Immunostainable inhibin alpha-subunit has been demonstrated in rat testes in a pattern consistent with localization in Sertoli cells. In the present study the distribution of alpha-subunit immunostaining was compared to those of beta-A- and beta-B-subunits. Immunostaining of alpha-subunit was present in the seminiferous epithelium of fetal, neonatal, pubertal, and adult rats as well as in Sertoli cells in culture. The distribution of inhibin beta-B-subunit immunostaining in this epithelium was consistent with Sertoli cell localization similar to that of the alpha-subunit. The predominant staining with antibodies against the beta-A-subunit was in nuclei of immature germ cells around the periphery of each seminiferous tubule. The most probable localization of this staining was in the nuclei of pachytene and zygotene spermatocytes. Specific immunostaining with beta-A-subunit antiserum was also evident in the seminiferous epithelium adjacent to the tubular lumen. Immunoreactive alpha- and beta-A-subunit staining was present in a Leydig cell line, and beta-A immunoreactivity was present in interstitial cells of neonatal rat testes. After hypophysectomy, inhibin alpha-subunit immunostaining decreased, beta-A-subunit staining did not change, and beta-B-subunit staining increased. We conclude that immunoreactive inhibin subunits are present in multiple cells in the testis and that the amounts of immunostainable subunits in the seminiferous epithelium are differentially regulated.

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-a (pla) 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 pIa-(l-26)-G-Y-per ml/10 6 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; 1-1000 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 X 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 X 10" 13 to x 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 17/3-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/10 6 cells-day (initial 24 h) to 40-50 fmol/10 6 cells-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. (Endocrinology 122: [717][718][719][720][721][722][723][724][725] 1988)

Circulating inhibin alpha concentrations were determined in infant, juvenile, and adult male rhesus monkeys with a RIA employing antisera to a synthetic fragment of the alpha-subunit of porcine inhibin. Binding of tracer, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-125I-Tyr, to antibody was inhibited by standard, [DSer1,Nle5]human inhibin alpha(1-25)-Gly-Tyr. and by plasma from adult male monkeys in a parallel fashion. Castration in adults resulted in a 5-fold decline in the levels of immunoreactivity in plasma. Mean (+/- SE) plasma inhibin alpha concentrations in infants and adults (322.9 +/- 51.9 and 460.1 +/- 43.9 pg/ml, respectively) were significantly higher (P less than 0.05) than those in juveniles (191.3 +/- 28.3 pg/ml). Moreover, initiation of puberty in juvenile males, 13-18 months of age, with a chronic (10- to 12-week) intermittent iv infusion of GnRH (0.1 microgram/min for 3 min every 3 h) resulted in a progressive rise in circulating inhibin alpha that plateaued, after 5 weeks of pituitary stimulation, at concentrations (343.9 +/- 38.2 pg/ml) comparable to those of infants and adults and twice those observed before initiation of the pulsatile infusion of GnRH. Circulating FSH concentrations increased during the first week of GnRH stimulation from 2.7 +/- 0.1 ng/ml before treatment to 6.0 +/- 1.2 ng/ml, where they remained for the duration of the experiment. Testosterone secretion during the initiation of precocious puberty occurred in discrete episodes that were robustly correlated with GnRH-induced LH discharges. In contrast, changes in circulating inhibin alpha concentrations over the 3-h interval between GnRH pulses were unremarkable. Activation of Sertoli and Leydig cells during initiation of puberty in the juvenile males, as reflected by circulating inhibin alpha and testosterone concentrations, respectively, occurred with similar time courses. At the time of orchidectomy, 10-12 weeks after initiation of GnRH treatment, testicular tissue was prepared for histological examination. In spite of a 2-fold gain in testicular weight and in hypertrophy of Sertoli cells in association with GnRH stimulation, maturation of the germinal epithelium did not progress past prophase I spermatocytes, and the number of these latter cells was meager. These findings indicate that the testis of the infant primate, like that of the adult, secretes significant amounts of inhibin, and that the quiescent Sertoli cell of the juvenile males may be readily provoked by appropriate gonadotropin stimulation into producing inhibin. The results also fail to provide evidence for the view that changes in circulating inhibin concentrations are robustly related, in an inve