Drying, Storing, and Reanimating Egg Germinal Vesicles to Preserve Fertility

项目摘要 保存雌性配子（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，治疗不孕症或延迟生育。然而，卵母细胞冷冻是复杂的 因为细胞体积大，含水量高，细胞骨架脆弱。储存这个细胞 和其他生物材料在超低温下也是昂贵的，这使得美国范围内的需求变得复杂， 更多的生物储存库来解决人类健康问题。我们的研究正在推进一种变革性的方式， 通过压缩、干燥、在零度以上的温度下储存来保存母体基因组， 使卵母细胞的细胞核（生殖囊泡，GV）而不是整个细胞复活。我们研究 家猫，一个模型，分享许多生理机制和基因组相似性， 人类近年来，我们已经证明：1）良好质量的卵母细胞可以重建 来自早期卵泡的有能力的GV被递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质被控制和维持在 细胞核，从而证实了保存分离的细胞核的价值;以及3）干燥的猫GV保留 存活数周，剩下的挑战是保持低而稳定的水分含量， 防止储存过程中的降解。迄今为止的研究结果引导我们采取了三个优先事项：1）干燥 的GV的中间水分水平结合生物稳定剂; 2）精确配制的 再水化方案;和3）产生具有未改变的表观遗传的胚胎和存活后代 模式.总的假设是：1）母体基因组和相关的核因子是 通过在更高（更适应）的温度下干燥GV，在室温下安全保存 与生物稳定剂结合时的含水量; 2）GV的关键成分已充分 储存后通过适当的再水化过程恢复;以及3）这些增强 促进GV在受体胞质中的存活和复活以及 重建的卵母细胞成熟，受精并成功发育成正常的胚胎， 体外/体内。考虑到我们最近的成果和我们的 获得新颖、先进的工具，包括用于干燥的新型受控环境室 和用于再水化的管状灌注系统以及计算转录组学/表观基因组学， 验证所得到的胚胎的存活能力。除了提供对弹性的更好理解之外， 卵母细胞的核，最终产品将告知GV储存在 生殖保健、生物医学模式的管理和建立低收入国家的替代办法， 成本存储库。

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.