PURIFICATION OF A HIGH-MOLECULAR-WEIGHT FOLLICLE-STIMULATING-HORMONE RECEPTOR-BINDING INHIBITOR FROM HUMAN FOLLICULAR-FLUID

In a previous study we reported the presence in human follicular fluid (hFF) of a FSH receptor-binding inhibitor (hFSH-BI) with FSH agonist activity, which was immunologically similar to FSH but could be distin guished from FSH on the basis of its greater stability in acid. We hav e now purified hFF-derived hFSH-BI after molecular sieving on Sephracr yl S-100 ion exchange chromatography using Diethyl-aminoethyl-cellulos e followed by polyacrylamide gel electrophoresis (PAGE). The purified hFSH-BI had a potency approximately 12,000-fold greater than that of d ialyzed hFF, based on its ability to inhibit the binding of [I-125]hFS H to its membrane receptor. The purified hFSH-BI also had FSH agonist activity, stimulating estradiol synthesis in cultured rat Sertoli cell s. Upon sodium dodecyl sulfate (SDS)-PAGE, hFSH-BI migrated as two ban ds of almost identical mobility, with an estimated mol wt of 57,000, c ompared with 30,000 for pituitary FSH run simultaneously. A monoclonal antibody to hFSH that also recognizes hFSH-BI was used for Western bl ot analysis of the SDS-PAGE fraction. The Western blot confirmed the d etection of two bands with very similar mobilities and estimated mol w t of 57,000, which were clearly distinguishable from that of immunolog ically reactive hFSH run in parallel. The hFSH-BI bands showed similar profiles upon cyanogen bromide cleavage and had indistinguishable ami no acid compositions. The amino acid composition of hFSH-BI was clearl y distinct from those of hFSH, hLH, hCG, and the alpha-subunit of huma n inhibin. Our studies confirm the presence in hFF of a unique agonist protein which is of potential importance in the regulation of gonadal function.