Collaborative Research: Cryopreservation of Zebrafish Oocytes by an Interdisciplinary Approach

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、器官发育和衰老的有价值的模型系统。成功的冷冻保存斑马鱼卵母细胞或胚胎是迫切需要的，以保持指数增长的突变体和转基因品系的数量。成功地储存斑马鱼配子或胚胎不仅可以显著降低成本，还可以避免与连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主要垫脚石，其中许多鱼类正在迅速减少。目前，鱼类精子的冷冻保存是可行的，尽管成功有限，而鱼类胚胎仍然是不可冷冻的，这是由于几个主要的障碍，如它们的大尺寸和多室结构，极低的膜透性水和冷冻保护化学品，以及对冷害和细胞内冰形成的敏感性。为了克服这些障碍，本项目提出采用多学科方法冷冻保存未受精的斑马鱼卵母细胞。这一建议预测，斑马鱼卵母细胞将比胚胎更适合冷冻保存，这是基于它们更小的尺寸，更简单的单细胞结构，以及更高的渗透性和对冷冻保护剂的耐受性。智力价值：这一项目是重要的和创新的，因为它提出解决一个基本的科学问题（即，冷冻保存鱼卵母细胞）通过结合工程（例如，模拟膜渗透性和细胞内冰形成，CPA加载/去除和冷却曲线的数学和实验优化）和细胞/分子生物学方法（例如，膜运输的分子操纵）。所提出的机制方法是新颖的，并且受到自然界中观察到的适应方案的启发（例如，生物体如青蛙、缓步动物、盐水虾、细菌和酵母的生存策略，它们能够适应极端条件，包括冷冻和干燥）;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，在哺乳动物细胞的冷冻保存和干燥过程中，糖的有益效果，而膜通透性屏障糖必须克服这种策略是有效的。初步研究表明，糖与低浓度常规渗透性冷冻保护剂的组合可以补偿前者的低渗透性，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渗透性冷冻保护剂的组合将保护斑马鱼卵母细胞免受冷冻相关的应力。本研究利用质量传递和非平衡相转化的工程模型，结合分子生物学技术，通过三个具体目标来验证中心假设：（1）验证斑马鱼卵母细胞渗透限制可以通过生物学、物理化学和工程学等多学科方法来克服的工作假设;（2）测试工作假设，即细胞内和细胞外糖与渗透性CPA的优化组合允许成功冷冻保存斑马鱼卵母细胞;（3）测试工作假设，即为了使存活率最大化，斑马鱼卵母细胞应该尽可能快地冷却而不引起有害的细胞内冰晶的形成。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，并导致斑马鱼卵母细胞成功冷冻保存的重大进展。本研究亦将推动低温生物学现象的生物热质传递模型的发展，以及生物热质传递过程的计算机辅助优化设计。本研究的更广泛影响将包括：（1）在本地高中开展教育推广活动，以斑马鱼为教学工具，唤起学生对科学和技术的好奇心/兴趣;（2）为K-12和本科生物工程学生开发教材;（3）为高中生创建暑期研究计划;（4）培养研究生和博士后研究员。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。拟议的教材和课程模块的开发将加强维拉诺瓦大学和其他大学的生物工程课程。

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.