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Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells
Journal of Experimental & Clinical Assisted Reproduction volume 2, Article number: 4 (2005)
Abstract
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.
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Acknowledgements
We thank K.-D. Schulz (Marburg, Germany) for providing the Ishikawa cell line, as well as H. G. Adelmann (Loughborough, UK) for constructive criticisms in advanced digital image processing and for kind provision of his Gauss bandpass and homomorphic filter plugins, and J. Huesing (Essen, Germany) for help with statistical analysis. The skilful technical assistance of K. Baden, B. Gobs, B. Maranca and D. Schuenke is gratefully acknowledged.
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Authors' contributions
CH conceived of the study and carried out the confocal laser scanning microscopical and electron microscopical investigations. MS provided expertise in small Rho GTPases and conducted biochemical experiments. HWD participated in the design of the study and helped to draft the manuscript. MT conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
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Heneweer, C., Schmidt, M., Denker, HW. et al. Molecular mechanisms in uterine epithelium during trophoblast binding: the role of small GTPase RhoA in human uterine Ishikawa cells . J Exp Clin Assist Reprod 2, 4 (2005). https://doi.org/10.1186/1743-1050-2-4
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DOI: https://doi.org/10.1186/1743-1050-2-4