A Bioassay for Inhibin Using Pituitary Cell Cultures

Abstract

A bioassay for inhibin based on the suppression of gonadotrophin releasing hormone (Gn-RH)-stimulated secretion of FSH by primary monolayer cultures of rat anterior pituitary cells is described. The cultures were exposed to standard or test materials for 3 days. The levels of FSH in the media were measured by radioimmunoassay after exposure for 6 h to a maximally stimulating concentration of Gn-RH (10 nmol/l). The standard was prepared from ovine testicular lymph. Several preparations of proteins from gonadal tissues or secretions suppressed the levels of FSH in parallel with the standard. The levels of LH were also reduced but higher doses of active material were required. Non-specificity from cell damage and inactivation of Gn-RH have been excluded. The secretion of gonadotrophins by the pituitary cells was also inhibited by androgens, but not in parallel with the standard and secretion of LH was affected more than that of FSH. Control lymph protein preparations from castrated sheep had no detectable activity. The assay was sensitive and had adequate precision and practicability. It has proved useful for monitoring preliminary steps in the purification of inhibin.

Peptides of postulated inhibin activity

“…This is in agreement with previous reports [21]. When inhibin activity of the 94-residue peptide and its Cterminal segment in a rat pituitary cell culture was reported [lo], no description was given of details on culture conditions, FSH and LH RIAs or number of experiments.…”

“…Anterior pituitary cells from adult male rats were prepared using digestion with trypsin [21] and diluted with the incubation medium [Dulbecco's modified Eagles medium (Flow Lab., UK), supplemented with 5% horse serum and 2.5% fetal calf serum (Flow Lab.)]. Aliquots (200 ~1) of about 150000 cells were cultured in multiwell plates (Falcon 3070) at 37°C in a water-saturated atmosphere of 95% sir/5% CO2 for 72 h before the addition of any test substance to the medium.…”

Inhibin production by bovine ovarian tissues in vitro and its regulation by androgens

“…Aliquants were then tested for their ability to inhibit LH-RH (10~8 M)-stimulated FSH secretion by cultured rat anterior pituitary cells during a 6-h incubation period. Inhibin activity of the cell culture medium was expressed relative to the potency of a standard reference preparation of inhibin derived from ovine testicular lymph which has been designated an arbitrary potency of 1 U/mg (Eddie, Baker, Higginson & Hudson, 1979). Regression analysis and parallel line assay statistics (Borth, 1976) were used to relate the inhibin activity of the culture medium to that of the inhibin standard.…”

An in-vivo method for estimating inhibin production by adult rat testes

“…Groups of adult rats were sham-operated or made bilaterally cryptorchid as previously described (de Kretser, Sharpe & Swanston, 1979 (Scott, Burger & Quigg, 1980) was validated for testicular extracts and used to measure inhibin activity as previously described (Au et ai, 1983). The inhibin potency of samples was assessed from the dose-dependent suppression of the FSH cell content parallel to a reference preparation derived from ovine testicular lymph and assigned a potency of 1 U/mg (Eddie, Baker, Higginson & Hudson, 1979). Rat testis extracts were prepared by homogenization at 4°C in Dulbecco's phosphate buffer pH 7-4 and centrifuged at 100 000 g for 1 h at 4°C and the supernatant was used for inhibin measurements.…”