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

项目摘要 保存雌配子(卵母细胞)的能力对于想要保持生育能力的女性来说至关重要。 癌症治疗前，治疗不孕不育或推迟生育。然而，卵母细胞冷冻是复杂的。 因为细胞体积大，水分含量高，细胞骨架脆弱。存储此单元格 和其他生物材料在超低温下也是昂贵的，这使美国范围内对 建立更多的生物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、在超零度温度下储存和保存母体基因组 复活卵母细胞的核(生发泡，GV)，而不是整个细胞。我们研究了 家猫，一种与家猫有许多生理机制和基因组相似性的模型 人类。近年来，我们已经证明：1)高质量的卵母细胞是可以重构的。 从早期卵泡获得合格的GV，并将其送入高质量的受体细胞质；2) GV的基本表观遗传标记和关键蛋白被控制和维持在 细胞核，从而证实了保存孤立核的价值；3)干燥的猫GV保留 生存能力几周，剩下的挑战是保持低和稳定的水分含量，以 防止在存储过程中降级。到目前为止，研究结果将我们引导到了三个优先事项：1)干燥 结合生物稳定剂的GV到中等湿度水平；2)精确的配方 再水化方案；以及3)用未改变的表观遗传学生产胚胎和可存活的后代 模式。总体假设是：1)母体基因组和相关的核因子是 通过将GV在更高的(更适宜的)温度下干燥，在室温下安全保存 与生物稳定剂结合时的水分含量；2)关键的GV成分完全 在储存后通过适当的复水过程恢复；以及3)这些增强 促进GV在受体细胞质中的存活和复活，以及 重建的卵母细胞成熟、受精并成功发育为正常胚胎 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，包括用于干燥的新的受控环境室 和用于再水化的管状灌流系统以及计算转录/表观基因组学 验证得到的胚胎的生存能力。除了提供对弹性的更好的理解 对于卵母细胞的核，最终产品将告知GV在 生殖健康保健、生物医学模式的管理以及建立更低成本-- 成本储存库。

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.