Hippo：一条新的通过调控卵巢干细胞的增殖和分化重塑及保护卵巢功能的信号通路

Efforts are being made to increase primordial follicle pool by regulating ovarian germline stem cells (FGSCs) proliferation and differentiation and to seek new preventive and curative ways in female infertility, especially premature ovarian failure patients, and aging women. Our recent studies have shown that the Notch signaling is an important signaling pathway in regulation of mammalian FGSCs proliferation and differentiation, but FGSCs regulation mechanism and its relationships between physiological and pathological recession still need to be further explored. Earlier we found that Hippo signaling pathway., which controls a variety of stem cell lines survive and Drosophila egg formation, was involved in the mouse ovary expression and regulation of FGSCs in proliferation. In this study we will use transgenic mice carrying Pou-5fl-EGFP and human ovarian tissue specimens. High/low Hippo signaling pathway expression of FGSCs by genetic engineering are implanted into the ovaries of SCID mouse model of infertility, observing the de novo follicle formation; By microinjection of high expression vectors Yap in the ovaries for further observations on the infertility modeling, fertility recovery in aging mice, and the protective effects of radiation damage to ovarian functions. The findings of this study will add new tactics on prevention and treatment of infertility patients and aging women.

通过调控卵巢生殖干细胞(FGSCs)的增殖分化，是增加原始卵泡池防治女性不孕症和卵巢早衰及衰老的关键手段。申请人最近研究表明，Notch是调控哺乳动物FGSCs增殖分化的重要信号通路，但对FGSCs的调控机制及其与卵巢功能的生理性和病理性衰退关系仍需深入探索。我们前期发现，调控多种干细胞生存并参与果蝇卵子形成的Hippo信号通路在小鼠卵巢中高表达并调控FGSCs的增殖。本研究利用携带Pou-5fl-EGFP的转基因小鼠和人体卵巢组织，拟通过分析Hippo信号通路在FGSCs增殖分化和衰老中的变化、作用及作用机理；通过基因工程手段使Hippo信号通路在FGSCs高/低表达后，将其移植到SCID不孕模型小鼠卵巢中，观察卵泡形成和发育及产生后代的影响；通过卵巢内微注射Yap高表达载体进一步观察对不孕模型和衰老小鼠卵巢功能的重塑及对放射损害卵巢功能的保护作用。为防治女性生育及相关问题提供新的策略。

Hippo信号通路是调控成体干细胞增殖分化及功能的重要信号通路，我们前期研究发现Hippo信号通路的主要效应因子能触发原始卵泡的启动。但Hippo信号通路是否参与卵巢生殖干细胞（OGSCs）功能的调控以及和生理性及病理性卵巢功能衰老的关系还有待进一步证实。本研究中，以 KM系小鼠及人卵巢皮层组织为实验对象：①分析Hippo信号通路的主要效应因子在卵巢表面上皮（ovarian surface epithelium, OSE）细胞中的表达及动态变化，这种变化与卵巢功能生理性衰老和病理性衰老的关系；②离体观察Hippo信号通路的主要效应因子YAP1在OGSCs增殖分化中的作用；③在体分析YAP1经卵巢内微注射对卵巢功能生理性衰老和病理性衰老的拯救作用。研究结果显示：①在不同年龄小鼠和病理性卵巢衰老模型小鼠OSE中存在Hippo信号通路相关分子与OGSCs标记因子（MVH与OCT4）共表达，而且两者的表达强弱和卵巢的功能状态呈现明显的时空相关性。②在不同年龄阶段人OSE中同样存在Hippo 信号通路相关分子与OGSCs标记因子（MVH与OCT4）共表达。同样两者的表达强弱和人卵巢的功能状态呈现明显的时空相关性。③分离得到的OGSCs能共表达MVH，OCT4，Nango，Fraglis， Stella等干细胞和生殖细胞特异性标记因子，OGSCs在转染YAP1过表达慢病毒载体后，MVH，OCT4的表达显著升高，而在转染YAP1shRNA的干扰慢病毒载体后，MVH，OCT4的表达则显著降低。④不孕模型小鼠在转染过表达YAP1后的30-75天期间，可以检测到卵巢内有卵泡再生，原始卵泡启动激活，子代出生率增加，同时伴有血清中E2和FSH水平上升，卵巢OSE细胞中YAP1与pYAP1表达和MVH与OCT4表达均明显增高；而生育旺盛期小鼠在转染YAP1的干扰慢病毒载体后的30-75天期间，检测到卵巢内卵泡数量下降，子代出生率减少，上述指标则呈现相反的变化。.以上结果提示，Hippo信号通路相关分子存在于OGSCs，并能调控OGSCs的体外增殖；Hippo信号通路可能通过调控OGSCs参与了卵巢功能的生理性衰老和病理性衰老过程；调控Hippo信号通路效应分子YAP1可在卵巢功能重塑中发挥重要作用。