STUDIES ON SPERM FERTILIZING ABILITY

精子受精能力的研究

• 批准号: 3312538
• 负责人: BARRY DOUGLAS BAVISTER
• 金额: $ 13.48万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-09-30 至 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3312538
• 关键词: SDS polyacrylamide gel electrophoresis; acrosome; affinity chromatography; cell cell interaction; cell communication molecule; dialysis; egg /ovum; fallopian tubes; fertilization; glycoproteins; graafian follicles; hamsters; high performance liquid chromatography; hybridomas; immunocytochemistry; laboratory mouse; microscopy; monoclonal antibody; physical separation; progesterone; protein purification; sperm; sperm capacitation

The overall objective of the proposed research is to provide new information on the regulation of sperm:egg interaction in mammals by components of the normal oviductal milieu. This information is needed for a more complete understanding of the way that gamete interactions are facilitated and coordinated at the site of fertilization in vivo. Increased knowledge of these regulatory mechanisms could assist the development of improved methods for amelioration of infertility in humans and for fertility control. Specific aims are (i) to identify molecules modulating terminal events of sperm:zona interactions and acrosome reactions, and to differentiate their sites of production (follicle or oviduct); (ii) to isolate and identify a heat labile oviductal factor that facilitates sperm penetration into zonae pellucidae, and to determine its mechanism of action; and (iii) to isolate and identify a heat stable factor present in the ovulated cumulus oophorus that facilitates acrosome reactions in free-swimming (i.e., non-zona bound) sperm. The research will use well-defined in vitro fertilization techniques to examine details of the regulatory mechanisms with golden hamster gametes. Hamster epididymal spermatozoa will be capacitated in vitro, then incubated for 60 or 90 min with living or salt-stored follicular or ovulated eggs. These eggs and/or sperm will be treated with impure or (later) purified heat labile and/or heat stable factors and the effects on sperm penetration of the zonae pellucidae will be evaluated; differential responses with these preparations will provide detailed information about the functions and mechanisms of action of the factors. Sperm acrosome reactions will be monitored using fluorescent monoclonal antibodies. The sites of production and of functional activities of these factors will be localized using polyclonal and monoclonal antibodies. Factors will be isolated by standard biochemical procedures, including SDS-polyacrylamide gel electrophoresis followed by western blotting, and identified using immunoenzymatic assay. Tests will show if the factors' actions are enzyme-like or ligand-like. The hypotheses will be tested that the factor(s) act as co-acrosome reaction inducing factors, i.e., in synergism with zonae pellucidae, and that the factors alter the physical characteristics of zonae pellucidae. Possible homology between some of the oviductal factors and the oviductal protein 'oviductin' will be examined. It will be determined whether the acrosome reaction inducing activity of the heat-stable factor can be ascribed to a macromolecule or to a ligand, such as progesterone, bound to a macromolecule. The results of these experiments will provide deeper insights into the functional roles of these naturally-occurring factors, and into their mechanisms of action in regulating events immediately preceding egg penetration.

这项研究的总体目标是提供新的 哺乳动物精子与卵子相互作用的调节信息， 正常输卵管环境的组成部分。 需要这些信息， 更全面地了解配子相互作用的方式 促进和协调在体内受精的网站。 增加对这些调节机制的了解， 改善人类不育症的改进方法的开发 和生育控制。 具体目标是（i）识别分子 调节精子的终末事件：精子相互作用和顶体 反应，并区分其生产部位（卵泡或 输卵管）;（ii）分离和鉴定热不稳定输卵管因子， 促进精子穿透透明带，并确定其 作用机制;和（iii）分离和鉴定热稳定因子 存在于排卵的卵丘中， 自由游动中的反应（即，非精子结合）精子。 这项研究将 使用定义明确的体外受精技术来检查 金黄地鼠配子的调节机制。 副附睾的 精子将在体外获能，然后孵育60或90 min 有活的或盐储存的卵泡或排卵的卵子。 鸡蛋和/或 精子将用不纯的或（后来）纯化的热不稳定的和/或 卵带热稳定因子及其对精子穿透的影响 将对透明科进行评价;对这些 准备工作将提供有关职能的详细信息， 这些因素的作用机制。 精子顶体反应将是 使用荧光单克隆抗体监测。 生产地点 和功能活动的这些因素将被本地化使用 多克隆和单克隆抗体。 因子将通过标准进行隔离 生化程序，包括SDS-聚丙烯酰胺凝胶电泳 然后进行蛋白质印迹，并使用免疫酶法鉴定。 测试将显示这些因子的作用是类似酶还是类似配体。 将检验因子作为辅助顶体的假设 反应诱导因子，即，与透明带有协同作用， 这些因素改变了透明带的物理特征。 某些输卵管因子与输卵管分泌物之间的可能同源性 将检查蛋白质“输卵管蛋白”。 将决定是否 热稳定因子的顶体反应诱导活性可以 归因于大分子或配体，例如黄体酮，与 一种高分子。 这些实验的结果将提供更深入的 深入了解这些自然发生的因素的功能作用， 并进入它们的作用机制， 在卵子进入之前。