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: 9983220
• 负责人: Pierre Comizzoli
• 金额: $ 48.97万
• 依托单位: SMITHSONIAN INSTITUTION
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9983220
• 关键词: Address; Advocacy; Animal Model; Biocompatible Materials; Biological; Biological Models; Biomedical Research; Breeding; Canis familiaris; Cell Nucleus; Cells; Clupeidae; Collection; Competence; Complex; Custom; Desiccation; Development; Disease; Embryo; Ensure; Epigenetic Process; Felis catus; Female; Fertility; Fertility Study; Fertilization in Vitro; Freezing; Genome; Genomics; Genotype; Germ Cells; Health; Hormonal; Human; In Vitro; Infertility; Knowledge; Meiosis; Metaphase; Methods; Modeling; Modification; National Institute of Child Health and Human Development; Non-Rodent Model; Nuclear; Oocytes; Outcome; Ovarian; Ovary; Ovum; Pattern; Perfusion; Physiological; Population; Process; Production; Proteins; Protocols documentation; Rehydrations; Reproduction; Reproductive Health; Reproductive Technology; Research; Resistance; Resources; Safety; Sampling; Secure; Skeleton; Specimen; Structure; System; Temperature; Testicular Tissue; Tubular formation; Vesicle; Water; Woman; biobank; cancer therapy; cold temperature; cost; egg; epigenomics; fertility preservation; folliculogenesis; health management; human disease; improved; improved functioning; in vivo; infertility treatment; innovative technologies; interest; mortality; novel; novel strategies; offspring; oocyte cryopreservation; oocyte quality; practical application; preservation; prevent; repository; reproductive; resilience; sperm cell; stressor; tool; transcriptomics

Project Summary The ability to preserve the female gamete (oocyte) is critical for women wanting to retain fertility before cancer therapy, treat infertility or delay reproduction. However, oocyte freezing is complex because of the cell’s large size, significant water content and fragile cellular skeleton. Storing this cell and other biomaterials at ultra-low temperature also is costly, which complicates the USA-wide need for more biorepositories to address human health issues. Our research is advancing a transformative way of preserving the maternal genome by compacting, drying, storing at supra-zero temperatures and reanimating the oocyte’s nucleus (germinal vesicle, GV) rather than the entire cell. We study the domestic cat, a model that shares many physiological mechanisms and genomic similarities with the human. In the recent years, we have demonstrated that: 1) good quality oocytes can be reconstructed with competent GVs from early stage follicles that are delivered into high quality, recipient cytoplasts; 2) fundamental epigenetic marks and key proteins of the GV are controlled and sustained within the nucleus, thereby confirming the value of preserving isolated nuclei; and 3) desiccated cat GVs retain viability for weeks, with the remaining challenge being to maintain a low and stable moisture content to prevent degradations during storage. Findings to date have guided us to three priorities: 1) desiccation of the GV to an intermediate moisture level combined with biostabilizing agents; 2) a precisely formulated rehydration protocol; and 3) production of embryos and viable offspring with unaltered epigenetic patterns. The overall hypotheses are: 1) the maternal genome and associated nuclear factors are securely preserved at room temperature by desiccating the GV at a higher (more accommodating) moisture content when combined with biostabilizing agents; 2) key GV components are fully recovered by an appropriate rehydration process after storage; and 3) these enhancements promote GV survival and reanimation in a recipient cytoplast as well as the ability of the reconstructed oocyte to mature, be fertilized and successfully develop into a normal embryo in vitro/in vivo. Likelihood of achieving the project’s priorities is high given our recent results and our access to novel, advanced tools, including new controlled environmental chambers for the desiccation and Tubular Perfusion Systems for rehydration as well as computational transcriptomics/epigenomics to validate the viability of resulting embryos. Besides providing an improved understanding of the resilience of the oocyte’s nucleus, end products will inform on the practicality and safety of GV storage in reproductive health care, management of biomedical models and alternative options in building lower- cost repositories.

项目概要 保存雌配子（卵母细胞）的能力对于想要保留生育能力的女性至关重要 在癌症治疗、治疗不孕症或延迟生育之前，卵母细胞冷冻很复杂。 因为细胞体积大、含水量高且细胞骨架脆弱。 和其他超低温生物材料的成本也很高，这使得美国范围内对生物材料的需求变得复杂 我们的研究正在推进一种变革性的方法来解决人类健康问题。 通过压实、干燥、在超零温度下储存和保存母体基因组 我们研究的是卵母细胞核（生殖囊泡，GV）而不是整个细胞的复活。 家猫，一种与家猫有许多生理机制和基因组相似性的模型 近年来，我们已经证明：1）可以重建高质量的卵母细胞。 来自早期卵泡的有能力的 GV 被输送到高质量的受体细胞质中 2) GV 的基本表观遗传标记和关键蛋白在 细胞核，从而证实保存分离细胞核的价值；3) 干燥的猫 GV 保留 数周的生存能力，剩下的挑战是保持低且稳定的水分含量 迄今为止的研究结果指导我们采取三个优先事项：1) 干燥。 与生物稳定剂结合使 GV 达到中等湿度；2) 精确配制； 再水化方案；3) 产生具有未改变的表观遗传的胚胎和可存活的后代 总体假设是：1）母体基因组和相关的核因子是。 通过在更高（更适宜）下干燥 GV，在室温下安全保存 与生物稳定剂结合使用时的水分含量；2)关键GV成分充分 储存后通过适当的再水化过程恢复；以及 3) 这些增强功能 促进受体细胞质中 GV 的存活和复活以及 GV 的能力 重建的卵母细胞成熟、受精并成功发育成正常胚胎 鉴于我们最近的结果和我们的研究成果，实现该项目优先事项的可能性很高。 获得新颖、先进的工具，包括用于干燥的新型受控环境室 和管式灌注系统用于补液以及计算转录组学/表观基因组学 除了提供对弹性的更好的理解之外，还验证所得胚胎的活力。 卵母细胞核的最终产品将告知 GV 储存的实用性和安全性 生殖保健、生物医学模型管理以及低水平建设的替代方案 成本存储库。