Localization and Trafficking Behavior of SPPL2A During Mouse Spermiogenesis

Signal peptide peptidase-like 2A (SPPL2A) is an intramembrane protease identified as a binding partner of intraflagellar transport protein 27 (IFT27), suggesting a potential role in sperm flagellum development. However, the localization and trafficking of SPPL2A during spermiogenesis remain unknown. Here, we generated an endogenous SPPL2A–mCherry knock-in mouse model using CRISPR/Cas9 to visualize SPPL2A in living male germ cells. Expression of the fusion protein was confirmed by Western blotting, and fertility, spermatogenesis, sperm morphology, and motility were evaluated. Live-cell fluorescence imaging, immunofluorescence, and single-particle tracking (SPT) were used to characterize SPPL2A localization and trafficking. Homozygous knock-in mice exhibited normal fertility, spermatogenesis, and sperm parameters, indicating that the mCherry fusion did not disrupt SPPL2A function. SPPL2A–mCherry was detected as punctate structures in developing germ cells and elongating sperm tails. Its localization changed dynamically during spermiogenesis and was closely associated with developing tail microtubules. Live-cell SPT demonstrated bidirectional movement of SPPL2A-containing particles within developing sperm tails. Mean-squared displacement analysis indicated predominantly confined diffusion with a confinement length scale of 153 ± 15 nm, while particle trajectories showed a slight bias away from the cell body. Particle mobility progressively decreased during sperm maturation, with highly mobile puncta in early spermatids becoming largely immobile in mature sperm. These findings establish an endogenous SPPL2A fluorescent knock-in mouse model and show that SPPL2A-containing particles exhibit intracellular movement within developing sperm tails during spermiogenesis. This model provides a valuable platform for investigating protein transport during sperm flagellum assembly.