Epididymal sperm maturation in mouse species with differing levels of sperm competition†

During epididymal transit, spermatozoa undergo crucial morphological, biochemical, and molecular modifications that enable the acquisition of motility, acrosomal remodeling, membrane reorganization, and chromatin stabilization, culminating in full maturation. This study investigated sperm motility, sperm head and acrosome remodeling, chromatin compaction (protamination), DNA integrity, and nuclear morphology in sperm collected from different epididymal regions (caput, corpus, and cauda) and the vas deferens in three closely related mouse species exhibiting varying levels of sperm competition. Using microscopy, fluorescent staining (Hoechst 33342, chromomycin A3), sperm chromatin structure assay (SCSA), and nuclear morphology software, we characterized these parameters. Results demonstrated a gradual acquisition of total and progressive motility from the caput onwards and a shift toward a more compacted acrosome, especially between corpus and cauda. Minor species-specific variations in nuclear shape and dimensions were observed. Chromatin compaction and DNA integrity significantly increased, evidenced by decreased chromomycin A3 positive cells and reduced DNA fragmentation indices (tDFI and HDS). A correlation between protamine status and DNA fragmentation appeared when data from all species were pooled. Mus musculus showed lower overall motility and a faster decline in loose acrosomes. M. spicilegus exhibited the most rapid sperm head compaction, whereas M. spretus had the highest sperm nuclear width and the lowest tDFI values. These findings reveal distinct changes in sperm maturation markers along the epididymis and suggest associations between these changes and sperm competition levels of these species. This enhances our understanding of sperm maturation mechanisms and may inform advances in reproductive technologies.