Figla通过调控c-KIT在窦前卵泡发育微环境中的作用及其机制研究

The development of preantral follicle influence the maintanence of ovarian reserve and the ART outcomes. Studies have found that many soluble factors secreted by primary oocyte facilitate the proliferation, inhibit the apoptosis of granular cells and increase the expression of Kitl in them. Our preliminary studies found that a transcription factor, Figla, increased the expression of c-KIT on the membrane of oocyte. Thus, we hypothesize that Figla facilitates the crosstalk between oocytes and granular cells and activates Kitl/c-KIT/PI3K/AKT pathway, which is important in influencing the microenvironment of preantral follicle development. This study plans to use conditional gene knockout mouse to construct ovulation induction model, in vitro ovary culture model and co-culture model with primary oocytes and granular cells, then study the influence of Figla on preantral follicle develpment via regulating c-KIT from the perspective of both single oocyte and entire ovary. The accomplishment of this study is hopefully to reveal the mechmisn of Figla in preantral follicle development and the pathophysiological process of premature ovarian failure. It is also hopefully to provide new therapeutic target for the patients with poor ovarian response or premature ovarian failure in IVF.

窦前卵泡的发育将影响卵巢储备的维持和ART结局。研究发现，初级卵母细胞分泌可溶性因子促进颗粒细胞增殖、高表达Kitl和抑制其凋亡。我们的前期研究发现，卵母细胞转录因子Figla可上调卵母细胞膜表面c-KIT的表达。我们推测，Figla可促进卵母细胞及颗粒细胞的“对话”，激活Kitl/c-KIT/PI3K/AKT通路，对窦前卵泡发育的微环境有至关重要的影响。本课题拟使用条件性诱导基因敲除小鼠，构建小鼠促排卵模型、卵巢体外培养模型和初级卵母细胞-颗粒细胞共培养模型，从单个卵母细胞的个体化角度和卵巢的整体角度两方面研究Figla通过调控c-KIT在窦前卵泡发育微环境中的影响。该课题的完成将有望揭示Figla在窦前卵泡发育中的作用机制，有望从Figla的基因调控角度揭示部分卵巢早衰的病理生理过程，并有望为IVF卵巢低反应性及卵巢早衰的病人提供新的治疗靶点。

始基卵泡向初级卵泡的发育是卵母细胞早期发育的关键环节，受到多种基因的调控。研究发现，FIGLA基因缺陷的小鼠在出生后卵巢结构异常，卵母细胞发育停滞在初级卵母细胞阶段，无法进一步形成初级卵泡，提示其在卵母细胞早期发育中的重要作用。本项目运用crisp-cas9技术构建了FIGLA基因敲除小鼠，比较了野生型和FIGLA基因敲除小鼠卵巢的表型的区别以及卵巢早期发育相关的基因Kit，Nobox，Pou5f，GDF9和Bmp15的区别，进一步证实了FIGLA是卵母细胞早期发育的关键调控因子。同时我们发现FIGLA和kit在小鼠卵巢中的表达有非常相似的时空特异性，提示FIGLA可能通过调控kit影响卵母细胞早期发育。为了进一步明确FIGLA作为转录调控因子如何发挥作用，我们通过Chip-seq技术，检测到和FIGLA结合的DNA片段中有50%以上的启动子序列。对这些启动子序列对应的基因进行KEGG和GO分析，我们发现FIGLA可能调控细胞周期和减数分裂中关键基因的转录。随后我们在FIGLA基因敲除小鼠的卵巢组织中证实了细胞周期相关基因E2F4，E2F7和减数分裂调控基因Ywhab受到抑制。该结果说明FIGLA可以和这些基因的启动子区域结合，并对其进行转录调控，揭示了FIGLA调控卵母细胞早期发育的新机制，为进一步研究FIGLA在卵母细胞早期发育中的作用提供了新的思路。