小鼠卵巢生殖干细胞分子标记的筛选及干细胞功能研究

In female mammals, whether there is postnatal neo-oogenesis has been vigorously debated for more than six decades. Recent in vitro studies suggested the existence of germ stem cells in postnatal ovaries of mouse, pig and human. However, it is unclear whether such germ stem cells are active under physiological conditions and play essential roles in sustaining normal ovarian function. Recently, we employed Oct4-driving in vivo lineage tracing to investigate if there are active germ stem cells in adult mouse ovaries. Our results demonstrated the existence of active germ stem cells and the germ stem cell-based primordial follicle replenishment. Based on the above progress, we next aim to identify the specific molecular markers of the ovarian germ stem cells and to understand the significance of germ stem cells in maintaining ovarian function. To this end, single cell RNA-Seq analysis will be utilized to identify the specific genes of germ stem cells in mouse. The germ stem cell-specific gene promoter driving Cre-ERT2 transgenic mouse line will be generated. One one hand, Cre-ERT2 transgenic mice are cross breed with Rosa26-Stop-YFP to establish new in vivo lineage tracing and investigate in detail the regenerative activity of germ stem cells under physiological conditions. On the other hand, Cre-ERT2 transgenic mice are cross breed with iDTR transgenic mice to specifically ablate germ stem cells and investigate the significance of germ stem cells in maintaining ovarian function. These studies are anticipated to clarify whether there is postnatal neo-oogenesis in mammals and how important the germ stem cells are to ovarian function. They can also provide new clues to understand and treat the premature ovarian failure in clinical.

经典理论认为哺乳动物在出生时卵巢已储备了终生所需的原始卵泡，出生后没有卵细胞的再生。但近10年，科学家从小鼠、人及猪的卵巢中分离并培养获得了生殖干细胞样细胞，移植到损伤卵巢中能发育形成有功能的卵细胞。这些研究结果提示哺乳动物出生后卵巢中可能存在生殖干细胞。但是，生理条件下体内是否真正存在卵巢生殖干细胞及其再生活动，且生殖干细胞的再生是否对卵巢功能的维持至关重要尚不清楚。我们前期通过Oct4基因驱动的体内世系示踪手段，观察到生理条件下小鼠卵巢内的确存在活跃的生殖干细胞及原始卵泡的再生现象。在此基础上，本项目拟通过单细胞RNA-Seq寻找小鼠卵巢生殖干细胞特异表达的基因，并建立特异基因驱动的体内示踪系统，对生殖干细胞在卵巢中的活动情况进行详细研究。另外，还将特异性清除生殖干细胞，观察对卵巢功能的影响，阐明生殖干细胞对卵巢正常功能维持的意义。结果有望重新认识哺乳动物雌性配子发生及卵巢衰退机制。

我们按研究计划，进行了三方面的内容研究：1) 寻找小鼠卵巢生殖干细胞特异表达的基因；2) 基于筛选到的特异标记基因建立新的Cre-ERT2转基因工具小鼠并研究卵巢生殖干细胞的再生活动及其对卵巢正常功能维持的意义；3) 在猕猴卵巢中初步检测是否也存在表达该特异基因的潜在生殖干细胞。目前，我们发现Sox2和Prdm14很可能标记潜在的卵巢生殖干细胞。并建立了Sox2-Cre-ERT2：Rosa26-Stop-eYFP示踪体系，通过示踪实验，初步认为为表达Sox2的潜在干细胞能够在生理条件下进行再生活动。我们也在幼年（1岁）和成年（5岁）猕猴的卵巢中检测了Sox2和Prdm14的表达情况，发现猕猴卵巢中也存在表达Sox2 和 Prdm14的未形成卵泡结构的生殖细胞。提示灵长类中可能也存在潜在的卵巢生殖干细胞。总体来说，按计划进行了研究，但由于动物实验花费时间较长，后续还需要继续完善。