Identification in human ovarian follicular fluid of proteins that share an epitope region unique to the extracellular domain of the follicle-stimulating hormone receptor.

Abstract

Recently we identified a unique region, residues 9-30 in the extracellular domain of the FSH receptor, capable of binding FSH but not LH or TSH. We have shown that polyclonal antibodies raised against this region specifically recognized intact FSH receptors present on plasma membranes of cultured rat Sertoli cells. In the present study, plasma membranes from human granulosa-lutein cells were solubilized and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by Western blot analysis. Antireceptor peptide antibody, but not preimmune serum control, recognized intact human FSH receptors, suggesting that human and rat FSH receptors share this unique N-terminus epitope region. Recent cloning studies have identified, in addition to full length receptor, the presence of FSH receptor-spliced messenger RNA variants, which encode receptor proteins with variable lengths of hydrophilic extracellular domains, but lacking transmembrane domains. Such proteins could theoretically represent secreted forms of the receptor. In this study, we used a polyclonal anti-FSH receptor (residues 9-30) peptide antibody to investigate whether FSH receptor-related soluble proteins might also be present in human ovarian follicular fluid (FF). In an enzyme-linked immunosorbent assay specific for the FSH receptor, antireceptor peptide antibody, but not preimmune serum serving as control, identified significant immunoreactivity in several human ovarian FF samples, suggesting that protein(s) present in FF share a common epitope with the extracellular domain of the FSH receptor. The apparent levels of FSH receptor-related activity in FF samples, expressed relative to the FSH receptor (residues 9-30) peptide, ranged from 31-55 ng/mL. Passing of the samples through 0.22-micron filters or subjecting the samples to high speed centrifugation did not alter the activity profiles of the samples ruling out effects due to contamination with plasma membranes from granulosa cells. When human FF samples were subjected to gel permeation chromatography, at least four distinct protein peaks were resolved, in the molecular mass range between 70-460 kilodaltons, each of which was recognized by the FSH receptor 9-30 peptide antibodies. Our results provide initial evidence for the presence in human ovarian FF of proteins sharing epitope with the extracellular domain of the FSH receptor and presumably derived from the granulosa cell. Since we have previously shown that the epitope region, represented by residues 9-30 in the extracellular domain of the FSH receptor specifically binds FSH, the proteins in human FF sharing this epitope may have functional significance.