成体罗非鱼雌性生殖干细胞鉴定及其在人工诱导已分化卵巢性逆转模型中转分化机制研究

The latest studies have revealed that germline stem cells (GSC) are present in adult vertebrate ovaries, which brought us into an epoch of female GSC. In our prophase research, germline stem-like cells were observed in the differentiated ovaries of three-month-old Nile tilapia (Oreochromis niloticus）,which could be successfully reversed to functional testes by aromatase inhibitor (Fadrozole) treatment. These data strongly suggest that female GSC might be present in the differentiated ovaries of Nile tilapia and possess the potential of transdifferentiation into male GSC. To identify female GSC in differentiated ovaries of Nile tilapia, mitotically active germline cells will be firstly examined by histological observation and BrdU incorporation method; then in vitro culture system of female GSC will be established and labeled with biological dye PKH26, which will be transplanted into female red tilapia sterilized by pre-treatment with busulphan to destroy the existing germ cell pools. Through transcriptome analysis from Nile tilapia gonads, related molecular markers for female and male GSC of Nile tilapia will be isolated by RT-PCR and 5', 3'-RACE, and then be characterized by qPCR, in situ hybridization and immunohistochemistry. As the above mentioned way, PKH26 labeled female GSC will be transplanted into male red tilapia to investigate the potential of female GSC of transdifferentiation into functional sperms. Based on a model in which differentiated ovary has been artificially induced to reverse to functional testis, migration and expression profiles of related molecular makers of female GSC will be investigated through BrdU incorporation method, in situ hybridization and immunohistochemistry; meanwhile, germination, proliferation, differentiation and expression profiles of related molecular markers of male GSC in the sex-reversed testis will also be investigated. Taken together, the potential and molecular mechanisms of female GSC of transdifferentiation will be elucidated. This work will promote the study of regulation mechanisms of differentiation of female GSC, and provide insight into molecular mechanisms of sex reversal of differentiated ovary.

研究证实，脊椎动物成体卵巢中依然存在雌性生殖干细胞（germline stem cells,GSC），开启了雌性GSC研究新纪元。我们在3月龄罗非鱼已分化卵巢中观察到雌性GSC样细胞,同时成功诱导其性逆转为功能性精巢。我们推测，已分化卵巢存在雌性GSC且能转分化为雄性GSC。本研究拟通过细胞增殖能力检测、细胞培养及细胞移植鉴定罗非鱼成体卵巢中雌性GSC；RT-PCR、qPCR、原位杂交、免疫组化等分离、鉴定雌、雄性GSC相关分子标记；细胞移植检测雌性GSC转分化形成功能性精子的能力，细胞增殖能力检测、原位杂交、免疫组化等检测人工诱导已分化卵巢性逆转模型中雌性GSC的迁移及相关分子标记的表达变化，及性逆转精巢组织中雄性GSC的发生、增殖、分化与相关分子标记的表达变化，探索雌性GSC转分化及其机制。该研究将推进雌性GSC及其分化调控机制的深入研究，同时为揭示已分化卵巢性逆转机制奠定基础。

生殖干细胞（germline stem cell,GSC）是动物体内唯一能向子代传递遗传信息的成体干细胞。对GSC的研究不仅可为体外进行生殖细胞发生过程研究提供新的技术平台，而且在治疗不育、基因治疗、转基因动物生产、珍稀物种遗传资源保护等方面具有重要意义。该项目开展了以下几方面研究：1）通过RT-PCR、免疫组化、原位杂交、启动子克隆及活性检测、细胞转染等方法，分离、鉴定了尼罗罗非鱼多能性标志分子oct4及GSC特异分子标志gfrα1，结果显示，罗非鱼oct4与哺乳类类似，主要表达于囊胚期多能性细胞、原始生殖细胞、各级卵细胞、精原细胞，其表达受到自身及sox2的正向调控，RA可诱导干细胞分化，同时可负向调控oct4的表达；gfrα1表达于胚胎干细胞、生殖干细胞； 2）通过RT-PCR、原核表达、免疫组化、原位杂交等方法，分离鉴定了罗非鱼生殖干细胞自我增殖及分化重要基因胶质细胞源神经因子gdnfa/b、lif及figla，并获得了纯化的原核可溶性表达的gdnfa/b及lif重组蛋白，对其在雌性生殖干细胞的增殖与分化方面的生物学作用进行了进一步研究；3）通过药物处理、细胞移植等实验，进行了罗非鱼生殖细胞的移植及检测，建立了罗非鱼生殖细胞移植的技术平台及检测体系。该项目研究成果不仅深化了我们对雌性GSC及其分化调控机制的认识，而且为后期硬骨鱼类生殖干细胞的培养、鉴定及其移植奠定了重要基础。本研究项目发表相关SCI论文4篇，中文核心期刊论文1篇，进一步的成果还在总结撰写中。