Genomic aspects of DNA damage induced by germplasm cryopreservation

生殖细胞的冷冻保存对医学、水产养殖和农业的有效性做出了重大贡献。特别是，它有助于建立濒危物种样本的生物库，并减少需要在实验室保存的转基因和突变研究动物的数量。这最后两个用途是特别重要的两栖动物在这个时候，生产大量的转基因和突变爪蟾系的技术现在被越来越多地使用，在野生环境中，两栖动物面临着巨大的压力，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物种特异性的，即使经过六十年的发展，也很少了解决定储存材料的活力和生育力的因素。该项目旨在研究冷冻损伤的基因组方面的模式物种（热带爪蟾）的精子，相信通过更好地了解的根本原因，解冻后生存和生育能力差，我们将能够开发的实际应用的遗传资源银行和提高其通用的成功在更广泛的物种。最近，我们专注于分析冷却和冷冻对精子核DNA的影响，这是一个严重缺乏关注的话题。我们证明：（1）尽管冷冻/解冻的泥鳅和斑马鱼精子具有受精能力，但随后的胚胎发育率低于用新鲜精子产生的相应胚胎，（2）将胚胎短暂暴露于DNA修复抑制剂会使精子冷冻保存的有害影响变得更糟，（3）当未冷冻精子（对照）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。在泥鳅和斑马鱼中，发育的主要失败发生在原肠胚阶段。我们现在将使用已建立的爪蟾模型在两栖动物中扩展这些实验。我们的目标是发现DNA损伤的“热点”是否发生，如果发生，这些是否与先前使用鱼类模型研究中发现的原肠胚形成和胚胎图案缺陷的发育失败直接相关。在该项目中，我们将确定分子标记和表型变化，表明生殖细胞DNA损伤水平影响发育潜力。在项目的最后部分，我们将使用非洲爪蟾模型和这些指标来测试新开发的冷冻保存技术的有效性，并随后完善这些技术。对于最终用户-生物实验室来说，从该项目中获得的数据将为开发适用于一系列濒危物种的低温保存方法提供信息。对于学生来说，除了接触到朴茨茅斯和剑桥大学的世界一流的研究设施外，他们还将学习经典的胚胎学，传统的分子和高通量技术，用于基因表达分析，功能方法的获得和丧失以及冷冻保存。我们期望这项工作的成果是：1）鉴定生殖细胞冷冻保存过程中DNA损伤的功能后果; 2）更好地理解这种DNA损伤与所产生胚胎的发育潜力之间的关系; 3）减少研究机构中非洲爪蟾的数量; 4）训练有素、多才多艺的博士后科学家。

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.