PRODUCTION OF EXTRACELLULAR-MATRIX, FIBRONECTIN AND STEROIDOGENIC ENZYMES, AND GROWTH OF BOVINE GRANULOSA-CELLS IN ANCHORAGE-INDEPENDENT CULTURE

A proportion of the granulosa cells from bovine antral follicles will survive, like stem cells, in anchorage-independent culture. To study t hese cells, bovine granulosa cells were isolated from medium-sized fol licles (3-5 mm), plated out (in aliquots of 2.5x10(4) viable cells) on to a 1 mL agar base, and overlaid with 1 mt of methycellulose solution in culture medium (control). The cells were cultured (14 days) and th en processed for histology (n = 14) or Western immunoblotting (n = 5). Under control conditions or after treatment with basic fibroblast gro wth factor (bFGF; 50 ng mL(-1)), a proportion of the granulosa cells d ivided to produce colonies; individual cells remained small. bFGF incr eased the number of cells harvested (15.8+/-7.3-foId, as measured indi rectly by the relative amount of the nuclear La antigen), increased th e average diameter of the colonies from 88.9+/-13.5 mu m to 136.5+/-4. 9 mu m and stimulated the production of fibronectin 5.7+/-1.5-fold (P < 0.05). An extracellular matrix, which has previously been shown to b e a basal lamina, was observed in 19.1% of the colonies (total of 350 colonies examined; n = 8 experiments). Cells treated with dibutyryl cA MP (1 mM) hypertrophied and had 50+/-28.7-fold and 102.6+/-55.8-fold h igher levels of cholesterol side-chain cleavage cytochrome P450 (P < 0 .001) and 3 beta-hydroxysteroid dehydrogenase (P < 0.01) respectively (n = 5). Thus, granulosa cells with characteristics of stem cells can divide and produce extracellular matrix, or be induced to differentiat e when in culture without anchorage.