Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581 Japan.
Mol Reprod Dev. 2011 Aug;78(8):611-20. doi: 10.1002/mrd.21352. Epub 2011 Jul 8.
Mammalian sperm flagella have filament-forming Tektin proteins (Tektin 1-5) reported to be involved in the stability and structural complexity of flagella. Male mice null for Tektin3 produce spermatozoa with reduced forward progression and increased flagellar structural bending defects. The subcellular localization of Tektin3 (TEKT3) in spermatozoa, however, has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT3 in flagella of rat spermatozoa, we performed extraction studies followed by immunoblot analysis, immunofluorescence microscopy, and immunogold electron microscopy. Extraction of sperm flagella from the cauda epididymis resulted in complete removal of axonemal tubulins, while TEKT3 was resistant to extraction with the same S-EDTA (1% SDS, 75 mM NaCl, 24 mM EDTA, pH 7.6) solution, suggesting that TEKT3 might be present in the peri-axonemal component and not directly associated with axonemal tubulins. Resistance to S-EDTA extraction might be due to disulfide bond formation during epididymal maturation since concentrations of DTT greater than 5 mM drastically promoted release of TEKT3 from flagella. Immunofluorescence microscopy and pre-embedding immunoelectron microscopy revealed that TEKT3 was predominantly associated with the surface of mitochondria and outer dense fibers in the middle piece. In addition, TEKT3 was found to be present at the equatorial segment region of the acrosome membrane in sperm heads. TEKT3 might not only work as a flagellar constituent required for flagellar stability and sperm motility but also may be involved in acrosome-related events, such as the acrosome reaction or sperm-egg fusion.
哺乳动物精子鞭毛含有丝状的 Tektin 蛋白（Tektin 1-5），据报道这些蛋白参与了鞭毛的稳定性和结构复杂性。Tektin3 基因敲除的雄性小鼠产生的精子向前运动能力降低，鞭毛结构弯曲缺陷增加。然而，Tektin3（TEKT3）在精子中的亚细胞定位在超微结构水平上尚未阐明。为了阐明 TEKT3 在大鼠精子鞭毛中的分子定位，我们进行了提取研究，随后进行了免疫印迹分析、免疫荧光显微镜和免疫金电子显微镜分析。从附睾尾部提取精子鞭毛会导致轴丝微管完全去除，而 TEKT3 抵抗相同的 S-EDTA（1%SDS、75 mM NaCl、24 mM EDTA、pH7.6）溶液提取，这表明 TEKT3 可能存在于轴丝周围的成分中，而不是直接与轴丝微管相关。抵抗 S-EDTA 提取可能是由于附睾成熟过程中形成二硫键所致，因为 DTT 浓度大于 5 mM 会大大促进 TEKT3 从鞭毛中释放。免疫荧光显微镜和预包埋免疫电子显微镜显示，TEKT3 主要与中段的线粒体表面和外致密纤维相关。此外，在精子头部顶体膜的赤道段区域发现了 TEKT3。TEKT3 不仅可能作为鞭毛稳定性和精子运动所必需的鞭毛成分发挥作用，而且可能参与顶体相关事件，例如顶体反应或精子-卵融合。
