Collaborative Research: Cryopreservation of Zebrafish Oocytes by an Interdisciplinary Approach

合作研究：通过跨学科方法冷冻保存斑马鱼卵母细胞

• 批准号: 1067601
• 负责人: Ali Eroglu
• 金额: $ 43.3万
• 依托单位: AUGUSTA UNIVERSITY RESEARCH INSTITUTE, INC.
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1067601&HistoricalAwards=false
• 关键词: Collaborative; Research; Cryopreservation; Zebrafish; Oocytes

The overall goal of the proposed research is to develop a novel cryopreservation technique for zebrafish oocytes. The zebrafish is a valuable model system for human diseases and other research areas such as embryogenesis, organ development, and aging. Successful cryopreservation of zebrafish oocytes or embryos is urgently needed to maintain exponentially growing numbers of mutant and transgenic lines. Successful banking of zebrafish gametes or embryos would not only significantly reduce costs, but also avoid risks of infection and genetic drift associated with continuous breeding. Furthermore, development of a successful cryopreservation technique for zebrafish oocytes would be a major stepping-stone towards conservation of other fish species, many of which are rapidly declining. Cryopreservation of fish sperm is currently practiced albeit with limited success, whereas fish embryos remain non-freezable due to several major obstacles such as their large size and multi-compartmental structure, extremely low membrane permeability to water and cryoprotective chemicals, and susceptibility to chilling injury and intracellular ice formation. To circumvent these obstacles, this project proposes to cryopreserve unfertilized zebrafish oocytes using a multidisciplinary approach. This proposal predicts that zebrafish oocytes will be more amenable to cryopreservation than embryos, based on their smaller size, much simpler single-cell structure, and higher permeability and tolerance to cryoprotectants.Intellectual merit: This project is significant and innovative because it proposes to tackle a fundamental scientific problem (i.e., cryopreservation of fish oocytes) by combining engineering (e.g., modeling membrane permeability and intracellular ice formation, mathematical and experimental optimization of CPA loading/removal and cooling profiles) and cell/molecular biology approaches (e.g., molecular manipulation of the membrane transport). The proposed mechanistic approach is novel and inspired by adaptation schemes observed in nature (e.g., survival strategies by organisms such as frogs, tardigrades, brine shrimp, bacteria, and yeast, which are able to adapt to extreme conditions including freezing and desiccation); a key adaptation is the accumulation of intra- and extracellular sugars. Recent studies have demonstrated beneficial effect of sugars during cryopreservation and desiccation of mammalian cells, whereas the membrane permeability barrier to sugars must be overcome for this strategy to be effective. Preliminary studies have demonstrated that the combination of sugars with low concentrations of conventional penetrating cryoprotectants can compensate for the low permeation of the former wile reducing the cytotoxicity of the latter. Thus, the central hypothesis of this project is that combination of intra- and extracellular sugars with small amounts of a penetrating cryoprotectant will protect zebrafish oocytes against freezing-associated stresses. Using engineering models of mass transport and non-equilibrium phase transformation, in combination with molecular biology techniques, this proposal will test the central hypothesis by pursuing three specific aims: (1) test the working hypothesis that osmotic limitations of zebrafish oocytes can be overcome by a multi-disciplinary strategy including biological, physico-chemical, and engineering approaches; (2) test working hypothesis that an optimized combination of intra- and extracellular sugars with a penetrating CPA permits successful cryopreservation of zebrafish oocytes; (3) test working hypothesis that to maximize viability, zebrafish oocytes should be cooled as rapidly as possible without causing formation of deleterious intracellular ice crystals. The interdisciplinary approach proposed here is expected to overcome the obstacles associated with cryopreservation of zebrafish germplasm, and lead to significant advances towards successful cryopreservation of zebrafish oocytes. The proposed work is also expected to lead to advances in the state-of-the-art of bioheat/mass-transfer modeling of cryobiological phenomena, and in computer-aided optimization of biothermal process design.Broader impacts of the proposed research will be attained by: (1) educational outreach activities in local high schools using zebrafish as a teaching tool to awaken curiosity/interest among the students in science and technology; (2) developing teaching materials for K-12 as well as undergraduate bioengineering students; (3) creating a summer research program for high school students; (4) training graduate students and postdoctoral fellows. The proposed outreach activities are expected to broaden and strengthen the ongoing partnership of the Medical College of Georgia with local high school and communities. The proposed development of educational materials and course modules will enhance the bioengineering curriculum at Villanova University and beyond.

本研究的总体目标是开发一种新的斑马鱼卵母细胞冷冻保存技术。斑马鱼是人类疾病和其他研究领域（如胚胎发生、器官发育和衰老）有价值的模型系统。目前迫切需要成功的冷冻保存斑马鱼卵母细胞或胚胎，以保持突变和转基因系的指数级增长。成功地储存斑马鱼配子或胚胎不仅可以显著降低成本，还可以避免与持续繁殖相关的感染和遗传漂变风险。此外，开发一种成功的斑马鱼卵母细胞冷冻保存技术将是保护其他鱼类物种的重要基石，其中许多物种正在迅速减少。鱼类精子的低温保存目前已经在实践中，尽管取得了有限的成功，而鱼类胚胎由于几个主要的障碍，如它们的大尺寸和多室结构，极低的膜对水和冷冻保护化学物质的渗透性，以及对冷伤和细胞内冰形成的敏感性，仍然不能冷冻。为了克服这些障碍，本项目建议采用多学科方法冷冻保存未受精的斑马鱼卵母细胞。该建议预测斑马鱼卵母细胞比胚胎更适合冷冻保存，基于它们更小的尺寸，更简单的单细胞结构，更高的渗透性和对冷冻保护剂的耐受性。智力优势：该项目具有重大意义和创新性，因为它提出通过结合工程（例如，膜通透性和细胞内冰形成建模，CPA加载/去除和冷却剖面的数学和实验优化）和细胞/分子生物学方法（例如，膜运输的分子操纵）来解决一个基本的科学问题（例如，鱼卵母细胞的冷冻保存）。提出的机制方法是新颖的，并受到自然界中观察到的适应方案的启发（例如，青蛙、缓步动物、盐水虾、细菌和酵母等生物的生存策略，它们能够适应包括冰冻和干燥在内的极端条件）；一个关键的适应是细胞内和细胞外糖的积累。最近的研究表明，糖在哺乳动物细胞的低温保存和干燥过程中具有有益的作用，然而，为了使这种策略有效，必须克服细胞膜对糖的渗透性屏障。初步研究表明，糖与低浓度的常规穿透性冷冻保护剂的结合可以弥补前者的低渗透，同时降低后者的细胞毒性。因此，该项目的中心假设是，细胞内和细胞外糖与少量穿透性冷冻保护剂的结合将保护斑马鱼卵母细胞免受与冷冻相关的压力。利用质量传递和非平衡相变的工程模型，结合分子生物学技术，本提案将通过追求三个具体目标来验证中心假设：(1)通过包括生物学、物理化学和工程方法在内的多学科策略来验证斑马鱼卵母细胞渗透限制可以克服的工作假设；(2)验证一个工作假设，即细胞内和细胞外糖与穿透性CPA的优化组合允许斑马鱼卵母细胞成功低温保存；(3)验证工作假设，即为了最大限度地提高卵母细胞的生存能力，应尽快冷却斑马鱼卵母细胞，以免形成有害的细胞内冰晶。本文提出的跨学科方法有望克服与斑马鱼种质冷冻保存相关的障碍，并在成功冷冻保存斑马鱼卵母细胞方面取得重大进展。所提出的工作也有望导致低温生物现象的生物热/传质建模的最新进展，以及生物热过程设计的计算机辅助优化。(1)利用斑马鱼作为教学工具，在当地高中开展教育外展活动，唤醒学生对科学技术的好奇心/兴趣；(2)编写中小学及本科生物工程专业教材；(3)为高中生创建暑期研究项目；(4)培养研究生和博士后。拟议的外联活动预计将扩大和加强格鲁吉亚医学院与当地高中和社区的现有伙伴关系。拟议的教育材料和课程模块的发展将加强生物工程课程在维拉诺瓦大学和超越。