Hypoxia and human placental development.
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DNA-Binding Proteins↗
Biomedical subjects
J D Aplin
Publications and source records attributed to J D Aplin.
Tissue interactions in the control of trophoblast invasion.
On the basis of observations made in an explant model of anchoring villus development, it is proposed that two discrete types of signal are required to trigger the entry of cytotrophoblast into the migratory extravillous lineage. The first is mediated by contact of the tip of the first trimester mesenchymal villus with decidual extracellular matrix, in response to which stem villous cytotrophoblast undergoes a proliferative burst and differentiates into cells of the cytotrophoblast column, anchoring peripheral villi to the uterus. Column stability is dependent on the interaction between extracellular fibronectin and integrin alpha 5 beta 1 expressed at the cell surface, both of which are upregulated in this pathway. Subsequently, cells detach in large numbers from the periphery of the column and become migratory infiltrative cells. This step is dependent on paracrine signalling from the mesenchymal cells that lie directly beneath the villous basement membrane. As the signal decreases with increasing distance from the placental anchoring villus, this mechanism might account for the fact that trophoblast invasion progresses no deeper than the inner myometrium.
Cell Adhesion Molecules↗
Studies of mesenchymal cells from 1st trimester human placenta: expression of cytokeratin outside the trophoblast lineage.
A fibroblast cell strain that expressed cytokeratins 8 and 18 was isolated from explanted first trimester placenta. Its properties were consistent with an origin in the villous fibroblast-myofibroblast lineage, including expression of vimentin, smooth muscle alpha-actin and fibroblast surface protein. The cells grew rapidly in vitro, and exhibited tightly aligned bipolar morphology at confluence, absence of multi-nucleated cells, lack of secreted chorionic gonadotrophin, and absence of HLA G, placental alkaline phosphatase and pregnancy-specific beta-1 glycoprotein (SP1). On these criteria it was concluded they were not trophoblasts. On the basis of their morphology, cytokeratin expression and absence of CD34 and endoglin it was concluded they were not endothelial cells. They lacked desmin and smooth muscle myosin and so were not vascular smooth muscle cells. Further mesenchymal cell isolates were studied to determine the generality of these findings. Phenotypic heterogeneity was a consistent characteristic, but cytokeratin-positive cells were always present both in first trimester and term strains. Desmin was absent from almost all the cells isolated using the protocols employed, despite its occurrence in a significant subpopulation of cells in the villous stroma. Cytokeratins 8 and 18 can be observed in the stromal compartment of both first trimester and term placental villi. Cytokeratin has hitherto been regarded as a highly reliable marker for cells of the trophoblast lineage in vitro. These observations suggest that care should be taken in characterization of placental cell isolates; trophoblasts should be identified by the presence of cytokeratin 7 in preference to cytokeratin 8/18. The functional significance of cytokeratin expression in placental mesenchymal cells remains to be established.
Biomarkers↗
MUC-1 glycosylation in endometrium: possible roles of the apical glycocalyx at implantation.
MUC-1 is a major epithelial apical surface glycoprotein in human endometrium. It has a large, extended and highly glycosylated ectodomain that contains keratan sulphate chains. MUC-1 is abundant at the luminal epithelial surface in the receptive phase, but keratan sulphate disappears at this time. MUC-1 has been shown experimentally to inhibit cell-cell interactions by steric hindrance of binding interactions mediated by receptors, including integrins and cadherins, so its high abundance at the time of implantation is unexpected. Here, various models for MUC-1 function in implantation are considered and its expression in different species compared. The possible evolutionary advantages of a maternal 'barrier' to implantation are discussed.
Animals↗
Development of cytotrophoblast columns from explanted first-trimester human placental villi: role of fibronectin and integrin alpha5beta1.
Human first-trimester floating mesenchymal villi explanted onto gels of collagen I or Matrigel were observed to undergo de novo development of anchoring sites. These consisted of cytotrophoblast columns that formed by proliferation of stem villous cytotrophoblast cells, as revealed by whole-mount and thin-section microscopy and incorporation of bromodeoxyuridine into DNA. Column formation occurred exclusively at the distal tips of the villi. No column formation was observed in tissue explanted onto agarose. On Matrigel, the developing columns penetrated downwards into the matrix, whereas on collagen I, cytotrophoblast sheets spread across the surface of the gel and merged to form a shell. The developing columnar cytotrophoblast up-regulated integrins alpha1beta1 and alpha5beta1 and produced an extracellular matrix containing oncofetal fibronectin, as in vivo. Function-blocking antibodies were used to investigate the role of the integrin-fibronectin interaction in anchoring villus development on collagen I. Antibodies to fibronectin and the integrin subunits alpha5 and beta1, added at 24 h, all changed the pattern of cytotrophoblast outgrowth. Anti-fibronectin caused cell rounding within the cytotrophoblast sheet and increased the population of single cells at its periphery. Anti-integrin alpha5 caused rounding and redistribution of cells within the outgrowth. In the presence of anti-integrin beta1, cell-collagen interactions within the sheet were destabilized, often leading to the appearance of an annulus of aggregated cells at the periphery. These results show that 1) mesenchymal villi retain the potential to form anchoring sites until at least the end of the first trimester, 2) adhesion to a permissive extracellular matrix stimulates cytotrophoblast proliferation and differentiation along the extravillous lineage, 3) integrin alpha5beta1-fibronectin interactions contribute significantly to anchorage of the placenta to uterine extracellular matrix. We suggest that as the developing placenta ramifies, new sites of anchorage form whenever peripheral villi contact decidua. This process is predicted to contribute to the stability of the placental-decidual interface.
Animals↗
BeWo choriocarcinoma cells produce laminin 10.
BeWo is a choriocarcinoma cell line that generates an extracellular matrix (ECM) rich in laminin and is a useful model for human trophoblast. Immunofluorescence with monoclonal antibodies demonstrates that BeWo ECM contains laminin subunits beta1 and gamma1. Immunoprecipitation from conditioned medium shows that the cells secrete two distinct laminin trimers both containing beta1 and gamma1 but with alpha subunits of approx. 400 and 450 kDa. The culture medium also contains a species thought to be beta1 gamma1 dimer. Immunoprecipitation with monoclonal antibody 4C7, previously thought to recognize the alpha1 subunit, isolates complexes containing only the smaller alpha subunit. A second complex containing the larger alpha subunit along with beta1, gamma1 and a 150 kDa polypeptide is precipitated from 4C7-depleted medium with an anti-(laminin 1) polyclonal antibody. Peptide sequencing demonstrates that the 4C7-reactive species is alpha5, which is present as two similarly sized polypeptides. mRNA species encoding laminin subunits alpha1, alpha5, beta1, beta2 and gamma1 are all present in the cells. These results demonstrate the secretion of a novel laminin isoform, laminin 10, the subunit composition of which is alpha5 beta1 gamma1. Laminin 1 is also produced. No evidence for the secretion of beta2-containing laminin isoforms could be derived despite the presence of beta2 mRNA. Analysis with reverse transcriptase-mediated PCR also showed the presence of laminin alpha5 in first-trimester placenta and decidua.
Antibodies, Monoclonal↗
Human endometrial MUC1 carries keratan sulfate: characteristic glycoforms in the luminal epithelium at receptivity.
MUC1 is a high molecular mass, highly glycosylated epithelial apical glycoprotein that has been shown to exhibit both adhesive and anti-adhesive properties. Its expression in human glandular endometrial epithelium is transcriptionally regulated with the highest levels in the mid secretory phase, the "receptive" period during which implantation occurs. We demonstrate that endometrial MUC1 carries highly sulfated lactosaminoglycan chains recognized by monoclonal antibody (Mab) 5D4, and the sialokeratan sulfate epitope recognized by Mab D9B1. These glycans are hormonally regulated in endometrium, and show increased abundance in the secretory phase, but detailed evaluation of their distribution shows important differences. The 5D4 epitope is abundant at the luminal epithelial surface until the implantation phase, when it disappears, first from patches of cells, then altogether. D9B1 binding sites are retained in the luminal epithelium at receptivity. These data show that endometrial MUC1 carries sulfated lactosaminoglycans. They identify the luminal epithelial compartment as a site of unique MUC1 glycosylation and independent regulation. Glycosylation and the negative charge associated with sialo- and sulfoglycans may be important in the regulation of embryo attachment.
Amino Sugars↗
Cyclic modulation of epithelial glycosylation in human and baboon (Papio anubis) endometrium demonstrated by the binding of the agglutinin from Dolichos biflorus.
Glycoconjugate expression in endometrial epithelium varies in a cycle-dependent manner in many species and is probably fundamental to the establishment of a receptive uterine environment for the implanting embryo. We have examined the distribution of N-acetyl galactosamine (GalNAc)-containing sequences bound by Dolichos biflorus agglutinin (DBA) in the cycling endometrium of the human and baboon (Papio anubis). In the human, DBA binding was absent in the proliferative and early secretory phases of the cycle, increasing thereafter both in glands and on the surface epithelium. In contrast, DBA binding in the baboon was maximal in the proliferative phase, diminishing to almost indistinguishable levels in the late secretory phase. Treatment of early to mid-secretory-phase baboons with the antiprogestin Onapristone caused staining to revert to the proliferative-phase pattern, confirming the importance of progesterone in the decrease in DBA staining. Investigations with other GalNAc-binding lectins indicated that epithelial GalNAc residues were present throughout the cycle. Variation in steroidally dependent 1,2 fucosyl transferase activity may therefore be responsible for the appearance of the new, DBA-binding, nonreducing terminal structure GalNAcalpha1,3(Fucalpha1,2)-Galbeta1,4GlcNAcbeta1++ + in the human during the mid-secretory phase and in the baboon during the late proliferative phase.
Acetylgalactosamine↗
Progesterone regulates glycosylation in endometrium.
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Animals↗
Adhesion molecules in implantation.
At implantation, trophectoderm attaches to the apical uterine luminal epithelial cell surface. Molecular anatomy studies in humans and mice, and data from experimental models have identified several adhesion molecules that could take part in this process: integrins of the alpha v family, trophinin, CD44, cad-11, the H type I and Lewis y oligosaccharides and heparan sulfate. The endometrial cell surface mucin MUC1 may play a role in both steric inhibition of attachment and selective glycan display. After attachment, interstitial trophoblast invasion occurs requiring a new repertoire of adhesive interactions with maternal extracellular matrix as well as stromal and vascular cell populations. Human anchorage sites contain columns of cytotrophoblasts in which self-attachment gives way progressively to adhesion to extracellular matrix and then interstitial migration. The beta 1 integrins are important during these later stages of implantation and placentation.
Animals↗
Sialyl-Lewis x and Sialyl-Lewis a are associated with MUC1 in human endometrium.
Endometrial epithelial cells express MUC1 with increased abundance in the secretory phase of the menstrual cycle, when embryo implantation occurs. MUC1 is associated with the apical surface of epithelial cells and is also secreted, being detectable in uterine fluid at elevated levels in the implantation phase. However, its physiological role is uncertain; it may either inhibit intercellular adhesion by steric hindrance or carry carbohydrate recognition structures capable of mediating cell-cell interaction. Here we show that endometrial epithelium expresses both Sialyl-Lewis x (SLex) and Sialyl-Lewis a (SLea), with a distribution and pattern of menstrual cycle regulation similar to that of MUC1. Using Western blotting and double determinant ELISA of uterine flushings, we demonstrate that SLex is associated with MUC1 core protein. The endometrial carcinoma cell lines HEC1A and HEC1B are shown to express MUC1 in a mosaic pattern, while three other cell lines express much lower amounts. HEC1A expresses both SLex and SLea while HEC1B expresses SLea only. Immunoprecipitation has been used to demonstrate that SLea is associated with MUC1 in HEC1B cells, and both SLex and SLea are associated with MUC1 in HEC1A cells.
Antibodies, Monoclonal↗
Abnormal expression of integrin alpha 6 beta 4 in cervical intraepithelial neoplasia.
We have used subunit-specific monoclonal antibodies (MAbs) and immunohistochemistry to examine the distribution of integrin alpha 6 beta 4 in normal ectocervical epithelium and various grades of cervical intraepithelial neoplasia (CIN). Antibodies were first characterised by immunoprecipitation from two surface-labelled tumour cell lines. Monoclonal antibody G71 was found to precipitate integrin beta 4 from BeWo but not T47D cells, while other anti-beta 4 antibodies precipitated beta 4 from both cell lines. Both G71 and an antiserum to the C-terminal peptide of beta 4 precipitated free beta 4 from surface-iodinated BeWo cells. Neither antibody recognised truncated beta 4 chains observed at approximately 160 kDa. These data suggest that different isoforms of beta 4 are expressed in different tumour cell lines, and that there may be a pool of beta 4 at the cell surface that is not complexed to alpha 6. In normal cervix, both the alpha 6 and beta 4 subunits occur at the basal surface of the basal cell layer. In CIN, the distribution is markedly altered, with strong expression of alpha 6 and beta 4 in the upper cell layers of the ectocervical epithelium. All 40 cases of CIN that were studied exhibited this alteration. Furthermore, the extent of extrabasal staining appeared to correspond with the grade of CIN. The form of integrin beta 4 recognised by antibody G71 also appears in the upper cell layers in CIN, but it shows a more restricted distribution than the normal isoform.
Amino Acid Sequence↗
Integrins beta 5, beta 3 and alpha v are apically distributed in endometrial epithelium.
Several adhesion molecules have been shown to occur at the surface of endometrial cells. One of these is the integrin alpha v subunit which associates with various beta chains including beta 5. We demonstrate the presence of integrin beta 5 polypeptide in human endometrial epithelial cells throughout the menstrual cycle using immunocytochemistry with monospecific antibodies, and at the mRNA level by thermal amplification from endometrial cDNA. Integrin beta 5 is also found in a population of bone marrow-derived cells. A notable feature of the distribution of the beta 5 subunit in the glandular and luminal epithelium is its apical localization, which may suggest an involvement in implantation. However, no evidence was found for regulated expression of epithelial beta 5. In mouse, the beta 5 subunit is found at both the apical and basal surface of epithelial cells and expression is essentially oestrous cycle-independent. Comparisons are made in both species with the distribution of the alpha v and beta 3 subunits which also localize to the apical epithelium.
Animals↗
MUC1 as a cell surface and secretory component of endometrial epithelium: reduced levels in recurrent miscarriage.
The mucin MUC1 is a large, highly glycosylated, hormonally regulated product of endometrial glandular and luminal epithelium with both cell surface-associated and secreted isoforms. The abundance of mRNA coding for MUC1 increases about sixfold from the proliferative to the early secretory phase (Hey et al., J. Clin. Endocrinol. Metab. 78:337-342, 1994). Immunohistochemical studies show intracellular deposits accumulating in the early secretory phase followed by the release of MUC1 into gland lumens. The apical surface of luminal epithelium is strongly immunopositive in the early secretory phase. We have used a two site ELISA to measure MUC1 in uterine flushings as a function of time after the luteinising hormone (LH) peak. Low levels of secretory MUC1 are observed before day LH+7, while values on days LH+7-LH+13 are much higher. Using semi-quantitative immunohistochemical methods we have shown that in women suffering recurrent spontaneous miscarriage, mid secretory phase levels of MUC1 core protein and mucin-associated glycans are reduced (Serle et al., Fertil. Steril. 62:989-996, 1994). Similarly, lower core protein levels are observed in uterine flushings after day LH+7 in these women. Reduced epithelial secretory function and a resultant change in uterine fluid composition are features of endometrium from recurrent miscarriage patients.
Abortion, Habitual↗
Epithelial-mesenchymal transition during trophoblast differentiation.
Embryo implantation in higher primates is mediated by trophoblast: in the earliest phases by syncytiotrophoblast, then by both cytotrophoblast and syncytiotrophoblast. In the course of placentation three main trophoblast populations can be identified: cytotrophoblast stem cells and two differentiated derivative cell types: the syncytiotrophoblast and the extravillous cytotrophoblast. The syncytiotrophoblast remains mainly epithelial while the extravillous cytotrophoblast undergoes an epithelial-mesenchymal transition (EMT), initially forming multilayered cell columns and then, in human, infiltrating deeply the maternal decidual stroma and blood vessels. Finally, some infiltrating cells differentiate further to become giant cells of the placental bed and myometrium. During the course of these events the extravillous cytotrophoblast acquires a distinct phenotype, losing some typical epithelial components (e.g. E-cadherin, integrin alpha 6 beta 4), but retaining others (e.g. cytokeratins). The signals that trigger this EMT are not well understood but its realisation is of critical importance for pregnancy success.
Animals↗
Decidual sialylation shows species-specific differences in the pregnant mouse and rat.
Biotinylated lectins from Sambucus nigra (SNA) and Maackia amurensis (MAA), which bind to alpha 2,6-linked and alpha 2,3-linked sialyl residues, respectively, were used as probes to study glycan terminal modifications associated with decidualization in the uterine stroma of pregnant rats and mice. Binding of lectins from Erythrina cristagalli (ECA), Phaseolus vulgaris (leukoagglutinin, L-PHA), Triticum vulgaris (WGA) and Bandeiraea simplicifolia (BSA-1B4) was also examined. Tissues from rats between day 5 and day 8 of gestation and mice between day 5 and day 7 of gestation were fixed in Bouin's solution and embedded in wax prior to lectin histochemistry. On day 7 in rats and day 6 in mice, there was a marked reduction in the binding of SNA in the subluminal decidua surrounding the implantation site. In rats, MAA binding to enlarged decidual cells around the implantation chamber was increased markedly, but there was no change in mice. In both species there was de novo binding of ECA in the SNA-negative area, suggesting that the loss of alpha 2,6-linked sialyl residues unmasks terminal N-acetyl lactosamine. These findings are consistent with previous evidence of a close structural and functional similarity between the artificially induced deciduoma and true decidua of rats and show identical changes to the glycosylation patterns previously found in differentiating rat deciduoma. In both species, therefore, decidua exhibits regionally specific terminal glycosylation. However, the species-specific expression of alpha 2,3-linked sialyl residues suggests distinct patterns of steroidally modulated sialyl transferase expression.
Animals↗
Laminins 2 and 4 are expressed by human decidual cells.
During pregnancy, the resident stromal cells of the endometrium differentiate to become decidual cells and produce a pericellular basement membrane. We used immunofluorescence and Western blotting with a panel of monoclonal Ab specific for various laminin subunits to examine the composition of decidual laminin. The stromal cell basement membrane contained subunits alpha 2 (M), beta 1 (B1), beta 2 (S), and gamma 1 (B2). Low levels of alpha 1 could also be detected. The glandular and vascular basement membranes of decidual tissue contained subunits alpha 1 (A), beta 1, and gamma 1. An extract was produced from decidual extracellular matrix. Western blots of nonreducing gels showed the presence of high molecular weight complexes containing alpha 2, beta 1, beta 2, and gamma 1. These data indicated that laminins 2 and 4 are coexpressed by decidual cells. Laminin 1 was present in the extract as a minor component. In contrast, cultured stromal cells expressed laminin 1 as the major secreted variant. Immunolocalization was carried out using tissue from various stages of the nonpregnant cycle. The alpha 2 chain polypeptide was absent in the proliferative phase of the cycle but present in late secretory phase in perivascular areas where predecidual differentiation occurs. Reverse transcriptase-PCR experiments confirmed the presence of alpha 2 chain mRNA in decidua but showed that this transcript is detectable throughout the nonpregnant cycle. The results showed that laminins 2 and 4 are hormonally regulated products of decidual cells. The composition of the vascular and epithelial basement membranes remained constant throughout the cycle.
Blotting, Western↗