptive phase and at least a subset of MUC1 oligosaccharides can carry selectin ligands under certain conditions (Hey et al., 1998). Moreover, preimplantation (four- to eight-cell stage) human embryos express selectins, although expression appears to decrease as these embryos approach the blastocyst stage (Campbell et al., 1995). Nonetheless, it remains possible that selectins or other mammalian lectins could participate in aspects of embryo attachment. Integrins and integrin ligands. There has been increasing interest in integrin cell adhesion molecules in the reproductive tract and significant data have accumulated to suggest their involvement in both fertilization and implantation (Bronson and Fusi, 1996; Sueoka et al., 1997). The integrins are among the best characterized of the immunohistochemical markers of uterine receptivity (Lessey, 1998). These glycoproteins serve as receptors for a variety of extracellular matrix ligands and act as modulators of cellular function through both attachment and signal transduction (Giancotti and Ruoslahti, 1999). The endometrium is an active site of integrin expression with both constitutive and cycle-dependent integrin expression (Lessey et al., 1992, 1994; Tabibzadeh, 1992). Decidualized endometrial stromal cells also display alteration of integrins at the time of implantation (Lessey et al., 1994; Ruck et al., 1994). At least three integrins appear to frame the window of implantation, being coexpressed on glandular epithelium only during cycle days 20 to 24, the putative window of implantation (Lessey et al., 1992). The apical pole of the luminal epithelium expresses both ␣v␤3 and ␣v␤5 (Aplin et al., 1996). The localization to the apical pole of the luminal epithelium suggests a role for these integrins in initial embryo– endometrial interaction (Lessey et al., 1996; Aplin et al., 1996). Similar findings of apical ␣v␤3 and ␣v␤5 integrin expression have been reported on the epithelial surface of blastocyst (Campbell et al., 1995; Sutherland et al., 1993). The placenta also undergoes dramatic programmatic expression of specific integrins, though this has been recently reviewed elsewhere (Damsky et al., 1994; Zhou et al., 1997).

The ␣v␤5 and ␣v␤3 integrins, along with the ␣5␤1 integrin recognize and bind to the tripeptide sequence Arg-Gly-Asp (RGD). The RGD sequence has been implicated in trophoblast adhesion to extracellular matrix and outgrowth (Armant et al., 1986; Yelian et al., 1995), and peptides containing this sequence can block mouse embryo attachment to human stromal cells, in vitro (Shiokawa et al., 1999). Recent studies have shown that implantation in the mouse can be significantly reduced by intrauterine injection of RGD peptides and monoclonal antibodies against the ␣v␤3 integrin, though it is not known at present where the perturbation occurs, on the embryo or endometrium (Illera et al., 2000). Integrin expression in the endometrium of other species has now been reported, including in the pig, baboon, goat, cow, and mouse (Burghardt et al., 1997; Fazleabas et al., 1997; Guillomot, 1999; Kimmons and MacLaren, 1999; Simo´n et al, 1998). Interesting differences in the integrins during the events of pregnancy may ultimately account for the varied types of attachment and invasion noted in the implantation process. The presence of integrins on the surface of the endometrium was initially thought to indicate their role as modulators of attachment, based on the receptor-mediated hypothesis of Yoshinaga (1989). Recent and evolving data using in vitro models of embryo– endometrial interaction suggest that binding of the embryo may not actually occur at the apical pole (Bentin-Ley et al., 1999). Rather, the apical interaction between embryo and maternal surface may be primarily one of matrix destabilization. Recent data from invasive melanoma cells, which also express the ␣v␤3 integrin, show that this integrin can bind to and activate matrix metalloproteinase 2 (Brooks et al., 1996). Thus, apical endometrial integrins may function to remove or digest the extracellular matrix surrounding the embryo and thereby destabilize the quiescent cellular phenotype. Since breakdown products of such digestion have been shown to stimulate cell motility (Biannelli et al., 1997), this may provide an explanation for the sudden shift from stationary to invasive cells that occurs at this time. Evidence from the baboon supports this hypothesis. In this species, ␣v␤3 appears 2 weeks later than that observed in the human (Fazleabas et al., 1997). In this species of primate, invasion is also delayed relative to that observed in the human. Trophinin, tastin, and bystin. Functional screening of a cDNA library derived from a human trophoblastic cell line has identified several novel proteins with adhesionpromoting activity. Trophinin is a 61-kDa protein with eight predicted transmembrane domains and is proposed to mediate homophilic cell adhesion (Fukuda et al., 1995). Trophinin is detected at the cell surface in cell lines and in both normal uterine epithelia and trophectoderm. Thus, this protein is positioned appropriately to mediate initial phases of blastocyst– uterine interactions. Trophinin requires the presence of a second cytoplasmic protein, tastin, to support adhesion; however, these proteins apparently do not directly interact with each other, but, rather, form part of a complex with cytoskeletal elements mediated by a

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third protein called bystin (Suzuki et al., 1998). All three proteins have been detected in both trophoblast and decidual cells at the human fetal–maternal interface as early as the sixth week of pregnancy with expression declining toward the end of the first trimester (Suzuki et al., 1999). It is not clear if these proteins also are localized to implantation sites in other species. Nonetheless, there is potential that these novel proteins may mediate early aspects of embryo– uterine epithelial interactions in humans and primates. Metalloproteases and their inhibitors. Many studies indicate an important role for metalloproteases and their inhibitors (TIMPs) in regulating aspects of the implantation process. Dramatic differences in the temporal and spatial patterns of expression of these molecules are observed both in uterine tissues and in trophoblast (Fisher and Damsky, 1993; Alexander et al., 1996; Das et al., 1997b). Expression is strongly influenced by physiologically relevant cytokines and hormones in both uterine cells (Huang et al., 1998; Schatz et al., 1999) and trophoblast (Librach et al., 1994; Meisser et al., 1999). Moreover, metalloprotease inhibitors administered in vitro or in vivo inhibit extracellular matrix degradation by trophoblast cells (Fisher et al., 1985; Behrendtsen et al., 1992; Rechtman et al., 1999) and severely impact formation of decidua (Alexander et al., 1996). None of these treatments nor any of the null mutations in metalloproteases or TIMPs have been reported to inhibit the initial aspects of implantation. Thus, it appears that the primary role that these activities play in the implantation process is in decidual tissue remodeling and regulation of trophoblast invasion. More recently, examination of the expression and function of disintegrins/ADAMs, cell surface proteins with both adhesion-modulating and proteolytic activities (Blobel, 1997), has revealed provocative patterns in both uterine (Olson et al., 1998) and trophoblast (Hurskainen et al., 1999) during the peri-implantation period. As null mutations are developed in genes encoding these proteins, critical assessment of their respective roles in implantation-related events will be possible.

GROWTH FACTORS, CYTOKINES, LIPID MESSENGERS, AND THEIR RECEPTORS The spatiotemporal expression of the EGF family of growth factors and their receptors (ErbBs) in the periimplantation uterus and embryo suggests that these growth factors serve as local mediators of embryo– uterine interactions during implantation (Das et al., 1997b). The EGF family includes EGF itself, transforming growth factor-␣ (TGF-␣), HB-EGF, amphiregulin (Ar), ␤-cellulin (BTC), epiregulin (Er), heregulins/neu-differentiating factors (NDFs), and cripto (Cohen, 1962; Derynck et al., 1984; Holmes et al., 1992; Shing et al., 1993; Shoyab et al., 1988; Toyoda et al., 1995). These molecules are synthesized as transmembrane proteins that are proteolytically processed to release the mature forms. Both the transmembrane and the mature

forms are biologically active (Massague and Pandiella, 1993). These ligands interact with the receptor tyrosine kinases of the erbB gene family for various signal transductions. The erbB family constitutes four receptor tyrosine kinases, erbB1, erbB2, erbB3, and erbB4. They share a common structural feature but differ in their ligand specificity and kinase activity (Heldin, 1995; Prigent and Lemoine, 1992; Peles and Yarden, 1993). All members of the EGF family except cripto can interact with erbB family members via homodimerization or heterodimerization (reviewed in Lim et al., 1997a). Thus, cross-talk between the receptor subtypes with various ligands can serve as a potential signaling mechanism. In mice, TGF-␣, HB-EGF, Ar, BTC, Er, and NDF-1 all are expressed in the uterus at the time of implantation (reviewed in Das et al., 1997a). TGF-␣ is indiscriminately expressed in the peri-implantation mouse uterus and its role in implantation is questionable because TGF-␣ null mice are apparently fertile (Bruce Mann et al., 1993; Luetteke et al., 1993); however, it is possible that the deficiency of TGF-␣ is compensated by other members of the EGF family. The Ar gene is induced in the uterine epithelium throughout day 4 and at the time of blastocyst attachment. It seems to be a more potent activator of EGF receptor (ErbB1) in the uterus than that in the blastocyst, implying a role in intrauterine signaling (Das et al., 1995). Er, BTC, and NDF-1 are induced in the luminal epithelium and stroma at the site of implantation (Das et al., 1997a; Reese et al., 1998). However, the significance of these growth factors in implantation is not known. HB-EGF with its expression pattern appears to be highly relevant to the implantation process (Das et al., 1994b). It is induced solely in the luminal epithelium surrounding the blastocyst 6 –7 h before the initial attachment of the blastocyst to the uterus. HB-EGF is not induced at the site of blastocyst during delayed implantation, but is rapidly induced by the termination of delayed implantation by E 2. This suggests that the blastocyst signals the luminal epithelial cells to express HB-EGF at the site of subsequent implantation and points toward a central role for HB-EGF in this process. In vitro experiments show that soluble HB-EGF can stimulate blastocyst proliferation, zona hatching, trophoblast outgrowth, and tyrosine phosphorylation of ErbB1 in mouse blastocysts. Furthermore, cells expressing the transmembrane form of HB-EGF can adhere to active, but not dormant, blastocysts (Raab et al., 1996). Using growth factor–toxin conjugates and egfr null blastocysts, it was recently shown that HB-EGF can interact with blastocyst ErbB4 and HS proteoglycan for the initiation of implantation (Paria et al., 1999b). Consistent with the findings in mice, HB-EGF is expressed in the human endometrium during the window of uterine receptivity for implantation (Leach et al., 1999; Yoo et al., 1997) and soluble HB-EGF is a potent growth factor for improving the development of in vitro-fertilized human embryos into blastocysts and zona hatching (Martin et al., 1998). ErbB1 and ErbB4 are expressed in mouse blastocysts

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(Paria et al., 1993b, 1999b); however, blastocysts with null mutation for egfr or erbB4 do not manifest obvious defects during the initial stage of implantation (Grassman et al., 1995; Threadgill et al., 1995), suggesting that the functions of these two receptors are redundant. The status of blastocysts with double mutations for egfr and erbB4 is not yet known. In addition to HB-EGF, the mouse uterus also expresses Er, BTC, and NDF-1 at the site of implantation that can interact with ErbB1 and ErbB4 (Lim et al., 1998). Thus, the contribution of these various ligands to implantation again could be redundant. In general, the expression of multiple ligands and multiple receptors of the EGF family might be a protective mechanism to ensure high probability of embryo development and implantation. Leukemia inhibitory factor (LIF). Leukemia inhibitory factor, a pleiotropic cytokine, is expressed at low levels in many different tissues and exhibits a multitude of biological actions including modulation of proliferation and differentiation (Hilton and Gough, 1991). A significant level of LIF is expressed in the endometrial glands of mice on day 1 (day 1 being the day of observance of the vaginal plug) of pregnancy and this expression declines by day 3 of gestation. A second burst of LIF expression occurs in uterine glands on day 4 of pregnancy, the day of implantation (Bhatt et al., 1991). On day 5, following implantation, LIF expression declines and is present at a low level throughout the rest of pregnancy. A key role for LIF in implantation was confirmed in a LIF-null mice (Stewart et al., 1992). Homozygous females lacking a functional LIF gene are viable and ovulate normally; however, blastocysts fail to implant. LIF also is expressed in pseudopregnant females, leading to the suggestion that its expression is under maternal control, possibly as a direct response to the increase in circulating E 2 levels that occurs on days 3– 4 of pregnancy (Bhatt et al., 1991; Shen and Leder, 1992). In humans, maximal LIF expression is observed during the P 4-dominated secretory phase of the menstrual cycle (Arici et al., 1995; Yang et al., 1996; Cullinan et al., 1996). LIF is expressed at a low level in the glandular epithelial and stromal cells of proliferative phase endometrium. Glandular epithelial staining increases significantly in the midsecretory phase and remains high until the end of the cycle. Hormonal regulation of LIF expression has been investigated in human endometrial stromal and glandular epithelial cells cultured in vitro. These studies indicate that, while E 2 or P 4 fails to enhance LIF expression, various cytokines and growth factors induce LIF expression in cultured endometrial cells. These studies suggest that LIF is not directly regulated by steroid hormones in the human endometrium (Arici et al., 1995; Senturk and Arici, 1998). Uterine LIF is proven to be essential for implantation and HB-EGF is also suspected as an important factor in implantation in the mouse. However, it is not yet known whether each of these factors acts independently or they work in concert. Based on published and our unpublished results, we hypothesize that uterine LIF is important for the preparation of the uterus and this preparation is required for

blastocyst activation and expression of uterine HB-EGF prior to the attachment reaction. This is consistent with the preliminary observation of loss of uterine expression of HB-EGF in LIF(⫺/⫺) mice (S. K. Dey et al., unpublished results). In turn, HB-EGF may interact with blastocyst HS proteoglycans and/or ErbBs in a paracrine/juxtacrine manner to modulate expression of COX-2 in the stroma at the sites of blastocyst apposition to initiate the attachment reaction and decidualization. This is consistent with initial results of loss of COX-2 expression in the stroma in the absence of HB-EGF expression in LIF(⫺/⫺) mice (S. K. Dey et al., unpublished results). Collectively, the results suggest that correct uterine expression of COX-2 is the convergent downstream signaling pathway for implantation. Colony-stimulating factor-1 (CSF-1). CSF-1, a hematopoietic growth factor, supports the growth and proliferation of mononuclear progenitor cells and promotes the proliferation of mature macrophages (Stanley and Heard, 1977). Interestingly, it was observed that CSF-1 mRNA and protein levels were markedly elevated in mouse uterus during pregnancy (Bartocci et al., 1986). In situ hybridization revealed that CSF-1 was localized exclusively to the luminal and glandular epithelial cells throughout gestation (Arceci et al., 1989). The functional role of this cytokine in implantation was studied in the osteopetrotic (op/op) mouse model. These animals harbor a naturally occurring null mutation in the CSF gene and exhibit decreased implantation rates and fetal viability (Pollard et al., 1991). Treatment of homozygotes with CSF-1 restored fertility, indicating an essential role played by this cytokine during this process (Wiktor-Jedrzejczak et al., 1991). CSF-1 mRNAs also are expressed in human endometrial glands during the midproliferative and midsecretory phases of the menstrual cycle (Pampfer et al., 1991); however, a correlation between abnormal CSF-1 expression and infertility has not yet been established. CSF-1 is the principal growth factor regulating the macrophage population in the uterus. Studies have shown that the number of uterine macrophages is reduced in CSF-1-less op/op mice. The macrophages become more rounded in appearance compared to those of normal mice (Pollard et al., 1998). Uterine CSF-1 may therefore be necessary to maintain normal macrophage population in the uterus during early pregnancy. These macrophages may also be stimulated by CSF-1 to produce cytokines that act on the trophoblast or other lymphoid cells in the uterus producing complex autocrine/paracrine loops (Pollard, 1990). Calcitonin. Calcitonin, a 32-amino-acid peptide hormone, has long been known to be synthesized and secreted primarily by the parafollicular C cells of the thyroid gland (Austin and Heath, 1981). Its most well-characterized physiological role is to regulate calcium levels in bone and kidney cells. Interestingly, recent studies revealed that calcitonin is transiently expressed in the rat uterine epithelium overlapping the window of implantation (Ding et al., 1994). In rats, calcitonin mRNA and protein expression increases by day 2 of gestation and reaches a peak on day 4,

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the day before implantation. On day 5, calcitonin expression starts to decline and by day 6 falls to below detection limits. P 4 is the primary inducer of calcitonin gene expression in rat endometrium while E 2 inhibits P 4-mediated calcitonin gene expression (Ding et al., 1994; Zhu et al., 1998a). The timing and location of calcitonin synthesis in the epithelium raise the possibility that calcitonin is secreted by the glands into the uterine lumen. Its principal function may be to regulate blastocyst implantation in an autocrine or paracrine manner. Recent studies have indeed shown that the administration of antisense oligodeoxynucleotides targeted specifically against calcitonin mRNAs, into the lumen of the preimplantation phase uterus, results in both suppression of calcitonin gene expression and a dramatic reduction in the number of implanted embryos (Zhu et al., 1998b). Further studies are necessary to decipher the functional role of calcitonin in embryo implantation. The synthesis of calcitonin mRNA and protein also has been monitored in the human endometrium at different days of the menstrual cycle (Kumar et al., 1998). Studies showed that calcitonin expression in human endometrium is temporally restricted to the epithelium of midsecretory phase of the cycle, which closely overlaps the putative window of implantation (Kumar et al., 1998). It also was observed that, as in rodents, P 4 regulates calcitonin expression in human endometrium. Calcitonin, therefore, emerges as a P 4-regulated potential marker of the receptive human endometrium. COX-2-derived prostaglandins. Prostaglandins (PGs) participate in various functions including modulation of vascular responses, cell proliferation, and differentiation. PGs are generated via cyclooxygenase (COX), the ratelimiting enzyme for the conversion of arachidonic acid into PGH 2, the common substrate for various PGs (Smith and DeWitt, 1996). COX exists in two isoforms, COX-1 and COX-2, encoded by separate genes. Gene targeting in mice has established distinct functions for these isoforms. While COX-1-deficient females are fertile with specific parturition defects, COX-2-deficient females have multiple reproductive failures that include defects in ovulation, fertilization, and implantation (Dinchuk et al., 1995; Langenbach et al., 1995; Lim et al., 1997). The uterine expression of COX-2 in an implantation-specific manner and defective implantation and decidualization in COX-2(⫺/⫺) mice establish that uterine COX-2 is essential for these processes (Chakraborty et al., 1996; Lim et al., 1997b). COX is present in both the endoplasmic reticular membrane and the nuclear envelope (Spencer et al., 1998), suggesting that PGs can exert their effects via different classes of receptors. Thus, PGs formed in the endoplasmic reticulum can exit cells and function via G-protein-coupled cell surface receptors (Negishi et al., 1995). In the mouse, cell surface receptors for PGE 2, PGF 2␣, PGD 2, and prostacyclin (PGI 2) are EP, FP, DP, and IP, respectively (Negishi et al., 1995). Further, EP receptors have four subtypes, EP 1, EP 2, EP 3, and EP 4, with different signal transductions (Ne-

gishi et al., 1995). In contrast, PGs produced via nuclear COX can exert their effects directly on the nucleus by activating peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptor superfamily (Mangelsdorf and Evans, 1995). The PPAR family members are PPAR␣, PPAR␥, and PPAR␦(␤) (Kliewer et al., 1994). PPAR␣ participates in lipid homeostasis and is activated by hypolipidemic drugs, fatty acids, leukotriene B 4, and PGI 2 agonists (Forman et al., 1997; Kliewer et al., 1997; Motojima et al., 1998). PGI 2 agonists, carbaprostacyclin and iloprost, also activate PPAR␦ (Forman et al., 1997; Kliewer et al., 1997; Lim et al., 1999a). PPAR␥ is primarily involved in adipocyte differentiation and terminal cell differentiation. The antidiabetic thiazolidinedione drugs and a metabolite of PGJ 2, 15-deoxy-⌬ 12,14-PGJ 2, act as ligands for PPAR␥ (Forman et al., 1995, 1997; Kliewer et al., 1997; Tontonoz et al., 1997). In contrast, information on the biological roles of PPAR␦ is very limited, although it is expressed in various embryonic and adult tissues (Braissant and Wahli, 1998; Braissant et al., 1996). PPARs modulate transcription by binding to sequence-specific PPAR response elements in the promoters of target genes, resulting in either transcriptional activation or suppression (Muerhoff et al., 1992; Tugwood et al., 1992). PPARs heterodimerize with retinoid X receptors (RXRs) for transcriptional regulation (Kliewer et al., 1992). In the presence of both PPAR- and RXR-selective ligands, synergistic activation of target genes occurs (Mukherjee et al., 1997; Schulman et al., 1998; Lim et al., 1999a). COX-2-derived PGE 2 and/or PGI 2 are potential candidates that can be involved in implantation working via EP and/or IP receptors. However, IP receptor is not detected in the uterus at the implantation site (Lim et al., 1999a) and IP-deficient mice are fertile (Murata et al., 1997), suggesting that IP receptor is not essential for implantation. Although EP receptor subtypes are expressed in the peri-implantation mouse uterus (Yang et al., 1997), the role of PGE 2 acting via EP 1, EP 2, and EP 3 subtypes in implantation appears to be insignificant, since mice null for these genes do not show implantation defects (Ushikubi et al., 1998; Kennedy et al., 1999). EP 4-deficient mice exhibit neonatal lethality and are thus uninformative for this function (Segi et al., 1998). Using COX-2(⫺/⫺) mice, it has recently been shown that COX-2-derived PGI 2 is the primary PG that is essential for implantation and decidualization and that the effects of PGI 2 are mediated by its activation of PPAR␦, demonstrating the first reported biologic function of this receptor. However, PGE 2 has a complementary role in PGI 2-induced implantation in COX-2-deficient mice (Lim et al., 1999).

SUMMARY AND FUTURE DIRECTIONS In recent years, there has been a large expansion of studies of the molecular basis of embryo implantation, not only in rodents, but also in a number of other species, including humans. Perhaps not surprising is the diversity of

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TABLE 3 Markers of Uterine Receptivity or Initial Embryo Attachment a Species Marker

Rodents

Rabbits

Domestic sp.

Primates

Humans

MUC1 (mucins) HB-EGF COX-2 Calcitonin ␣v, ␤3 integrins

⫺ ⫹ ⫹ ⫹ ⫺

⫺ ? ? ? ?

⫺ ⫺ ? ? ⫹

⫺ ? ⫹ ⫹ ?

⫹c ⫹ ? ⫹ ⫹

b

a The presence or absence of the various markers in uterine epithelium at the receptive phase is indicated by ⫹ or ⫺, respectively. A question mark indicates that information for the corresponding species is not available. For further discussion, see text. b Only at implantation sites. c Implantation sites have not been examined.

strategies and molecular players implicated in this complex and crucial biological process. Nonetheless, a focus on events occurring during the initial interaction of trophectoderm and the uterine epithelia and stroma at implantation sites indicates that some common themes emerge, at least from the maternal side. Many of these concepts are summarized based on studies in the mouse in Table 2. Several patterns emerge. Some uterine factors are strongly expressed early in the preimplantation period, but are lost prior to the time of implantation, e.g., MUC-1 and EGF. Others reappear around the time of implantation in response to either hormonal or embryo-derived cues, e.g., LIF, amphiregulin, cyclooxygenase-2, and HB-EGF. Activation of some of these genes appears to be transient; however, some persist and expression may spread from the immediate implantation site into surrounding decidua, e.g., cyclooxygenase-2 and HB-EGF. Expression of several key markers of the early implantation process has been extended to other species (Table 3). Loss of MUC1, either locally or throughout the uterine epithelium, appears to be a general principle, one potential exception being humans. Enhanced expression of several other genes in uterine epithelium has been observed during the receptive phase or at implantation sites in multiple species. These include genes encoding growth factors/hormones (HB-EGF, calcitonin), enzymes producing lipid hormones (cyclooxygenase2), or adhesion-promoting cell surface receptors (␣v␤3 integrins, HB-EGF). As additional work is performed in other mammals, it will be possible to determine which of these markers are species-specific or represent well-conserved responses. Information is also beginning to accumulate on another class of molecules that appear only in the uterus during the refractory stage, i.e., if implantation does not occur. These genes include TNF-␣ as well as endometrial bleeding-associated factor (ebaf; TGF␤4), a human orthologue of the mouse gene lefty (Kothapalli et al., 1997). Many questions remain. We still know relatively little about the molecular events critical for embryo conversion to an attachment-competent state, including what factors

embryos secrete that may trigger uterine events. While interferon-␶ is an example of such a factor produced by many ruminants, it is not clear if similar systems exist in other species. In the case of humans and primates, it is possible that gonadotropins have direct, early, and, perhaps even local actions on uterine cells (Fazleabas et al., 1999). Lipid hormones or even peroxide-based compounds may be involved as well. Mouse mutants undoubtedly will provide powerful tools to sort out many of these events; however, while we have known for over 10 years that LIF-null mice have severe implantation defects, we have yet to identify the downstream events impacted by LIF. Integrating the endocrinology, cell, molecular, and developmental biology to understand the process of embryo implantation will provide significant challenges in years to come.

ACKNOWLEDGMENTS The authors appreciate the assistance of Sheila Larson and Sharron Kingston in preparation of the manuscript and the many helpful comments of the members of their respective labs. This work was supported by NIH grants (HD29963 to D.D.C., HD34760 to I.B., HD29968 to S.K.D., HD299661 to A.T.F., HD34824 to B.A.L.) as part of the National Cooperative Program on Markers of Uterine Receptivity and by HD10342 to A.C.E.

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