Inhibin Production by Primary Sertoli Cell-Enriched Cultures: Regulation by Follicle-Stimulating Hormone, Androgens, and Epidermal Growth Factor*

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Abstract
Inhibin levels in the serum-free media of primary Sertoli cell-enriched (SCE) cultures were studied as a function of time and hormonal treatment. SCE cultures were established from 20-day-old rats and maintained in SF media supplemented with insulin, transferrin, epidermal growth factor (EGF), and bacitracin. Radioimmunoassayable inhibin was measured using an antibody directed against a synthetic porcine inhibin-.alpha. (PI.alpha.) and measured using this same synthetic peptide as well as a highly purified ovine inhibin standard. Results of these determinations are expressed in terms of a synthetic peptide as femtomoles of pI.alpha.-(1-26)-G-Y-per ml/106 cells. Inhibin levels (.+-.SEM) that accumulated at this rate in media from control cultures were 184.9 .+-. 6.1 and 167.4 .+-. 5.2 on days 0-4 and 4-8, respectively. When FSH (oFSH 17; 1-100 ng/ml) was added, a dose-dependent increase in inhibin levels was significant at all time points beyond the initial 24 h. The simultaneous addition of 2 .times. 10-7 M testosterone (T) with low doses of FSH suppressed the inhibin response to FSH, but when T was combined with 300 ng/ml FSH, there was no effect of T on inhibin levels compared to FSH alone, regardless of time in culture. In spite of the modest effect of T, androstenedione (A; 2 .times. 10-13 to 2 .times. 10-5 M) produced a dose-dependent suppression of inhibin levels. This inhibition was also observed at all doses of FSH. The action of A was not due to its conversion to estrogens, as 17.beta.-estradiol had no effect on inhibin production by SCE cultures in either the presence or absence of FSH. The effect of EGF, a component of the basal serum-free medium, was next examined; it produced a 1.5-fold higher level of inhibin (188.7 .+-. 9.5) compared to cultures without EGF (135.6 .+-. 5.0). When SCE cultures were plated with FSH plus EGF, the stimulation of inhibin levels was additive. We conclude that in Sertoli cell cultures established from immature rats (1) the accumulation of inhibin in medium declines from 90 fmol/106 cells .cntdot. day (initial 24 h) to 40-50 fmol/106 cells .cntdot. day (over the first 48 h) and continues to accumulate in the medium for 8 days of culture; (2) FSH regulates the production of inhibin by Sertoli cells; the best dose response is observed over a 3-day period; (3) has a more marked suppressive effect on inhibin accumulation than does T; and (4) EGF stimulates basal inhibin production, and its effects are additive with those of FSH.