FXYD1 participates in regulating sperm motility, capacitation, and acrosome reaction via associations with NKA/NCX1, ion homeostasis, membrane potential, and acrosin activity, with distinct localization in mouse and human sperm

Abstract Background FXYD1 plays a significant role in diverse biological processes and diseases. However, whether FXYD1 regulates sperm function is unknown. Methods We investigated the effects of FXYD1 on motility, capacitation, and the acrosome reaction, as well as the potential mechanisms underlying these effects. Results FXYD1 localization changed dynamically during sperm maturation: it was present at the acrosome in the caput epididymis and then transitioned to the head hook in the cauda epididymis. Treatment with the anti-FXYD1 antibody resulted in a significant decrease in the percentage of motile and progressive sperm, as well as reduced straightness (STR) and linearity (LIN). Furthermore, antibody perturbation suppressed capacitation and the acrosome reaction. Mechanistically, we demonstrated that FXYD1 interacted with acrosin, and antibody treatment significantly decreased acrosin activity. FXYD1 also interacted with Na + -K + -ATPase (NKA); blocking FXYD1 increased NKA activity and decreased intracellular sodium concentration ([Na + ] i ). Concurrently, the FXYD1 perturbation induced a profound change in the membrane potential through interaction with the sodium-calcium exchanger 1 (NCX1) and decreased intracellular calcium ([Ca²⁺] i ) levels. Intracellular signaling analysis revealed that FXYD1 is phosphorylated at serine 68 (S68) by PKA and PKC, and its inhibition altered global protein tyrosine and serine phosphorylation. Finally, unlike in mice, FXYD1 in human sperm was localized to centrioles. Conclusion FXYD1 is involved in sperm function, particularly motility, capacitation, and the acrosome reaction by interacting with NKA, NCX1, and acrosin, thereby influencing ion homeostasis ([Na⁺] i , [Ca²⁺] i , and Vm), enzymatic activity, and downstream phosphorylation cascades. This study contributes to the understanding of FXYD1’s biological roles in sperm physiology and provides novel insights into the mechanisms by which FXYD1 regulates sperm function.