鸡Rab23介导SLIT1/ROBOs信号通路调控卵泡选择与分化的分子机制

Egg production performance is depended by the numbers of ovarian prehierarchal follicle recruited, dominant follicles selection, differentiation and hierarchy establishment. Our group previous studies had proved that SLIT/ROBO signal pathway plays a pivotal role in this process. Moreover, Rab23 as a member of the GTPase family, and its downstream potential effectors (Gli2/3, PAK1/2) have unexpectedly been found to be implicated in regulation trough the signal transduction. Threfore, we postulate that a possible SLIT/ROBO signaling pathway transduced by Rab23 may be involved in regulation of follicle selection and differentiation. However, the exact roles and molecular mechanism of SLIT/ROBO-Rab23 inn the regulation remains poorly understood currently. This project is aimed to reveal the biological effects of Rab23, Gli2/3 and other related factors transduced by SLIT1/ROBOs signaling pathway using the methods of immunofluorescence, co-immunoprecipitation, GTPase activity detection, flow cytometry assay, in vivo bioluminescence imaging system comprehensively. The purpose of this studies lies in providing evidences for the roles of Rab23-Gli2/3 and Rab23-PAKs mediating the SLIT/ROBO signaling in granulosa cell proliferation, differentiation and apoptosis　in prehierarchal follicle development. Furthermore, this work is expected to elucidate the mechanism of the SLIT1/ROBOs-Rab23 signaling pathway in hen prehierarchal follicle selection and differentiation, and supply useful data for further studies of the follicles development and the genetic mechanism of egg laying performance.

鸡的产蛋性能决定于卵巢中被募集的前等级卵泡数目、优势卵泡选择、分化和等级化建立等发育过程。本项目组前期研究证实，SLIT/ROBO信号在此过程中发挥关键调控作用；且意外发现，小GTPase家族成员Rab23及下游潜在的效应分子Gli2/3、PAK1/2均与该信号转导密切相关。故推测，SLIT/ROBO信号可能存在由Rab23介导的通路参与了卵泡选择与分化的发育调控过程，但具体功能和分子机制目前尚不清楚。本项目拟综合运用免疫荧光、免疫共沉淀、酶活性检测、流式细胞检测、小动物活体成像技术等方法，揭示Rab23、Gli2/3等分子在SLIT1/ROBOs信号转导中的生物学效应，解析Rab23-Gli2/3和Rab23-PAKs介导该信号在前等级卵泡颗粒细胞增殖、分化和凋亡过程中的作用，旨在阐明该通路调控前等级卵泡选择与分化的分子机制，为深入探究鸡卵泡发育和产蛋性状形成的遗传机理提供依据。

卵泡的生长发育是一个非常复杂而又高度协调的生理过程，此过程不仅受到下丘脑-垂体-性腺轴（HPG axis）内分泌激素的控制，而且受到卵泡内多种旁分泌和自分泌因子等的共同调节。近年来有研究表明，小GTPase蛋白Rab23可能在该过程中发挥重要作用，然而，其具体作用机制尚不清楚。本项目研究通过免疫荧光、免疫共沉淀、过表达、靶向干扰、EDU等技术揭示了Rab23的下游效应分子Gli2、PAK1/2、PAK1、ERK1/2之间的调控关系，以及Rab23不同表达状态下其上下游因子和细胞增殖、分化、凋亡的变化。在此基础上阻断srGAPs-CDC42信号通路、再分别阻断Rab23-PAK1-ERK1/2和Rab23-Gli2通路，确定了由Rab23所介导的Rab23-PAK1-ERK1/2和Rab23-Gli2通路对鸡前等级卵泡颗粒细胞增殖、分化和凋亡的生物学效应与影响，进一步证实Rab23通过PAK1-ERK1/2和Rab23-Gli2调控卵泡选择与分化的作用机制。再通过靶向干扰SLIT1基因，阐明两通路在调控卵泡选择、分化和凋亡过程中的相互协同关系。最后在活体水平上进行试验，进一步阐明Rab23介导SLIT1/ROBOs信号通路调控卵泡选择与分化的作用机制。本项目揭示了SLIT1/ROBOs-Rab23以及其下游信号通路调控前等级卵泡选择与分化生长发育的分子机制，此结果可为深入研究维持卵泡募集、优势卵泡选择的遗传机理提供理论依据，为深入探究鸡卵泡发育和产蛋性状形成奠定分子基础，具有重要的科学意义和应用前景。