Mechanism for the regulation of sperm flagellar motility by ubiquitin/proteasome system

泛素/蛋白酶体系统参与短寿命蛋白的降解和细胞内几个重要过程的调控。它在受精过程中也起着重要的作用，如精卵相互作用和精子运动的调节。本研究以鲑科鱼类和海鞘为实验材料，从多个方面揭示了该系统对精子运动的调控机制。首先，除了与精子蛋白酶体系统密切相关的动力蛋白轻链外，我还鉴定了一种新的15 kDa蛋白，其磷酸化受蛋白酶体调节。该蛋白质被证明位于鞭毛的基部。第二，蛋白酶体系统被证明是由钾通道施加的膜超极化的上游调节。第三，进行研究以揭示泛素化蛋白是否存在于广泛的生物体中。结果表明，鲑鱼精子中不含可检测到的泛素化蛋白，而海鞘精子中含有大量的泛素化蛋白。在海鞘精子中，泛素定位于精核和精子囊之间的区域。这些表明，泛素不参与精子功能的一般方面，如精子活力，但在海鞘特异性过程中，最有可能在受精时观察到的线粒体歧视。最后通过对玻璃海鞘中表达基因的分析，分离出了几个泛素/蛋白酶体系统的基因。到目前为止分析的12，000个EST包含几种蛋白质泛素化酶和26 S蛋白的组分。这些cDNA为进一步研究精子细胞泛素/蛋白酶体系统的功能提供了有力的工具。

Ubiquitin/proteasome system is involved in the degradation of short life-time proteins and the regulation of several important intracellular processes. It also plays important roles in the process of fertilization, such as sperm-egg interaction and the regulation of sperm motility. In this research several approach were conducted to reveal the mechanism for the regulation of sperm motility by this system, with salmonid fish and ascidian as experimental materials. First, apart from a dynein light chain which is closely related to sperm proteasome system, I have identified a novel 15 kDa protein of which phosphorylation is regulated by proteasome. The protein is shown to be located at the base of the flagellum. Second, the proteasome system is shown to be upstream regulated by membrane hyperpolarization exerted by a pottasium channel. Third, research was conducted to reveal if ubiquitinated proteins were present in broad range of organisms. It was shown that salmonid fish sperm contain no detectable ubiquitinated protein, however ascidian sperm show high content in ubiquitinated proteins. In ascidian sperm, ubiquitin is localized in the region between sperm nucleus and mitochondrion. These suggest that ubiquitin is involved not in the general aspect of sperm function such as sperm motility but in ascidian-specific process, most likely in mitochndrial discrimination observed at fertilization. Finally several genes for ubiquitin/ proteasome system have been isolated by overall anaysis of genes expressed in Ciona testis. The 12,000 ESTs so far analyzed contain several enzymes for protein ubiquitination and components of 26 S proteins. These cDNAs would surely provide a powerful tools for further research on the function of ubiquitin/ proteasome system in sperm cells.