精卵融合是哺乳动物繁殖成功的关键过程之一，但对其分子机制知之甚少，只在精子和卵子上分别鉴定出若干影响精卵膜融合的蛋白，但这些蛋白都没有融合素或者SNARE蛋白的结构特点，不可能是其中某一种蛋白聚合引发膜融合，应该是通过相互作用介导精卵膜融合。在精子上的8种精卵融合相关蛋白中目前只探明IZUMO1能与TSSK6结合，尚无其它精卵融合相关蛋白之间相互作用的研究报道。申请人前期实验发现绒山羊PDIA3能与TSSK6、CRISP2、IZUMO1等相互作用，提示精子上的精卵融合相关蛋白之间存在复杂的相互作用，因此本项目拟用免疫共沉淀、酵母双杂交、GST Pull-down等技术全面鉴定绒山羊精子上的精卵融合相关蛋白之间的相互作用，并找到介导其相互作用的结构域或者基序。在明确某对蛋白相互作用的基础上，进一步观察其在顶体反应前后精子上的共定位，最后归纳出它们介导精卵膜融合的相互作用模式.

Sperm-egg fusion is one of the key steps in mammalian fertilization. However, the mechanisms by which acrosome-reacted spermatozoa bind to oolemma and the subsequent fusion with the oolemma remain unknown except that a dozen of proteins with putative roles in sperm-oocyte fusion have been identified both on sperm and egg, among which eight are located on the sperm, including IZUMO1, CRISP1, CRISP2, PDIA3, SPACA3(SLLP1), Equatorin, SPESP1 and TSSK6, whereas CD9 and some unknown GPI-anchored proteins are on the oocyte. These proteins all do not have stereotypical features of a fusogen or SNARE protein, thus could not mediate membrane fusion directly. They may function coordinately with each other during the process of sperm-egg fusion. Therefore it is urgently to clarify the interaction patterns among these proteins for elucidating the molecular mechanisms of mammalian sperm-egg fusion. However there are only a few reports that IZUMO1 can bind to TSSK6 on sperm. Our lab had found that cashmere goat PDIA3 can bind to TSSK6, CRISP2, IZUMO1, suggesting there are complicated interactions among these gamete fusion related proteins on sperm. Therefore we design this project to identify and characterize the potential interaction and the domain or motifs that mediate the interaction among all the eight proteins on sperm using Co-IP analyses, yeast two-hybrid and GST Pull-down techniques. The colocalization on sperm pre- and post acrosomal reaction of those protein pairs with interaction will also be determined for helping draw interaction patterns.