Two-directional trafficking of the IFT25 protein in the developing mouse sperm flagella.

Intraflagellar transport 25 (IFT25) is a component of the IFT-B complex. In mice, even though this IFT 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 IFT in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse IFT25-GFP knock-in (KI) mouse model using the CRISPR/cas9 system, with the mouse IFT25 protein fused with a GFP tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-IFT25 and anti-GFP antibodies showed that the IFT25-GFP fusion protein was highly abundant only in the testis, which is consistent with the endogenous IFT25 protein. Examination of localization of the IFT25-GFP 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 KI mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of IFT25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching (FRAP). Kymograph and FRAP analyses demonstrate the transport of IFT25-GFP within the developing tail demonstrate no apparent preference for trafficking towards 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 IFT25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.