PURIFICATION OF LOW MW FSH BINDING INHIBITOR

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The presence of a low molecular weight factor in ovarian follicular fluid which inhibits the binding of FSH to receptor suggests local modulation of gonadotropin action. Such modulation would be expected to play a significant role in the control of folliculogenesis and selection of follicles for ovulation. The specific aims of this project are: a) to purify this factor from bovine follicular fluid and b) determine its chemical identity, mechanisms of action and pharmacological potential. Bovine follicular fluid will be ultrafiltered to obtain a desalted fraction containing components of molecular weights between 500 and 5,000. Purification of the putative regulatory peptide from this fraction will be accompanied by gel filtration, ion-exchange and reverse-phase (RP-HPLC) liquid chromatography. Purity will be determined by RP-HPLC, thin-layer chromatography and mass spectroscopy. Chemical identity (composition and sequence) of the purified factor will be accomplished by manual Edman degradation and mass spectroscopy. Mechanism of action will be examined by studying: 1) effects of purified inhibitor on the binding of FSH to receptor, 2) binding of radiolabeled inhibitor to gonadal (sertoli and granulosa cells) tissue and 3) ability of inhibitor to affect other responses to FSH in cultured gonadal (sertoli and granulosa) cells (i.e., agonist vs antagonist activity). Finally, the pharmacologic potential will be investigated by examining the efficacy of the purified inhibitor in blocking FSH actions in vivo. The long term objectives of this project are to identify a naturally occurring inhibitor of FSH and to provide information regarding its nature, pharmacologic potential as a contraceptive agent and usefulness in basic research on the action of FSH. These studies will be relevant to understanding human ovarian function and dysfunction since a local modulator of FSH action is a potential agent or target for the regulation of folliculogenesis and human fertility.