| Budget Amount *help |
¥3,960,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥360,000)
Fiscal Year 2007: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2006: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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.
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