INTERACTION OF GONADOTROPINS WITH OVARIAN CELLS

促性腺激素与卵巢细胞的相互作用

• 批准号: 3447968
• 负责人: Ellen Rorke
• 金额: $ 5.53万
• 依托单位: BOSTON CITY HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-01 至 1986-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3447968
• 关键词: cell free system; chemical structure function; chorionic gonadotropin; density gradient ultracentrifugation; gel filtration chromatography; hormone regulation /control mechanism; luteinizing hormone; ovary; pseudopregnancy; receptor mediated endocytosis; tissue /cell culture

In preliminary studies, a markedly different apparent rate of LH and hCG metabolism within cells of the pseudopregnant rat ovary was observed. Since LH and hCG have markedly different plasma half lives and metabolic clearance rates, in vitro studies using cultured rat granulosa cells need be udertaken. The studies in this proposal are designed to better define the subcellular metabolism of hCG, LH and hybrid molecules of complementary subunits of LH and hCG. The differential effects of LH and hCG on receptor turnover and internalization will also be examined. Internalized hormone will be examined by a variety of techniques including sucrose gradient ultracentrifugation, immunoreactivity, bioactivity, gel filtration, receptor binding and binding to concanavalin A. To better understand if the level of glycosalation of the two gonadotropins is responsible for potential observed differences, hybrid LH/hCG molecules will be studied in the in vitro granulosa culture system. Subcellular sites to which gonadotropin associates following internalization will be determined by classical subcellular fractionation techniques. Membrane fractions will be identified by specific enzymes associated with subcellular organelles. In separate but related studies ovarian LH/hCG receptor turnover rates will be examined using a density shift technique. Ovarian cells will be exposed to amino acids enriched in the dense isotopes 2H, 13C, 15H. Newly synthesized receptor will be "heavier" than pre-existing receptor binding sites. Scatchard analysis of normal and dense receptor will be performed to assure that the dense amino acids do not alter receptor affinity for gonadotropin. Using the density shift technique, the differential rates of receptor biogenesis and down regulation will be examined in response to LH and hCG since those two hormones appear to have markedly different capacities for inducing down regulation. Moreover, this technique will permit assessment of differential rates of receptor synthesis and catabolism in response to hormonal interaction.

在初步研究中，LH和hCG的表观速率明显不同， 观察假孕大鼠卵巢细胞内的代谢。 由于LH和hCG具有明显不同的血浆半衰期和代谢产物， 清除率，使用培养的大鼠颗粒细胞的体外研究需要 被征服 本提案中的研究旨在更好地定义 hCG、LH和互补的杂合分子的亚细胞代谢 LH和hCG的亚基。 促黄体生成素和人绒毛膜促性腺激素对受体的不同作用 还将审查人员更替和内部化问题。 内化激素 将通过各种技术进行检查，包括蔗糖梯度 超离心，免疫反应性，生物活性，凝胶过滤， 受体结合和与伴刀豆球蛋白A的结合。 为了更好地理解如果 两种促性腺激素的糖基化水平负责 潜在的观察到的差异，混合LH/hCG分子将在 体外颗粒层培养系统。 亚细胞位点， 内化后的促性腺激素相关物质将由以下因素确定 经典的亚细胞分离技术。 膜组分将是 由与亚细胞器相关的特定酶鉴定。 在 单独但相关的研究卵巢LH/hCG受体转换率将 使用密度偏移技术检查。 卵巢细胞将暴露于 富含高密度同位素2 H、13 C、15 H的氨基酸。 新合成 受体将比预先存在的受体结合位点“更重”。 将对正常和致密受体进行Scatchard分析，以确保 密集的氨基酸不会改变受体对 促性腺激素 利用密度位移技术， 受体的生物发生和下调将在对LH的反应中进行检查 和hCG，因为这两种激素似乎有显着不同， 诱导下调的能力。 此外，这项技术将 允许评估受体合成的差异速率， 激素相互作用的反应。