Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture

The regulation of pig theca cell steroidogenesis was studied by the develop ment of a physiological serum-free culture system, which was subsequently e xtended to investigate potential theca-granulosa cell interactions. Theca c ells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150 x 10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis we re examined. The purity of the theca cell preparation was verified biochemi cally and histologically. Go-cultures contained 50 x 10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa c ells only were supplemented with 'physiological' doses of androstenedione o r 100 ng ml(-1). Oestradiol production by cocultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured sep arately. Oestradiol and androstenedione production continued throughout cul ture. High plating density decreased steroid production (P < 0.01). LH incr eased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and t he sensitivity of the cells increased with time in culture. Oestradiol prod uction was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Go-cultures produced more oestradiol th an the sum of oestradiol synthesized by theca and granulosa cells cultured. separately (P < 0.001), irrespective of the androstenedione dose. This ser um-free culture system for pig theca cells maintained in vivo steroidogenes is and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by L H and IGF-I, respectively. Theca-granulosa cell interactions stimulated oes tradiol synthesis and this interaction was mediated by factors additional t o the provision of thecal androgen substrate to granulosa cells.