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

Abstract

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.

Chromosomal integrity and DNA damage in freeze‐dried spermatozoa

“…4). DNA damage in sperm induced by heat-stress was not detected using the neutral comet assay, but it was detected using the comet assay with the A/N protocol [46]. The type of damage induced directly in sperm DNA would be not the DSBs that are responsible for the induction of chromosome breaks resulting in chromosomal aberrations.…”

“…PCD is induced just after freeze-drying. In contrast, ACD arises during post-freeze-drying storage [46]. The types of PCD and ACD reviewed here are DNA damage and/or aberrant chromatin remodeling [46,47].…”

“…The origin of PCD can be assessed using single cell gel electrophoresis assay (comet assay) [46]. The comet assay is a well-known technique used to detect DNA damage in situ.…”

“…In contrast, ACD arises during post-freeze-drying storage [46]. The types of PCD and ACD reviewed here are DNA damage and/or aberrant chromatin remodeling [46,47]. Currently, it is unknown whether PCD and ACD can include numerical chromosome aberrations because no studies have focused on these types of aberrations in embryos derived from freeze-dried spermatozoa.…”

Genome Banking for Vertebrates Wildlife Conservation

“…High-temperature storage, however, might be damaging to DNA integrity according to some (Kaneko & Nakagata, 2005;Hochi et al, 2008) but not all Klooster et al, 2011) researchers. These differences may be due to differences in the drying technique or related to differences between species Klooster et al, 2011;Kusakabe & Tateno, 2011).…”