Abstract

Ionizing radiation impairs male fertility, but the ultrastructural lesions linking radiation to sperm dysfunction remain unclear. The sperm annulus, a ring structure essential for flagellar integrity, is a potential but unresolved target. Here, we integrate synchrotron X-ray ptychography, cryogenic soft X-ray tomography, electron microscopy, and confocal imaging for cross-scale visualization of radiation-induced injury. Ptychography enables quantitative imaging of the annulus at ∼40-nm resolution, bridging light and electron microscopy. In irradiated mice, we find annulus fracture, loss of cytomembrane coverage, and disruption at the midpiece-principal piece junction. These defects worsen during maturation, correlating with reduced motility and increased abnormalities. Protein analyses show down-regulation of SEPT12 and AKAP4, indicating compromised annulus and fibrous sheath. Cryogenic tomography confirms ultrastructural deterioration in the axoneme, fibrous sheath, and mitochondria. These findings identify the sperm annulus and fibrous sheath as primary radiation targets, defining a structural basis for radiation-induced male infertility. This multimodal approach offers a generalizable strategy for nanoscale 3D cellular architecture analysis.