Deconstructing the Mammalian Sperm’s Molecular Motor: Structural Insights into Flagellar Architecture and Maturation

Abstract

Sperm cells are highly specialized for fertilization, navigating complex environments through distances exceeding 1,000 times their own length. This thesis investigates the molecular motor of mammalian sperm, the flagellum, examining its architecture, mechanical reinforcement, and maturation processes. The mammalian sperm flagellum comprises four segments: the connecting piece (neck), midpiece, principal piece, and endpiece. Its core is the axoneme, a conserved "9+2" microtubule-based motor system. Mammalian sperm are distinguished by accessory structures including the mitochondrial sheath, outer dense fibers (ODFs), and fibrous sheath, which provide rigidity and torque for swimming in viscous media. The study employed cryo-electron tomography (cryo-ET) to visualize cells in frozen-hydrated, near-native states, avoiding artifacts that arise in classical electron microscopy approaches. Cryo-focused ion beam (cryo-FIB) milling enabled imaging of thick sperm cells by creating transparent lamellae (<200 nm). Specialized computational workflows for tilt series acquisition and sub-tomogram averaging achieved near-atomic resolution within intact cells. Comparative analysis of pig, horse, and mouse sperm revealed several novel findings: First, the proximal centriole in pig sperm was asymmetric with unequal microtubule triplet lengths. The distal centriole featured splayed-open doublet microtubules arrayed asymmetrically around central singlets, non-canonical structures unprecedented in mammalian sperm. Second, spatial regulation of ODF-axoneme coupling was discovered: ODFs connect directly to the axoneme in the principal piece for force transmission but remain detached in the midpiece, presumably enabling flexibility for high-amplitude bends during hyperactivation. Two mechanisms for doublet-to-singlet transition at the tail tip and conserved microtubule capping plugs were also identified. At near-atomic resolution, single-particle cryo-EM identified over 60 proteins decorating axonemal doublet microtubules. Microtubule inner proteins (MIPs) form elaborate luminal ornamentation. In bovine sperm, testis-specific Tektin-5 forms a massive sickle-shaped supercomplex within the A-tubule, likely reinforcing the flagellum against mechanical stress. TEKTL1 and the Mn-motif were established as universal microtubule-binding and stabilization features. Maturation occurs during transit through the epididymis. Caput (immature) sperm possess functional but inhibited axonemal machinery. Nearly half their outer dynein arms remain locked in inactive post-power stroke conformations versus the primed pre-power stroke states dominating mature cauda sperm. Seahorse flux analysis surprisingly revealed declining oxidative phosphorylation and glycolysis during maturation, suggesting more efficient energy management rather than increased ATP production. Structural insights into the cytoplasmic droplet—a migrating organelle involved in maturation—revealed complex vesicular networks with densities potentially involving SYPL1 and ALOX15 proteins managing degradation and trafficking. This multi-scale structural blueprint links microscopic anatomy to atomic-scale protein interactions, providing foundations for understanding male infertility and identifying novel contraceptive targets.