OOCYTE CRYOPRESERVATION IN RARE BIOMEDICAL MODELS

描述（由申请人提供）：候选人将在接受培训的同时产生关于卵母细胞低温生物学的学术知识。由于许多罕见的猫基因型繁殖能力差，配子冷冻保存结合体外胚胎生产和受体雌性移植可以帮助繁殖和管理这些种群。目的：1)研究卵母细胞的膜生物物理特性；2)表征和减轻两步冷冻造成的冷冻损伤；3)将新开发的卵母细胞冷冻保存方案应用于珍稀动物生物医学模型，以辅助种群管理。由于这项技术具有从垂死动物身上“拯救”配子的潜力，因此重点将放在未成熟的卵母细胞上。低温保护剂暴露的影响，直接冷却和保持在零度以下的温度，然后直接投入液氮将被表征。评估目标包括生物物理膜特性、细胞内冰的形成、细胞骨架的完整性、细胞质膜和与积云细胞的相互作用，以及最终在体外和体内的发育能力。研究结果将应用于野外模型，同时利用对选定野生野外物种的独特访问，以更快地了解机制。这种基础和比较的方法提供了一个多学科的培训机会，使候选人能够在生物物理学、低温生物学和卵母细胞体外培养方面成为一名具有专业知识的生理学家。研究的好处将是深入了解未成熟猫卵母细胞的生物物理特性，以及调节其在冷却-冷冻-解冻和发育成可存活的胚胎和后代的能力的因素。实际的好处将是另一种工具，用于解决管理繁殖受到损害的小种群这一始终困难的任务。与公共卫生的相关性：动物模型对于研究通过生物医学研究了解和改善人类状况的相关机制至关重要。这种多学科的方法将提供新的卵母细胞冷冻保存方案，用于管理作为人类疾病生物医学模型的猫菌落。

DESCRIPTION (provided by applicant): The Candidate will receive training while producing scholarly knowledge about felid oocyte cryobiology. Since many rare cat genotypes reproduce poorly, gamete cryopreservation combined with in vitro embryo production and transfer in recipient females can help to reproduce and manage these populations. Objectives are to: 1) investigate membrane biophysical properties of the oocyte; 2) characterize and mitigate cryoinjuries caused by 2-step freezing; and 3) apply a newly developed oocyte cryopreservation protocol to rare felid biomedical models to assist in population management. Because of the potential of this technology for 'rescuing' gametes from dying animals, the focus will be on the immature oocyte. Impact of cryoprotectant exposure, direct cooling and holding at a subzero temperature followed by direct plunging into liquid nitrogen will be characterized. Evaluation targets include biophysical membrane properties, intracellular ice formation, integrity of the cytoskeleton, cytoplasmic membrane and interactions with cumulus cells, and eventually developmental competence in vitro and in vivo. Findings will be applied to felid models while taking advantage of unique access to selected wild felid species to more rapidly understand mechanisms. This fundamental and comparative approach provides a multidisciplinary training opportunity that will allow the Candidate to advance as a physiologist with expertise in biophysics, cryobiology and in vitro culture of oocytes. The research benefit will be a thorough understanding of the biophysical properties of the immature cat oocyte and the factors regulating its ability to survive cooling-freezing-thawing and to develop into a viable embryo and offspring. The practical benefit will be another tool for addressing the always-difficult task of managing small populations of reproductively compromised felids. Relevance to public health: Animal models are essential to studying mechanisms pertinent to understanding and improving the human condition through biomedical research. This multidisciplinary approach will provide new oocyte cryopreservation protocols useful to the management of cat colonies used as biomedical models for human diseases.