Integrin trafficking regulates adhesion to fibronectin during differentiation of mouse peri‐implantation blastocysts

Abstract

Trophoblast cells of the peri-implantation blastocyst differentiate from a polarized epithelium, the trophectoderm, into invasive cells having an apical surface occupied by integrins that mediate adhesion to the extracellular 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 differentiation was unaffected by transcriptional inhibition with alpha-amanitin, 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 only when added during the first 24 h of blastocyst culture, indicating that proteins required for adhesion to fibronectin were synthesized at least 24 h before blastocyst differentiation was completed. Since blastocyst differentiation did not appear to be regulated temporally by gene expression, the possible role of protein trafficking was investigated 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 fibronectin 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 alpha llb beta 3, but not alpha4 which recognizes the lllCS site. Trafficking of alpha 5 beta 1, and possibly other integrins, to the apical surface of trophoblast cells appears to be a critical step in the differentiation of the mouse blastocyst to an invasive phenotype.

A new microfluidic concept for successfulin vitroculture of mouse embryos

“…3). This assay mimics the natural mechanism of adhesion of trophoblasts on the blastocysts to the endometrium which is regulated by interaction between the integrins naturally expressed on endometrial cells and on the apical surface of competent blastocysts [59][60]. By assessing the extension and adhesion of the hatched blastocysts it is possible to evaluate their developmental potential.…”

Forced Suppression of let-7a-5p in Mouse Blastocysts Improves Implantation Rate

“…Previous research has demonstrated that Integrin-b3 is a target of let-7a in the human HeLa cell line and in mouse blastocysts (11). Integrins are a family of transmembrane adhesion receptors and are primarily responsible for regulating the interactions between the trophoblasts and the extracellular matrix molecules of the endometrium (31). They are heterodimers composed of non-covalently associated α and β subunits that engage with extracellular matrix proteins and couple to intracellular signalling and cytoskeletal complexes (32).…”

Embryo–epithelium interactions during implantation at a glance

“…Integrins αvβ3, α5β1 and α6Bβ1 are present in the early and late mouse blastocyst, whereas the α2, α6A and α7 subunits appear in the late blastocyst, and α1 appears only in outgrowths (Sutherland et al, 1988(Sutherland et al, , 1993; see also Box 2). In mice, binding of fibronectin, vitronectin or OPN to the blastocyst stimulates the trafficking of integrins to the mouse trophectodermal surface and the assembly of focal contact-like structures, as indicated by the presence of the focal adhesion markers vinculin and talin (Chaen et al, 2012;Schultz and Armant, 1995;Schultz et al, 1997;Yelian et al, 1995). Integrin signalling modulates trophoblast adhesion through the activation of phospholipase Cγ to initiate phosphoinositide signalling and intracellular Ca 2+ mobilisation Wang et al, 2007).…”