Abstract
Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine-the axoneme-but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.