Localization and Trafficking Behavior of SPPL2A During Mouse Spermiogenesis.
6 affiliations
- 1Department of Environmental Health and Occupational Medicine, Academy of Nutrition and Health, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Public Health, Wuhan University of Science and Technology, Wuhan 430065, China.
- 2Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI 48201, USA.
- 3Department of Physics and Astronomy, Wayne State University, Detroit, MI 48201, USA.
- 4Barber Center for Multiscale Systems Biology, Wayne State University, Detroit, MI 48201, USA.
- 5Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
- 6Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI 48201, USA.
Abstract
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.
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BibTeX
@article{zheng2026localization,
title = {{Localization and Trafficking Behavior of SPPL2A During Mouse Spermiogenesis.}},
author = {Cheng Zheng and Wei Li and Shahnaz Parveen and Anuradha Yadav and Kristina Bennett and Jie Xu and Jifeng Zhang and Ling Zhang and Christopher V Kelly and Zhibing Zhang},
journal = {Cells},
year = {2026},
month = sep,
doi = {10.3390/cells15191773},
url = {https://europepmc.org/article/MED/42882472},
note = {CC BY},
}
RIS
TY - JOUR
TI - Localization and Trafficking Behavior of SPPL2A During Mouse Spermiogenesis.
AU - Cheng Zheng
AU - Wei Li
AU - Shahnaz Parveen
AU - Anuradha Yadav
AU - Kristina Bennett
AU - Jie Xu
AU - Jifeng Zhang
AU - Ling Zhang
AU - Christopher V Kelly
AU - Zhibing Zhang
PY - 2026
DA - 2026/09/29
JO - Cells
DO - 10.3390/cells15191773
UR - https://europepmc.org/article/MED/42882472
AB - 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.
SN - 2073-4409
ER -