ANKEF1 is a key axonemal component essential for murine sperm motility and male fertility

Sperm motility is essential for male fertility and depends on the structural integrity of the sperm axoneme, which features a canonical '9 + 2' microtubule arrangement. This structure comprises nine outer doublet microtubules (DMTs) that are associated with various macromolecular complexes. Among them, the nexin-dynein regulatory complex (N-DRC) forms crossbridges between adjacent DMTs, contributing to their stabilization and enabling flagellar bending. In this study, we investigated Ankyrin repeat and EF-hand domain containing 1 (ANKEF1, also known as ANKRD5), a protein highly expressed in the sperm axoneme. We found that ANKEF1 interacts with DRC5/TCTE1 and DRC4/GAS8, two key components of the N-DRC, and these interactions occur independently of calcium regulation. Male Ankef1^-/- mice exhibited impaired sperm motility and infertility. Cryo-electron tomography revealed a typical '9 + 2' axoneme structure with intact DMTs in Ankef1 null sperm; however, the DMTs showed pronounced morphological variability and increased structural heterogeneity. Notably, ANKEF1 deficiency did not alter ATP levels, reactive oxygen species levels, or mitochondrial membrane potential. These findings suggest that ANKEF1 may attenuate the N-DRC's mechanical buffering-akin to a 'car bumper'-between adjacent DMTs, thereby compromising axonemal stability under high mechanical stress during vigorous flagellar beating.