Isolation of nine different biologically and immunologically active molecular variants of bovine follicular inhibin

Abstract

A combination of immunoaffinity chromatography, SDS-PAGE, and electroelution was used to simultaneously isolate 0.36-4.65 mg of nine different molecular forms of inhibin (proα(c)-29 kDa; fully processed 34 kDa; and large inhibin forms 49, 53, 56, 77, 88, 110, and > 160 kDa) from 0.675 L of bovine follicular fluid (bFF). Each inhibin form, except proα(c), cross-reacted with inhibin α(c)1-26- and β(A)82-114- subunit- directed antibodies during immunoblot analysis. Proα(c) cross reacted only with α-subunit antibodies. The inhibin forms consisted of 22-, 29-, 49-, or 58-kDa α subunits and 17- or 58 kDa β subunits. During cultures of ovine pituitary cells, a 5-ng/ml dose of each inhibin form (except proα(c)) suppressed basal accumulation of FSH 30% to 50% but increased GnRH-induced LH release 40% to 248%. The various inhibin forms cross-reacted in a parallel fashion with standard curves generated during homologous and heterologous RIAs but with markedly different relative immunopotencies. In the RIAs, proα(c) cross reacted 3- to 18-fold more than the fully processed inhibin form. The fully processed and the seven different large forms of inhibin cross reacted with different relative immunopotencies in a two-site dimer- specific ELISA. We concluded that 1) a combination of immunoaffinity extraction, SDS-PAGE, and electroelution simultaneously isolated relatively large amounts of highly enriched preparations of nine different molecular forms of immunologically and biologically active inhibin from bFF; 2) eight different dimeric forms of bovine inhibin may regulate both basal FSH and GnRH-induced LH secretion by the pituitary gland, and 3) eight or nine different molecular forms of inhibin cross-react with different relative immunopotencies in the two-site dimer specific assay or RIAs.