BASAL LAMINA AND OTHER EXTRACELLULAR-MATRIX PRODUCED BY BOVINE GRANULOSA-CELLS IN ANCHORAGE-INDEPENDENT CULTURE

Bovine granulosa cells from 3-7 mm follicles were cultured without anc horage in soft agar/methylcellulose solution for 14 days, with or with out 50 ng/ml basic fibroblast growth factor. The granulosa cells divid ed to form colonies of cells. These were analysed by light and electro n microscopy, immunohistochemistry and Western immunoblotting. In appr oximately 20% of the colonies extracellular matrix was clearly visible at the light-microscope level. Ultrastructurally the matrix resembled a basal lamina 30-100 nm thick and was composed of tangled fibres or cords. Unidentified spherical structures of less than 50 nm diameter w ere sometimes present and attached to this basal lamina. The basal lam ina of follicles had similar features, except that the basal lamina pr oduced in vitro was a large aggregate of many convoluted layers. The c ells produced collagen type IV and the cellular form of fibronectin. I ntercellular areas not associated with basal lamina were identified. R uthenium red staining revealed these areas to be rich in proteoglycan granules. Free granules were clustered near the cell surface, and the lumina of these areas were rich in fibres decorated with ruthenium red . This material did not resemble follicular fluid of antral follicles. Thus, granulosa cells in anchorage-independent cultures have a follic ular cell morphology and secrete two distinct extracellular matrices, one similar to the follicular basal lamina.