Drying, Storing and Reanimating Egg Germinal Vesicles to Preserve Fertility

描述（由申请人提供）：长时间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。尽管精子在冷冻中相对容易存活，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，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）被分离并单独干燥作为基因组保存的潜在手段时会发生什么。具体来说，我们将确定一个干燥的生发囊泡（来自卵巢内收集的任何卵母细胞）是否可以再水化，然后放入一个新鲜的（先前去核的）供体卵母细胞中，以复活和恢复原始的母体基因组。然后，“重建”的卵母细胞可以在体外培养并与合适的精子受精，以产生所需基因型的可存活胚胎。一般的假设是，在室温下干燥和储存可以保持生发囊泡的结构和功能的完整性。之所以在家猫身上进行生发囊泡的研究，是因为：(1)家猫是人类健康和繁殖的重要模型，(2)我们的初步数据很广泛，很有希望。拟议项目的一个重要组成部分是在干燥前将生发囊泡压实至小直径，这将确保在干燥或储存期间获得最佳保护，同时允许通过注射轻松转移到代孕卵母细胞中。我们的目标是首次研究压实、干燥和室温储存对生殖囊泡的影响，包括其重建卵母细胞的能力，卵母细胞可以进行减数分裂、受精和胚胎发育至足月（即产生健康的幼体）。早期数据显示：(1)体外压实是生发囊泡对抗应激源的良好保护，(2)生发囊泡向受体卵母细胞的转移是成功的，(3)存在糖的干燥保护核含量。该项目挑战了现有的关于如何在一个从未被探索过的领域（生发囊泡保存和储存）保存女性生育能力的范例，但它显示出强大的前景。这种创新的策略可以避免传统的低效率、破坏性和昂贵的全卵母细胞液氮冷冻储存所带来的并发症。所要求的研究也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，包括调节生殖囊泡发育能力的机制。

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.