Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC 20013-7012, USA.
Biol Reprod. 2013 Jun 6;88(6):139. doi: 10.1095/biolreprod.113.108472. Print 2013 Jun.
The study explored a novel approach for preserving the maternal genome without the entire oocyte by air-drying the cat germinal vesicle (GV) in the presence of the disaccharide trehalose. Specifically, we examined GV structure and function after desiccation, storage at 4 °C (up to 32 wk), and rehydration including the ability to resume meiosis after injection into a fresh, conspecific cytoplast. In experiment 1, DNA integrity was similar to fresh controls after 1 and 4 wk storage in the presence of trehalose, but was more fragmented at later time points (especially after 32 wk). Nuclear envelope integrity was sustained in >90% of oocytes stored for 0, 4, or 16 wk regardless of protective treatment. In experiment 2, compacted, air-dried GVs were stored for 2 or 4 wk, rehydrated, and injected into fresh cytoplasts. After culture for 24 h in vitro, up to 73% of oocytes reconstructed with desiccated GVs preserved in trehalose resumed meiosis compared to 30% of those dried in the absence of the disaccharide. At each storage time point, trehalose presence during air-drying was advantageous for resumption of meiosis, with >20% of oocytes completing nuclear maturation to metaphase II. This demonstrates a potential for preserving the female genome using the GV alone and for multiple weeks after desiccation. Trehalose enhanced the process by retaining the ability of a dried and rehydrated GV to resume communication with the surrounding cytoplasm of the recipient oocyte to permit reaching metaphase II and likely sustain subsequent embryo development.
本研究探索了一种新的方法，即在存在二糖海藻糖的情况下，通过风干猫的生发泡（GV）来保存母体基因组，而无需整个卵母细胞。具体来说，我们研究了干燥、4°C 储存（长达 32 周）以及复水后的 GV 结构和功能，包括在注入新鲜同种胞质体后恢复减数分裂的能力。在实验 1 中，在海藻糖存在的情况下，DNA 完整性在 1 周和 4 周的储存后与新鲜对照组相似，但在稍后的时间点（尤其是在 32 周后）更为碎片化。核膜完整性在 0、4 或 16 周储存的卵母细胞中保持在>90%以上，无论是否进行了保护处理。在实验 2 中，压缩、风干的 GV 在 2 或 4 周内储存，复水并注入新鲜胞质体。在体外培养 24 小时后，与在没有二糖的情况下干燥的卵母细胞相比，用保存在海藻糖中的干燥 GV 重建的卵母细胞中有高达 73%恢复减数分裂，而在没有二糖的情况下干燥的卵母细胞中有 30%恢复减数分裂。在每个储存时间点，在空气干燥过程中存在海藻糖有利于恢复减数分裂，有超过 20%的卵母细胞完成核成熟至中期 II。这表明单独使用 GV 并在干燥后数周内保存雌性基因组具有潜力。海藻糖通过保留干燥和复水的 GV 与受体卵母细胞的周围细胞质恢复通讯的能力来增强该过程，从而允许达到中期 II，并可能维持随后的胚胎发育。
