FoxG1对热应激湖羊卵泡颗粒细胞凋亡的调控及分子机制研究

Heat stress is the major cause of reduced animal reproduction and lost profits. Hu sheep is well-known as a humidity and heat-stress tolerant, year-round estrus sheep breed which could be used as the animal model of heat stress tolerant research to lessen the profits loss caused by heat stress. Our previous studies showed that transcription factor FoxG1 was highly expressed in the tissues of hypothalamus-pituitary-ovarian axis of Hu sheep, and the expression could be significantly increased by cute heat stress. Moreover, FoxG1 was located in follicular granulosa cells, follicular membrane cells, and luteal cells in the ovaries of Hu sheep. We speculate that FoxG1 may play vital roles in the apoptosis of follicular granulosa cells induced by heat stress. The following research will be carried out to prove this hypothesis. Firstly, the expression level, phosphorylation, and acetylation of FoxG1 in different follicles will be detected, and the relationship between FoxG1 expression and the apoptosis as well as hormone secretion of follicular granulosa cells under heat stress will be analyzed to find out the function of FoxG1 in follicular granulosa cells. Secondly, FoxG1-over-expressed and FoxG1-silenced granulosa cells will be heat-stress treated and high throughout sequenced to find out the key genes in FoxG1 regulating apoptosis and hormone secretion. Luciferase carriers of the selected key gene promotor and the FoxG1 carrier will be co-transfected to the follicular granulosa cells to confirm the functions and cell signal pathways of FoxG1 on regulating the genes induced by heat stress. This study will improve our knowledge of the molecular basis involved in humidity and heat-stress tolerance of Hu sheep and provide idea for genetic alteration of heat-stress tolerant Hu sheep.

热应激严重降低动物繁殖性能。湖羊是耐湿热和四季发情的绵羊品种，阐明并利用其耐湿热机制有利于降低热应激造成的经济损失。申请人研究发现，急性热应激可上调湖羊下丘脑、垂体和卵巢组织中FoxG1基因的表达；FoxG1在湖羊卵巢的卵泡颗粒细胞、卵泡膜细胞和黄体细胞中呈细胞特异性表达，推测FoxG1可能参与调控热应激引起的卵泡颗粒细胞凋亡和卵泡闭锁。本项目首先研究热应激条件下FoxG1在湖羊不同卵泡（大、中、小/健康、闭锁）中的时空表达及磷酸化和乙酰化修饰模式，以及其与卵泡颗粒细胞凋亡和激素合成的关系，探讨FoxG1在热应激引起的湖羊卵泡颗粒细胞凋亡中的作用。然后，在体外培养的卵泡颗粒细胞中干扰或过表达FoxG1，通过高通量测序等筛选和验证FoxG1调控热应激湖羊卵泡颗粒细胞凋亡和功能的关键基因及信号通路，以期揭示湖羊耐湿热和夏季发情的分子机制，为湖羊种质资源的利用提供理论依据。

热应激严重降低动物繁殖性能，给畜牧业带来巨大损失。湖羊是耐湿热和四季发情的绵羊品种，滩羊与湖羊共同起源于蒙古羊，长期的选择条件下，其表现出与湖羊差异较大的热应激不耐受，利用湖羊卵泡颗粒细胞（granulosa cells，GCs）的体内外试验和湖羊与滩羊的热应激耐受比较试验，探索叉头转录因子FoxG1在湖羊耐湿热机制中的作用。利用免疫组织化学方法研究表明：FoxG1在下丘脑、垂体、卵巢、睾丸、背腰肌、腿肌、瘤胃组织中均有细胞特异性表达；构建了FoxG1真核表达载体，利用脂质体介导法将其在湖羊GCs过表达，合成SiRNA干扰湖羊GCs中FoxG1的表达，利用流式细胞仪测定GCs的凋亡，利用CCK8试剂盒测定GCs的增殖，结果表明：FoxG1转录因子可促进热应激状态下湖羊的GCs的增殖。总之，热应激条件下，FoxG1可能通过在下丘脑—垂体—生殖系统（卵巢/睾丸）中高表达，促进GCs的增殖和提升其分泌E2和P4等生殖激素的功能，从而提升湖羊的热应激耐受能力。该研究结果揭示了湖羊耐湿热和夏季发情的分子机制，为湖羊种质资源的利用提供理论依据，有利于降低热应激造成的经济损失。