Pituitary Binding of3H-Labeled Sertoli Cell Factorin Vitro: A Potential Radioreceptor Assay for Inhibin*

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Abstract
Binding of partially purified, 3H-labeled Sertoli cell factor (SCF) to rat anterior pituitary homogenates was tissue specific, saturable, time and temperature dependent, and competitively inhibited by unlabeled SCF. The binding of [3H]SCF to rat anterior pituitary in vitro and its potential use as a radioreceptor assay for SCF and other inhibin preparations were considered. [3H]SCF was synthesized by rat Sertoli cells cultured in the presence of [3H]leucine (5 .mu.Ci/ml) and was then partially purified by Sephadex gel filtration and high pressure liquid chromatography. The purified [3H]SCF had a specific activity of .apprx. 20,000 dpm/.mu.g protein and was biologically active in pituitary cell cultures. The binding was carried out in 0.5 ml buffer, containing 1 pituitary equivalent and, wherever appropriate, various unlabeled competing substances. The binding was optimal at pH 7.4 and was decreased by pretreatment of [3H]SCF with trypsin (0.25%; 37.degree. C; 30 min) or heat (100.degree. C; 10 min). Storage of the pituitary glands at -20.degree. C for several months and differences in animal age did not affect total binding per pituitary, but the amount of radioactivity bound per mg pituitary tissue declined progressively between 18-90 days of age. Over 90% of the bound [3H]SCF was competitively inhibited by excess unlabeled SCF and several other inhibin preparations of testicular origin: high MW fraction of ram testis fluid (MW > 10,000), low MW fraction of ram testis fluid (MW < 5,000), ovine testicular lymph and aqueous rat testicular extract. The degree of inhibition was dose dependent, and except for the low MW fraction of ram testis fluid, the displacement curves were parallel (slope, 0.95). Various noninhibin substances tested [rat androgen-binding protein, bovine [leuteinizing hormone], BSA [bovine serum albumin], native GnRH [gonadotropin releasing hormone] or GnRH agonist analogs D-Ser-(tBu)6-des-Gly10-GnRH-N-ethylamide [Buserelin], and D-Ala6-des-Gly10-GnRH-N-ethylamide] did not significantly compete for the [3H]SCF-binding. The binding ability correlated well with inhibin biological activity in vitro. SCF-binding sites in the rat anterior pituitary interact with several different inhibin preparations of testicular origin but appear to be distinct from GnRH-binding sites. The pituitary binding may be used as a rapid radioreceptor assay for SCF and various other inhibin preparations.