Molecular structure of sperm flagella in mammal

本研究旨在结合利用差异显示和酵母双杂交技术克隆的Tektin4和Spetx-1基因，阐明哺乳动物精子鞭毛的分子结构。我们用免疫电子显微镜揭示了Tektin4存在于外密纤维(ODF)的表面，而Spetex-1是卫星纤维的组成部分。此外，我们还发现Tektin2和Tektin5分别定位于ODF和线粒体鞘内表面。我们还发现，与Tektin4一样，GCL-2也与ODF的皮质相关。我们通过免疫印迹分析从纯化的大鼠精子制备的分离样品，即质膜、轴丝、ODF和纤维鞘，对这些分子的定位进行了生化研究。结果表明，所有这些分子都在6M尿素提取组分(ODF和ODF相关组分)中检测到。当Tektin4和Spetex-1共转染COS-7细胞时，发现这两个分子共定位于细胞细胞核周围的斑片状结构中，这表明两个分子在细胞内可以相互作用。最后，我们进行了酵母双杂交筛选，以分离与Tektin2和Tektin5相互作用的分子，从而分离出几个新的分子。通过对这些分子的研究，将为理解哺乳动物精子鞭毛的分子结构奠定基础。

This study is aimed to elucidate the molecular architecture of mammalian sperm flagella with special reference to Tektin4 and Spetx-1 that have been cloned by differential display and yeast two-hybrid technique. We have disclosed by immnunoelectron microscopy that Tektin4 is present at the surface of outer dense fiber (ODF) and Spetex-1 is a component of satellite fibrils. In addition, we showed that Tektin2 and Tektin5 were localized around ODF and the inner surface of mitochondrial sheath, respectively. We also found that Gcl-2 is associated with the cortex of ODF, like Tektin4. We biochemically studied the localization of these molecules by immunoblot analyses using fractionated samples prepared from purified rat spermatozoa, i.e., plasma membrane, axoneme, ODF, and fibrous sheath. The outcome revealed that all of these molecules were found to be detected in 6M urea-extracted fraction (ODF and ODF-related fractions). When Tektin4 and Spetex-1 were co-transfected into COS-7 cells, the two molecules were found to be co-localized in patch-like structures around nuclei of the cells, the observation suggesting that the two molecules could interact each other in the cells. Finally, we performed yeast two-hybrid screening to isolate molecules that interact with Tektin2 and Tektin5, which resulted in isolation of several novel molecules. Through study of these molecules, we will make a progress for understanding the molecular architecture of mammalian sperm flagella.