JUNO在山羊精卵融合中的作用及与CD9的相关关系研究

In 2014, Nature confirmed that Izumo1-Juno interaction is essential for sperm-egg fusion. At present, it is not clear whether Cd9 is involved in this process. No study has tested the function of JUNO and CD9 protein on goat oocytes. Firstly, this project intends to get goat JUNO protein by expression and purification in vitro. Then we will test its binding ability with IZUMO1 in vitro. Secondly, after inhibition of goat oocyte JUNO protein expression or activity, we will detect the sperm binding assays, ability of fertilization. We will illuminate the role of JUNO on goat sperm-egg fusion through interaction with the sperm protein IZUMO. Meanwhile, after ICSI, IVF, parthenogenetic activation, the variations of JUNO and cortical granules distribution will be observed by immunofluorescence, ensuring a mechanism for the JUNO blocking to polyspermy. Finally, by inhibiting CD9 or JUNO expression or activity, we will observe the variations distribution for each other and confirming the interaction between CD9 and JUNO by immunoprecipitation. Our study of the mechanism of JUNO protein in goat sperm-egg fusion will provide the theoretical basis for the improvement of goat embryo production in vitro.

2014年《自然》杂志证明小鼠卵质膜Juno蛋白与精子Izumo1蛋白的结合是精卵融合的先决条件。而目前对于同样影响精卵融合的Cd9是否参与此过程并未明确。鉴于目前山羊卵子JUNO与CD9蛋白的功能均未有明确报道。因此，首先本项目拟通过体外表达纯化山羊JUNO蛋白，研究其与IZUMO1在体外的结合能力；其次通过抑制山羊卵JUNO蛋白表达或活性，检测其对精子的黏着能力、受精能力等指标的影响，进一步阐明JUNO蛋白在山羊精卵融合中是与IZUMO1蛋白结合而产生对精子的黏着作用；同时通过免疫荧光观察IVF、ICSI、孤雌激活前后JUNO与皮质颗粒在质膜上的分布变化，明确其在抑制多精受精中的作用；最后通过分别抑制CD9与JUNO表达或活性，观察对彼此表达分布的影响，并利用蛋白质免疫共沉淀验证二者在精卵融合中的关系。最终阐明JUNO蛋白在山羊精卵融合中的作用机制，为完善羊体外胚胎生产体系提供理论依据