Maintenance of oestradiol production and expression of cytochrome P450 aromatase enzyme mRNA in long-term serum-free cultures of pig granulosa cells

Studies were carried out to investigate the conditions required for mainten ance of aromatase activity and expression in long-term cultures of pig gran ulosa cells. Cells from large (> 2 mm) and small (less than or equal to 2 m m) follicles were cultured at 37 degrees C with 5% CO2 in McCoys 5a medium supplemented with 0.1% (w/v) BSA, testosterone (100 mu g l(-1)), insulin (1 0 mu g l(-1)) and long R3 insulin-like growth factor I (IGF-I) (100 mu g l( -1)). Cells were cultured with five concentrations of USDA pFSH-I-2 (0-100 mu g l(-1)) for 48, 96 or 144 h with or without fetal calf serum (FCS). The number of cells and oestradiol, progesterone and inhibin production were m easured. In marked contrast to oestradiol production from cells cultured in plates precoated with FCS, 1 mu g FSH l(-1) was optimal for the maintenanc e of high oestradiol production by granulosa cells from large follicles aft er 144 h of serum-free culture. Culture with FCS promoted cell proliferatio n, reduced oestradiol production, and supported FSH-dependent (P < 0.01) in creased progesterone and inhibin production indicating cellular luteinizati on. Northern blot analysis of total RNA from cells cultured with 1 mu g FSH l(-1) detected 2.5 and 1.8 kb transcripts encoding aromatase cytochrome P4 50 (P450(arom)) and cholesterol side-chain cleavage cytochrome P450 (P450(s cc)), respectively. Transcript expression was hormone sensitive, irrespecti ve of the presence of FCS. High concentrations of FSH (100 mu g l(-1)) stim ulated expression of P450(scc), but inhibited P450(arom) expression as the cells luteinized after 144 h of culture. This serum-free system, which main tains the aromatase enzyme complex, is fundamental if physiologically relev ant observations are to be made of the mechanisms regulating follicle hiera rchy development from long-term cultures of pig cells.