Drying, Storing and Reanimating Egg Germinal Vesicles to Preserve Fertility

描述(申请人提供)：长期保存精子和卵子的能力对于保持生育能力、解决不育症和延长个体的生殖寿命至关重要。尽管精子在冷冻中存活相对容易，但卵子(卵母细胞)的保存更为复杂，因为细胞体积大，水分含量高，细胞骨架脆弱。目前保存卵母细胞的方法远远不是最理想的，因此有必要探索新的方法。这项提议研究了未成熟的卵母细胞核，也被称为生发泡，作为一种潜在的基因组保存手段，被分离并单独干燥时会发生什么。具体地说，我们将确定干燥的生发泡(来自卵巢内收集的任何卵母细胞)是否可以重新水化，然后放入新鲜的(先前去核的)供体卵母细胞中，以重振和恢复原始的母体基因组。然后，“重建”的卵母细胞可以与合适的精子进行体外培养和受精，以产生所需基因的可存活胚胎。一般的假设是，在室温下干燥和储存可以保持生发泡的结构和功能的完整性。生发泡将在家猫身上进行研究，因为(1)该物种是人类健康和生殖的重要模型，(2)我们的初步数据是广泛的和非常有希望的。拟议项目的一个重要元素是在干燥前将生发泡压缩成小直径，这将确保在干燥或储存期间获得最佳保护，同时允许通过注射到替代卵母细胞中轻松转移。我们的目标是首次研究压实、干燥和室温储存对生发泡的影响，包括其重建卵母细胞的能力，该卵母细胞可以经历减数分裂、受精和胚胎发育到足月(即产生健康的幼崽)。早期的数据表明：(1)体外压实是生发泡抵抗应激源的良好保护剂，(2)生发泡移植到受体卵母细胞是成功的，(3)在糖存在下干燥可以保护核含量。该项目挑战了关于如何在一个从未被探索但显示出强大前景的领域(生发泡保存和储存)保护女性生育能力的现有范例。这一创新的策略可以避免传统的低效、破坏性和昂贵的整个卵母细胞在液氮中冷冻所带来的并发症。所需的研究还将大大增加关于卵母细胞核和细胞质之间复杂相互作用的学术知识，包括调节生发泡发育能力的机制。 公共卫生相关性(由申请人提供)：正如将描述的那样，卵母细胞生发泡的干燥、储存和复活代表着保存人类生育能力的巨大潜力。这种方法还可以广泛、实际地应用于更有效、更经济地保存、繁殖和管理用于研究人类疾病的稀有动物模型。有效地保持这些独特的基因类型的能力使人们能够通过转译生物医学研究了解改善人类健康的机制。

DESCRIPTION (provided by applicant): The ability to preserve spermatozoa and eggs for a long period of time is critical for retaining fertility, addressing infertility and lengthening the reproductive life span of individuals. Although spermatozoa survive freezing with relative ease, egg (oocyte) preservation is more complex because of the cell's large size, significant water content and fragile cellular skeleton. Current methods to preserve the oocyte are far from optimal, so there is a need to explore novel approaches. This proposal examines what happens when the immature oocyte nucleus, also known as the germinal vesicle, is isolated and dried alone as a potential means of genome preservation. Specifically, we will determine if a desiccated germinal vesicle (from any oocyte collected within the ovary) can be rehydrated and then placed into a fresh (previously enucleated) donor oocyte to reanimate and recover the original maternal genome. The 'reconstructed' oocyte then could be cultured and fertilized in vitro with appropriate spermatozoa to produce a viable embryo of a desired genotype. The general hypothesis is that desiccation and storage at room temperature can preserve the structural and functional integrity of the germinal vesicle. The germinal vesicle will be studied in the domestic cat because (1) this species is an important model for human health and reproduction, and (2) our preliminary data are extensive and highly promising. An important element of the proposed project is compacting the germinal vesicle to a small diameter before drying, which will ensure optimal protection during desiccation or storage while permitting easy transfer by injection into a surrogate oocyte. Our objective is to examine, for the first time, the impact of compaction, desiccation and storage at room temperature on the germinal vesicle, including its ability to reconstitute an oocyte that can undergo meiosis, fertilization and embryo development to term (i.e., production of healthy young). Early data have revealed that (1) in vitro compaction is an excellent protector of the germinal vesicle against stressors, (2) germinal vesicle transfer into a recipient oocyte is successful and (3) desiccation in the presence of sugar safeguards nuclear content. The project challenges existing paradigms about how to preserve female fertility in an area (germinal vesicle preservation and storage) that has never been explored, but shows strong promise. This innovative strategy could circumvent the complications associated with conventional inefficient, damaging and costly freeze-storage of whole oocytes in liquid nitrogen. The required studies also will substantially increase scholarly knowledge about complex interactions between the oocyte's nucleus and cytoplasm, including the mechanisms regulating developmental competence of the germinal vesicle. PUBLIC HEALTH RELEVANCE (provided by the applicant): As will be described, the drying, storage and reanimation of the oocyte's germinal vesicle represent an enormous potential for preserving human fertility. This approach also could have widespread, practical application to more effective, economical preservation, propagation and management of rare animal models used to study human diseases. The ability to efficiently perpetuate these unique genotypes allows understanding mechanisms to improve human health through translational biomedical research.