Molecular structure of sperm flagella in mammal

本研究旨在阐明哺乳动物精子鞭毛的分子结构，特别以通过差异展示和酵母双杂交技术克隆的Tektin4和Spetx-1为例。我们通过免疫电镜发现Tektin4存在于外层致密纤维（ODF）表面，Spetex-1是卫星原纤维的一个组分。此外，我们发现Tektin2和Tektin5分别位于ODF周围和线粒体鞘内表面。我们还发现Gcl-2和Tektin4一样与ODF皮层相关。我们利用纯化大鼠精子（即质膜、轴突、ODF和纤维鞘）制备的分离样品，通过免疫印迹分析，从生化角度研究了这些分子的定位。结果显示，所有这些分子都被发现在6M尿素提取部位（ODF和ODF相关部位）中检测到。将Tektin4和Spetex-1共转染到COS-7细胞中，发现这两个分子在细胞核周围的斑块状结构中共定位，这表明这两个分子在细胞中可以相互作用。最后，我们进行酵母双杂交筛选，分离出与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，我们将进一步了解哺乳动物精子鞭毛的分子结构。

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.