The effects of DNA damage caused by sperm cryopreservation in Xenopus

Until recent years sperm fitness has been measured using traditional parameters such as motility and morphology. Given the importance of the sperm genome in the subsequent development of the organism, more focus has now been placed on measuring DNA quality of spermatozoa. This study investigated genomic aspects of cryoinjury in the spermatozoa of the model organism Xenopus. The membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation. A dye exclusion assay revealed that plasma membrane integrity in both species decreased after freezing, more so in X. laevis spermatozoa than X. tropicalis. The sperm chromatin dispersion test showed that for both X. tropicalis and X. laevis activated, frozen spermatozoa produced the highest levels DNA fragmentation compared to all fresh samples and frozen, non-activated samples. Immediately after thawing, the most relevant for fertilisation use, there was a significant increase in DNA fragmentation in frozen, activated X. laevis samples. DNA damage in frozen, activated X. tropicalis samples was not detected until after four hours of incubation at room temperature. However, the DNA fragmentation dynamics over twenty-four hours suggested reduced DNA stability and cryptic DNA damage. 3-aminobenzamide (3-AB), a potent DNA repair inhibitor, was used to treat embryos and thus reveal the effect of damaged sperm DNA on development. Comparisons were made between control embryos derived from fresh spermatozoa and frozen spermatozoa. Results showed a decrease in the survival percentage of treated embryos derived from cryopreserved sperm compared to controls. Furthermore, there was an increased occurrence of posterior and gastrula defects in these embryos. Further analysis of these embryos revealed altered expression of gastrula markers, in particular Fgf8. These may be candidates for genes that undergo DNA damage in cryopreserved spermatozoa. Transcriptome analysis comparing 3-ABtreated embryos derived from frozen and fresh spermatozoa revealed that there were subsets of genes whose expression was consistently altered by cryopreservation; some of these genes are important at the mid-blastula transition and gastrula stage of X. tropicalis development. Physical mapping of the differentially expressed genes (DEGs) onto the X. tropicalis chromosomes revealed particular regions contained more DEGs compared to others, showing non-random distribution of genes affected by sperm cryopreservation. These data will guide future studies of amphibian sperm preservation enhancing conservation and lab animal resource biobanking.