Estrogen and progesterone secretion by isolated cultured porcine thecal and granulosa cells.

Abstract

Porcine follicular granulosa and theca interna cells were cultured singly and in combination. Both cell types were isolated from a pool of the same proestrous large follicles. Cultures were maintained for 8 days in culture medium 199 enriched with 10% calf serum and the culture medium was changed every second day. In half of the cultures, testosterone, in a concentration of 150 ng/ml (0.5 × 10−7 M), was added to the culture medium. The conditioned culture media subsequently were assayed for estrogen, progestogen and androgen using radioimmunoassay. Unless stated otherwise, data are expressed as pg/105 cells/48 h in the case of estrogen and androgen and ng/105 cells/48 h in the case of progesterone. Each culture was replicated 4 times and the data shown is the mean ± SEM of 4 determinations. Theca interna cells cultured alone secreted more estrogen 473 ± 43.9 pg than granulosa cells which secreted 34 ± 3.4 pg, (P<0.001). Mixed cultures of theca and granulosa secreted the largest amount of estrogen, 754 ± 5.8 pg, (P<0.001) versus theca alone and (P<0.001) versus granulosa alone. Theca and granulosa cultured alone secreted 81.8 ± 4.7 pg and 40.9 ± 1.6 pg androgen, respectively, and the mixed culture of theca and granulosa secreted 110 ± 5.7 pg androgen. These differences were true also if the secretion rate was estimated over the entire 8-day culture period. Granulosa cells cultured alone secreted the greatest amount of progesterone and the elevated secretion rate was maintained during the entire 8-day culture period (1222 ± 125, 1033 ± 79, 894 ± 43, 234 ± 26 ng per culture/day on Days 2,4,6 and 8, respectively). After 48 h granulosa cells secreted 113.9 ± ng progesterone, whereas thecal cells secreted only 0.7 ng progesterone/105 cells. The addition of testosterone to the culture medium brought about a marked increase of estrogen secretion not only by theca interns cells but also by granulosa and by granulosa plus thecal cell cultures as well. Testosterone also led to an increase in progesterone secretion by granulosa cells cultured alone and by theca cultured alone. In cultures of theca plus granulosa, addition of testosterone led to suppression (P<0.001) of progesterone secretion in comparison with granulosa cells cultured alone.