Ganglioside inhibition of attachment and differentiation of cultured rat granulosa cells: interactions with fibronectin.

Abstract

The involvement of fibronectin in the attachment and differentiation of rat granulosa cells, cultured in a chemically defined medium, was investigated using the inhibitory properties of mixed brain gangliosides (MBGs) and highly purified disialoganglioside, GD1a. MBGs inhibited cell attachment to plastic culture surfaces in a concentration-dependent manner, with 0.1 mmol l-1 causing significantly decreased attachment between 0.5 and 24 h of incubation. Inhibition of attachment to a fibronectin-coated substratum was even greater. The inhibitory effect of MBGs was not caused by binding to the cell surface, but instead the inhibitory factor(s) were adsorbed on a surface of immobilized human plasma fibronectin, thereby preventing cell attachment to this surface. The inhibitory action of MBGs was also neutralized by the addition of soluble fibronectin. Furthermore, at least one component of MBGs, detected chemically following thin-layer chromatography, was directly shown to bind to human fibronectin. MBGs inhibited to varying degrees the follicle-stimulating hormone(FSH)-dependent responses: augmentation of cellular protein content, production of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and progestins (progesterone + 20 alpha-hydroxypregn-4-en-3-one + pregnenolone), and induction of aromatase activity. These inhibitory activities of MBGs could not be eliminated by adsorption on immobilized fibronectin or reversed by addition of soluble fibronectin, thus distinguishing these actions from the early inhibition of cell attachment. FSH-dependent responses were also inhibited by GD1a, while responses to stimulation by dibutyryl cyclic AMP plus 3-isobutyl-1-methyl xanthine were less affected by this ganglioside. These results suggest that gangliosides inhibit attachment of granulosa cells in culture by binding to fibronectin, whereas the inhibition of FSH-dependent differentiation occurs by other modes of action that are unrelated to the effects on cell adhesion.