BIOCHEMISTRY OF SPERM-EGG INTERACTION AND FERTILIZATION

这项研究的目的是获得基础知识。 精子-卵子相互作用的细胞生物学和生物化学。 海胆和鲍鱼之所以被使用，是因为它们不是哺乳动物， 却表现出与哺乳动物配子惊人的相似之处 互动。精卵的细胞生物学和生物化学 相互作用是最鲜为人知的基本生物学之一。 流程。通过这项研究获得的基本知识可能会被使用 在新的避孕方法的未来发展中 哺乳动物。在海胆中，这一建议侧重于相互作用。 与ARI(卵冻的精子膜受体)的结合 从蛋冻中提纯的顶体反应诱导糖蛋白)。 50%的REJ与人类多囊肾病同源 蛋白PKD1.关于Rej如何调节精子离子通道的研究可能 有助于了解这一重大的人类疾病。在……里面 鲍鱼，这个项目的重点是卵黄信封 溶解卵子的精子蛋白--赖氨酸的配基 非酶机制的包膜。这项研究可能有助于 了解哺乳动物精子利用的生化机制 穿透卵透明带。海上的具体目标 海胆精子主要有：1.鉴定精子膜蛋白 与210 kDa的蛋冻受体(Rej)相关。2.至 克隆、测序和鉴定与之相关的蛋白质 雷杰。3.确定各蛋白质模块的功能。 雷杰。4.鉴定顶体反应诱导剂(ARI) 结合精子的海胆卵冻。5.隔离和 鉴定与Rej结合的ARI寡糖。6. 使用我们克隆自的Synaxin和Synaptopagmin 精子，以确定与精子胞吐相关的蛋白质 顶体小泡。鲍鱼配体的具体目标是 溶血素为：7.研究其生化和超微结构。 卵黄包膜配体(VE-L)，用于溶素。要确定 同种和异种组合的亲和力 赖氨酸和VE-L。8.克隆、测序和鉴定 VE-L的基因序列。9.确定溶素是否与 VE-L是以VE-L的蛋白质或碳水化合物成分为基础的。 10.将VE-L切割成保留的最小片段 对赖氨酸的亲和力。分离并鉴定这一片段。 使与此片段络合的赖氨酸共结晶以进行x射线检查 衍射分析。11.测定赖氨酸的哪些残留量 对其特定物种的卵子溶解负责 卵黄蛋白外膜(VE)。12.确定通过何种机制 赖氨酸溶解VE。它是基于立体专门化吗 切断氢键，还是疏水相互作用？

The objective of this research is to gain fundamental knowledge of the cell biology and biochemistry of sperm-egg interaction. Sea urchins and abalone are used because they are nonmammalian, yet exhibit striking similarities to mammalian gamete interaction. The cell biology and biochemistry of sperm-egg interaction is one of the least understood fundamental biological processes. Basic knowledge gained by this research may be used in the future development of novel methods of contraception in mammals. In sea urchins this proposal focuses on the interaction of REJ (the sperm membrane receptor for egg jelly) with ARI (the acrosome reaction inducing glycoprotein purified from egg jelly). 50% of REJ is homologous to the human polycystic kidney disease protein PKD1. Study of how REJ regulates sperm ion channels may aid in the understanding of this significant human disease. In abalone, this project focuses on the egg vitelline envelope ligand for lysin, the sperm protein which dissolves the egg envelope by a nonenzymatic mechanism. This study may aid in understanding the biochemical mechanism mammalian sperm use to penetrate the egg zona pellucida. The specific aims for sea urchin sperm are: 1. To identify sperm membrane proteins associated with the 210kDa receptor for egg jelly (REJ). 2. To clone, sequence and characterize the proteins associated with REJ. 3. To determine the functions of the protein modules of REJ. 4. To characterize the acrosome reaction inducer (ARI) from sea urchin egg jelly which binds sperm REJ. 5. To isolate and characterize the oligosaccharide of ARI which binds to REJ. 6. To use syntaxin and synaptotagmin, which we have cloned from sperm, to identify proteins associated with exocytosis of the acrosomal vesicle. The specific aims for the abalone ligand for lysin are: 7. To study the biochemistry and ultrastructure of the vitelline envelope ligand (VE-L) for lysin. To determine the affinities of homospecific and heterospecific combinations of lysins and VE-Ls. 8. To clone, sequence and characterize the cDNA for the VE-L. 9. To determine if the binding of lysin to VE-L is based on protein or carbohydrate components of the VE-L. 10. To cleave the VE-L into the smallest fragment retaining affinity for lysin. To isolate and characterize this fragment. To cocrystallize lysin complexed with this fragment for x-ray diffraction analysis. 11. To determine which residues of lysin are responsible for its species-specific dissolution of the egg vitellin envelope (VE). 12. To determine the mechanism by which lysin dissolves the VE. Is it based on the stereospecific severing of hydrogen bonds, or hydrophobic interaction?