The factors contributing to a fertile endometrium receptive to the presenting embryo remain unclear despite continual advances in the field of reproductive medicine1. In most animals, it is thought that the endometrium undergoes a series of changes in response to ovarian steroids leading to a period called the window of implantation2,3. Prior to and subsequent to this phase, the uterus is refractory to embryo attachment. In humans, the window of implantation starts approximately six days after ovulation (Day 20) and usually lasts for four days4,5. Associated with the onset of receptivity is the appearance of transient (over 24–48 hours) apical epithelial cellular protrusions called pinopodes6. These coincide with an increase in epithelial secretory activity and dilatation of the glands, prominence of epithelial subnuclear vacuoles and cessation of mitotic activity in the epithelial cells7. Coincident with the conversion of the endometrial surface from a non-receptive to a receptive state are alterations in the expression of cell surface molecules. The MUC1 mucin is thought to have a pivotal role in embryo attachment and implantation, and is likely to have an impact upon the expression and function of other adhesion molecules, such as the integrins, cadherins (CADs) and CD44.
Mucins are glycoproteins with a high carbohydrate content (exceeding 50% by weight) and a threonine/serine rich peptide core serving as a scaffold for the addition of simple and mainly acidic oligosaccharides8–11. Fourteen human mucins have been cloned and partially characterised to date12,13. Most of the currently known MUC species can be categorised as: membrane bound (MUC1, MUC3, MUC4, MUC12 and MUC13) or secretory (MUC2, MUC5AC, MUC5B, MUC6 and MUC7). Only MUC1 mucin, and to a lesser extent MUC6, have been demonstrated in the human endometrium14,15.
The structure of the MUC1 gene became accessible by the cloning and sequencing of full-length cDNA derived from different human carcinoma cell lines16–20. The gene is located on chromosome 1q21–24 and spans between 4 and 7 kilobases of genomic DNA, depending on the size of the variable number tandem repeat region (VNTR) of 60 base pair units. Four regions can be identified spanning seven exons: the N-terminal region containing a hydrophobic signal sequence, the VNTR (on exon 2), a C-terminal region consisting of a transmembrane sequence and a sequence encoding the cytoplasmic tail (Fig. 1).
Three isoforms of the MUC1 mucin have been shown to be generated by alternative splicing of the primary MUC1 transcript: MUC1, MUC1/Y and MUC1/SEC21. MUC1 is a 200 to 500 kDa protein which contains the VNTR domain, flanked by short regions containing degenerate repeat peptides, the transmembrane and the cytoplasmic domain. The MUC1/Y isoform, is devoid of the VNTR, spans the cell membrane and serves as a binding partner for the secreted MUC1/SEC, which contains a tandem repeat array but no transmembrane domain21,22. Post-translationally, the MUC1 core protein is modified by extensive glycosylation, particularly within the VNTR domain, which carries five potential O-glycosylation sites per repeat (three threonines, two serines)19. Several rounds of recycling are necessary to fully sialylate the mucin23. Accordingly, membrane-associated MUC1 comprises a mixture of glycoforms.
MUC1 protein is normally expressed on the apical surfaces of epithelial cells in the mammary and salivary glands, oesophagus, stomach, pancreas, bile ducts and the respiratory and urogenital tracts24,25. It behaves both as an anti-adhesion molecule, acting by steric hindrance preventing the formation of cell–cell contacts mediated by E-CAD, and as an adhesion molecule binding β-catenin and presenting carbohydrates as ligands for selectin-like molecules26–29. Putative functions for MUC1 include lubrication, cell protection, cell signalling and cell adhesion, as well as an important role in tumour progression and metastasis10,24,25,28–40. Differences between normal and cancer-associated mucin have been attributed to differences in glycosylation, as well as changes in cell membrane distribution41. In breast cancer, for example, instead of long polylactosamine-type side chains, there is a predominance of short sialylated oligosaccharides42,43.
Studies in humans and other animals have strongly suggested that endometrial MUC1 mucin expression has an important role in embryo implantation2,3,45,44. In the mouse, Muc-1 is hormonally regulated with endometrial expression being reduced during the receptive phase suggesting that loss of this glycoprotein may be important for embryo interaction with the epithelial layer46,47. Moreover, it has been shown that uterine epithelial cells isolated from Muc-1 null mice have an increased capacity to bind embryos compared with their wild-type counterparts48,49. Selective enzymatic removal of mucins from the apical surface of wild-type uterine epithelium has a similar effect and cells transfected with Muc-1 bind to mouse blastocysts much less efficiently than those that are Muc-1 null49. Nonetheless, there are species differences in MUC1 expression during the receptive phase of implantation. In the rabbit and pig, MUC1 mucin expression actually increases during the receptive phase and MUC1 is only reduced at the site of blastocyst attachment50,51. Similar to the rabbit and pig, MUC1 mucin is expressed in human glandular and luminal endometrial epithelium throughout the menstrual cycle with maximal expression during the implantation window52. The amount of MUC1 mRNA increases in endometrial tissue from the proliferative to the secretory phase. However, this has not been evaluated in well defined human endometrial samples nor correlated with pregnancy outcome. Besides, although full-length MUC1 appears to remain at the surface of receptive luminal epithelium, the glycoform pattern of MUC1 differs from that of the proliferative phase53–55. Localised alterations in the pattern of uterine MUC1 glycan expression, as a result of variations in the size or number of oligosaccharide chains on the core molecule, could potentially allow the endometrium to become selectively adhesive to the presenting embryo56. Alternatively, it has been suggested that the embryo itself induces paracrine changes in MUC1 expression at the implantation site57. Furthermore, two important differences distinguish the mouse MUC1 homologue from its human counterpart. The mouse Muc-1 mucin accounts for less than 10% of the total complement of mucins found in mouse uterine epithelia, and structurally, the mouse and human MUC1 genes differ due to the size of the VNTR which carries most of the O-glycosylation sites. The mouse sequence contains 16 repeats, whereas in the human, this varies from 20 to 12558,59. Highly polymorphic genetic variation is therefore a characteristic of the human MUC1 gene and the size of the MUC1 VNTR has been recently suggested to determine whether successful implantation occurs60.
The integrins are a large family of transmembrane glycoproteins expressed on all cell types that are involved in cell–cell and cell–extracellular matrix (ECM) interactions61–64. They are heterodimers consisting of non-covalently associated different α- (120–180 kDa) and β-subunits (90–110 kDa), and there are in excess of 20 recognised functional heterodimers. Each integrin subunit has a large extracellular domain, a single membrane spanning region and a short cytoplasmic domain65 (Fig. 2). The αβ associations determine the binding specificities of the integrin heterodimers for their various ligands. In general, members of the β2 subfamily are mostly involved in cell–cell adhesion, whereas integrins in the β1 and β3 subfamilies have been found to mediate cell adhesion to ECM components66. An individual integrin commonly recognises several distinct ECM proteins although some, such as the fibronectin receptor α5β1, only interact with a single protein66,67. Often, cells express multiple integrins capable of interacting with a particular ECM protein60,67. Thus, many integrins are not specific for individual ligands. This may seem paradoxical but the integrins do not bind to their ligand unless they have been activated to undergo a conformational change.
In relation to the endometrial surface, the integrins are essential to the function of the continuous polarised epithelial layer68. In addition, the discovery that specific integrin molecules are expressed by different endometrial components during the proliferative and luteal phases of the menstrual cycle has also led to speculation that integrins may participate in embryo attachment and implantation69–71. At least 14 integrin subunits are found in the human endometrium. In particular, the coexpression of three integrins, α1β1, α4β1 and αvβ3 is associated with the window of implantation to the presenting embryo72–75. The α1β1 integrin is expressed on glandular epithelium between Days 15 and 28 of the menstrual cycle, and the α4β1 integrin appears on the glandular epithelium for the first time just after ovulation, disappearing on Day 24. Yet, as these two integrins are expressed in the glandular epithelium and not the luminal epithelium, it is unlikely that they are directly involved in blastocyst adhesion. The αvβ3 integrin is, however, expressed on the luminal epithelium, with maximum representation on Day 24, coinciding with the rise in progesterone. The αvβ3 integrin is capable of binding to a wide variety of extracellular components, including vitronectin, fibronectin, oncofetal fibronectin and osteopontin, and may facilitate endometrial–embryonic attachment via a common bridging ligand. Oncofetal fibronectin is present on the invading human trophoblast and osteopontin is present on the uterine epithelium76. It has thus been postulated that this integrin acts as a tether between the two cell types. Furthermore, recent studies demonstrate that individual human blastocysts up-regulate β3 expression in cultured endometrial epithelial cells, suggesting that the embryo may regulate its own ability to implant77.
Integrin expression in the mouse uterus as a function of the stage of the reproductive cycle has not been studied, yet information has been derived from ‘knockout’ mice in which 19 of the known 26 integrin subunits have been deleted78,79. Deletion of the β1, αv and α4 subunits resulted in embryonic death by Days 5, 10 and 11 respectively, but the blastocyst was still able to attach to the endometrium prior to embryonic death78,80–83. Interestingly, loss of β3 does not affect viability, possibly due to the resulting up-regulation of β1 which may act as a substitute.
Cadherins are a rapidly expanding family of calcium-dependent adhesion molecules (CAMs)84–88. At least 80 members of the CAD superfamily have been shown to be expressed within a single mammalian species89. The classical CADs, such as E- (epithelial) and P- (placental) CAD, are integral membrane glycoproteins, between 723 and 748 amino acids long, which generally promote cell adhesion through homophilic interactions. They have been shown to regulate epithelial, endothelial and neural cell adhesion, with different CADs expressed on different cell types84. The structures of the CADs are similar, being composed of two to five extracellular repeats of approximately 110 amino acids (EC1–EC5), a single hydrophobic domain that traverses the plasma membrane and two cytoplasmic domains. The calcium binding motifs, DXNDN, DXD and LDRE, are interspersed throughout the extracellular domains (Fig. 3). The first extracellular domain (EC1) contains the classical cadherin adhesion recognition (CAR) sequence, His–Ala–Val (HAV). Linear synthetic peptides containing the CAR sequence, such as FHLRAHAVDINGNQV and LRAHAVDING, and antibodies directed against the CAR sequence have been shown to inhibit CAD-dependent processes84,90–93. Atypical CADs, such as K- (kidney) and OB- (osteoblast) CADs, are distinguished from classical CADs by not possessing the CAR sequence, but still possessing the characteristic calcium binding repeats94. Despite the variability of the extracellular domain between different CADs, all the members of this family possess a highly conserved cytoplasmic domain which functions as a binding site for catenins mediating binding to the cytoskeleton95. Cleavage of the molecule by metalloproteinases may result in the withdrawal of this non-specific supporting mechanism, leading the cell to apoptosis93.
Atypical and classical CADs have been identified in the human endometrium. K-CAD is strongly expressed in the glandular epithelium during the proliferative phase and decreases after ovulation96. OB-CAD expression in the glandular epithelium remains unchanged throughout the menstrual cycle, yet interestingly, its expression in the stromal cells appears to be inversely proportional to that of K-CAD96. E- and P-CAD, which promote calcium-dependent cell–cell adhesion, are found in the luminal and glandular endometrial epithelium throughout the menstrual cycle94,97,98. E-CAD is up-regulated by progesterone and is found on the trophoblast and it has thus been suggested that it may be involved in the initial attachment of the embryo99,100. E-CAD also appears to have an important role in the mouse as transgenic mice lacking the gene fail to form trophectoderm and to implant101. However, P-CAD has not been shown to be regulated by progesterone and is not present on human trophoblasts102. As a result, the exact function of P-CAD in the human remains unclear. Similar to the human, the precise involvement of P-CAD in the mouse is controversial as the uterus is unaffected in P-CAD null mice103,104.
CD44 is a transmembrane protein with many isoforms that are expressed in a cell-specific manner and differentially glycosylated105–108. The CD44 gene consists of 20 exons, only 10 of which are normally expressed, encoding standard form CD44. The additional 10 exons, encoding extracellular regions, are only expressed by alternative splicing of the nuclear RNA. Theoretically, the number of different exon combinations could exceed 100, but only about 30 isoforms (denoted CD44v_) have been identified to date106. Whereas CD44s is quite widely distributed in normal tissues, CD44 isoforms are found predominantly in tumour cells109. The major ligand for CD44 is hyaluronan, but it also binds to fibronectin, collagen and sulphated proteoglycans. Although specific ligands have not been identified for the variant region, it is remarkably hydrophobic and could thus mediate additional binding properties.
In rabbits, CD44 appears to be expressed most strongly in the luminal uterine epithelium during the pre-implantation phase, and its expression is thought to be triggered by embryonic signalling110. In humans, CD44 and its isoforms are expressed in the endometrial epithelium and in stromal cells both during the cycle and in pregnancy111. In the proliferative phase, CD44s is sporadically found on the endometrial glandular cells but is more prevalent in the stroma, particularly on infiltrating lymphocytes112. In the secretory phase, CD44s expression is much stronger in the glandular epithelium but is unchanged in the stroma. The endometrium also expresses some of the larger isoforms that arise through alternative splicing (such as CD44v3, v6, v8–v10) and some of these are thought to give rise to mature glycoproteins that contribute to the endometrial glycocalyx in the secretory phase113,114. It has further been suggested that the function of CD44 on the endometrium epithelium is to interact with chondroitin sulphate-bearing proteoglycans expressed on the embryo during the early stages of implantation, but this seems unlikely as CD44 has never been demonstrated on the luminal cell surfaces.
CEACAM1 (CD66a, BGP, C-CAM) is an adhesion molecule belonging to the carcinoembryonic antigen (CEA) family. It is the human homologue of the adhesion molecule cell-CAM (C-CAM) of the rat and it has been suggested to function as a ligand for E-selectin115–117. In contrast to most of the genes of the CEA family, the CEACAM1 gene codes for cytoplasmic domain containing sequence motifs involved in signal transduction, which has been shown to associate with pp60 c-src9118–120. It has been shown to be normally expressed at the apical pole of most epithelial cells, including the apical pole of the endometrial surface and glandular epithelia121. To date, there have been no studies determining cyclical variations in CEACAM1. However, on the maternal side of the maternal–fetal interface, CEACAM1 has been demonstrated on epithelial cells of pregnancy endometrium as well as in small endometrial vessels, while it is absent from decidual cells122. On the fetal side, CEACAM1 is strongly expressed by the extravillous (intermediate) trophoblast at the implantation site, as well as by extravillous trophoblast cells with invasive phenotype in primary culture122. Given its specific expression pattern, CEACAM1 can be a useful marker for extravillous intermediate trophoblast and might be functionally implicated in mediating trophoblast/endometrial interactions.