PURIFICATION OF LOW MW FSH BINDING INHIBITOR

卵泡液中低分子因子的存在 抑制FSH与受体的结合提示局部调节 促性腺激素的作用。这样的调制预计会起到 在卵泡发生的控制和卵泡的选择中的重要作用 用于排卵的卵泡。该项目的具体目标是：a) 从牛卵泡液中纯化该因子并测定其 化学特性、作用机制和药理潜力。 牛卵泡液将被超滤，以获得脱盐部分 含有分子量在500到5000之间的组分。 从这一组分中提纯可能的调节肽将是 伴随着凝胶过滤、离子交换和反相(RP-HPLC) 液体色谱学。纯度将用反相高效液相色谱法测定，薄层 层析和质谱学。化学特性(成分和 纯化因子的序列)将由人工Edman完成 降解和质谱学。行动机制将由以下机构研究 研究：1)纯化的抑制剂对FSH结合的影响 受体，2)放射性标记的抑制剂与性腺的结合(Sertoli和 颗粒细胞)组织和3)抑制物影响其他 培养的性腺(支持和颗粒)细胞对FSH的反应(即， 激动剂与拮抗剂活性)。最后，药理上的潜力将 通过检测纯化的抑制剂在 在体内阻断FSH的作用。这个项目的长期目标是 鉴定一种自然产生的促卵泡激素抑制剂，并提供 关于它的性质，药理作用作为一种 避孕剂及其在促性腺激素作用基础研究中的应用。 这些研究将与了解人类卵巢功能和 功能障碍，因为FSH作用的局部调节者是一个潜在的或 调节卵泡发生和人类生育能力的靶标。

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.