Genomic aspects of DNA damage induced by germplasm cryopreservation

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Cryopreservation of germ cells makes major contributions to the effectiveness of medicine, aquaculture and agriculture. In particular it contributes to the biobanking of samples from endangered species and to the reduction of the number of transgenic and mutant research animals that need to be held in laboratories. These last two uses are particularly important for amphibia at this time, the technology for producing large numbers of transgenic and mutant Xenopus lines is now being used increasingly and, in the wild, amphibia are under great pressure with around one-third of species under threat. The current technologies required for gamete and embryo cryopreservation are largely species-specific and, even after six decades of development, there is little understanding of the factors that determine the viability and fertility of the stored materials. This project aims to investigate genomic aspects of cryoinjury in the spermatozoa of a model species (Xenopus tropicalis) in the belief that by better understanding the fundamental reasons for poor posthaw survival and fertility we will be able to develop the practical applications of genetic resource banking and improve its generic success across a wider range of species. Recently we have focused on analysing the effects of chilling and freezing on sperm nuclear DNA, a topic that has seriously lacked attention. We showed that: (1) while the frozen/thawed loach and zebrafish spermatozoa were competent at fertilization, subsequent embryonic developmental rates were poorer than the corresponding embryos produced with fresh spermatozoa, (2) that the deleterious effects of sperm cryopreservation were made worse by briefly exposing embryos to DNA repair inhibitors, and (3) when the embryos produced from non-frozen spermatozoa (controls) were exposed to DNA repair inhibitors, there was no detrimental effect on development. These data supported the hypothesis that sperm DNA was damaged by the freezing process. The DNA repair inhibitors showed that the embryos have a limited intrinsic ability to rescue the damaged DNA. In both loach and zebrafish the major failures in development occurred at the gastrula stage. We will now extend these experiments in an amphibian using the established Xenopus model. We aim to discover whether 'hotspots' of DNA damage occur and if they do, whether these can be directly related to the developmental failure at gastrulation and embryonic patterning defects found in the previous studies using fish models. During the project we will identify both molecular markers and phenotypic changes that indicate the level of germ cell DNA damage necessary to affect developmental potential. In the final part of the project we will use the Xenopus model and these indicators to test the efficacy of newly developed cryopreservation techniques and subsequently to refine these techniques. For the ZSL, the end-user, the data obtained from this project will inform the development of cryopreservation methods to be applied across a range of endangered species. For the student, in addition to being exposed to world-class research facilities at both Portsmouth and the ZSL, they will learn classical embryology, traditional molecular and high throughput techniques for gene expression analysis, gain and loss of function methods and cryopreservation. We expect the outputs from this work to be 1) identification of the functional consequences of DNA damage during germ cell cryopreservation 2) better understanding of the relationship between this DNA damage and the developmental potential of the resulting embryos 3) a reduction in the number of Xenopus held in research facilities and 4) a well-trained and versatile post-doctoral scientist.