Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth

Abstract

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits alpha 3, alpha 5, alpha IIb, alpha v, beta 1 and beta 3, but not alpha 4, all bound to trophoblast cells. Antibodies raised against either the beta 1 or beta 3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, alpha 3 beta 1, alpha 5 beta 1, alpha IIb beta 3, and alpha v beta 3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation.

Decreased expression of fibroblast growth factor-4 and associated dysregulation of trophoblast differentiation in mouse blastocysts exposed to high D-glucose in vitro

“…It has been shown to promote the attachment of mouse blastocysts and outgrowth of the trophoblast cells in vitro [18]. Several receptors of the integrin family are expressed by the mouse blastocyst which recognize a specific site of fibronectin and their interaction is supposed to mediate trophoblast cell migration [19]. Other ECM molecules (specially, laminin) are found in the endometrium and have been used as in vitro substrates for studies of blastocyst outgrowth.…”

Adhesion molecules in implantation

“…Subunit α3 appears from the eight-cell stage, while α2, α6A and α7 all appear at the late blastocyst stage. Localization has been reported of integrins α1, α3, α5, αIIb, βv, β1 and β3 in trophoblast outgrowths in vitro (Sutherland et al, 1993;Yelian et al, 1995). Both αv and β1 integrins can be found in focal adhesions at the spreading margins of trophoblast.…”

“…Correspondingly, hatched blastocysts readily attach and outgrow in an integrin-dependent process on a variety of surfaces containing ligands found in the endometrial stroma of pregnancy. These include fibronectin, collagens, laminins, entactin, vitronectin, thrombospondin and Matrigel (basement membrane-like) matrix (Armant et al, 1986;O'Shea et al, 1990;Stephens et al, 1995;Yelian et al, 1995). In mice, local maternal epithelial retraction and apoptosis follow attachment (Denker, 1990), whereas in humans, evidence from attachment experiments in vitro suggests protrusive penetration of trophoblast through the epithelium (Lindenberg et al, 1986).…”

Secreted phosphoprotein 1 (osteopontin) is expressed by stromal macrophages in cyclic and pregnant endometrium of mice, but is induced by estrogen in luminal epithelium during conceptus attachment for implantation

“…While a clear role for avb3 in mouse implantation has not been defined, it is possible that its ligation, perhaps to luminal Spp1, leads to apical translocation of a5b1 for stable conceptus attachment through fibronectin (Sutherland et al 1993, Wang et al 2000, Armant 2005. Indeed, avb3, a5b1, and aIIbb3 appear in focal adhesions formed in trophoblast cells during outgrowth on fibronectin (Sutherland et al 1993, Yelian et al 1995, Wang et al 2000. Further, trophoblast binding is attenuated by antibodies to the subunits av and b3 (Schultz & Armant 1995, Illera et al 2000, Rout et al 2004.…”

“…Indeed, avb3, a5b1, and aIIbb3 appear in focal adhesions formed in trophoblast cells during outgrowth on fibronectin (Sutherland et al 1993, Yelian et al 1995, Wang et al 2000. Further, trophoblast binding is attenuated by antibodies to the subunits av and b3 (Schultz & Armant 1995, Illera et al 2000, Rout et al 2004. Finally, it appears that unoccupied avb3 transmits a positive cellular death signal and ligand interaction with this integrin protects cells from apoptosis (Zhao et al 2005).…”