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

基本信息

批准号：
10464007
负责人：
Pierre Comizzoli
金额：
$ 8.9万
依托单位：
SMITHSONIAN INSTITUTION
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-15 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10464007
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

项目摘要

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.

项目摘要保持女配子（卵母细胞）的能力对于想要保持生育能力的女性至关重要在癌症治疗之前，治疗不育症或延迟繁殖。但是，卵母细胞冷冻很复杂由于细胞的尺寸较大，水分含量明显和脆弱的细胞骨架。存储这个单元格在超低温度下的其他生物材料也很昂贵，这使美国范围内的需求复杂化更多的生物疗法来解决人类健康问题。我们的研究正在推动一种变革性的方式通过压实，干燥，在上零温度下储存来保存母体基因组复活卵母细胞的核素（生发囊泡，GV），而不是整个细胞。我们研究家猫，该模型具有许多物理机制和基因组相似性人类。近年来，我们证明：1）可以重建高质量的卵母细胞从早期阶段的胜任的GV中，可以将其送入高质量，受体细胞体； 2） GV的基本表观遗传标记和关键蛋白受到控制和维持核，从而确认保留分离的核的值； 3）被摧毁的猫GV保留可行性数周，剩下的挑战是保持低和稳定的水分含量防止存储期间降解。迄今为止的调查结果已指导我们三个优先事项：1）干燥 GV达到中间水分水平，结合了生物稳定剂； 2）精确配制补液方案； 3）生产胚胎和可行的后代，并具有不变的表观遗传模式。总体假设是：1）母体基因组和相关的核因子是通过在较高的（更多住宿）将GV置于室温下在室温下牢固保存与生物稳定剂结合使用水分含量； 2）关键GV组件完全存储后通过适当的补液过程恢复； 3）这些增强功能促进受体细胞体以及能力的GV生存和复活的能力重建的卵母细胞成熟，被施肥并成功发展为正常胚胎体内/体内。鉴于我们最近的成绩以及我们访问新颖的高级工具，包括新的受控环境室用于补液的管状灌注系统以及计算转录组学/表观基因组学验证产生的胚胎的生存能力。除了提高对弹性的了解在卵母细胞的核中，最终产品将告知GV存储的实用性和安全性生殖卫生保健，生物医学模型的管理以及建立较低的替代选择成本存储库。