Radial spokes (RSs) are conserved multimolecular structures attached to the axonemal microtubule doublets and are essential for the motility control of both cilia and sperm flagella. CFAP91, an RS3 protein, is implicated in human male infertility, yet its molecular function remains poorly understood. Here, we demonstrate that Cfap91 knockout (KO) mice exhibit impaired sperm flagellum formation and male infertility. Using a transgenic rescue model expressing FLAG- and BioID2-tagged CFAP91, we reveal that CFAP91 immunoprecipitates with RS3 proteins CFAP251 and LRRC23, whose localization is disrupted in Cfap91 KO sperm flagella. In addition, proximity labeling in mature spermatozoa identifies EFCAB5 as a sperm-specific CFAP91-proximal component. We show that Efcab5 KO males exhibit reduced sperm motility and fertility. Our findings establish CFAP91 as an essential scaffolder of RS3 assembly and EFCAB5 as a sperm-specialized movement regulator, advancing understanding of axonemal specialization in mammalian spermatozoa and its relevance to male infertility.
© 2025. The Author(s).