Serum inhibin concentrations rise throughout normal male and female puberty.

Using a newly developed, sensitive, and specific RIA, we measured the serum concentrations of inhibin, together with those of FSH, LH, and sex steroids, throughout puberty in 99 boys and 102 girls attending a suburban Melbourne school. Serum inhibin levels rose from a geometric mean level of 161 U/L (range, 87-310; 67% confidence interval) at stage I puberty in boys to 442 U/L (range, 300-626) at stage V, while corresponding values in girls were 97 U/L (range, 46-204) and 231 U/L (range, 187-372), respectively. Serum inhibin concentrations were strongly correlated with age and serum FSH, LH, testosterone, and estradiol; all hormones increased in parallel in both boys and girls. After adjustment for age, the partial correlation coefficients remained significant only for testosterone in the boys. We hypothesize that gonadal inhibin production is stimulated by rising gonadotropin levels during pubertal development.

A rapid, sensitive and reliable assay for inhibin bioactivity.

A rapid 2-day quantitative assay for inhibin bioactivity based on FSH secretion from pituitary cells of immature female rats is described. The bioassay exhibited steeper slopes, improved precision and greater (fourfold) sensitivity compared with a previously established pituitary FSH cell content assay. Whole pituitary glands were used for the preparation of pituitary cells and the method for cell dispersion required a single enzymatic treatment with trypsin. Cells (180,000 viable cells per well) were dispensed into culture media containing inhibin and incubated for 48 h. Media were removed and assayed for FSH by radioimmunoassay. Using a ram rete testis fluid preparation as standard the inhibin dose-response curves of 25 consecutive experiments showed indices of precision of -0.08(mean)[range -0.04 to -0.17] and Finney's G values of 0.017[0.003-0.06]. The mean ED40 was 0.17 units of inhibin activity per well with interassay variation of 16.2% at this point of the dose-response curve. The assay had a practical capacity of 400 wells, permitting the measurement of dose-response curves of at least 40 unknowns with three dose points and triplicate wells per dose. The assay is specific for inhibin-containing preparations from several animal species. Overall, the assay is simple, precise, and sensitive, indicative of its applicability to the measurement of inhibin samples with low inhibin bioactivity and to the screening of large numbers of fractions during inhibin purification.

Inhibin in individual ovine follicles in relation to diameter and atresia.

Inhibin activity was measured by bioassay in follicular fluid of 99 individual ovine follicles ranging from 1 . 4 to 6 . 8 mm diameter (used to calculate volume) and in various stages of atresia. Treatment of samples before assay with charcoal concentrations of greater than 1 mg/ml resulted in significant loss of inhibin activity. The inhibin content of follicular fluid from individual follicles varied with follicular fluid volume but not with the degree of atresia, as assessed by morphological criteria. Inhibin concentration was not related to atresia, but was correlated with follicular fluid volume. However, aromatase activity in granulosa cells and oestradiol-17 beta concentration of follicular fluid, considered to be good indices of atresia, were highly correlated with both inhibin content and concentration in follicles greater than or equal to 3 . 5 mm diameter. Inhibin in ovine follicular fluid shows marked variation between follicles and it is suggested that this reflects a combination of the number and activity of granulosa cells within the follicle and the exit rate of inhibin from the follicle.

The specificity of inhibin bioassay using cultured pituitary cells.

It has been established that the inhibition of the cellular content of FSH in cultured pituitary cells can be used as a sensitive and precise bioassay for inhibin. During studies on the inhibin content of human plasma, FSH suppression was noted to occur together with LH suppression. Further studies revealed that where combined FSH and LH suppression occurred, cytological changes in the pituitary cells suggestive of toxicity were found, indicating non-specific effects of these substances. Charcoal treatment or gel filtration of seminal plasma removed or separated the toxic substances from the inhibin activity, the latter characterized by FSH suppression parallel to the standard preparation, no LH suppression and no light-microscopic changes in pituitary cells. It is recommended that careful evaluation of all inhibin bioassay systems should be undertaken to detect substances producing non-specific effects and additional guidelines for the assessment of specificity are suggested.

Ovarian and peripheral blood inhibin concentrations increase with gonadotropin treatment in immature rats.

The effects of PMSG treatment on ovarian and circulating inhibin concentrations in immature female rats has been examined. Sixty-four hours after injection of 10, 20 or 40 IU of PMSG the animals were anesthetized with ether; ovaries, uteri and blood from the abdominal aorta were collected. Steroid-free extracts of ovary and serum samples were prepared and assayed quantitatively for inhibin activity in an in vitro bioassay system. PMSG treatment elevated (p less than 0.001) both uterine and ovarian wt, and ovarian and peripheral concentrations of inhibin. A dose-related increase occurred ovarian wt, and in peripheral and ovarian content of inhibin. Ovarian inhibin concentration increased with dose of PMSG until the highest dose, where a significant decline and luteinization were seen. Peripheral FSH levels were significantly lowered at all doses of PMSG treatment; in contrasts, LH was significantly elevated, due to cross-reaction of PMSG in the LH assay. These results show that both ovarian and circulating levels of inhibin are related to the degree of gonadotropic stimulation, supporting the view that inhibin is involved in folliculogenesis and in the feedback regulation of FSH.

A simple and rapid in vitro bioassay for inhibin.

This report describes a quantitative bioassay for inhibin which can be used to monitor purification and establish the physiological role of this hormone in spermatogenesis and folliculogenesis. Anterior pituitary cells from adult male Sprague-Dawley rats were dispersed and precultured for 18 h before the addition of inhibin-containing materials to the culture medium. After a further 72 h in culture, the medium was removed by aaspiration, and the cells were lysed releasing their intracellular hormone into RIA buffer. Cell content of FSH was reduced in inhibin-containing preparations, but without changes in LH, GH, and PRL concentrations. The inhibin dose-response curve, based on the inhibition of FSH in 15 experiments using an ovine testicular lymph preparation as a standard, had indices of precision between -0.032 and -0.098, and Finney's g ranged from 0.003--0.025. The interassay variability ranged from 15.0--16.9%. The assay had a practical capacity of 300--400 wells, which permitted the measurement of dose-response curves of 15 or more unknowns with quadruplicate wells per dose. The potency of unknown preparations was calculated with reference to the inhibin standard, which had a designated potency of 1 U/mg. Preparations showing nonparallelism were excluded. This assay presented advantages over those described previously, since it showed specificity of response to FSH and accommodated a large number of samples without loss of precision or reproducibility. It is therefore suitable for measurement of the inhibin-like activity in some biological fluids.