Interactions between follicle-stimulating hormone and growth factors in modulating secretion of steroids and inhibin-related peptides by nonluteinized bovine granulosa cells

The aim was to investigate potential interactions between FSH and intraovar ian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E-2), and progesterone (P-4) by bovin e granulosa cells cultured under conditions in which a nonluteinized FSH-re sponsive phenotype is maintained. Cells from 4- to 6-mm follicles were cult ured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10(-7) M), and effects of ovine FSH (0.037-3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2-50 ng/ml) and epidermal growth factor (EGF; 0.1-10 ng/ml). Medium was ch anged every 48 h and cultures ended after 144 h, when cell number was deter mined. Between 48-96 h and 96-144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E-2 (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses f or each of the above hormones, whereas P-4 output was maximal (3-fold) at t hese doses. FSH promoted a slight increase in cell number (similar to1.7-fo ld; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of i nh A (8-fold), act A (41-fold), FS (12-fold), and E-2 (18-fold); this was a ccompanied by modest increases (P < 0.01) in P-4 output (similar to2.5-fold ) and cell number (similar to2-fold). Whereas FSH enhanced inh A, act A, FS , and E-2 secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold incr ease in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E- 2. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A: FS ratio (similar to3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-f old; P < 0.001), suggesting that both factors selectively raise activin "to ne" and that this could be a key requirement for FSH and IGF-induction of f ollicular E-2 production. This hypothesis was reinforced by the finding tha t addition of FS, to reduce the act A:FS ratio and sequester secreted activ in, markedly suppressed (P <less than> 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E-2 output.