PARENT SESSION
Implantation & Early Development

REORGANIZATION OF THE ACTIN CYTOSKELETON DURING FIBRONECTIN-MEDIATED UP REGULATION OF TROPHOBLAST ADHESION IN THE PERI-IMPLANTATION MOUSE BLASTOCYST.

Armant, D. Randall¹, Wang, Jun¹, ¹

ABSTRACT- Blastocyst implantation requires optimal interaction between trophoblast cells and the endometrial extracellular matrix. The apical surfaces of newly formed mouse trophoblast cells do not adhere to fibronectin (FN) until they develop to an adhesion-competent stage. However, the apical surfaces of adhesion-competent trophoblast cells express strong FN-binding activity only after exposure to FN for 60 min. Ligand-mediated up regulation of FN-binding activity is blocked by cytochalasin D, suggesting a role for cytoskeletal reorganization in achieving strong integrin-mediated adhesion during implantation. The intracellular integrin-binding protein, paxillin, was detected near the apical surface of trophoblast cells by immunofluorescent staining and confocal microscopy. Detergent extraction prior to fixation of blastocysts cultured in the absence of FN markedly reduced paxillin staining, suggesting a limited association between integrin complexes and filamentous actin. Detergent sensitivity increased progressively during the first 30 min after FN exposure, but as strong FN-binding activity was achieved at 60 min, paxillin became predominantly detergent resistant. Paxillin dissociation from microfilaments may reflect cytoskeletal reorganization, leading after 60 min to the tethering of an integrin complex containing paxillin to actin fibers. Blastocysts treated with FN for 60 min were unable to bind FN in the presence of 50 g/ml cytochalasin D, demonstrating that integrin complexes need to associate with the cytoskeleton in order to mediate trophoblast adhesion. The requisite cytoskeletal reorganization may be dependent on small rho-like GTPases that can be inhibited in preimplantation embryos by treatment with C3 transferase. FN-mediated up regulation of adhesion was blocked and integrin-mediated intracellular Ca²⁺ signaling was prevented in blastocysts pretreated for 1 h with 5 g/ml C3 transferase. We conclude that the conversion of trophoblast integrins from weak to strong binding requires rho-like GTPase activity that mediates the reorganization of linkages between filamentous actin and protein complexes associated with the cytoplasmic domains of integrins. Supported by NIH grant HD36764

KEY WORDS: implantation , extracellular matrix, cytoskeleton, adhesion