Ovine anterior pituitary production of follistatin in vitro

Rat pituitary cells express messenger RNA for the activin-binding protein, follistatin (FS), and rat and bovine pituitary cell cultures secrete FS into the medium. In the present study, a previously validated, heterologous RIA for ovine FS was employed to investigate FS synthesis, secretion, and regulation in cultures of ovine anterior pituitary cells. The validity of the RIA was confirmed by the finding that FS immunoreactivity in ovine pituitary cell culture-conditioned medium diluted in parallel with purified bovine FS, and fractionation of the conditioned medium resulted in the coelution of activin-binding activity with the FS immunoactivity. The concentration of endogenous ovine FS achieved in the culture medium (0.08-0.6 nM) was in the range over which bovine FS suppresses FSH secretion in these cultures (IC50 = 0.5 nM). To characterize the relationship between endogenous FS and FSH secretion, dispersed ovine pituitary cells were preincubated with 10% fetal bovine serum for 2 days, then cultured between days 2-5 in the presence of a chemically defined serum substitute. Under these conditions, FS was continuously secreted at a rate of 12.1 +/- 1.8 ng/10(6) cells.day (mean +/- SEM; n = 18), whereas FSH was secreted at 64 +/- 13 ng/10(6) cells.day (n = 7). The secretion of FS and FSH changed in a reciprocal way as culture conditions were altered either by maintaining exposure of the cells to fetal bovine serum or by plating the cells at a 6- to 10-fold higher seeding density. Under the latter circumstance, for instance, FS secretion during the 3-day test period decreased to 47 +/- 14% (n = 10) and FSH secretion increased to 137 +/- 6% (n = 6) of the respective values in cultures of dispersed cells. FS secretion was increased nearly 3-fold (P < 0.05) in a dose-dependent manner by continuous exposure of ovine pituitary cells between days 2-5 to recombinant human activin A (1-10 nM), which concomitantly increased FSH secretion. Recombinant human inhibin A (0.003-10 nM); the synthetic glucocorticoids, RU28362 and dexamethasone (each 1-100 nM); the sex steroids, testosterone (1-100 nM), 17 beta-estradiol (0.001-5 nM), and progesterone (4-2500 nM); and the vitamin A derivative, retinoic acid (0.3-32 microM), each inhibited FSH secretion from these cultures, but only the last agent significantly (P < 0.05) increased FS secretion. Inhibin prevented the stimulation of FSH secretion by activin A without affecting its stimulation of FS secretion.