title: Production of extracellular matrix, fibronectin and steroidogenic enzymes, and growth of bovine granulosa cells in anchorage-independent culture.
abstract: A proportion of the granulosa cells from bovine antral follicles will survive, like stem cells, in anchorage-independent culture. To study these cells, bovine granulosa cells were isolated from medium-sized follicles (3-5 mm), plated out (in aliquots of 2.5 x 10(4) viable cells) onto a 1 mL agar base, and overlaid with 1 mL of methycellulose solution in culture medium (control). The cells were cultured (14 days) and then processed for histology (n = 14) or Western immunoblotting (n = 5). Under control conditions or after treatment with basic fibroblast growth factor (bFGF; 50 ng mL-1), a proportion of the granulosa cells divided to produce colonies; individual cells remained small. bFGF increased the number of cells harvested (15.8 +/- 7.3-fold, as measured indirectly by the relative amount of the nuclear La antigen), increased the average diameter of the colonies from 88.9 +/- 13.5 microns to 136.5 +/- 4.9 microns and stimulated the production of fibronectin 5.7 +/- 1.5-fold (P < 0.05). An extracellular matrix, which has previously been shown to be a basal lamina, was observed in 19.1% of the colonies (total of 350 colonies examined; n = 8 experiments). Cells treated with dibutyryl cAMP (1 mM) hypertrophied and had 50 +/- 28.7-fold and 102.6 +/- 55.8-fold higher 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 differentiate when in culture without anchorage.
authors.affiliation: Department of Medicine, Flinders University of South Australia, Bedford Park, Australia.
authors.forename: R J
authors.initials: RJ
authors.lastname: Rodgers
authors.forename: C A
authors.initials: CA
authors.lastname: Vella
authors.forename: H F
authors.initials: HF
authors.lastname: Rodgers
authors.forename: K
authors.initials: K
authors.lastname: Scott
authors.forename: T C
authors.initials: TC
authors.lastname: Lavranos
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002417:Cattle; D002448:Cell Adhesion; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D003470:Culture Media; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005353:Fibronectins / Q000096:biosynthesis*; D006107:Granulosa Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D013256:Steroids / Q000096:biosynthesis*
chemical_list: D003470:Culture Media; D005353:Fibronectins; D013256:Steroids; D016222:Fibroblast Growth Factor 2; D003994:Bucladesine; D015096:3-Hydroxysteroid Dehydrogenases; D002786:Cholesterol Side-Chain Cleavage Enzyme