Genomic aspects of DNA damage induced by germplasm cryopreservation

生殖细胞的超低温保存对医药、水产养殖和农业的有效性有着重要的贡献。特别是，它有助于对濒危物种的样本进行生物库管理，并有助于减少需要在实验室保存的转基因和突变研究动物的数量。这最后两种用途对两栖动物来说尤其重要目前，生产大量转基因和突变非洲爪哇品系的技术正在越来越多地被使用，在野外，两栖动物面临着巨大的压力，大约三分之一的物种受到威胁。目前配子和胚胎冷冻保存所需的技术在很大程度上是特定物种的，即使经过60年的发展，人们对决定存储材料的活力和繁殖力的因素也知之甚少。该项目旨在研究模式物种(热带非洲爪哇)精子冷冻损伤的基因组方面，相信通过更好地了解解冻后存活率和繁殖力差的根本原因，我们将能够开发遗传资源库的实际应用，并提高其在更广泛物种中的仿制成功。最近，我们重点分析了冷冻和冷冻对精子核DNA的影响，这是一个严重缺乏关注的话题。结果表明：(1)当冷冻/解冻的泥鳅和斑马鱼精子具有受精能力时，随后的胚胎发育率低于用新鲜精子产生的相应胚胎；(2)短期暴露于DNA修复抑制剂的胚胎会加剧精子冷冻的有害影响；(3)当未冷冻的精子产生的胚胎暴露于DNA修复抑制剂时，对发育没有不利影响。这些数据支持了精子DNA在冷冻过程中受损的假设。DNA修复抑制剂表明，胚胎拯救受损DNA的内在能力有限。泥鳅和斑马鱼的主要发育失败都发生在原肠胚期。现在，我们将使用已建立的非洲爪哇模式将这些实验扩展到两栖动物中。我们的目标是发现DNA损伤的“热点”是否发生，如果发生，这些损伤是否与先前使用FISH模型进行的研究中发现的原肠发育失败和胚胎模式缺陷直接相关。在项目期间，我们将确定分子标记和表型变化，以表明影响发育潜力所需的生殖细胞DNA损伤水平。在项目的最后部分，我们将使用Xenopus模型和这些指标来测试新开发的冷冻保存技术的有效性，并随后改进这些技术。对于ZSL这一最终用户来说，从该项目获得的数据将为开发适用于一系列濒危物种的超低温保存方法提供参考。对于学生来说，除了接触朴茨茅斯和ZSL的世界级研究设施外，他们还将学习经典胚胎学、传统分子和高通量基因表达分析技术、功能得失方法和冷冻保存。我们预计这项工作的成果将是：1)确定生殖细胞冷冻期间DNA损伤的功能后果；2)更好地了解这种DNA损伤与所产生胚胎的发育潜力之间的关系；3)减少研究机构中保存的非洲爪哇数量；4)培养训练有素的博士后科学家。

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.