Introduction: This project aims to investigate the nature of cryo-induced DNA damage in the spermatozoa of the model species Xenopus tropicalis. We have turned to Xenopus as a tool to achieve this because the genome is available and we aim to identify specific genomic regions that are most affected during sperm cryopreservation.
Materials and methods: The sperm chromatin dispersion (SCD) test was used to show DNA damage and the potent DNA repair inhibitor 3-aminobenzamide (3-AB) was used to reveal the effects of sperm DNA damage in whole embryos produced by IVF. Morphological assessment and in situ hybridization was used to analyse the phenotypes. Biochemical techniques were used to validate genes, particularly gastrula markers which were identified as potential hotspots for DNA damage.
Results and discussion: Single and double stranded DNA breakage was induced by sperm cryopreservation. In fresh sperm samples 6.06% showed fragmentation compared to 17.65% in cryopreserved sperm samples. Furthermore, the dynamics of DNA damage revealed the chromatin is less stable following the freezethaw process. This is demonstrated in vivo by treating embryos derived from cryopreserved sperm with 3-AB. Over 65% of these embryos exhibit developmental abnormalities, mostly grastrula defects, compared to 17% abnormal embryos without 3-AB. Based on the observed phenotypes fibroblast growth factor 8 (Fgf8), a gene necessary for proper gastrulation, was identified as one of the genes affected most by cryopreservation. By better understanding cryo-induced damage we will be able to develop the practical applications of genetic resource banking and improve its generic success across a wider range of species.
02 - 04 Sep 2014
World Congress of Reproductive Biology