Characterization of chromosomal damage accumulated in freeze-dried mouse spermatozoa preserved under ambient and heat stress conditions

Structural chromosome aberrations and DNA damage generated in freeze-dried mouse spermatozoa were investigated. Freeze-dried sperm samples were preserved at 4, 25 and 50°C for short duration (1 day to 2 months) and at 25°C for long duration (2 years). The spermatozoa were injected into mouse oocytes to analyse the chromosomes of the zygotes at the first cleavage metaphase. Chromosome break of the chromosome-type aberrations was the most common type of structural chromosome aberrations observed in all freeze-dried samples. The frequency of chromatid exchanges rapidly increased in freeze-dried spermatozoa preserved at 50°C for 1-5 days. The frequency of chromatid-type aberrations (break and exchange) gradually increased in freeze-dried spermatozoa preserved at 25°C for up to 2 months. Alkaline comet assay revealed significant migration of damaged DNA accumulated in freeze-dried spermatozoa preserved at 50°C for 3 days and 25°C for 2 years. However, no DNA damage was detected using the same sperm samples by neutral comet assay, which can detect mostly DNA double-strand breaks in cellular DNA. These results suggest that DNA single-strand breaks were accumulated in freeze-dried spermatozoa preserved under ambient or heat conditions, and then chromatid-type aberrations, especially the chromatid exchanges, were formed via post-replication repair system in zygotes.