Cell-cell junctions during transepithelial trophoblast invasion in a human in vitro implantation model

Embryo implantation refers to the embedding of an embryo in the uterine endometrium. It starts with the attachment of the outer embryo layer to the endometrial surface involving interactions between two types of one-layered epithelia, i.e. the embryonic trophoblast and the endometrial epithelium. Transepithelial migration occurs next, during which trophoblast cells cross the endometrial barrier. Trophoblast cells subsequently invade the stromal compartment. Implantation is highly species-specific. Human implantation differs from other types of invasion involving cell-cell interaction between the apical sides of two polarized epithelia. Although implantation is a prerequisite for a successful pregnancy, our understanding of the exact molecular and cellular mechanism is limited in human because of the obvious ethical and technical constraints. Suitable in vitro models are therefore essential to study this process. The aim of this work was to meet this need and to improve currently available models by creating a physiological mechanophysical 3D environment to study the attachment and transepithelial invasion of the embryonic trophoblast through the endometrial epithelium. I first established a model of luminal endometrial epithelium using a hormone-responsive human endometrial adenocarcinoma-derived cell line and tested the effects of substrate stiffness on epithelial morphology and polarization. Our results revealed that the cells were responsive to stiffness-derived mechanical cues resulting in differences in the cell polarization. Subsequently, I generated spheroids from an immortalized human trophoblast cell line to mimic size and shape of the embryonic trophoblast. The attachment and penetration of the trophoblast through the endometrial monolayer could then be examined. Using this setup, I showed that substrate stiffness scaled with spheroid attachment. To find out whether cell-cell junctions are involved in trophoblast adhesion and invasion, I studied the localization and rearrangements of all three major cell-cell adhesion types at the invasion site. This included the barrier-forming tight junctions, the actin-anchoring adherens junctions and intermediate filament-anchoring desmosomes. Fluorescent dyes were used to separately label the trophoblast and endometrial epithelium. By immunofluorescence microscopy, I was able to detect heterologous junctions between trophoblast and endometrium. Electron microscopy provided additional evidence for the presence of the different junction types at the invasion site. To further validate their relevance, I used primary endometrial cells instead of the adenocarcinoma-derived endometrial cell line. Immunostaining not only confirmed that all three cell-cell junction types were formed between trophoblast and endometrium but also revealed a reinforcing feedback effect on junction formation between trophoblast cells after contact with the endometrial epithelium. Finally, I tested the functionality of the heterologous junctions during the attachment-invasion process. To assess the functionality of tight junctions, I performed impedance measurements at the invasion site. An increase in impedance could be detected during spheroid attachment and invasion, indicating the maintenance and even enforcement of a tightly sealed tight junction network at the invasion site. Next, I investigated the role of desmosomes in transepithelial invasion by using blocking antibodies against the desmosomal cadherin desmoglein 2. The interference with desmoglein 2-mediated adhesion resulted in a slowdown of transepithelial trophoblast invasion.Taken together, I established and validated a new model of human embryo attachment and invasion. Using this model, I was able to show that heterologous junctions are involved in this process. The results highlight the importance of cell mechanics to understand and model the biological processes occurring during human embryo implantation.