Evidence that the bovine ovary secretes large amounts of monomeric inhibin alpha subunit and its isolation from bovine follicular fluid

Analysis of bovine follicular fluid (FF) using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with a sensitive immunoblotting procedure resolved several components that were immunoreactive with an antiserum directed against the n-terminus of the alpha subunit of human inhibin (hI alpha(1-32]. Under non-reducing conditions, three intensely stained bands having apparent Mr values of 116,000, 44,000 and 25,000 were present, whilst under reducing conditions only two intensely stained bands (Mr 43,000 and 21,000) were detected. The Mr 44,000 and 25,000 immunoreactive forms (non-reducing conditions) were also demonstrated in bovine utero-ovarian vein and peripheral venous plasma after subjecting samples (40 ml) to immunoaffinity concentration using Sepharose beads coupled to anti-hI alpha(1-32), SDS-PAGE and immunoblotting. The same approach revealed the presence of the smaller (Mr 25,000) form in bovine granulosa cell-conditioned culture medium (GCCM). Gel-permeation chromatography (Sephacryl S-200), immunoaffinity chromatography (Sepharose-anti-hI alpha(1-32] and reversed-phase high-performance liquid chromatography (RP-HPLC; C18 and C8 columns) were employed to isolate from bFF (30 ml, 19.5 g protein) 750 micrograms protein which appeared essentially homogeneous by RP-HPLC and SDS-PAGE and had an Mr of 25,000 (non-reducing conditions)/21,000 (reducing conditions), identical to that of the immunoreactive component of lowest Mr found in bovine FF, utero-ovarian vein plasma, peripheral plasma and GCCM. The isolated material was highly immunoreactive with antisera against both hI alpha(1-32) and purified Mr 32,000 bovine inhibin but was devoid of biological activity when tested in a rat pituitary cell inhibin bioassay. Amino-terminal analysis revealed an amino acid sequence (residues 1-14) identical to that reported elsewhere for the alpha subunit (Mr 20,000/21,000) of bovine inhibin. In conclusion, the present study has revealed that the bovine ovary secretes considerable quantities of monomeric inhibin alpha subunit. The unexpected presence of this material in peripheral blood is likely to hinder attempts to obtain physiologically relevant data on circulating levels of inhibin in cattle using conventional radioimmunoassays.