STUDIES ON SPERM FERTILIZING ABILITY

拟议研究的总体目标是提供新的 调节精子的信息：哺乳动物中卵子的相互作用 正常输卵管环境的成分。此信息需要用于 更完整地理解配子相互作用的方式 在体内受精部位促进和协调。 增加对这些监管机制的了解可以帮助 改善人类不孕症的改进方法的发展 也是为了控制生育。具体目标是(I)识别分子 精子末端事件的调控：透明带相互作用和顶体 反应，并区分它们的产生部位(卵泡或 输卵管)；(Ii)分离和鉴定一种不耐热的输卵管因子 促进精子穿透透明带，并确定其 作用机制；以及(Iii)分离和鉴定热稳定因子 存在于排卵的卵丘卵丘中，促进顶体 自由游动(即不受透明带限制的)精子的反应。这项研究将 使用定义明确的体外受精技术检查细节 金黄地鼠配子的调控机制。仓鼠附睾体 精子在体外获能，然后孵化60或90分钟 有活的或盐储存的卵泡或排卵卵的。这些鸡蛋和/或 精子将用不纯净或(稍后)纯净的热不稳定和/或 热稳定因素及其对精子穿透性的影响 将对透明科进行评估；对这些不同的反应 准备工作将提供有关职能的详细信息和 这些因素的作用机制。精子顶体反应将是 使用荧光单抗进行监测。生产现场 以及这些因子的功能活动的本地化使用 多克隆抗体和单抗。因素将通过标准进行隔离 生化程序，包括十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法 然后进行免疫印迹，并用免疫酶法进行鉴定。 测试将显示这些因子的作用是类似酶的还是类似配体的。 这些假设将被检验，即因子(S)充当协同顶体 反应诱导因素，即与透明带的协同作用，以及 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的可能同源性 将对蛋白质“输卵管蛋白”进行检测。将确定是否将 顶体反应诱导活性的热稳定因子可以 属于大分子或配体的，如黄体酮 一种大分子。这些实验的结果将提供更深层次的 对这些自然因素的功能作用的洞察， 并研究他们在立即规范事件方面的行动机制 在卵子穿透之前。

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.