INHIBIN - STUDIES OF STORED AND SECRETED FORMS BY BIOSYNTHETIC LABELING AND IMMUNODETECTION IN CULTURED RAT GRANULOSA-CELLS

The biosynthesis of inhibin in rat granulosa cells was studied by biosynthetic labeling, immunoblotting, and immunocytochemical techniques. Granulosa cells from immature hypophysectomized estrogen-treated rats were cultured in the presence of [35S]cysteine. Both conditioned media and cell extracts were subjected to immunoprecipitation with an antibody directed against the N-terminal 26 amino acids of the .alpha.-chain of porcine inhibin (pI.alpha.1-26), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Treatment with FSH (100 ng/ml) and .DELTA.4-androstenedione (10-7 M) increased the secretion of 35S-labeled inhibin immunoreactivity by 2.6-fold over that in control cultures treated with androstenedione alone. The radiolabeled inhibin had mol wt (Mr) values of 45,000 and 30,000. Upon reduction, the 45,000 Mr polypeptide remained (with increased apparent Mr of 49,)00), but the 30,000 Mr species disappeared with the concomitant appearance of two bands with 18,000 and 11,000 Mr. Competition studies with pI.alpha.1-26 confirmed that these polypeptides were all related to inhibin. Furthermore, immunoblotting with an antibody directed against the porcine inhibin .beta.-A chain (pI.beta.A81-113) indicated that the 11,000 Mr peptide was the inhibin .beta.-A chain. Extracts of cells treated with FSH contained only a high Mr .alpha.-related species (Mr, 41,000 nonreduced; 49,200 reduced). The inhibin .alpha. antibody was also used to immunocytochemically stain cultured granulosa cells. Cells that had been treated with FSH or the adenyl cyclase activator forskolin (3 x 10-5M), but not untreated cells, exhibited positive staining. These results indicate that granulosa cells synthesize and store inhibin .alpha.-chain precursor with 49,000 Mr. Although some of the high Mr .alpha.-form was secreted, the majority of the .alpha.-subunit was processed to the 18,000 Mr form and dimerized with the 11,000 Mr .beta.-chain to form the mature inhibin dimer immediately before secretion. The cultured granulosa cells may provide a model for future studies on the hormonal regulation of inhibin .alpha.- and .beta.-gene expression as well as subunit dimerization and secretion.