Blastocyst implantation: the adhesion cascade

seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol. 11, 2000: pp. 77–92 doi: 10.1006/scdb.2000.0154, available online at http://www.idealibrary.com on

Blastocyst implantation: the adhesion cascade Susan J. Kimber∗ and Catherine Spanswick

capable of undergoing a transition to an invasive phenotype. However, implantation of such a blastocyst can only take place during a defined period of less than 24 h, the ‘window of receptivity’.1 This is defined by the sequential actions on the endometrium of progesterone followed by a small increase in estrogen. The uterus then becomes refractory to implantation and a transferred embryo cannot implant.2, 3 In women implantation can occur over about 5 or 6 days,4 apparently, and secretory phase estrogen may not be needed.5 The lumenal epithelium (LE) lining the uterine cavity appears to be the site of receptive sensitivity, because the hormone-regulated restriction on attachment and invasion of the intact uterus is abolished if the LE is broken or absent,6 or during ectopic implantation. Implantation occurs by a number of steps in which TE undergoes a series of distinct interactions. It is inititiated by close proximity between TE and LE: apposition, promoted by closure of the uterine lumen.7 This is followed by firmer adhesion. The intimate association between the two cell types has been captured in electron micrographs.8 The initial period may be a transient event, as in the mouse and human, with invasive trophoblast penetration, or effectively last throughout pregnancy in ruminants with epithelial–chorial placentation (see Bowen and Burghardt, this volume). Electron micrographs capturing murine trophoblast cells squeezing between LE cells illustrate the next step.8, 9 When trophoblast cells contact lateral or basal LE surfaces small desmosomelike membrane specializations have been visualized.8 Passage through the underlying basement membrane is facilitated by decidual-cell-mediated breakdown of this structure in advance of trophoblast penetration.10, 11 Blastocyst and uterus must differentiate in synchrony so that they provide the precise molecular repertoire required for adhesive interaction between TE and LE. Engagement of cell adhesion molecules leads to transduction of cytoplasmic signals which trigger the next steps in implantation involving phenotypic change. Since adhesion must be transient,

This review covers the sequence of cell adhesion events occurring during implantation of the mammalian embryo, concentrating on data from mouse and human. The analogy is explored between initial attachment of trophoblast to the uterine lining epithelium and that of neutrophils to the endothelial lining of blood vessels at sites of inflammation. The possible role of various carbohydrate ligands in initial attachment of the blastocyst is reviewed. The evidence for subsequent stabilization of cell adhesion via integrins or the trophinin-tastin complex is discussed. Key words: cell-adhesion / implantation / blastocyst / carbohydrate / integrin c 2000 Academic Press 

Introduction SUCCESSFUL IMPLANTATION OF THE mammalian embryo is an absolute requirement for furtherance of the species, and has been safeguarded by the utilization of multiple molecular mechanisms and a high level of redundancy. Implantation of the blastocyst takes places in the uterus at about midnight on day 4 of pregnancy in mice. The blastocyst has differentiated an outer cell layer the trophectoderm (TE, the first epithelium of the body) surrounding a fluid filled cavity. Within the TE a clump of cells, the inner cell mass (ICM), will form the fetus. It is the TE which interacts with the uterus at implantation and subsequently forms the placenta (Cross, this volume). From rodents it is known that prior to implantation the TE must become activated (trophoblast) and

From the School of Biological Sciences, University of Manchester, 3.239 Stopford Building, Oxford Rd, Manchester M13 9PT, UK. *Corresponding author. c 2000 Academic Press 1084–9521 / 00 / 000077+ 16 / $35.00 / 0

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Analogy with neutrophil–endothelial interaction

the molecular environment must evolve rapidly to facilitate penetration of trophoblast through the LE and into the stroma.

The analogy has been made between interaction of leukocytes with endothelial cells at sites of inflammation and that of the blastocyst with LE at implantation.16, 25 However, the blastocyst moves through the luminal fluid under very gentle flow conditions, while neutrophils experiences shear stress even in capillaries and venules. In damaged or infected tissues neutrophils adhere to activated vascular endothelium. Initial adhesion is mediated by selectins, transmembrane proteins with terminal C-type, calcium dependent lectin domains.26 Under conditions of flow they induce the slowed rolling motion of leukocytes preceding attachment, by low affinity, specific, carbohydrate binding.27 Although shear stress does not apply in the uterus, it is likely that trophoblast interaction with LE proceeds stepwise starting with a ‘tethering’ step which leads to a sequence of further adhesive interactions. Once rolling, leukocytes adhere by low avidity interactions between β2 integrins and immunoglobulin super-family endothelial receptors. Binding-induced increase in avidity leads to firmer adhesion, which is followed by extravazation,27 with morphological similarities to trophoblast penetration of LE. Unfortunately, current in vitro models for TE–LE interactions cannot accurately reflect the changing time course of adhesive events, so the sequential nature of TE–LE interactions is still to be documented.

Changes in the LE in preparation for implantation Molecules involved in adhesion between LE and trophoblast would be predicted to be under the control of ovarian steroids which regulate receptivity. The simplest model to account for receptivity is for new adhesive molecules to be synthesized and expressed on the apical LE membrane with counter-receptors appearing on the trophoblast, but new endometrial RNA or protein synthesis is not required,112 arguing against de novo expression in LE. Rather, adhesive components appear to be unmasked, modified or relocated from baso-lateral aspects to the apical region of the cell. Apical epithelial surfaces are normally nonadhesive, yet during implantation, apical interaction between TE and LE occurs. This suggests that the transition of the pre-receptive to receptive uterus requires fundamental changes in epithelial cell organization.13–16 Apical–basal polarity of LE becomes less marked, cells flatten and lose microvilli and the composition of apical and basal surfaces becomes similar. There is a redistribution of normally basal molecules to apical or lateral locations, e.g. syndecan in mice,17 α6 integrin in human18 and lateral to apical redistribution of cadherin in rat.19 Changes in LE cell–cell adhesion molecules reflect this phenotypic transition. Estrogen induces E-cadherin degradation on d4.5 of pregnancy;20 there is down-regulation and redistribution of desmosomal proteins (I Illingworth, D Garrod, G Ireland and S Kimber, submitted) and a change in distribution and complexity of LE tight junction particle networks.21, 22 Thus the postovulatory and receptive LE cells differ in phenotype. The ability of human epithelial cell lines to support embryo attachment correlated with their reduced polarity and apical expression of normally baso-lateral cell adhesion molecules.23 Since initial apposition and adhesion occur universally among mammals, a change in LE organization at implantation may be general, irrespective of differences in control mechanisms and subsequent trophoblast behaviour. So what do we know about the molecules involved? Targeted gene deletion has led to the identification of surprisingly few genes with an implantation phenotype,24 perhaps because of the importance of implantation being successful. Therefore, our knowledge of the key molecules comes from less direct approaches.

Molecular basis of initial attachment Carbohydrates and their receptors Carbohydrate chains can extend into the extracellular space by, in the case of mucins, as much as a micron, well beyond the projection of proteins, suggesting that the embryo first contacts oligosaccharide chains of the glycocalyx as it approaches the LE.28 This prediction is supported by the observation of a 0.2–0.7 µm space between LE and TE surfaces of blastocysts attached to cultured uterine strips,29 although whether this occurs in vivo is not clear. Fucosylated Carbohydrates and Ligands at Implantation Carbohydrate–selectin interaction initiates tethering of neutrophils and P-selectin has been detected on cleaving human embryos30 and L-selectin on murine blastocysts but not morulae31 (R Stones, D Bloor and 78

Blastocyst implantation: the adhesion cascade

can function in attachment.55 Since Le-y glycolipid binds selectively to H-type-1 and -2 chain glycolipids, blastocyst Le-y might bind H-type-1 on apical LE. Direct carbohydrate–carbohydrate interactions have been reported to mediate adhesion.56, 57 Uterine injection of an anti-H-type-1 mAb failed to block implantation, but it is possible that this antibody did not recognize/block all sites on the LE.

S Kimber, unpublished). However, mutant mice which lack each of the selectins have been produced and show normal embryonic development and implantation and breed normally.32–34 Gene deletion experiments may be misleading because of the possibility of compensatory upregulation of other members of a gene family, or the redundancy of molecules. Thus the significance of blastocyst selectin expression remains unclear. The major selectin ligand α(2–3)sialylLe-x is expressed by murine LE during the receptive period31 and blastocysts31, 35 as well as by human LE.36 Another fucosylated sugar, the H-type-1 antigen (Fucα1-2Ga1β1-3GIcNAcβ1-) may be an initial attachment ligand. Expression of this antigen on murine LE is estrogen-dependent and controlled by an α1-2 fucosyltransferase (α1-2FT).16, 37–41 In vivo, attachment of blastocysts to LE is inhibited by a pentasaccharide carrying H-type-1, or a monoclonal antibody (mAb) that recognizes it.42 H-type-1 is expressed uniformly at the apical LE cell surface up to day 4 of pregnancy and so could interact with the TE at attachment.25 Its disappearance between day 5 and day 616, 43 may contribute to the refractory phase when implantation cannot occur. Binding sites for H-type-1 are first detected on abembryonic TE of hatching blastocysts44–46 (which first contacts the LE in mice) suggesting the presence of receptor(s) on the trophoblast. Zona-enclosed embryos would not normally have access to LE-bound H-type-1, but its presence in uterine fluid up to day 3 of pregnancy could lead to competitive inhibition of blastocyst receptors which might contribute to the inability of transferred blastocyst to implant until the uterus becomes receptive.47 Additionally, H-type-1 on LE may be unavailable to trophoblast receptors up to the normal time of implantation. It is not known if hatched human blastocysts carry H-type-1 receptors, but the carbohydrate is expressed on human LE48, 49 as well as that of rat,50 bovine and caprine (Bowen and Burghardt, this volume). The Le-y carbohydrate antigen (Fucα1-2Galβ14[Fucα1-3]GlcNAcβ1-) is also a contender for an attachment molecule. It is present on the murine blastocyst surface51, 52 and LE37, 43, 53 as well as human LE.48, 54 Lumenal injection of mAb to Le-y specifically blocked implantation, but only between 87 h and 93 h post coitum, just before the normal period of attachment.53 Pre-treatment of blastocysts with mAb inhibited implantation after transfer.55 In co-culture experiments pre-treatment of either blastocysts or LE with mAb to Le-y inhibited attachment, suggesting that Le-y on both surfaces

Galectins Galectins are a group of lectins (10 to date) that share structural similarity and exhibit specificity for α-galactosides.58, 59 They occur inside cells, in association with the cell membrane or in the extracellular matrix.60–62 Both galectin 1 and galectin 3 are expressed by hatching and implanting blastocysts on the TE surface but not the ICM.63–65 They can bind Htype-1 epitope66 and so could act as TE receptors for it.28 This is supported by the promotion of attachment of blastocysts on LE cells cultured in medium containing galectin-1 protein (Blissett, Poirier, Ripoche Timmons, Bardsley and Kimber, submitted). Alternatively, galectins are major non-integrin laminin binding proteins67, 68 and might interact with LE or blastocyst laminin during implantation. For instance, it has been shown that galectin-1 can selectively modulate interaction between α7β1 integrin and laminin during skeletal muscle differentiation.69 α7 integrin mRNA is present from the blastocyst stage in mice,70 but the protein distribution is not known. α7β1 seems to be involved in later invasion of trophoblast (see below), interacting with laminin via its E8 domain. Mutant mice lacking either the galectin-1 or galectin3 gene, or both, are viable and fertile and embryos implant successfully.65, 71, 72 The double mutants tell us that redundancy between galectins 1 and 3 cannot account for the lack of phenotype in the absence of the single galectin. Blastocysts also express galectin5 in TE and ICM28, 72 . So, since several members of the galectin family are expressed by trophoblast at implantation, they may function interchangeably, thus safeguarding this critically important process. Heparan sulfate proteoglycan Heparan sulfate proteoglycan (HSPG) is another possible contender for initiating the implantation adhesion cascade since mouse embryos will attach to HSPG binding proteins isolated from uterine epithelial cells (reviewed in73 ). A basement membrane form of HSPG, perlecan, surrounds the murine blastocyst after hatching and expression of mRNA and protein 79

S. J. Kimber and C. Spanswick

correlates with acquisition of attachment competence.74, 75 It is not known if perlecan is expressed by hatched human blastocysts, but attachment of labelled JAR cells (human choriocarcinoma-derived trophoblast cell line) to RL95 monolayers (human endometrial adenocarcinoma line) occurs by an HSPG/heparin-dependent mechanism.76 RL-95 cells, which grow with a relatively unpolarized phenotype, are one of the few human epithelial cell lines that allow trophoblast adhesion at their apical surface.23, 77 They express heparin/HSPG-interacting protein (HIP), non-covalently associated with the external membrane. HIP supports HS-dependent attachment of JAR cells,78 so it could bind trophoblast HSPG which might act as a bridge between trophoblast and LE (see below and Figure 1). It binds perlecan79 suggesting a HIP–perlecan interaction might be involved in human trophoblast attachment. This is supported by strong HIP expression by human endometrial epithelium throughout the menstrual cycle.80 In the first trimester, strong HIP reactivity is found at points of villus cytotrophoblast attachment and cytotrophoblast invasion of endometrium where high perlecan is also present. It is enriched on cytotrophoblast which has penetrated maternal blood vessel walls indicating involvement in trophoblast invasion. Two pieces of evidence support this: firstly antibodies to HIP block JAR cells invasion into Matrigel but not attachment to Matrigel or perlecan. Secondly, in pre-eclampsia, infiltration of trophoblast through arterial walls is compromized and HIP expression is very low.79 HIP is expressed by murine LE81 and might act as a ligand in this species. However, heparin-binding EGF-like growth factor (HB-EGF), induced by the embryo in apical LE in the implantation chamber, also interacts with HSPG.82 N-CAM found on murine blastocyst83 can bind HSPG84, 85 as well and is expressed by cleaving human embryos30 (blastocysts were not examined). Both P- and L- but not E-selectin also interact with endothelial HS chains,86 an intriguing finding in the light of possible blastocyst selectin expression (above).

(Paria et al, this issue). Interaction of HB-EGF with either of these molecules might contribute to initial TE adhesion. HB-EGF also promotes trophoblast outgrowth in vitro, and zona-hatching indicating a multifunctional interaction. So its binding to blastocyst surface HSPG/EGF-R may act both directly in cell adhesion and secondarily, through signals transduced via ErbB4, to affect other trophoblast functions.89, 90 In human endometrium HB-EGF mRNA is highest just prior to the opening of the implantation window, and then declines. In the expected receptive phase the protein is expressed at the apical surface of the LE,91 as observed in mice adjacent to the embryo. So it could also act as attachment ligand in the human. CD-44 Other carbohydrate binding molecules such as CD-44 could potentially be involved in TE adhesion although the evidence is circumstantial. CD-44 consists of a family of alternatively spliced polymorphic membrane glycoproteins, involved in cell–cell and cell-matrix interactions. Unspecified isoforms are expressed both by pre-implantation human embryos (but not first trimester trophoblast) and endometrial epithelium.92, 93 CD-44 isoforms could form bridging ligands interacting with the abundant sialylated and sulfated carbohydrates on the apical surface of human LE.36, 94, 95 Alpha2-3 sialylated sugars also increase apically on murine LE at the time of implantation.31 Other potential CD-44 ligands are present both in endometrium and blastocyst95 (see below), osteopontin being arguably the most important.

Masking of adhesive ligands for the blastocyst Large glycosylated mucins, such as Muc-1, on the LE membrane have extensive core proteins and charged carbohydrate chains and could mask adhesive ligands95 as shown for cell lines.96, 97 In mice Muc-1 integral membrane protein in LE is down-regulated by progesterone at implantation98, 99 as similarly demonstrated in vitro for pigs100, 101 (Bowen and Burghardt, this issue). Steroidal control of Muc-1 is species dependent. In rabbits and primates although it is stimulated by progesterone, it is still selectively reduced adjacent to the implanting rabbit blastocyst and in baboon in LE but not glands.102, 103 Removal of its ectodomain by cleavage as occurs in vitro 104 might allow trans-binding between other adhesive molecules on TE and LE. Null mice are fertile, though suffer

Heparin binding EGF-like growth factor HB-EGF, a member of the epidermal growth factor family, binds to HSPG as well as EGF receptors:87 both are present on the hatching blastocysts74, 88 (Paria et al, this issue). The unprocessed transmembrane form of HB-EGF is induced specifically on LE at the implantation site and interacts with the ErbB4 EGF-receptor and HSPG on blastocyst TE82, 89, 90 80

Blastocyst implantation: the adhesion cascade

Continued interaction with LE

from reproductive tract infections. In human, MUC-1 is strongly expressed during the receptive phase, at the epithelial apical cell surface on microvilli and cilia and in uterine secretions105, 106 As well as having anti-infective properties, it may act as a selective barrier to prevent adhesion of substandard blastocysts to LE.95, 107 In this way it might contribute to the recognized greater success rate of implantation in mice compared with human, reflected in the higher frequency of pregnancy for mouse embryo transfer compared with human IVF replacement. However, human cleavage stage embryo replacement is generally to the uterus, a non-physiological environment for early embryos. Furthermore, the human embryos are a selected, potentially compromized, population. Interestingly, in patients with recurrent miscarriage MUC1 is less abundant, suggesting a reduction in the barrier, which might allow implantation of embryos with reduced developmental potential.106, 107 In women, carbohydrate structures such as keratan sulfate, associated with implantation success, are carried by MUC-1,94, 106, 108 as are potential selectin ligands like sialyl Le-x and sialyl Le-y.36 It is possible that special properties of human MUC-1 render it an initial adhesion molecule with carbohydrate groups functioning as tethering agents interacting with trophoblast sugar-binding molecules. At the same time it might sterically block interaction with other CAMs on substandard blastocysts which may lack lectin-type receptors. A specific ectodomain of MUC1, though highly expressed in most proliferative phase endometrial epithelium, is drastically reduced in mid-secretory phase,108, 109 but restored by keratinase or neuraminidase treatment. This suggests that specific glycosylation occurs at the expected time of implantation to mask the epitope and reinforces the possibility that lectin-like trophoblast molecules may interact first with temporally regulated sialylated or sulfated sugars expressed on large mucins. Recently another mucin, Muc-4, has also been shown to express on rat apical LE and to be steroidally regulated in the uterus but not other segments of the reproductive tract.11, 110 Expression disappears at receptivity in pregnant animals and was shown to be stimulated by estrogen, an effect antagonized by progesterone. Thus this mucin may also modulate interaction between TE and apical LE.

Once the embryo is tethered, possibly by a low binding-strength link, other molecules probably establish firmer contact with LE in preparation for its penetration. Some of the best candidates are integrin cell adhesion molecules and their ligands,95, 112 and the trophinin-tastin-bystin complex.113, 114 Trophinin-tastin-bystin complex Cell adhesion between a human trophoblast cell line and an endometrial adenocarcinoma cell line has been shown to occur through a unique intrinsic membrane protein, trophinin, but only when complexed to a cytoplasmic protein, tastin.113, 114 Trophinin and tastin do not bind directly to one another but both bind an intermediate protein bystin, also expressed by both cell lines.115 Functional complex formation is facilitated by interaction of these molecules with cytokeratins 8 and 18, suggesting the assembly of a true functional adhesion complex. Trophinin appears to mediate calcium-dependent homophilic binding and its structure, with its 69 serine/threonine-rich decapeptide repeats has been likened to a mucin.95 In mice, trophinin is specifically expressed by the blastocyst and uterus between 3.5 and 5.5 days post coitum. Null mice have been produced by homologous recombination in ES cells and early results indicate that the lack of trophinin is lethal around the implantation period. However, survival and birth of a proportion of trophinin null mice suggests that, as for other adhesion systems, there may be overlap of function and some compensation by other molecules. An alternative explanation is that maternal trophinin may persist and be used at the blastocyst stage.114 Parallel endometrial expression of trophinin and tastin occurs in human: they are absent in proliferative phase but appear at day 16/17, in early secretory phase, at the apical endometrial epithelial surface. Then they disappear rapidly during the mid/late secretory period and when implantation should occur they are apparently absent from epithelium but present in uterine mucus. However trophinin is present later at the utero-placental interface at 6–7 weeks of human pregnancy.114 Naturally the implantation site in a conception cycle has not been examined, but it would be interesting to know if this adhesion complex is retained in LE specifically adjacent to the blastocyst. Certainly, at the macaque implantation site trophinin expression is observed at the apposed apical surfaces of trophoblast and LE113 . Similarly it is present on the apical sur81

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face of rhesus monkey blastocyst TE, predominantly at the embryonic pole which first contacts the LE in primates.

available for adhesion of human embryos to LE (as in the mouse), as may other integrins (α3β1, α6β4, αvβ5 and theoretically αvβ1). A number of ligands for integrins are also expressed at or by the blastocyst stage. In the mouse these include fibronectin,123 laminin and entactin-1124, 125 and type-IV collagen126 but apart from laminin they are predominantly in the ICM and developing basement membrane of TE. In 8-cell + bovine embryos, fibronectin, and in human morulae laminin, is expressed at the cell surface.122, 127 Osteopontin is also expressed by human cytotrophoblast and mRNA levels are stimulated by progesterone.128 Laminin, HSPG and thrombospondin are detected around the external cell surface of the murine blastocyst.74, 125, 129 Laminin and osteropontin might be anchored by αvβ3, while thrombospondin can interact with both αvβ3 and HSPG core protein.130, 131 HSPG also has a number of other potential ligands on LE or trophoblast (above). Theoretically, any of these ECM molecules could act as a bridge, binding the TE to the luminal surface. However, fibronectin and vitronectin null mouse embryos implant normally132, 133 and these molecules are probably not present on the apical LE. The best candidates for a bridging function are osteopontin, embryonic laminin and HSPG. Osteopontin is also expressed by human endometrial epithelium, particularly apically and with highest levels in the secretory phase glands.134 If ECM components expressed by the embryo or LE are to act as bridging type molecules, the LE surface must carry appropriate receptors. A number of β1 integrins, α1β1, α2β1, α3β3, α5β1 and α6β1, have been detected on human endometrial epithelial cells and α1β1, α4β1, and α5β1 are also expressed by stromal cells.135–139 Most of these proteins show little menstrual cycle regulation with the exception of α1β1 and α4β1. Expression of α4β1 is highest in glandular epithelium between mid-proliferative and mid-secretory phase. However, α1β1 shows possible implantationphase-related changes: its expression is restricted to early- and mid-secretory phase in epithelium and in stroma is only expressed in the pre-decidual stage. At the mRNA level all integrins examined (α2, α3, α4, α5, α6, αv, β1, β2, β3 and β5) were shown to increase in the secretory phase-suggesting differential control of transcription and translation.140 αv integrin protein is expressed in the endometrium139, 141 and a potential β subunit partner, β5, is expressed by LE and stroma but not regulated at the protein level.141 Higher levels of integrin β6 protein in secretory phase LE have been reported.142 Another partner, β3, appears abruptly on

Integrins A number of α and β integrin subunits are expressed continuously from the fertilized murine egg through to the peri- and post-implantation period, including α5, α6B, αv, β1 and β3,70 while other integrins are regulated in the embryo. In particular α2, α7 and α6A mRNAs are detected from the fully expanded blastocyst stage and α3, α2 and α1 can be detected as protein from the stage of trophoblast outgrowth in vitro.70, 116, 117 Therefore, several β1 and αv integrin heterodimers may be expressed at the time of blastocyst attachment (Table 1) and αvβ3, α5β1 and α6β1 are present at the cell surface.70 Identified ligands for the different heterodimers are shown in Table 2. Of the blastocyst integrins, α5β1 and α6β1 integrins are not regulated and are expressed between ICM cells and on the cavity, rather than the external TE surface70 of early expanded blastocysts. This makes them unlikely candidates for adhesion to LE. However, as differentiation of the TE continues in culture, α5β1 does translocate to the apical surface of abembryonic TE which first contacts the uterus and generates the primary trophoblast118, 119 (see Cross, this issue). This translocation regulates fibronectin binding. Integrin translocation is regulated by ligation of the blastocyst calcitonin receptor and receptor-mediated Ca2+ signalling and is a prerequisite for attachment and outgrowth on fibronectin.118, 119 This exemplifies the crucially important signalling function of cell adhesion molecule engagement. In contrast, αvβ3 can be demonstrated on the external surface of TE and later, in vitro, in focal contacts of spreading trophoblast.120 It is therefore in a position to mediate initial adhesion and/or subsequent invasion. Integrin αvβ3 interacts with fibronectin, vitronectin, tenascin, osteopontin and thrombospondin and with a cryptic domain of laminin (Table 2). All are found in the trophoblast adhesion/invasion pathway. Furthermore, function-blocking antibodies to αvβ3 in utero have been reported to reduce the number of implantation sites,121 though a full publication has not been presented. Human pre-implantation embryos have also been shown to express a number of integrin subunits and at the blastocyst stage these include α3, αv, β1, β3 and β530, 120 (D Bloor and Kimber, in preparation). So far there is little evidence for developmental regulation at the protein level. However, trophoblast αvβ3 heterodimers may be 82

Blastocyst implantation: the adhesion cascade

Table 1.

Expression Of Adhesion Molecules Potentially Involved In Trophoblast Adhesion Cascade

Cell adhesion molecule α1β1 α2β1 α3β1 α3β3 α4β1 α5β1 α6β1 α6β4 α7β1 αvβ1 αvβ3 αvβ5 αIIβ3

Mouse Blastocyst

+ +

+ +

N-CAM

+

P-selectin L-selectin

+u

+

+?

↑ +r ↓

+ + + +? +? + +?

+

+

+→− −→+

+ +

+u

+ + +→− +r

+ +

+ +

+? +

+

Laminin Thrombospondin Osteopontin

↑

+? + +

+

+ + + +

Human endometrial epithelium

+ +

+

Galectin-1 Galectin-3 Galectin-5 Le-y H-type-1

1st trimester trophoblast

+

+

+

CD-44 Perlecan HIP Muc-1 HB-EGF

Human/primate Blastocyst

+

E-cadherin Cadherin-11

Trophinin/ tastin

Mouse Luminal epithelium

+p

+

+

+ +

+ + +

+ + +

+ +

+

+?

+ +

+

r: restricted expression; p: primate; ?: requires confirmation due to low numbers or weak staining or inferred integrin heterodimer; u: Stones and Kimber unpublished data; ↑: increasing expression on subset of cells; ↓: decreasing expression on subset of cells. For references refer to text. Modified from181 with permission.

null mice do not appear to have any reproductive defects suggesting that it does not play a major role. Some evidence of αvβ3 expression related to fertility comes from the absence of β3 as well as α1 and α4 in menopausal endometrium.143 Integrin β3 is also absent or expressed late in delayed endometrial dif-

the GE on day 19 of the menstrual cycle at the initiation of the window of receptivity for implantation, but slightly later on the LE.137, 138 Integrin αvβ3 has many potential ligands (Table 2). As well as those mentioned above it binds vitronectin which may be present on the trophoblast. However, vitronectin homozygous 83

S. J. Kimber and C. Spanswick

Table 2. α1β1 α2β1 α4β1 α6β1 α8β1 α9β1 αvβ1 αvβ3 αvβ5 αvβ6 αvβ8

Extracellular matrix ligands of β1- and αv-family integrins α3β1 α5β1 α7β1

COL COL

LM LM

PE FN

LM FN COL LM

FN FN FN FN

VN FB FB FB

VN VN VN

TN TN vWF vWF

TN

OST

TSP

PE

PEC

TN VN

Collagen, COL; laminin, LM; fibronectin, FN; fibrinogen, FB; vitronectin, VN; von Willebrand factor, vWF; tenascin, TN; osteopontin, OST; thrombospondin, TSP, perlecan, PE; Platelet endothelial cell adhesion molecule −1, PEC. Integrins known to recognize the RGD motif found in most ligands are underlined. Based on: Sugimori et al 1997; Haas and Plow, 1994; Craig et al 1995; Liaw et al 1995; Humphries, 1990. Modified from181 with permission.

ferentiation in cases of unexplained infertility144, 145 and β3 is lacking in endometriosis.146 Another ligand for αvβ3 is oncofetal fibronectin, expressed by human trophoblast.147 Most importantly for initial TE–LE interaction, αvβ3 is expressed apically in both mouse and human endometrial epithelium, as is another αv partner β5 which can also bind fibronectin and vitronectin.141 Although none of αv, β3 and β5 appear to be hormonally regulated in mouse LE141 their apical location would allow a role in strengthening the initial interaction between TE and LE. It is also possible that αv heterodimers are masked100 prior to the period of receptivity in mice.

outgrowth of trophoblast on single 2D extracellular matrix substrates for mouse, or invasion of human cytotrophoblast into 3D matrix substrates in vitro. This has been considered to resemble the threedimensional invasion in utero, but care must be taken in interpreting observations on single substrates and particularly in 2D systems. Cultured trophoblast may upregulate appropriate matrix receptors according to environment. In vitro, chondroitin sulfate proteoglycan appears to block interaction with collagen155 and could have an adhesion moderating effect in vivo. Mouse trophoblast invades endometrial ECM156 and human and primate blastocysts attach to and invade into EHS matrix157–159 and adhere to fibronectin and laminin160 Protease production, particularly of metalloproteases (MMPs), is an important moderator of invasion/outgrowth.161–163 One of these, MMP-2, also binds αvβ3 to facilitate directed cell migration.164 Trophoblast is likely to be adaptable and capable of reacting to and migrating on a number of substrates. This responsiveness is exemplified by the reaction to fibronectin. Murine blastocysts bind by their abembryonic pole to the cell binding fragment of fibronectin attached to microspheres and adhesion is inhibited by soluble fibronectin or antibodies to αv, α5, β1 or β3 integrin subunits. Interaction with fibronectin is required to induce translocation of integrin to the trophoblast cell surface thereby achieving adhesion.118, 165 As murine trophoblast outgrowth proceeds in vitro, α1, α6A and α7 integrin become trophoblast specific.71 In combination with β1 these integrins form laminin and collagen receptors (Table 2). They

Later events in adhesion–invasion Laminin and collagen-IV disappear from the LE basement membrane before trophoblast reaches the basal LE cell surface11, 12 so they are unavailable for trophoblast interaction here. However, decidualization leads to increased secretion by stromal cells of laminin, entactin, type-IV collagen and HSPG (all trophoblast substrates). ECM is rearranged into a pericellular matrix layer around the cells in human, or into patches in mice and there is decreased production of fibronectin by mouse decidua.107, 148–150 These molecules are available for adhesion and migration of trophoblast late in the adhesion cascade but also as a source of bound growth factor151, 152 and induction of differentiation.153, 154 Evidence that all these ECM components actually function in trophoblast interactions has come from adhesion and 84

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are expressed during invasion in vivo suggesting a switch to this group of integrins for this phase. Murine trophoblast adheres to and spreads on both the P1 fragment of laminin via its RGD sequence and the E8 fragment, independent of RGD and possibly via its IKVAV cell recognition domain known to bind trophoblast.70, 166 The P1 fragment, recognized by αvβ3, seemed to be cryptic in intact laminin and the E8 fragment is thought to facilitate trophoblast invasion in vivo.70 Antibodies against one E8 receptor, α6β1, failed to block outgrowth and in the absence of available antibodies against α7β1, the latter was predicted to be a major outgrowth receptor for mouse trophoblast on laminin. However, α7 null mice do not appear to have defective implantation167 though redundancy, involving other integrins is possible. Mice with a null deletion in the integrin α1 gene also implant and develop normally, as do those null for αv (a candidate for initial adhesion), though most die at mid-gestation from placental failure.168, 169 Other α subunits may compensate for loss of α1 or αv integrins at implantation. Galactosyl transferase expressed on secondary trophoblast (from ectoplacental cone) was reported to function in their migration on laminin in vitro 170 suggesting that carbohydrate chains of laminin may influence later invasion of trophoblast. These experiments also suggest that secondary trophoblast uses multiple interaction mechanisms during invasion. Anti-β1 integrin antibodies prevent mouse trophoblast adhesion and outgrowth on fibronectin116, 165 and adhesion of human cytotrophoblast to fibronectin or laminin.173 Outgrowth of mouse trophoblast on human decidual cells is also inhibited by anti-β1 treatment of decidual cells.171, 172 Involvement of this integrin in implantation is supported by the reduced trophoblast invasion in β1−/−mouse embryos.174 β1 integrin null trophoblast invades through LE but only poorly into decidua.174–176 The major defect has been ascribed to failure in the β1−/− ICM because trophoblast outgrowth on fibronectin and vitronectin appeared normal. Lack of ICM signals may account for the limited invasion. However, β1−/− trophoblast did not outgrow on laminin (a likely substrate in vivo) and anti-β1 antibodies block outgrowth of wild-type embryos on laminin. So β1 integrins may be involved in invasion of the laminin-enriched decidua. In the human the changing pattern of integrin subunits expressed by different populations of cytotrophoblast, at different states of differentiation and invasion, implies a complex sequence of interactions with different ECM molecules.158, 177, 178 The

phenomenon of evolving integrin expression during differentiation and invasion of the trophoblast, or integrin switching,158, 177 may reflect changes in available matrix in utero, but in vitro evidence suggests that it is pre-programmed. In utero, α6 integrin is restricted to cytotrophoblast stem cells and lost on invasion, while invasive, differentiating cytotrophoblast upregulates α5β1 and α1β1. The same pattern of changing human cytotrophoblast integrin expression is seen on cells invading EHS matrix.158 Antibody inhibition of α1β1 interaction with laminin and collagen-IV inhibited invasion while antibody to α6β1, the fibronectin receptor, accelerated invasion. This suggests the regulated counterbalancing of adhesive and migration-promoting machinery in invasive cytotrophoblast, driven by integrin cell-surface expression which controls the interactions between laminin/collagen and their receptors (adhesion) and fibronectin–integrin binding (migration). The lack of a normal cytotrophoblast integrin differentiation programme in pregnancies with pre-eclampsia179 where infiltration of the arterial system and endovascular remodelling is restricted180 supports this idea.

Conclusion A summary model for the adhesion cascade which drives implantation of the embryo is outlined in Figure 1. This is simplified to allow representation of a complex series of interactions. The major masking substances in the preceptive uterus are depicted as mucins (Muc-1) associated with the apical LE surface. However, these could equally be other membrane-bound mucins (Muc-4?) or molecules secreted into the uterine fluid which, by binding to the cell surface, mask blastocyst or LE receptors. Carbohydrate ligands on LE are shown as the initiators of attachment by their (low avidity?) tethering function. However, TE HSPG might be involved in interacting with HB-EGF on the LE, or possibly HIP, in this initial phase. Once tethered, the proximity of cell surface proteins facilitates firmer adhesion mediated by trophinin–trophinin trans-interaction or αv family integrins probably interacting with ECM components at the trophoblast–LE interface. To fulfil their function as bridging molecules ECM molecules will need at least two accessible (integrin?) binding sites, or they may bridge by forming complexes with other ECM components at the trophoblast–LE interface (see also Bowen and Burghardt, this issue). Integrin αvβ3 is a prime candidate for a functional 85

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Figure 1. Diagramatic representation of the series of interactions between the TE/trophoblast and LE and subjacent stroma. Potential roles of cell adhesion molecules at each stage are indicated. (1) Pre-receptive endometrium: desmosomes distributed along lateral LE cell surfaces and non-adhesive apical cell surface. (2) Receptive endometrium and initial embryo attachment: reorganization of lateral LE adhesion complexes accompanies apical carbohydrate ligand engagement to tether blastocyst, αv integrins now becoming available for binding. (3) Stabilization of initial attachment: αv integrin mediated adhesion involving bridging ligands shown but trophinin homophilic binding also probably functional. (4) Potential signalling through cell adhesion ligand receptor interaction. (5) Penetration of LE and interaction with extracellular matrix via β1 integrins (basement matrix degraded). (6) Invasion into stroma: continuing β1 integrin activity and trophoblast signalling. Modified from181 with permission.

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integrin receptor on both trophoblast and apical LE. This is particularly because its ligands are found at this interface in mouse and human (laminin, osteopontin) and it shows appropriate cyclic regulation in human LE. The absence of an implantation phenotype in the αv knock-out embryo precludes a unique function. However, αvβ5 might also promote adhesion by interaction with human trophoblast oncofetal fibronectin, or vitronectin. The relaxation of polarity in the LE during the window of receptivity suggests that appropriate ECM substrates become available on the lateral aspect of LE cells to act as substrates for trophoblast invasion. The clearance of laminin and type-IV collagen from the LE basement membrane in mice would allow direct interactions with stromal matrix components from the point that trophoblast leaves the lateral surface of LE. Finally, interaction with decidual matrix appears to be driven by β1 integrins in human with the up-regulation of cytotrophoblast α5β1 and α1β1. β1 integrins similarly become important from an early stage in stromal invasion in mice. The search for molecules that mediate the stages of implantation has identified several strong candidates. However, the scarcity of implantation phenotypes observed after gene deletion of adhesion molecules either individually or as serveral members of a family, emphasizes that in most cases different molecular mechanisms work in parallel to drive trophectoderm adhesion and invasion allowing compensation when one mechanism fails. Following initial TE–LE adhesion there are important species differences in detailed cellular architecture of implantation and placentation (see, e.g. Bowen and Burghardt, this issue) indicating that various anatomical strategies have been successful during evolution in establishing a functional transporting unit between fetus and mother. The lack of molecular systems that are specific to particular implantation functions (the trophinin complex may prove to be one exception), suggests a relatively weak evolutionary drive to eliminate surplus mechanisms. This may help to account for the observed redundancy, which confers the added evolutionary benefit of securing implantation success in the presence of random mutations affecting the function of individual components.

by the Wellcome Trust, the BBSRC and a MRC studentship to CS.

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