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Adhesion molecules and implantation
- Implantation mechanisms: insights from the sheep


The external surface of the trophoblast and endometrial LE cells is composed of a glycoprotein coat, or glycocalyx. Biochemical changes occur in the composition or distribution of the trophoblastic glycocalyx during the process of blastocyst attachment in the ewe (Guillomot et al. 1982), but little information is available on the relative glycoprotein composition of the apical membrane of either the trophectoderm or endometrial LE. Recently, several candidate adhesion factors that mediate blastocyst implantation under the influence of progesterone have emerged in the sheep. As the hormone of pregnancy, progesterone plays a pivotal and indisputable role in the establishment and maintenance of pregnancy in mammals. In a number of mammalian uteri, progesterone receptors (PR) are expressed in the endometrial epithelia and stroma during the early to midluteal phase, allowing direct regulation of a number of genes by progesterone via activation of the PR. However, continuous exposure of the endometrium to progesterone negatively regulates PR expression in the endometrial epithelium. Expression of PR protein is not detectable in endometrial LE and GE in sheep after days 11 and 13 of pregnancy respectively (Spencer & Bazer 1995). Furthermore, PR expression is detected only in the endometrial stroma and myometrium throughout most of gestation in the ovine uterus (Spencer et al. 2004). The paradigm of loss of PR in uterine epithelia immediately before implantation is common across mammals (Carson et al. 2000, Spencer et al. 2004). Thus, regulation of endometrial epithelial function during the peri-implantation period may be dependent on loss of epithelial cell PR and/or be directed by specific factors produced by PR-positive stromal cells (Spencer & Bazer 2002, Spencer et al. 2004). The loss of the PR by the endometrial epithelium can be directly correlated with reduced expression of certain genes, such as the antiadhesive protein MUC1. Furthermore, PR loss in the endometrial GE appears to be required for the onset of expression of other genes during pregnancy, such as galectin-15, osteopontin and ovine uterine serpins (or uterine milk proteins) (Spencer et al. 2004), several of which are discussed further below.

As the blastocyst approaches the endometrial LE, it encounters the glycocalyx. One component of the glycocalyx is MUC1, a large, transmembrane mucin glycoprotein expressed at the apical surface of a variety of reproductive tract epithelia (Brayman et al. 2004). MUC1 is particularly abundant on the microvilli and cilia that extend from the apical cell surface of the endometrial LE. The extracellular domain of MUC1 contains a very large amount of glycans (Aplin & Hey 1995). In fact, the core protein is 120–220 kDa, but with glycosylation it can be over 400 kDa. In both humans and rodents, the expression pattern of the glycoproteins MUC1 and MUC4 on uterine LE may control the accessibility of trophoblast integrin receptors to their ligands by sterically blocking cell–cell and cell–extracellular matrix (ECM) adhesion and access of conceptus trophoblast to uterine LE, due to their extensive glycosylation and extended extracellular structure (Carson et al. 2000, Burghardt et al. 2002). The implantation adhesion cascade in sheep is initiated after down-regulation of MUC1, and this is coincident with loss of PR from uterine epithelium (Johnson et al. 2001). Immunoreactive MUC1 expression by LE decreases at days 9–17 of early pregnancy in normal (Johnson et al. 2001) and UGKO (Gray et al. 2002) ewes. This pattern of MUC1 expression contrasts with that in rabbits and humans, in which there is an overall increase in MUC1 expression during the receptive phase under the influence of progesterone; however, MUC1 is locally reduced at implantation sites, via the activity of cell-surface proteases that are triggered by the blastocyst or mediated by paracrine signals from blastocysts (Carson et al. 2000, Brayman et al. 2004). Regardless of the mechanisms by which MUC1 is downregulated, removal of this antiadhesive barrier is hypothesized to be necessary to expose other glycoproteins involved in the adhesion between trophoblast and LE. Given that the mucins contain a large number of glycans that can be potentially recognized by the blastocyst or secreted animal lectins, they may also be involved in the apposition phase of implantation (Aplin & Hey 1995, Brayman et al. 2004).

Glycosylated cell adhesion molecule 1 (GlyCAM-1)
GlyCAM-1 is a sulfated glycoprotein secreted by the endothelium that mediates leukocyte–endothelial cell adhesion (Lasky et al. 1992). GlyCAM-1 is a member of the mucin family of glycoproteins, with approximately 70% of the native molecular mass composed of O-linked carbohydrates found in two serine/threonine-rich domains (Rosen 1993). This mucin glycoprotein is expressed predominantly at the luminal surface of high endothelial venules of peripheral and mesenteric lymph nodes. As illustrated in Fig. 3Go, GlyCAM-1 functions as a carbohydrate ligand for the lectin domain of leukocyte cell-surface selectin (L-selectin) in the lymphoid system (Rosen 1993). Ligation of L-selectin by GlyCAM-1 activates ß1 and ß2 integrins and promotes firm adhesion to fibronectin (Hwang et al. 1996, Giblin et al. 1997). In humans, trophoblast L-selectin appears to mediate interactions with the uterine epithelium that may be critical to establishing human pregnancy (Genbacev et al. 2003). The temporal and spatial patterns of GlyCAM-1 in the uterus of cyclic and pregnant ewes implicate GlyCAM-1 as a potential regulator of implantation (Spencer et al. 1999a). In cyclic ewes, GlyCAM-1 expression increases in the endometrial LE and superficial GE between days 1 and 5 and then decreases between days 11 and 15. In pregnant ewes, GlyCAM-1 in the LE and superficial ductal GE is low on days 11 and 13, increases on day 15 and is abundant on days 17 and 19. Immunoreactive GlyCAM-1 is also detected in the conceptus trophoblast on days 13–19. In pregnant ewes, the relative amount of immunoreactive GlyCAM-1 in uterine flushings is low on days 11 and 13, but abundant on days 15 and 17. Thus, a GlyCAM-1-like protein may be a secretory product of the endometrial epithelium and/or conceptus trophoblast. Patterns of distribution observed for immunoreactive GlyCAM-1-like protein in the endometrial epithelium, combined with proposed functions for lymphoid GlyCAM-1, suggest that this mucin glycoprotein may be involved in conceptus–maternal interactions during the peri-implantation period of pregnancy in sheep.

Galectins are proteins with a conserved carbohydrate recognition domain (CRD) that bind ß-galactosides, thereby cross-linking glycoproteins as well as glycolipid receptors on the surface of cells and initiating biologic responses (Cooper 2002, Yang & Liu 2003) (Fig. 3Go). Recently, a new galectin family member, galectin-15, was discovered in the endometrium of sheep by gene expression profiling to investigate the peri-implantation pregnancy defect in UGKO ewes (Gray et al. 2004). Ovine endometrial galectin-15 contains a conserved carbohydrate recognition domain (CRD) that binds ß-galactosides, but the carbohydrate-binding specificity for each galectin appears to be different (Cho & Cummings 1995). In addition to a conserved CRD, galectin-15 also contains predicted cell attachment sequences (LDV and RGD) that could mediate binding to integrins in ECM proteins (Kimber & Spanswick 2000, Wang & Armant 2002). Other galectins bind integrins as well as fibronectin, laminin and MUC16/CA-125, because these proteins are modified with ß-galactoside sugars (Cooper 2002, Seelenmeyer et al. 2003, Yang & Liu 2003). Functional studies of other galectins have implicated these proteins in cell growth, differentiation and apoptosis as well as in cell adhesion, chemoattraction and migration (Barondes et al. 1994, Hughes 2001, Yang & Liu 2003).

In the ovine uterus, galectin-15 mRNA was detected only in the endometrial LE and superficial ductal GE (Gray et al. 2004), which are the primary sites of blastocyst apposition and adhesion. In endometria of cyclic and pregnant ewes, galectin-15 mRNA was not detected before day 10, appeared and then increased 13-fold between days 10 and 14, and then noticeably decreased between days 14 and 16 in cyclic, but not pregnant, ewes. Immunoreactive galectin-15 protein was concentrated near and on the apical surface of the luminal and superficial ductal epithelia and localized within discrete cytoplasmic structures of conceptus trophectoderm. In uterine flushings, galectin-15 was present at low levels on days 10 and 12, but was abundant on days 14 and 16 of pregnancy. Progesterone induced and IFN{tau} increased galectin-15 mRNA in the endometrium. Thus, galectin-15 and Wnt7a are the only genes currently known to be increased by IFN{tau} in the endometrial LE of the ovine uterus (Choi et al. 2001, Kim et al. 2003, Gray et al. 2004).

The temporal and spatial alterations in galectin-15 mRNA and protein in endometrial LE and lumen of the ovine uterus during pregnancy, combined with the functional aspects of galectin-15 and its family members, make it a strong candidate for a mediator of conceptus–endometrial interactions during implantation. Therefore, the proposed extracellular role of galectin-15 in the uterine lumen is functionally to bind and cross-link ß-galacto-sides on glycoproteins, such as mucins, integrins, fibronectin, laminin and other glycoproteins and glycolipids, thereby allowing it to function as a heterophilic cell adhesion molecule bridging the blastocyst and the endometrial LE. The biologic responses of the trophoblast to galectin-15 may also include migration, proliferation and differentiation, which are critical for successful conceptus implantation.

Interestingly, galectin-15 appears to be the 14K protein from sheep endometrium initially characterized as a progesterone-modulated protein associated with crystalline inclusion bodies in uterine epithelia and conceptus trophoblast (Kazemi et al. 1990). The 14K protein was originally identified as a component of conceptus-conditioned culture medium and uterine flushes (Salamonsen et al. 1984). Release of the 14K protein was attributed to the cellular breakdown of conceptuses in culture (Kazemi et al. 1990). Immunogold electron microscopy revealed that within trophoblast, the 14K protein was localized to large, membrane-bound rhomboidal or needle-shaped crystal structures. Thus, it was suggested that the protein was secreted by the endometrial epithelia, taken up by the conceptus from uterine histotroph, and deposited as crystals (Kazemi et al. 1990). These crystals are first observed in the sheep trophoblast on day 10 and then increase in number and size between days 10 and 18 of pregnancy (Wintenberger-Torres & Flechon 1974). Indeed, the crystals exhibit a lattice periodicity of about 20 nm in day-14 blastocysts. Similar progesterone-induced crystal proteins are present in endometrium and conceptus trophoblast of many mammals, including rabbit, mouse, pig and human (Nakao et al. 1971, Calarco & Szollosi 1973, Daniel & Chilton 1978, Daniel & Kennedy 1978, Hoffman & Olson 1984, Hernandez & Baum 2002). However, the crystals are generally absent in blastocysts derived in vitro or in parthenotes (Daniel & Kennedy 1978, Talbot et al. 2000). Accordingly, galectin family members are likely to be expressed in the endometrium of many mammals to facilitate conceptus–endometrial interactions. Although the biologic role of galectin-15 crystals in the conceptus is not known, the intracellular roles of other galectins include modulation of cell growth, differentiation and apoptosis through functioning as pre-mRNA splicing factors and interacting with specific intracellular ligands such as Ras and Bcl-2 (Hernandez & Baum 2002, Liu et al. 2002).

Integrins comprise a family of heterodimeric intrinsic transmembrane glycoprotein receptors that mediate cellular differentiation, motility and adhesion (Giancotti & Ruoslahti 1999). They play a dominant role in interactions with ECM to transduce cellular signals in uterine epithelial cells and conceptus trophoblast (Johnson et al. 2001, Burghardt et al. 2002, Johnson et al. 2003a). The central role of integrins in the implantation adhesion cascade is to bind ECM ligand(s) to cause cytoskeletal reorganization, stabilize adhesion, and mediate cell migration, proliferation and differentiation through numerous signaling intermediates (Giancotti & Ruoslahti 1999). Altered expression of integrins is correlated with several causes of infertility (Lessey 1998), null mutations of several integrins leads to peri-implantation lethality (Hynes 1996), and functional blockade of selected integrins reduces the number of implantation sites (Illera et al. 2000). During the peri-implantation period of pregnancy in ewes, integrin subunits {alpha} (v, 4, 5) and ß (1, 3 and 5) are constitutively expressed on the apical surfaces of both conceptus trophoblast and endometrial LE (Johnson et al. 2001). These integrin subunits are detected at the apical surfaces of the LE and GE and on conceptus trophoblast; expression of these integrins is constitutive and not influenced y pregnancy or presence of the conceptus. In the sheep, receptivity to implantation does not appear to involve changes in either temporal or spatial patterns of integrin expression, but may depend on expression of other glycoproteins and ECM proteins, such as galectin-15, OPN and fibronectin, which are ligands for heterodimers of these integrins (Johnson et al. 2003a, Gray et al. 2004). In species such as pig, mouse and humans, interactions between specific integrins and ECM proteins frame the putative window of implantation (Carson et al. 2000, Burghardt et al. 2002, Lessey 2002).

Osteopontin (OPN)
OPN is a member of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family of genetically related ECM proteins recognized as key players in a number of diverse processes such as bone mineralization, cancer metastasis, cell-mediated immune responses, inflammation, angiogenesis and cell survival (Sodek et al. 2000, Johnson et al. 2003a). OPN has also been linked to pregnancy (Johnson et al. 2003a). Microarray profiling identified OPN as the most highly upregulated ECM adhesion molecule in human endometrium that is receptive to implantation (Carson et al. 2002, Kao et al. 2002). Multiple integrin receptors for OPN are present on trophoblasts and LE of humans and domestic animals, some of which increase during the peri-implantation period (Lessey et al. 1994, Bowen et al. 1997, Johnson et al. 2001). Ovine and porcine trophoblast and LE cells show evidence of integrin receptor activation and cytoskeletal reorganization in response to OPN binding in vitro (Johnson et al. 2001, Garlow et al. 2002), and polymerized OPN has high tensile strength when simultaneously binding receptors on different cells during adhesion and matrix assembly (Goldsmith et al. 2002). Finally, disruption of the OPN gene in OPN-null and OPN heterozygote mice decreases reproductive success at midgestation, and OPN-null embryos are significantly smaller than wild-type counterparts at term (Weintraub et al. 2004).

OPN has been detected in epithelia and in secretions of many tissues, including the uterus (Johnson et al. 2003a). OPN binds to integrin heterodimers ({alpha}vß1, {alpha}vß3, {alpha}vß5, {alpha}vß6, {alpha}vß8, {alpha}4ß1, {alpha}5ß1 and {alpha}8ß1) via its Arg-Gly-Asp (RGD) sequence, and to {alpha}4ß1 and {alpha}9ß1 by other sequences to promote cell adhesion, spreading and migration (Fig. 3Go). In sheep, OPN is also a component of histotroph secreted from endometrial GE into the uterine lumen during pregnancy. During the peri-implantation period of pregnancy in sheep, OPN mRNA is expressed only by the endometrial glands, is first detected in some glands of some ewes by day 13, and is present in all glands by day 19 (Johnson et al. 1999b). Progesterone induces expression of OPN in the endometrial glands, and this induction is associated with a loss of PR in the GE (Spencer et al. 1999b, Johnson et al. 2000). The 45 kDa form of OPN is present in greater amounts in uterine flushings from pregnant than cyclic ewes (Johnson et al. 1999a, 2001). The 45 kDa fragment of OPN has greater binding affinity for {alpha}vß3 integrin than the native 70 kDa form (Senger et al. 1996). Evidence suggests that secreted OPN binds integrin receptors expressed on conceptus trophoblast and endometrial LE, where it can stimulate changes in proliferation, migration, survival, adhesion and remodeling of the conceptus as it elongates, apposes and adheres to the LE. OPN is hypothesized to serve as a bifunctional bridging ligand that mediates the adhesion between LE and trophoblast essential for implantation and placentation (Johnson et al. 1999a, 2003a,Johnson et al. b).

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