Drying, Storing and Reanimating Egg Germinal Vesicles to Preserve Fertility

干燥、储存和复活卵子胚泡以保持生育能力

• 批准号: 8235845
• 负责人: Pierre Comizzoli
• 金额: $ 37.04万
• 依托单位: SMITHSONIAN INSTITUTION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8235845
• 关键词: Address; Animal Model; Animals; Area; Biological Preservation; Biomedical Research; Breeding; Caliber; Cell Nucleus; Cells; Characteristics; Chemicals; Collection; Companions; Competence; Complex; Cytoplasm; Data; Desiccation; Development; Disaccharides; Disease; Elements; Embryo; Embryonic Development; Ensure; Felis catus; Female; Fertility; Fertilization; Freezing; Genome; Genotype; Germ Cells; Glass; Health; Hormonal; Human; Immobilized Enzymes; In Vitro; Inbreeding; Individual; Infertility; Injection of therapeutic agent; Institution; Knowledge; Life; Liquid substance; Longevity; Meiosis; Methods; Modeling; Nitrogen; Nuclear; Oocytes; Organism; Ovary; Plants; Population; Production; Rehydrations; Relative (related person); Reproduction; Research; Resistance; Science; Skeleton; Staging; Structure; Technology; Temperature; Time; Trehalose; Vesicle; Water; Woman; aged; cancer therapy; cost; cost effective; egg; experience; feral; human disease; improved; in vivo; innovation; interest; intraovarian; laboratory cat; novel strategies; practical application; prepuberty; prevent; public health relevance; reconstitution; reproductive; sperm cell; stressor; sugar

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.

描述（由申请人提供）：长期保存精子和卵子的能力对于保持生育能力、解决不孕症和延长个体的生殖寿命至关重要。尽管精子相对容易在冷冻中存活，但卵子（卵母细胞）的保存更为复杂，因为细胞体积大、含水量高且细胞骨架脆弱。目前保存卵母细胞的方法远非最佳，因此需要探索新的方法。该提案研究了当未成熟卵母细胞核（也称为生发囊泡）被单独分离和干燥作为基因组保存的潜在手段时会发生什么。具体来说，我们将确定是否可以将干燥的生发囊泡（来自卵巢内收集的任何卵母细胞）再水化，然后放入新鲜的（先前去核的）供体卵母细胞中以复活并恢复原始母体基因组。然后，“重建”的卵母细胞可以在体外培养并与适当的精子受精，以产生所需基因型的可行胚胎。一般假设是室温下的干燥和储存可以保持生发囊泡的结构和功能完整性。将在家猫中研究生发囊泡，因为（1）该物种是人类健康和繁殖的重要模型，（2）我们的初步数据广泛且前景广阔。该项目的一个重要组成部分是在干燥前将生发囊泡压缩至小直径，这将确保在干燥或储存期间提供最佳保护，同时允许通过注射到代孕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囊泡的影响，包括其重建卵母细胞的能力，该卵母细胞可以进行减数分裂、受精和胚胎发育至足月（即产生健康年轻）。早期数据显示：（1）体外压实是生发囊泡抵抗应激的极好保护器，（2）生发囊泡成功转移到受体卵母细胞中，（3）在糖存在下干燥可以保护核内容物。该项目挑战了关于如何在一个从未被探索过但显示出强大前景的领域（生殖囊保存和储存）中保护女性生育能力的现有范例。这种创新策略可以避免与液氮中整个卵母细胞冷冻储存的传统低效、破坏性和昂贵相关的并发症。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，包括调节生发囊发育能力的机制。 公共健康相关性（由申请人提供）：正如将要描述的，卵母细胞生发囊泡的干燥、储存和复活代表了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。有效地延续这些独特基因型的能力使我们能够了解通过转化生物医学研究改善人类健康的机制。