Li Wei, Niu Changmin, Yap Yi Tian, Li Tao, Zheng Cheng, Goswami Mariska, Kandiraju Sanjana, Dhikhirullahi Opeyemi, Xu Jie, Zhang Jifeng, Kelly Christopher V, Zhang Zhibing
Department of Physiology, Wayne State University, Detroit, MI, USA.
School of Nursing School of Public Health, Yangzhou University, Yangzhou, Jiangsu, China.
Biol Reprod. 2025 Feb 14;112(2):309-318. doi: 10.1093/biolre/ioae171.
Intraflagellar transport 25 is a component of the intraflagellar transport 25-B complex. In mice, even though this intraflagellar transport component is not required for cilia formation in somatic cells, it is essential for sperm formation. However, the intracellular localization of this protein in male germ cells is not known given no reliable antibodies are available for histologic studies, and the dynamic trafficking in the developing sperm flagella is not clear. To examine localization of the protein in male germ cells and further investigate the mechanism of intraflagellar transport in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse intraflagellar transport 25-green fluorescent protein knock-in mouse model using the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 system, with the mouse intraflagellar transport 25 protein fused with a green fluorescent protein tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-intraflagellar transport 25 and anti-green fluorescent protein antibodies showed that the intraflagellar transport 25-green fluorescent protein fusion protein was highly abundant only in the testis, which is consistent with the endogenous intraflagellar transport 25 protein. Examination of localization of the intraflagellar transport 25-green fluorescent protein in isolated germ cells revealed that the fusion protein was present in the cytoplasm of spermatocytes and round spermatids and a strong signal was present in the developing sperm flagellar. The homozygous knock-in mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of intraflagellar transport 25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching. Kymograph and fluorescence recovery after photobleaching analyses demonstrate the transport of intraflagellar transport 25-green fluorescent protein within the developing tail demonstrate no apparent preference for trafficking toward and away from the cell body. The speed of trafficking depends on the stage of sperm development, ranging from highly mobile unrestricted diffusion initially, mobile punctate structures in developing sperm, and immobile punctate structures in mature sperm. Our studies demonstrate that mouse intraflagellar transport 25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.
鞭毛内运输蛋白25是鞭毛内运输蛋白25 - B复合体的一个组成部分。在小鼠中，尽管该鞭毛内运输蛋白成分并非体细胞纤毛形成所必需，但对精子形成却是至关重要的。然而，鉴于没有可靠的抗体可用于组织学研究，该蛋白在雄性生殖细胞中的细胞内定位尚不清楚，并且其在发育中的精子鞭毛中的动态运输情况也不明确。为了研究该蛋白在雄性生殖细胞中的定位，并进一步探究精子形成过程中鞭毛内运输的机制，特别是研究该蛋白的动态运输情况，我们使用成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列相关蛋白9系统构建了一种小鼠鞭毛内运输蛋白25 - 绿色荧光蛋白基因敲入小鼠模型，其中小鼠鞭毛内运输蛋白25与C端的绿色荧光蛋白标签融合。分析了三个独立的品系。使用抗鞭毛内运输蛋白25和抗绿色荧光蛋白抗体进行的蛋白质印迹分析表明，鞭毛内运输蛋白25 - 绿色荧光蛋白融合蛋白仅在睾丸中高度丰富，这与内源性鞭毛内运输蛋白25一致。对分离的生殖细胞中鞭毛内运输蛋白25 - 绿色荧光蛋白的定位检查显示，融合蛋白存在于精母细胞和圆形精子细胞的细胞质中，并且在发育中的精子鞭毛中有强烈信号。纯合基因敲入小鼠的精子发生、生育能力和精子参数均正常。对发育中的鞭毛内鞭毛内运输蛋白25的扩散分析表明，通过光漂白后荧光恢复显示存在移动和固定部分。动态光密度扫描图和光漂白后荧光恢复分析表明，发育中的尾部内鞭毛内运输蛋白25 - 绿色荧光蛋白的运输显示出向细胞体和远离细胞体运输时没有明显偏好。运输速度取决于精子发育阶段，最初是高度移动的不受限制的扩散，发育中的精子中有移动的点状结构，成熟精子中有固定的点状结构。我们的研究表明，小鼠鞭毛内运输蛋白25沿着发育中的精子鞭毛在两个方向上移动，这可能对功能性精子形成至关重要。
