In vitro Studies on Endometrial Adhesiveness for Trophoblast: Cellular Dynamics in Uterine Epithelial CellsThie M. · Denker H.-W.
Institut für Anatomie, Lehrstuhl für Anatomie und Entwicklungsbiologie, Universitätsklinikum Essen, Germany
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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.
© 2002 S. Karger AG, Basel
- Albers, A., M. Thie, H.P. Hohn, H.-W. Denker (1995) Differential expression and localization of integrins and CD44 in the membrane domains of human uterine epithelial cells during the menstrual cycle. Acta Anat 153: 12–19.
Anderson, T.L., S.M. Sieg, G.D. Hodgen (1988) Membrane composition of the endometrial epithelium: Molecular markers of uterine receptivity to implantation; in Iizuka, R., K. Semm (eds): Human Reproduction. Int Congr Ser No 768. Amsterdam, Excerpta Medica, pp 513–516.
Anderson, T.L., J.A. Simon, G.D. Hodgen (1990) Histochemical characteristics of the endometrial surface related temporally to implantation in the non-human primate (Macaca fascicularis); in Denker, H.-W., J.D. Aplin (eds): Trophoblast Invasion and Endometrial Receptivity. Novel Aspects of the Cell Biology of Embryo Implantation. Trophoblast Research. New York, Plenum Medical Book, vol 4, pp 273–284.
- Aoki, R., M.N. Fukuda (2000) Recent molecular approaches to elucidate the mechanism of embryo implantation: Trophinin, bystin, and tastin as molecules involved in the initial attachment of blastocysts to the uterus in humans. Semin Reprod Med 18: 265–271.
- Aplin, A.E., A. Howe, S.K. Alahari, R.L. Juliano (1998) Signal transduction and signal modulation by cell adhesion receptors: The role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol Rev 50: 197–263.
- Aplin, J.D. (1999) MUC-1 glycosylation in endometrium: Possible roles of the apical glycocalyx at implantation. Hum Reprod 14(suppl 2): 17–25.
- Aplin, J.D., M. Meseguer, C. Simon, M.E. Ortiz, H. Croxatto, C.J. Jones (2001) MUC1, glycans and the cell-surface barrier to embryo implantation. Biochem Soc Trans 29: 153–156.
- Armant, D.R., J. Wang, Z. Liu (2000) Intracellular signaling in the developing blastocyst as a consequence of the maternal-embryonic dialogue. Semin Reprod Med 18: 273–287.
- Aurrand-Lions, M., C. Johnson-Leger, C. Lamagna, H. Ozaki, T. Kita, B.A. Imhof (2002) Junctional adhesion molecules and interendothelial junctions. Cells Tissues Organs 172: 152–160.
- Bagchi, I.C., Q. Li, Y.P. Cheon (2001) Role of steroid hormone-regulated genes in implantation. Ann NY Acad Sci 943: 68–76.
- Benoit, M., H.E. Gaub (2002) Measuring cell adhesion forces with the atomic force microscope at the molecular level. Cells Tissues Organs 172: 174–189.
- Bentin-Ley, U., T. Horn, A. Sjögren, S. Sörensen, J. Falck Larsen, L. Hamberger (2000) Ultrastructure of human blastocyst-endometrial interactions in vitro. J Reprod Fertil 120: 337–350.
Binnig, G., C.F. Quate, C. Gerber (1986) Atomic force microscope. Phys Rec Lett 56: 930.
- Bishop, A.L., A. Hall (2000) Rho GTPases and their effector proteins. Biochem J 348: 241–255.
Böving, B.G. (1963) Implantation mechanisms; in Hartmann, C.G. (ed): Mechanisms Concerned with Conception. New York, Pergamon Press, pp 321–396.
- Boyer, B., A.M. Valles, N. Edme (2000) Induction and regulation of epithelial-mesenchymal transitions. Biochem Pharmacol 60: 1091–1099.
- Burghardt, R.C., G.A. Johnson, L.A. Jaeger, H. Ka, J.E. Garlow, T.E. Spencer, F.W. Bazer (2002) Integrins and extracellular matrix proteins at the maternal-fetal interface in domestic animals. Cells Tissues Organs 172: 202–217.
- Calderwood, D.A., S.J. Shattil, M.H. Ginsberg (2000) Integrins and actin filaments: Reciprocal regulation of cell adhesion and signaling. J Biol Chem 275: 22607–22610.
- Carson, D.D., I. Bagchi, S.K. Dey, A.C. Enders, A.T. Fazleabas, B.A. Lessey, K. Yoshinaga (2000) Embryo implantation. Dev Biol 223: 217–237.
- Carson, D.D., M.M. DeSouza, R. Kardon, X. Zhou, E. Lagow, J. Julian (1998a) Mucin expression and function in the female reproductive tract. Hum Reprod Update 4: 459–464.
- Carson, D.D., M.M. DeSouza, E.G. Regisford (1998b) Mucin and proteoglycan functions in embryo implantation. Bioessays 20: 577–583.
- Chervenak, J.L., N.P. Illsley (2000) Episialin acts as an antiadhesive factor in an in vitro model of human endometrial-blastocyst attachment. Biol Reprod 63: 294–300.
- Clark, E.A., J.S. Brugge (1995) Integrins and signal transduction pathways: The road taken. Science 268: 233–239.
- Cooper, J.A. (1987) Effects of cytochalasin and phalloidin on actin. J Cell Biol 105: 1473–1478.
Denker, H.-W. (1970) Topochemie hochmolekularer Kohlenhydratsubstanzen in Frühentwicklung und Implantation des Kaninchens. I. Allgemeine Lokalisierung und Charakterisierung hochmolekularer Kohlenhydratsubstanzen in frühen Embryonalstadien. II. Beiträge zu entwicklungsphysiologischen Fragestellungen. Zool Jb 75: 141–308.
Denker, H.-W. (1986) Epithel-Epithel-Interaktionen bei der Embryo-Implantation: Ansätze zur Lösung eines zellbiologischen Paradoxons. Verh Anat Ges 80, Anat Anz Suppl 160: 93–114.
Denker, H.-W. (1990) Trophoblast-endometrial interactions at embryo implantation: A cell biological paradox; in Denker, H.-W., J.D. Aplin (eds): Trophobloast Invasion and Endometrial Receptivity. Novel Aspects of the Cell Biology of Embryo Implantation. Trophoblast Research. New York, Plenum Medical Book, vol 4, pp 3–29.
- Denker, H.-W. (1993) Implantation: A cell biological paradox. J Exp Zool 266: 541–558.
Denker, H.-W. (1994) Endometrial receptivity: Cell biological aspects of an unusual epithelium. Ann Anat 176: 53–60.
- Denker, H.-W. (2000) Structural dynamics and function of early embryonic coats. Cells Tissues Organs 166: 180–207.
- Denker, H.-W., M. Thie (2001) The regulatory function of the uterine epithelium for trophoblast attachment: Experimental approaches. Ital J Anat Embryol 106(suppl 2): 291–306.
- Drubin, D.G., W.J. Nelson (1996) Origins of cell polarity. Cell 84: 335–344.
- Enders, A.C., A.G. Hendrickx, S. Schlafke (1983) Implantation in the rhesus monkey: Initial penetration of endometrium. Am J Anat 167: 275–298.
- Enders, A.C., A. Lopata (1999) Implantation in the marmoset monkey: Expansion of the early implantation site. Anat Rec 256: 279–299.
- Enders, A.C., R.A. Mead (1996) Progression of trophoblast into the endometrium during implantation in the western spotted skunk. Anat Rec 244: 297–315.
Enders, A.C., S. Schlafke (1977) Surface coats of the mouse blastocyst and uterus during the preimplantation period. Anat Rec 180: 31–46.
- Evenas, J., A. Malmendal, S. Forsen (1998) Calcium. Curr Opin Chem Biol 2: 293–302.
- Fei, G., W. Peng, C. Xin-Lei, H. Zhao-Yuan, L. Yi-Xun (2001) Apoptosis occurs in implantation site of the rhesus monkey during early stage of pregnancy. Contraception 64: 193–200.
- Florin, E.L., V.T. Moy, H.E. Gaub (1994) Adhesive forces between individual ligand-receptor pairs. Science 264: 415–417.
Galan, A., R. Herrer, J. Remohi, A. Pellicer, C. Simon (2000a) Embryonic regulation of endometrial epithelial apoptosis during human implantation. Hum Reprod 15(suppl 6): 74–80.
- Galan, A., J.E. O’Connor, D. Valbuena, R. Herrer, J. Remohi, S. Pampfer, A. Pellicer, C. Simon (2000b) The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion. Biol Reprod 63: 430–439.
- Geiger, B., A. Bershadsky (2001) Assembly and mechanosensory function of focal contacts. Curr Opin Cell Biol 13: 584–592.
- Geiger, B., A. Bershadsky, R. Pankov, K.M. Yamada (2001) Transmembrane crosstalk between the extracellular matrix – cytoskeleton crosstalk. Nat Rev Mol Cell Biol 2: 793–805.
- Giancotti, F.G., E. Ruoslahti (1999) Integrin signaling. Science 285: 1028–1032.
- Glasser, S.R., J. Mulholland (1993) Receptivity is a polarity dependent special function of hormonally regulated uterine epithelial cells. Microsc Res Tech 25: 106–120.
Hay, E.D. (1990) Epithelial-mesenchymal transitions. Semin Dev Biol 1: 347–356.
- Hay, E.D. (1995) An overview of epithelio-mesenchymal transformation. Acta Anat 154: 8–20.
- Hoffman, L.H., V.P. Winfrey, G.L. Blaeuer, G.E. Olson (1996) A haptoglobin-like glycoprotein is produced by implantation-stage rabbit endometrium. Biol Reprod 55: 176–184.
- Hohn, H.-P., H.-W. Denker (2002) Experimental modulation of cell-cell adhesion, invasiveness and differentiation in trophoblast cells. Cells Tissues Organs 172: 218–236.
- Hohn, H.-P., M. Linke, H.-W. Denker (2000) Adhesion of trophoblast to uterine epithelium as related to the state of trophoblast differentiation: In vitro studies using cell lines. Mol Reprod Dev 57: 135–145.
- Illingworth, I.M., S.J. Kimber (1999) Demonstration of oestrogenic control of H-type-1 carbohydrate antigen in the murine endometrial epithelium by use of ICI 182,780. J Reprod Fertil 117: 89–95.
John, M.J., M. Linke, H.-W. Denker (1993) Quantitation of human choriocarcinoma spheroid attachment to uterine epithelial cell monolayers. In Vitro Cell Dev Biol 29A: 461–468.
- Kimber, S.J. (2000) Molecular interactions at the maternal-embryonic interface during the early phase of implantation. Semin Reprod Med 18: 237–253.
- Kimber, S.J., I.M. Illingworth, S.R. Glasser (1995) Expression of carbohydrate antigens in the rat uterus during early pregnancy and after ovariectomy and steroid replacement. J Reprod Fertil 103: 75–87.
- Kimber, S.J., S. Lindenberg (1990) Hormonal control of a carbohydrate epitope involved in implantation in mice. J Reprod Fertil 89: 13–21.
- Kimber, S.J., C. Spanswick (2000) Blastocyst implantation: The adhesion cascade. Semin Cell Dev Biol 11: 77–92.
- Kuramoto, H., S. Tamura, S.Y. Notake (1972) Establishment of a cell line of human endometrial adenocarcinoma in vitro. Am J Obstet Gynecol 114: 1012–1019.
- Lampelo, S.A., A.P. Ricketts, D.W. Bullock (1985) Purification of rabbit endometrial plasma membranes from receptive and non-receptive uteri. J Reprod Fertil 75: 475–484.
- Lessey, B.A. (1998) Endometrial integrins and the establishment of uterine receptivity. Hum Reprod 13: 247–258.
- Lessey, B.A. (2000) Endometrial receptivity and the window of implantation. Baillières Best Pract Res Clin Obstet Gynaecol 14: 775–788.
- Lessey, B.A., A.J. Castelbaum, S.W. Sawin, J. Sun (1995) Integrins as markers of uterine receptivity in women with primary unexplained infertility. Fertil Steril 63: 535–542.
- Li, H.Y., S.P. Chang, C.C. Yuan, H.T. Chao, H.T. Ng, Y.J. Sung (2001) Nitric oxide induces extensive apoptosis in endometrial epithelial cells in the presence of progesterone: Involvement of mitogen-activated protein kinase pathways. Mol Hum Reprod 7: 755–763.
- Liu, S., J. Julian, D.D. Carson (1998) A peptide sequence of heparin/heparan sulfate (HP/HS)-interacting protein supports selective, high affinity binding of HP/HS and cell attachment. J Biol Chem 273: 9718–9726.
- Lopata, A (1996a) Implantation of the human embryo. Hum Reprod 11(suppl 1): 175–184.
- Lopata, A. (1996b) Blastocyst-endometrial interaction: An appraisal of some old and new ideas. Mol Hum Reprod 2: 519–525.
- Ludwig, M., W. Dettmann, H.E. Gaub (1997) AFM imaging contrast based on molecular recognition. Biophys J 72: 445–448.
- Luxford, K.A., C.R. Murphy (1992) Changes in the apical microfilaments of rat uterine epithelial cells in response to estradiol and progesterone. Anat Rec 233: 521–526.
- Martin, J.C., M.J. Jasper, D. Valbuena, M. Meseguer, J. Remohi, A. Pellicer, C. Simon (2000) Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression. Biol Reprod 63: 1370–1376.
- Merritt, J.E., W.P. Armstrong, C.D. Benham, T.J. Hallam, R. Jacob, A. Jaxa-Chamiec, B.K. Leigh, S.A. McCarthy, K.E. Moores, T.J. Rink (1990) SK&F 96365, a novel inhibitor of receptor-mediated calcium entry. Biochem J 271: 515–522.
- Meseguer, M., J.D. Aplin, P. Caballero-Campo, J.E. O’Connor, J.C. Martin, J. Remohi, A. Pellicer, C. Simon (2001) Human endometrial mucin MUC1 is up-regulated by progesterone and down-regulated in vitro by the human blastocyst. Biol Reprod 64: 590–601.
Morris, J.E., S.W. Potter (1990) An in vitro model for studying interactions between mouse trophoblasts and uterine cells; in Denker, H.-W., J.D. Aplin (eds): Trophoblast Invasion and Endometrial Receptivity. Novel Aspects of the Cell Biology of Embryo Implantation. Trophoblast Research. New York, Plenum Medical Book, vol 4, pp 51–69.
- Murphy, C.R. (2000) The plasma membrane transformation of uterine epithelial cells during pregnancy. J Reprod Fertil Suppl 55: 23–28.
- Nelson, W.J. (1992) Regulation of cell surface polarity from bacteria to mammals. Science 258: 948–955.
- Niklaus, A.L., C.R. Murphy, A. Lopata (1999) Ultrastructural studies of glycan changes in the apical surface of the uterine epithelium during pre-ovulatory and pre-implantation stages in the marmoset monkey. Anat Rec 255: 241–251.
- Niklaus, A.L., C.R. Murphy, A. Lopata (2001) Characteristics of the uterine luminal surface epithelium at preovulatory and preimplantation stages in the marmoset monkey. Anat Rec 264: 82–92.
- Olson, G.E., V.P. Winfrey, P.E. Matrisian, S.K. NagDas, L.H. Hoffman (1998) Blastocyst-dependent upregulation of metalloproteinase/disintegrin MDC9 expression in rabbit endometrium. Cell Tissue Res 293: 489–498.
- Pampfer, S., I. Donnay (1999) Apoptosis at the time of embryo implantation in mouse and rat. Cell Death Differ 6: 533–545.
- Paria, B.C., H. Lim, S.K. Das, J. Reese, S.K. Dey (2000) Molecular signaling in uterine receptivity for implantation. Semin Cell Dev Biol 11: 67–76.
Pattillo, R.A., A. Ruckert, R. Hussa, R. Bernstein, E. Delfs (1971) The JAR cell line – continuous human multihormone production and controls. In Vitro 6: 398.
- Perret, S., P. Dockery, B.J. Harvey (2001) 17β-Oestradiol stimulates capacitative Ca2+ entry in human endometrial cells. Mol Cell Endocrinol 176: 77–84.
- Raboudi, N., J. Julian, L.H. Rohde, D.D. Carson (1992) Identification of cell surface heparin/heparan sulfate binding proteins of a human uterine epithelial cell line (RL95). J Biol Chem 267: 11930–11939.
- Radmacher, M., R.W. Tillmann, M. Fritz, H.E. Gaub (1992) From molecules to cells – imaging soft samples with the AFM. Science 257: 1900–1905.
- Rhode, L.H., D.D. Carson (1993) Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell line (JAR) to a human uterine epithelial cell line (RL95). J Cell Physiol 155: 185–196.
- Rief, M., M. Gautel, F. Oesterhelt, J.M. Fernandez, H.E. Gaub (1997) Reversible unfolding of individual titin Ig-domains by AFM. Science 276: 1109–1112.
Rugar, D., P.K. Hansma (1990) Atomic force microscopy. Phys Today 43: 23–30.
- Salamonsen, L.A., E. Dimitriadis, L. Robb (2000) Cytokines in implantation. Semin Reprod Med 18: 299–310.
- Savagner, P. (2001) Leaving the neighborhood: Molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays 23: 912–923.
- Schlafke, S., A.C. Enders (1975) Cellular basis of interaction between trophoblast and uterus at implantation. Biol Reprod 12: 41–65.
- Selam, B., U.A. Kayisli, N. Mulayim, A. Arici (2001) Regulation of Fas ligand expression by estradiol and progesterone in human endometrium. Biol Reprod 65: 979–985.
- Simon, C., F. Dominguez, J. Remohi, A Pellicer (2001) Embryo effects in human implantation: Embryonic regulation of endometrial molecules in human implantation. Ann NY Acad Sci 943: 1–16.
- Sjaastad, M.D., W.J. Nelson (1997) Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium. Bioessays 19: 47–55.
- Tabibzadeh, S. (1998) Molecular control of the implantation window. Hum Reprod Update 4: 465–471.
- Terry, V., T.J. Shaw, C.D. Shorey, C.R. Murphy (1996) Actin-binding proteins undergo major alterations during the plasma membrane transformation in uterine epithelial cells. Anat Rec 246: 71–77.
Thie, M., H.-W. Denker (1997) Endometrial receptivity for trophoblast attachment: Model studies using cell lines; in Motta, P.M. (ed): Microscopy of Reproduction and Development. A Dynamic Approach. Rome, Antonio Delfino Editore S.r.l., pp 241–249.
- Thie, M., P. Fuchs, S. Butz, F. Sieckmann, H. Hoschützky, R. Kemler, H.-W. Denker (1996) Adhesiveness of the apical surface of uterine epithelial cells: The role of junctional complex integrity. Eur J Cell Biol 70: 221–232.
- Thie, M., B. Harrach-Ruprecht, H. Sauer, P. Fuchs, A. Albers, H.-W. Denker (1995) Cell adhesion to the apical pole of epithelium: A function of cell polarity. Eur J Cell Biol 66: 180–191.
Thie, M., C. Heneweer, L.H. Kruse, M. Schmidt, H.-W. Denker (2002) Function of Rho protein in the process of trophoblast adhesion – in vitro studies on signalling mechanisms in human uterine epithelial RL95–2 cells. Verh Anat Ges 97, Ann Anat Suppl 184: 172–173.
- Thie, M., P. Herter, H. Pommerenke, F. Dürr, F. Sieckmann, B. Nebe, J. Rychly, H.-W. Denker (1997) Adhesiveness of the free surface of a human endometrial monolayer for trophoblast as related to actin cytoskeleton. Mol Hum Reprod 3: 275–283.
- Thie, M., R. Röspel, W. Dettmann, M. Benoit, M. Ludwig, H.E. Gaub, H.-W. Denker (1998) Interaction between trophoblast and uterine epithelium: Monitoring of adhesive forces. Hum Reprod 13: 3211–3219.
Thiery, J.P., D. Chopin (1999) Epithelial cell plasticity in development and tumor progression. Cancer Metastasis Rev 18: 31–42.
- Tinel, H., H.-W. Denker, M. Thie (2000) Calcium influx in human uterine epithelial RL95-2 cells triggers adhesiveness for trophoblast-like cells. Model studies on signalling events during embryo implantation. Mol Hum Reprod 6: 1119–1130.
- Triggle, D.J. (1999) The pharmacology of ion channels: With particular reference to voltage-gated Ca2+ channels. Eur J Pharmacol 375: 311–325.
- von Rango, U., I. Classen-Linke, C.A. Krusche, H.M. Beier (1998) The receptive endometrium is characterized by apoptosis in the glands. Hum Reprod 13: 3177–3189.
- Wang, J., D.R. Armant (2002) Integrin-mediated adhesion and signaling during blastocyst implantation. Cells Tissues Organs 172: 190–201.
- Way, D.L., D.S. Grosso, J.R. Davis, E.A. Surwit, C.D. Christian (1983) Characterization of a new human endometrial carcinoma (RL95-2) established in tissue culture. In Vitro 19: 147–158.
Wild, M.K., K. Lühn, T. Marquardt, D. Vestweber (2002) Leukocyte adhesion deficiency II: Therapy and genetic defect. Cells Tissues Organs, 172: 161–173.
- Wodarz, A. (2001) Cell polarity: No need to reinvent the wheel. Curr Biol 11: R975–978.
- Yeaman, C., K.K. Grindstaff, M.D. Hansen, W.J. Nelson (1999a) Cell polarity: Versatile scaffolds keep things in place. Curr Biol 9: R515–517.
- Yeaman, C., K.K. Grindstaff, W.J. Nelson (1999b) New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol Rev 79: 73–98.
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