INTEGRIN TRAFFICKING REGULATES ADHESION TO FIBRONECTIN DURING DIFFERENTIATION OF MOUSE PERIIMPLANTATION BLASTOCYSTS

Trophoblast cells of the peri-implantation blastocyst differentiate fr om a polarized epithelium, the trophectoderm, into invasive cells havi ng an apical surface occupied by integrins that mediate adhesion to th e extracelluar matrix. Blastocyst differentiation was assessed during serum-free culture using a fibronectin-binding assay with intact mouse blastocysts. Fibronectin binding activity became elevated during a 24 -h ''window'' after approximately 72 h of culture. Blastocyst differen tiation was unaffected by transcriptional inhibition with alpha-amanit in, however, exposure of cavitating morulae to the drug significantly delayed the onset of maximal fibronectin-binding activity. Inhibition of de novo protein synthesis with cycloheximide delayed development on ly when added during the first 24 h of blastocyst culture, indicating that proteins required for adhesion to fibronectin were synthesized al least 24 h before blastocyst differentiation was completed. Since bla stocyst differentiation did not appear to be regulated temporally by g ene expression, the possible role of protein trafficking was investiga ted using the inhibitor, brefeldin A. Brefeldin A caused a reversible, dose-dependent decrease in fibronectin-binding activity when added to the culture medium between 48 and 72 h of culture. During the period of brefeldin A sensitivity, alpha(5) beta 1 integrin, a major fibronec tin receptor, translocated to the apical surface of trophoblast cells, as determined by immunohistochemistry and confocal microscopy. Mouse blastocysts expressed other integrins that recognize the central cell- binding domain of fibronectin, including the alpha(v) integrins and al pha(llb)beta(3), but not alpha(4) which recognizes the IIICS site. Tra fficking of alpha(5) beta(1), and possibly other integrins, to the api cal surface of trophoblast cells appears io be a critical step in the differentiation of the mouse blastocyst to an invasive phenotype. (C) 1997 Wiley-Liss, Inc.