PURIFICATION OF FSH BINDING INHIBITORS

从猪体中分离纯化了两种与受体结合的FSH抑制剂 卵泡液。有不同的物质具有相反的 对FSH反应细胞的影响；一个(58,000 mW的蛋白质)是 刺激性(促卵泡刺激素激动剂)，另一种(复杂糖肽)抑制 (FSH拮抗剂)。本项目的具体目标是：1)获得 足够数量的高纯度缓蚀剂，用于化学和 生物学研究，2)化学鉴定这两种抑制剂，3) 开发用于研究抑制剂的特定探针，4)识别 两种抑制剂的作用机制，以及5)确定它们的作用机制 体内细胞定位。 提出的纯化方法是根据以往的分离经验提出的 猪卵泡液中的两种抑制剂，化学成分研究 低分子量缓蚀剂及高分子量缓蚀剂的抗原性。 作用机制研究包括受体结合，体外(Sertoli 细胞培养)和体内实验，旨在确定这些 抑制剂本身与FSH受体相互作用，如果是这样的话，是否或 抑制物不能与FSH所涉及的相同受体表位相互作用 有约束力的。FSHβ亚基或高分子FSH合成肽片段 针对这些合成肽的结合抑制剂和抗体将是 作为特定的探针产生，以促进行动机制的研究。 免疫组织化学研究将被用来定位抑制剂在 活着。 长期目标包括评估心脏的生理意义。 抑制剂。这些抑制剂可能被证明是局部的 (旁分泌/自分泌)在受体水平上起作用的调节剂 调节促性腺激素对性腺功能的调控。这样的行动意味着 生理研究将因局部的代偿性变化而复杂化 作为任何一种因素的浓度的调制器在实验上 被操纵了。实现本提案的目标将有助于 这一长期目标是通过提供关于蜂窝 抑制剂的本地化和开发特定的探针。获得的数据 希望通过这个项目提高我们对FSH的认识 结构/功能关系。此外，这个项目可能会导致 提高了对正常性腺功能(如生育能力)的了解。自.以来 对当地监管机构生物合成的异常控制可能是基础 某些形式的人类不孕症(例如，高促性腺激素减退症)，新的 对这些疾病的病因学的洞察也可能获得。

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.