Purification of a high molecular weight follicle-stimulating hormone receptor-binding inhibitor from human follicular fluid.

Abstract

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 distinguished from FSH on the basis of its greater stability in acid. We have now purified hFF-derived hFSH-BI after molecular sieving on Sephracyl S-100 ion exchange chromatography using Diethyl-aminoethyl-cellulose followed by polyacrylamide gel electrophoresis (PAGE). The purified hFSH-BI had a potency approximately 12,000-fold greater than that of dialyzed hFF, based on its ability to inhibit the binding of [125I]hFSH to its membrane receptor. The purified hFSH-BI also had FSH agonist activity, stimulating estradiol synthesis in cultured rat Sertoli cells. Upon sodium dodecyl sulfate (SDS)-PAGE, hFSH-BI migrated as two bands of almost identical mobility, with an estimated mol wt of 57,000, compared with 30,000 for pituitary FSH run simultaneously. A monoclonal antibody to hFSH that also recognizes hFSH-BI was used for Western blot analysis of the SDS-PAGE fraction. The Western blot confirmed the detection of two bands with very similar mobilities and estimated mol wt of 57,000, which were clearly distinguishable from that of immunologically reactive hFSH run in parallel. The hFSH-BI bands showed similar profiles upon cyanogen bromide cleavage and had indistinguishable amino acid compositions. The amino acid composition of hFSH-BI was clearly distinct from those of hFSH, hLH, hCG, and the alpha-subunit of human inhibin. Our studies confirm the presence in hFF of a unique agonist protein which is of potential importance in the regulation of gonadal function.

Manipulation of Human Ovarian Function: Physiological Concepts and Clinical Consequences*

“…In addition, a high molecular weight FSH receptor binding inhibitor was partially purified from human follicle fluid by the same group of investigators (216). However, these proteins have never been fully characterized, and the physiological relevance remains uncertain (for review see Ref.…”

Aromatase mRNA expression in individual follicles from polycystic ovaries

“…There are several proteins in follicular fluid with the potential to inhibit oestradiol production such as a high molecular weight FSH receptor binding inhibitor (Lee et al, 1993), inhibin-α subunit precursor (Schneyer et al, 1991), insulin-like growth factor binding proteins (IGFBPs) (Ui et al, 1989) epidermal growth factor (EGF) (Mason et al, 1990) and tumour necrosis factor-α (TNF-α) (Rice et al, 1996). No data are available demonstrating that the physiological concentrations of these peptides that are present in follicular fluid can significantly inhibit oestradiol production in PCOS.…”

“…Our observation that a small proportion of PCOS follicles begin to have elevated aromatase stimulating bioactivity but do not appear to respond to the FSH is consistent with the concept that PCOS follicles contain inhibitors of FSH action. Several molecules have been suggested as potential inhibitors including a high molecular weight FSH receptor binding inhibitor (Lee et al, 1993), IGFBP (Ui et al, 1989), EGF (Mason et al, 1990) and TNF-α (Rice et al, 1996). There do not appear to be alterations in the concentrations of FSH receptor inhibitory activity in PCOS women relative to regularly cycling women (Schipper et al, 1997).…”

Granulosa Cell Apoptosis Induced by a Novel FSH Binding Inhibitory Peptide From Human Ovarian Follicular Fluid

“…Reichert and colleagues purified FSHBI from bovine, porcine, and human follicular fluid (Darga and Reichert 1978;Sluss and Reichert 1984;Lee et al 1990). …”