【作者】 王大辉；

【导师】 杨万喜； 邵建忠；

【作者基本信息】 浙江大学 ， 细胞生物学， 2012， 博士

【摘要】 东方蝾螈(Chinese fire-belly newt, Cynops orientalis)是国家级珍稀保护动物,在发育生物学、进化生物学和保护生物学等领域具有重要科研价值。但是,近些年由于野外人为过度抓捕和蝾螈栖息地遭到破坏,东方蝾螈野生资源日益枯竭。东方蝾螈日益减少是否由于环境的恶化导致其野外生殖能力下降是我们非常关心的科学问题,深入了解该物种精子形成过程以及精子质量控制机制是解决上述问题的关键。精子形成过程是一个高度协调的、复杂的动态过程,精子形成过程通常有三大重要的生物学事件发生：顶体发生、细胞核形变和精子尾部形成。精子形成过程的顺利进行和高质量精子的产生对于东方蝾螈的物种繁衍至关重要。本论文以东方蝾螈精子发生作为模式系统,从精子结构和生精细胞凋亡模式到精子形态建成的动力学机制等角度,对该物种功能性精子形成过程和机制进行了比较深入的探索,旨在解决上述科学问题。1.p53在生精细胞凋亡过程中的作用p53是一个重要的癌症抑制因子,它在DNA修复、细胞凋亡等过程中发挥关键性作用。动物精子发生过程中普遍存在生精细胞凋亡现象,而p53在这个过程中作为重要角色发挥功能。基于已有的科学研究成果,我们总结了精子发生过程中p53的表达模式、外界压力对生精细胞中p53基因和蛋白表达模式的影响、p53缺失或者表达异常对雄性生殖能力的影响以及p53参与生精细胞凋亡过程的分子机制。本综述为更为深入了解和研究p53这一重要癌症抑制因子在动物精子发生过程的重要作用及其机制提供很好的参考。2.东方蝾螈精子超微结构我们利用扫描电镜和透射电镜方法研究了东方蝾螈成熟精子的超微结构。实验结果表明,成熟精子包括头部、颈部和尾部三个部分。精子头部包括顶体、顶体下锥、穿孔器和细胞核等结构。颈部比头部稍短,连接头部和尾部。尾部主要包括中段、主段和末段,中段和主段主要由轴纤维、波动膜、轴丝和边缘丝等结构组成,末段由轴丝和边缘丝或者只有轴丝构成。3.东方蝾螈精子发生过程中生精细胞凋亡模式以及p53、Apafl、Caspase3和Caspase7的作用东方蝾螈作为重要两栖类动物是研究生精细胞凋亡机制的良好动物模型,因为东方蝾螈不但对温度敏感,对饥饿有超强的忍耐力,而且因其皮肤很薄对水环境重金属污染非常敏感,而温度、饥饿、重金属等是研究生精细胞凋亡常用的手段。本研究中,我们利用基因克隆、Realtime RT-PCR, TUNEL, Caspase酶活检测等方法研究了东方蝾螈受到外界环境和内部生理压力后,生精细胞发生凋亡过程中p53、Apafl、Caspase3和Caspase7的作用。研究结果表明,热休克、冷休克、重金属暴露等诱导的生精细胞凋亡过程是通过启动内源性和外源性细胞凋亡通路并最终激活Caspase3实现的,Apafl很可能参与了这些凋亡过程。而饥饿压力诱导的生精细胞凋亡可能主要通过外源性细胞凋亡路径来实现,p53可能没有参与到各种环境压力和生理压力诱导的细胞凋亡过程。东方蝾螈精子发生过程中高频率的精细胞和成熟精子凋亡现象可能是由东方蝾螈精子形态特点以及特殊生殖策略导致的。4.东方蝾螈精子形成过程中KIFC1和Nup62的功能探索KIFC1作为重要的驱动蛋白在动物精子形成过程中发挥重要作用。我们利用基因克隆、蛋白原核表达、抗体制作、免疫荧光和免疫电镜等方法,研究了东方蝾螈精子形成过程中KIFC1的作用。结果表明,KIFC1和Nup62重要功能结构域以及蛋白三级结构在物种进化过程中非常保守。东方蝾螈精子形成过程中,KIFC1可能通过运输高尔基体来源的膜泡物质参与了精子顶体发生过程,并且在Nup62和微管协同作用下参与了精细胞核的形变拉伸以及核质之间的物质运输过程。更多还原

【Abstract】 The Chinese fire-belly newt, Cynops orientalis, is rare amphibian in China, which has great scientific value because of its important status in developmental biology, evolutionary biology and conservation biology. However, its wild resource decreased sharply these years caused by human over-hunting and their habitant being destroyed. Whether it resulted from the weakened male reproductive capability in the wild was the spotlight of our research. To solve this problem, we must understand the spermiogenesis and the mechanism of sperm quality controlling in Cynops orientalis. Spermiogenesis is a highly coordinated and complex process, in which acrosome biogenesis, nuclear shaping and tail formation commonly occurred. A smooth spermiogenesis and high-quality sperm production were quite essential for the reproductive performance of Cynops orientalis. In our researh, using spermatogenesis of Cynops orientalis as our research model, we firstly studied the sperm ultrastructure and the germ cell apoptosis, and then investigated the dynamic mechanism of the sperm morphogenesis. Eventually, we will obtain a good elucidation of the mechanism of spermiogenesis of Cynops orientalis.1. The functions of p53 in germ cell apoptosis during spermatogenesis. p53 is an important tumor suppressor, which plays pivotal functions in DNA repair and cell apoptosis. Germ cell apoptosis commonly occurred during spermatogenesis, and p53 proved to be involved in this process. Based on previous research, we firstly summarized the expression pattern of p53 during spermatogenesis and the influence of environmental stress on its gene and protein expression. Then we discussed the effect of knock out and exotic expression of p53 on male reproductive performance. Finally the molecular mechanism of the germ cell apoptosis regulated by p53 was elucidated. This review can serve as a good reference for further investigation of the performance of p53 in spermatogenesis of Cynops orientalis.2. The sperm ultrastructure of Cynops orientalis Scanning electron microscopy and transmission electron microscopy were conducted to investigate the sperm ultrastruture of Cynops orientalis, and the result showed that the sperm had head, neck piece and tail. The head mainly contained the acrosome, acrosomal cone, perforatorium and nucleus. The neck was shorter than the head, which just linked the head and the tail. The tail consisted of three segments, the midpiece, the major piece and the terminal piece. The midpiece and major piece was mainly composed of axial fibre, undulating membrane, axenome and marginal fibre. But in the terminal piece of the tail, marginal fibre and axenome or only axenome existed.3. Germ cell apoptosis in spermatogenesis of Cynops orientalis and the functions of p53, Apafl, Caspase3 and Caspase7. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation and sensitive skin to heavy metal pollution. A series of methods, like gene cloning, qRT-PCR, TUNEL and caspase activity analysis, were performed in our research, and we analyzed the functions of p53, Apafl, Caspase3 and Caspase7 in germ cell apoptosis induced by various stress that included heat shock, cold shock, cadmium exposure and starvation. And we found that severe germ cell apoptosis triggered by heat shock, cold exposure and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apafl may be involved in this process without elevating its gene expression. Starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 probably didn’t participate in stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence of apoptotic spermatid and sperm probably resulted from sperm morphology and unique reproduction policy of Chinese fire-belly newt, Cynops orientalis.4. The functions of KIFC1 and Nup62 during the spermiogenesis of Cynops orientalis. KIFC1 is an important C-terminal kinesin, which played critical roles in spermiogenesis. In our study, gene cloning, protein prokaryotic expression, antibody production, immunofluoresence and immuno-electron microscopy were performed to investigate the functions of KIFC 1 and Nup62 during the spermiogenesis of Cynops orientalis. We found that KIFC1 and Nup62 were highly conserved in their functional subdomain and protein 3-D structures. KIFC1 probably participated in the acrosome biogenesis by delivering Golgi-originated vesicles, and in cooperation with Nup62, KIFC1 was likely involved in nuclear elongation of spermatid and vesicle transportation between nucleus and cytoplasm.更多还原