The Mechanism Of Germ Cell Apoptosis During Spermatogenesis Of Cynops Orientalis And The Functions Of KIFC1 In Spermiogenesis

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.