Michael Thie

Interactions between trophoblast and uterine epithelium: monitoring of adhesive forces.

At embryo implantation, it is postulated that the initial contact between blastocyst and maternal tissues is by adhesion of the trophoblast to the uterine epithelium. This cell-to-cell interaction is thought to be critical for implantation, although the actual adhesive forces have never been determined. In the present study, the atomic force microscope (AFM) was used to study the adhesion between human uterine epithelial cell lines (HEC-1-A; RL95-2) and human trophoblast-type cells (JAR). Specific interaction forces of these epithelia via their apical cell poles were determined on the basis of approach-and-separation cycles. For this purpose, the AFM tip was functionalized with JAR cells, then brought to the surface of uterine epithelial monolayers and was kept in contact for different periods of time (ms, 1, 10, 20, 40 min). The approach force curves displayed repulsive interactions for both HEC-1-A and RL95-2 cells. However, RL95-2 cells (with a smooth surface structure and a thin glycocalyx) showed lower values of the repulsive regime than HEC-1-A cells (with a rough surface structure and a thick glycocalyx). After having overcome repulsive interactions, the initial contact was followed by adhesive interactions. For contact times of 20 and 40 min, RL95-2 cells, but not HEC-1-A cells, showed specific JAR binding, i.e. the separation force curves displayed repeated rupture events in the range of 1-3 nN with a distance between 7-15 microm and, thereafter, a final rupture event at a distance of up to 45 microm. These features point to the formation of strong cell-to-cell bonds. Collectively, these studies provide the first definition of interaction forces between the trophoblast and the uterine epithelium, and are consistent with the hypothesis that an RL95-2-like architecture of uterine epithelial cells, i.e. an non-polarized phenotype, is essential for apical adhesiveness for the human trophoblast.

Differential Expression and Localization of Integrins and CD44 in the Membrane Domains of Human Uterine Epithelial Cells During the Menstrual Cycle

Human uterine epithelium displays a distinctly polarized organization with basal, lateral, and apical plasma membrane domains. Although nonadhesive throughout most of the menstrual cycle, uterine epithelial cells allow attachment of trophoblast cells to their apical pole during embryo implantation. Development of the receptive state might involve expression of cell adhesion molecules and/or redistribution of such molecules with respect to their localization at the basal, lateral, and apical membrane domains of cells. Expression and distribution of alpha 1-, alpha 3-, alpha 5-, alpha 6-, beta one-, beta 3- and beta 4-integrin subunits as well as of CD44 were examined in the luminal epithelium of human endometrium by immunohistochemistry in different phases of the menstrual cycle. The luminal epithelium was found to express alpha 1-, alpha 3-, alpha 6-, beta 1-, beta 4-integrin subunits and CD44. alpha l6-Integrin subunits and CD44 displayed cycle dependency. The alpha 6-integrin subunits were detected in the basal membrane domains in all phases. However, in correlation with increasing expression during the secretory phase of cycle, these subunits newly appeared in the lateral membranes of epithelial cells. CD44 showed increased expression in the secretory phase but was always restricted to the lateral membranes. The conspicuous behavior of alpha 6-integrin subunits and CD44 is discussed with respect to its possible functional significance for embryo implantation, and in relation to a hypothesis postulating that steroid-controlled master genes direct the acquisition of the receptive state of the luminal uterine epithelium by changing elements of the apicobasal polarity of these cells.

In vitro Studies on Endometrial Adhesiveness for Trophoblast: Cellular Dynamics in Uterine Epithelial Cells

Initiation of embryo implantation involves adhesion of trophoblast cells to the epithelial lining of the endometrium. The mechanisms regulating the adhesive properties of the uterine epithelium for trophoblast during initiation of human embryo implantation, however, are still incompletely understood. We report here on model studies that we have performed in our laboratory, and in particular on certain methodological approaches that seem to yield new insight into basic mechanisms involved. Of central interest is the ability of the uterine epithelium to develop an adhesion competence at its apical cell pole. This confronts us with a cell biological paradox in that adhesion must be established at the pole which in simple epithelia is typically specialized to resist adhesion. Gain of apical adhesion competence by uterine epithelial cells should be related to cellular rearrangements, i.e. a modulation of their apicobasal cell polarity. Here, we used monolayer-cultured uterine epithelial RL95-2 cells as an in vitro model for the human receptive uterine epithelium. We demonstrated that formation of stable cell-to-cell bonds between the free (apical) pole of these cells and attaching trophoblast (modelled by JAr cells) depends on a number of structural and functional peculiarities that RL95-2 cells have in contrast to other uterine epithelial cells (HEC-1-A cells) which resist attachment via this cell pole. RL95-2 cells were shown to lack tight junctions and to exhibit only rudimentary adherens junctions and a non-polar organization of the actin cytoskeleton. Using the atomic force microscope in a force spectroscopy mode, we exactly defined the time dependence of adhesive interactions between RL95-2 cells and trophoblast, measured the pressure force needed to initiate this process, and screened the buildup of the adhesive forces between the binding partners. A dynamic interaction between the actin cytoskeleton and integrins (a prerequisite for functional activity of integrins) was shown to be an important aspect of the adhesive properties of RL95-2 cells. In addition, at least two types of calcium channels in the plasma membrane of RL95-2 cells seem to play a role in activation of a variety of calcium-sensitive response mechanisms including adhesiveness for trophoblast, i.e. diltiazem-sensitive channels seem to contribute to the initiation of JAr cell binding and SKF-96365-sensitive channels to participate in a feedback loop that controls the balance of bonds. By extrapolation, these data suggest an active role of the uterine epithelium in the process of embryo implantation which we are just beginning to understand in terms of its cell biology.

Epithelial-mesenchymal transition in colonies of rhesus monkey embryonic stem cells : A model for processes involved in gastrulation

Rhesus monkey embryonic stem (rhES) cells were grown on mouse embryonic fibroblast (MEF) feeder layers for up to 10 days to form multilayered colonies. Within this period, stem cell colonies differentiated transiently into complex structures with a disc‐like morphology. These complex colonies were characterized by morphology, immunohistochemistry, and marker mRNA expression to identify processes of epithelialization as well as epithelial–mesenchymal transition (EMT) and pattern formation. Typically, differentiated colonies were comprised of an upper and a lower ES cell layer, the former growing on top of the layer of MEF cells whereas the lower ES cell layer spread out underneath the MEF cells. Interestingly, in the central part of the colonies, a roundish pit developed. Here the feeder layer disappeared, and upper layer cells seemed to ingress and migrate through the pit downward to form the lower layer while undergoing a transition from the epithelial to the mesenchymal phenotype, which was indicated by the loss of the marker proteins E‐cadherin and ZO‐1 in the lower layer. In support of this, we found a concomitant 10‐fold upregulation of the gene Snail2, which is a key regulator of the EMT process. Conversion of epiblast to mesoderm was also indicated by the regulated expression of the mesoderm marker Brachyury. An EMT is a characteristic process of vertebrate gastrulation. Thus, these rhES cell colonies may be an interesting model for studies on some basic processes involved in early primate embryogenesis and may open new ways to study the regulation of EMT in vitro.

Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells

Background Embryo implantation requires that uterine epithelium develops competence to bind trophoblast to its apical (free) poles. This essential element of uterine receptivity seems to depend on a destabilisation of the apico-basal polarity of endometrial epithelium. Accordingly, a reorganisation of the actin cytoskeleton regulated by the small GTPase RhoA plays an important role in human uterine epithelial RL95-2 cells for binding of human trophoblastoid JAR cells. We now obtained new insight into trophoblast binding using human uterine epithelial Ishikawa cells. Methods Polarity of Ishikawa cells was investigated by electron microscopy, apical adhesiveness was tested by adhesion assay. Analyses of subcellular distribution of filamentous actin (F-actin) and RhoA in apical and basal cell poles were performed by confocal laser scanning microscopy (CLSM) with and without binding of JAR spheroids as well as with and without inhibition of small Rho GTPases by Clostridium difficile toxin A (toxin A). In the latter case, subcellular distribution of RhoA was additionally investigated by Western blotting. Results Ishikawa cells express apical adhesiveness for JAR spheroids and moderate apico-basal polarity. Without contact to JAR spheroids, significantly higher signalling intensities of F-actin and RhoA were found at the basal as compared to the apical poles in Ishikawa cells. RhoA was equally distributed between the membrane fraction and the cytosol fraction. Levels of F-actin and RhoA signals became equalised in the apical and basal regions upon contact to JAR spheroids. After inhibition of Rho GTPases, Ishikawa cells remained adhesive for JAR spheroids, the gradient of fluorescence signals of F-actin and RhoA was maintained while the amount of RhoA was reduced in the cytosolic fraction with a comparable increase in the membrane fraction. Conclusion Ishikawa cells respond to JAR contact as well as to treatment with toxin A with rearrangement of F-actin and small GTPase RhoA but seem to be able to modify signalling pathways in a way not elucidated so far in endometrial cells. This ability may be linked to the degree of polar organisation observed in Ishikawa cells indicating an essential role of cell phenotype modification in apical adhesiveness of uterine epithelium for trophoblast in vivo.

Human Uterine Epithelial RL95-2 Cells Reorganize Their Cytoplasmic Architecture with Respect to Rho Protein and F-Actin in Response to Trophoblast Binding

Embryo implantation is initiated by interaction of trophoblast with uterine epithelium via the apical cell poles of both partners. Using spheroids of human trophoblastoid JAR cells and monolayers of human uterine epithelial RL95-2 cells to simulate this initial interaction, we previously demonstrated that formation of stable cell-to-cell bonds depends on actin cytoskeleton (F-actin) and small GTPases of the Rho family, most likely RhoA. In this study, we determined the apical as well as the basal distribution of these proteins by fluorescence confocal microscopy before and after binding of JAR spheroids. We focussed on changes in cytoplasmic organization with respect to apicobasal polarity of RL95-2 cells. Before binding of spheroids, significantly higher fluorescence signals of RhoA [37 ± 6 grey scale values (gsv)] and of F-actin (41 ± 3 gsv) were found in the basal region of RL95-2 cells as compared to the apical pole (RhoA: 24 ± 3 gsv, F-actin: 28 ± 2 gsv). After binding of JAR spheroids, this apicobasal asymmetry was inverted (RhoA: 55 ± 10 gsv apical vs. 25 ± 3 gsv basal; F-actin: 108 ± 17 gsv apical vs. 57 ± 7 gsv basal). Inactivation of Rho GTPases in RL95-2 cells by Clostridium difficile toxin A leads to a loss of their apical adhesion competence, as previously published. Here, we observed a uniform distribution of RhoA and F-actin between apical and basal region rather than an asymmetric one in toxin A-treated cells. These data suggest that activation of Rho GTPases and coordinated rearrangement of F-actin within uterine epithelial cells in response to trophoblast binding are part of a generalized structural and functional reorganization of the cytoplasm. This involves not only the immediate contact zone (apical) but also the opposite (basal) cell pole and may be a critical element of uterine epithelial reactions during transition between trophoblast adhesion and transmigration.

Epithelial-mesenchymal transition in Rhesus monkey embryonic stem cell colonies: a model for processes involved in gastrulation?

A characteristic feature of embryonic stem (ES) cells is their ability to give rise to differentiated cell types that are derived from all three primary germ layers. In the embryo of higher vertebrates, formation of mesoderm and definitive endoderm (gastrulation) occurs at the primitive streak through a spatially highly ordered process of cell ingression, combined with epithelial-mesenchymal transition (EMT). With respect to ES cell differentiation in vitro, however, germ layer derivative formation has not been studied in much detail, and data on any degree of spatial order that may be attained here are lacking. In the investigations to be reviewed here, rhesus monkey ES cells (line R366.4) were grown on mouse embryonic fibroblast feeder layers for up to 10 days during which time they formed multilayered disc-like colonies with an upper epithelial and a lower mesenchymal cell layer. Processes of epithelialization as well as EMT were studied by transmission electron microscopy, immunohistochemistry combined with confocal laser scanning microscopy, and marker mRNA expression (in situ hybridization, RT-PCR). It was found that under the culture conditions used most of the ES cell colonies developed transitorily a central pit where the epithelial upper layer cells underwent an EMT-like process and appeared to ingress to form the lower, mesenchymal layer, accompanied by appropriate changes of morphology and molecular markers. Similarities and differences in comparison with gastrulation/primitive streak formation in vivo are briefly discussed, as are ethical implications with respect to human ES cells. It is concluded that this rhesus ES cell colony system may be an interesting in vitro model for studies on some basic processes involved in early embryogenesis such as EMT/gastrulation and may open new ways to study the regulation of these processes experimentally in vitro in nonhuman primates.