Sugars as Novel Cryoprotectants for Primate Oocytes

描述（由申请人提供）：该提案的总体目标是开发一种新的灵长类卵母细胞保存技术。卵母细胞是独特的全能细胞，为研究基础发育生物学提供了特殊的前景，在包括核重编程和胚胎干细胞衍生在内的许多领域都得到了成功的探索。因此，成功储存不同模式物种的卵母细胞将极大地促进科学、医疗技术和濒危物种保护的进步。使用非人类灵长类卵母细胞开发并彻底测试的冷冻保存方法不仅将促进濒危灵长类动物的基础研究和保护，而且还将成为用于不孕症治疗的人类卵母细胞可靠保存方案的主要踏脚石。为此，我们提出了一种基于自然界生存方案的机械新方法，其中青蛙、缓步动物和丰年虾等多种生物体可以在冷冻或几乎完全干燥的情况下生存。糖的积累对于它们的生存策略极其关键。最近的研究清楚地表明，通过克服哺乳动物细胞膜对糖的渗透性屏障，体细胞的冷冻存活能力得到改善。因此，我们假设当存在于细胞内部和外部时，糖通过其优异的玻璃形成能力以及与亚细胞成分的直接相互作用来保护卵母细胞免受冷冻诱导的损伤。我们还假设糖与传统渗透性冷冻保护剂的组合以及新型糖基冷冻介质的开发将进一步改善冷冻存活率。我们将使用具有不同玻璃化转变温度和分子大小的胞内和胞外糖系统地测试这些假设，然后将最有效的糖与传统冷冻保护剂结合起来。接下来，利用水运输和细胞内冰形成的耦合机制模型，我们将建立在糖/冷冻保护剂存在下卵母细胞冷冻的最佳热力学途径。最后，我们将测试冷冻卵母细胞足月的发育能力。我们的研究将有助于了解卵母细胞冷冻生物学的基本方面以及糖的冷冻保护机制。我们预测，拟议的研究将通过规避与传统方案相关的许多困难，为灵长类动物卵母细胞提供可靠的保存技术。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a novel preservation technique for primate oocytes. Oocytes are unique totipotent cells that offer exceptional prospects for studying fundamental developmental biology, as successfully explored in many areas including nuclear reprogramming and derivation of embryonic stem cells. Therefore, successful banking of oocytes from different model species would enormously contribute to the advancement of science, medical technologies, and conservation of endangered species. A cryopreservation method developed and thoroughly tested using non-human primate oocytes would not only facilitate basic research and conservation of endangered primates but would also be a major stepping-stone towards a reliable preservation protocol for human oocytes for infertility treatment. To this end, we propose a mechanistic novel approach based on survival schemes in nature where a variety of organisms such as frogs, tardigrades, and brine shrimp can survive freezing or almost complete drying. Accumulation of sugars is extremely critical for their survival strategy. By overcoming the permeability barrier of mammalian cell membranes to the sugars, recent studies have clearly demonstrated improved cryosurvival of somatic cells. Therefore, we hypothesize that when present both inside and outside cells, sugars protect oocytes against freezing-induced injury by their excellent glass formation abilities and direct interactions with subcellular components. We also hypothesize that combinations of sugars with conventional penetrating cryoprotectants and development of a novel sugar-based freezing medium will further improve cryosurvival. We will test these hypotheses systematically using intra- and extracellular sugars with different glass transition temperatures and molecular sizes, and then combining the most effective sugars with conventional cryoprotectants. Next, using a coupled mechanistic model of water transport and intracellular ice formation, we will establish an optimal thermodynamic pathway for oocyte freezing in the presence of sugar/cryoprotectant. Finally, we will test developmental capacity of cryopreserved oocytes to term. Our studies will contribute to understanding fundamental aspects of oocyte cryobiology as well as cryoprotective mechanisms of sugars. We predict that the proposed research will result in a reliable preservation technique for primate oocytes by circumventing many of the difficulties associated with conventional protocols.