Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were c ultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibron ectin-coated plates with or without androstenedione to define the time cour se profile of oestradiol and progesterone secretion, and the possible modul ator role of androstenedione and fibronectin during FSH-induced rabbit gran ulosa cell differentiation. Every 48 h, cultures were photographed and samp les of medium were collected and assayed by ELISA for oestradiol and proges terone. FSH increased oestradiol secretion in a dose-dependent manner. Andr ostenedione augmented FSH-stimulated oestradiol secretion, and led to a dec rease in secretion of oestradiol with time in culture. FSH stimulated proge sterone secretion in a dose-dependent manner. This was increased by androst enedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH -stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fi bronectin prevented this decrease, without affecting oestradiol or progeste rone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodi fferentiation and reaggregation of granulosa cells. Androstenedione appeare d to act synergistically with FSH to promote steroidogenesis. Fibronectin s ustained progesterone secretion during differentiation.