Molecular structure of sperm flagella in mammal

本研究的目的是阐明哺乳动物精子鞭毛的分子结构，特别是参考Tektin 4和Spetx-1已被克隆的差异显示和酵母双杂交技术。我们用免疫电镜观察发现，Tektin 4存在于外致密纤维（ODF）的表面，Spetex-1是卫星纤维的组成部分。此外，我们发现Tektin 2和Tektin 5分别定位于ODF周围和线粒体鞘的内表面。我们还发现Gcl-2与ODF的皮质相关，如Tektin 4。我们使用从纯化的大鼠精子制备的分级样品，即，质膜、轴丝、ODF和纤维鞘。结果显示，所有这些分子被发现在6 M尿素提取的级分（ODF和ODF相关级分）中被检测到。当Tektin 4和Spetex-1共转染COS-7细胞时，发现这两种分子共定位于细胞核周围的斑块状结构中，这一观察结果表明这两种分子在细胞中可以相互作用。最后，我们进行了酵母双杂交筛选，以分离与Tektin 2和Tektin 5相互作用的分子，从而分离出几种新分子。通过对这些分子的研究，将为进一步了解哺乳动物精子鞭毛的分子结构打下基础。

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.