Hedgehog信号受体PTC1、CDO、BOC和GAS1在小鼠精卵融合中的作用和机理

对哺乳动物精卵融合分子机理的研究已陷入僵局。在目前已知的10多种精卵融合相关蛋白中只有精子的IZUMO1与卵子的JUNO为小鼠精卵融合所必需，但仅有IZUMO1和JUNO只能介导细胞粘附，并不能介导细胞融合，说明还需精子或卵子上其他蛋白的协助。哺乳动物Hedgehog(HH)信号有四种受体，PTC1、CDO、BOC和GAS1。申请人前期研究发现小鼠BOC既能与IZUMO1也能与JUNO结合。最近有报道小鼠GAS1影响受精，提示HH受体可能参与精卵融合。因此本项目拟用间接免疫荧光技术鉴定小鼠配子上有哪几种HH受体；用体外受精技术鉴定哪种HH受体参与受精；用酵母双杂交和免疫共沉淀技术鉴定参与受精的HH受体与何种已知精卵融合相关蛋白有相互作用；最后探明HH受体与相互作用的蛋白在配子上是否共定位。项目为揭示精卵融合的分子机制、精卵融合与受精卵激活的关联以及人类不孕不育的部分病因提供新的资料。

Research on molecular mechanism of mammalian sperm-egg fusion has reached an impasse. Among a dozen proteins which had been verified to be, only the interaction between IZUMO1 of sperm and JUNO of egg was confirmed to be essential for mouse sperm-egg fusion. However, the interaction between IZUMO1 and JUNO alone could rather mediate somatic cell binding than fusion, suggesting there must be other proteins assisting them to fufill the membrane fusion. Mammalian hedgehog (HH) signaling molecules have four receptors, PTC1, CDO, BOC and GAS1. Our lab found BOC could bind both to IZUMO1 and to JUNO in rat. Recently it was also reported that GAS1 might be involved in mouse fertilization. These works implied that the HH receptors might be a new kind of sperm-egg fusion related proteins. So, this application project aims to verify 1) which HH receptor is on sperm or egg by indirect immunofluorescence technique, and 2) which is involved in sperm-egg fusion by in vitro fertilization, and 3) the interaction modes of the implicated HH receptors to the IZUMO1, JUNO and other known sperm-egg fusion related proteins by yeast two hybrid(Y2H) and co-immunoprecipitation(Co-IP) system, as well as 3) the co-localization of the implicated HH receptors with the interacted proteins on sperm and egg, for finding new sperm-egg fusion related proteins and new clues to human infertility,.and bring new insights into understanding the molecular mechanism of mammalian sperm-egg fusion and the relationship of gamate fusion with egg activation.