Two inhibitors of FSH binding to receptor have been purified from porcine follicular fluid. There are distinct substances which have opposite effects on FSH responsive cells; one (a 58,000 MW protein) being stimulatory (FSH agonist), the other (a complex glycopeptide) inhibitory (FSH antagonist).
The specific aims of this project are: 1) to obtain sufficient quantities of highly purified inhibitors for chemical and biological study, 2) to chemically identify the two inhibitors, 3) to develop specific probes for use in studying the inhibitors, 4) to identify the mechanism of action of both inhibitors, and 5) to identify their cellular localization in vivo. Purification methods proposed are based on previous experience isolating both inhibitors from porcine follicular fluid, chemical composition studies of low MW inhibitor and antigenic characterization of high MW inhibitor. Mechanism of action studies include receptor binding, in vitro (Sertoli cell culture) and in vivo experiments designed to determine if these inhibitors interact with receptor for FSH per se, and, if so, whether or not the inhibitors interact with the same receptor epitopes involved in FSH binding. Synthetic peptide fragments of FSH beta subunit or high MW FSH binding inhibitor and antibodies against these synthetic peptides will be generated as specific probes to facilitate mechanism of action studies. Immunohistochemical studies will be used to localize the inhibitors in vivo. Long term goals include evaluation of the physiological significance of the inhibitors. These inhibitors may prove to represent local (paracrine/autocrine) regulators which act at the level of receptor action to modulate FSH control of gonadal function. Such action implies that physiological studies will be complicated by compensatory changes in local modulators as concentrations of any one factor are experimentally manipulated. Achieving the goals of the present proposal will facilitate this long term goal by providing information regarding the cellular localization of inhibitors and by developing specific probes. Data obtained by this project is expected to improve our understanding of FSH structure/function relationships. Furthermore, this project may lead to improved understanding of normal gonadal function (e.g., fertility). Since aberrant control of the biosynthesis of local regulators might underlie some forms of human infertility (e.g., hypergonadotropic hypogonadism), new insights into the etiology of these diseases may also be obtained.
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