Regulation of cytoskeletal proteins involved in cell contact formation during differentiation of granulosa cells on extracellular matrix

Abstract

The organization and the expression of cytoskeletal proteins involved in determining cell contact and shape were analyzed in granulosa cells during their differentiation on extracellular matrix (ECM)-coated culture plates. Rat granulosa cells from preovulatory follicles displayed an epithelial shape on ECM and formed multilayered aggregates with numerous gap junctions between neighboring cells. These cells had few actin cables and often only a diffuse pattern of actin and a low amount of vinculin in very thin focal adhesion sites. In contrast, cells grown on plastic formed a monolayer of flat cells with a reduced number of gap junctions but with numerous stress fibers and abundant large vinculin-containing focal contacts. On ECM, the cells were stimulated to produce high levels of progesterone, while only trace amounts of the steroid accumulated in cells on plastic dishes. Two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cells revealed a dramatic decrease in vinculin, α-actinin, and actin synthesis in cells grown on ECM, as compared to cells grown on plastic, while the synthesis of the tubulins and of the intermediate filament protein vimentin remained unchanged. RNA blot analysis showed a marked decrease in actin mRNA levels in cells from ECM plates, while the level of tubulin mRNA remained essentially unchanged. It is concluded that the differentiation of granulosa cells on ECM in vitro is associated with changes in cell shape and cell contacts and that such changes in cell morphology are accompanied by simultaneous alterations in the organization and expression of cytoskeletal proteins that are involved in determining these cellular structures.