OOCYTE CRYOPRESERVATION IN RARE BIOMEDICAL MODELS

描述(由申请人提供)：应聘者将接受训练，同时产生关于猫类卵母细胞冷冻生物学的学术知识。由于许多稀有猫科动物的繁殖能力很差，配子冷冻保存结合受体雌性的体外胚胎生产和移植可以帮助繁殖和管理这些种群。目的是：1)研究卵母细胞的膜生物物理性质；2)表征和减轻二步冷冻引起的冷冻损伤；3)将新开发的卵母细胞冷冻保存方案应用于稀有猫科动物生物医学模型，以辅助种群管理。由于这项技术有可能从濒临死亡的动物中拯救配子，因此重点将放在未成熟的卵母细胞上。将描述低温保护剂暴露、直接冷却和在零度以下保持，然后直接插入液氮的影响。评价指标包括生物膜的性质、细胞内冰的形成、细胞骨架的完整性、细胞质膜和与卵丘细胞的相互作用，以及最终的体外和体内发育能力。这些发现将应用于猫科动物模型，同时利用对选定野生猫科动物物种的独特访问，以更快地了解机制。这种基本和比较的方法提供了一个多学科的培训机会，使应聘者能够成为一名拥有生物物理学、低温生物学和卵母细胞体外培养专业知识的生理学家。这项研究的好处将是彻底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-解冻过程中存活并发育成可存活胚胎和后代的因素。实际的好处将是另一个工具，用于解决管理繁殖能力受损的少量猫科动物这一始终困难的任务。与公共卫生相关：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是必不可少的。这种多学科的方法将提供新的卵母细胞冷冻保存方案，有助于管理用作人类疾病生物医学模型的猫克隆。

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.