Akt1s1基因在小鼠精原干细胞自我更新与维持中的作用机制

Spermatogonial stem cells (SSCs) play important functions for the development of male gamete and the propagation of animal species. Mammalian spermatozoa are developed from SSCs through the process of spermatogenesis that is composed of mitosis, meiosis and spermiogenesis. In addition, SSCs have been an ideal model system for the study of adult stem cells. However, the molecular mechanisms of SSC self-renewal and differentiation remain largely unclear. Our previous studies using cell sorting methods isolated developing SSCs at various stages in mice and compared their differential gene expression profiles. Numerous genes were found to highly enrich in SSCs, including akt1s1, of which the expression is about 12 times higher than that of somatic cells. Recent studies also showed that growth factor GDNF emitted from sertoli cells can elicit its function through SSC receptor Gfra1/cRet and Akt signaling pathway; in addition, SSC marker gene plzf regulates SSC self-renewal and growth by negatively regulating mTORC1 activity and Gfra1 expression in SSCs. Thus to uncover the function of akt1s1 is important for the understanding of SSC regulation. Through the analyses of akt1s1 expression and its protein interactions in SSCs, the molecular mechanisms of its functions will be dissected. Using gene modifications, such as shRNA and mutagenesis methods, the functional roles of akt1s1 will be studied in cultured SSC colonies. Further more, by applying mouse genetics, the physiological function and molecular mechanisms of akt1s1 will be analyzed in conditional knockout mouse model. The proposed research will not only help to decipher the molecular circuitry of SSC self-renewal and differentiation, but will also help to find novel means to treat relevant reproductive diseases.

精原干细胞对精子细胞发育和动物物种的繁衍起着重要的作用。作为成体干细胞，精原干细胞的自我更新与分化的调控机制尚不清楚。在对不同发育期小鼠精原干细胞基因表达的对比分析中，我们发现多个在精原干细胞中高表达的基因，其中akt1s1的表达比体细胞高约12倍。AKT1S1是AKT和mTORC1信号通路的中介，近期研究发现两者在精原干细胞的自我更新与维持中起重要作用，其分子机制有待深入研究。本项目通过详细分析akt1s1在小鼠精原干细胞中的表达及其蛋白相互作用，揭示akt1s1的作用机制；利用精原干细胞体外培养系统中对akt1s1的敲减和变异，分析akt1s1对精原干细胞自我更新与维持的影响；结合对akt1s1在精原干细胞中的条件敲除小鼠的分析，阐明akt1s1生物学功能的分子机制。通过这些研究，我们希望对精原干细胞自我更新与分化的调控机理作深入的理解，为寻找有效治疗相关生殖疾病的方法奠定基础。

精原干细胞是动物物种繁衍和精子细胞发育的基础。做为成体干细胞，精原干细胞自我更新与分化的平衡对动物维持正常生精功能和生育力起着重要作用。在小鼠中，精原干细胞受周边环境因子，包括睾丸中体细胞分泌的激素及生长因子的影响，有序完成自我更新、增殖、减数分裂及分化生长。 已知GDNF、PI3K/AKT及mTORC1信号通路对调节精原干细胞的维持和增殖生长有调节作用，但其中的分子机制尚不十分清楚。在前期研究中，我们发现AKT和mTORC1信号通路中的AKT1S1在未分化精原干细胞中的表达相比于分化的精原细胞高约12倍。果蝇中的研究表明，AKT1S1的缺失导致mTORC1活力的过表达，动物个体及卵母细胞的生长得以增强。在哺乳动物中，AKT1S1在不同组织和细胞中表现出调节作用的差异，其对生殖细胞的作用尚不清楚。本项目通过分析Akt1s1在小鼠精原干细胞中的表达及蛋白相互作用，结合基因敲除小鼠模型，旨在揭示AKT1S1在精原干细胞中的生物学功能和作用机制。初步研究显示，AKT1S1在不同年龄小鼠睾丸中维持稳定的表达水平，基因敲除小鼠的整体生长状态和雄性小鼠生育力未有明显变化，但发育早期小鼠中精原干细胞相较于野生型对照有一定增加。AKT1S1对所处细胞中信号通路因子的表达和修饰亦表相出组织间差异。不同于胰脏及脑组织，在睾丸中，mTORC1下游S6K磷酸化及上游RAPTOR、RICTOR蛋白的表达上调，而4EBP1未有明显变化，表明精原干细胞受PI3K/AKT和mTORC1信号调节的特异性。这些结果为深入理解精原干细胞自我更新与分化的调控机理，寻找有效治疗相关生殖疾病的新靶点奠定了基础。