Elliott Gloria D, Lee Pei-Chih, Paramore Elisha, Van Vorst Matthew, Comizzoli Pierre
1Department of Mechanical Engineering and Engineering Sciences, University of North Carolina at Charlotte, Charlotte, North Carolina.
2Smithsonian Conservation Biology Institute, National Zoological Park, Washington, District of Columbia.
Biopreserv Biobank. 2015 Jun;13(3):164-71. doi: 10.1089/bio.2014.0078.
The ability to compact and inject the cat germinal vesicle (GV) into a recipient cytoplast allows exploration of a new fertility preservation strategy that avoids whole oocyte freezing. The objective of the present study was to understand the impact of water loss and storage time on GV DNA integrity. Immature cat oocytes were exposed to 1.5 M trehalose for 10 min before microwave-assisted dehydration for 0, 5, 10, 15, 20, 25, 30, or 40 min. Oocytes then were rehydrated to assess chromatin configuration and the incidence of DNA fragmentation (TUNEL assay). The moisture content progressively decreased (p<0.05) from 1.7 to 0.1 gH2O/gDW over the first 30 min, but did not decrease further (p>0.05) after 40 min. Chromatin configuration was unaffected (p>0.05) over time. The percentage of GVs with DNA fragmentation was unaltered (p>0.05) from 0 to 30 min of treatment (range, 6.1%-12%), but increased (p<0.05) to 32.5% after 40 min. Next, the influence of storage at two different supra-zero temperatures after 30 min of drying was investigated. Oocyte-loaded, microwave-treated filters were individually sealed in Dri-Shield moisture barrier bags and stored at 4°C or ambient temperature for 0 to 8 weeks. Moisture contents gradually decreased (p<0.05) from 0.12 to 0.10 gH2O/gDW after 8 weeks of storage at 4°C or ambient temperature. The percentage of GVs with DNA fragmentation more than doubled (p<0.05) from 0 (14.3%) to 2 days (30.0%-33.0%), but remained stable (p>0.05) thereafter (1 through 4 weeks, 25.0%-35.0%). Collective results demonstrate the feasibility of using microwave processing to dehydrate the mammalian GV to a moisture content that is nonlethal and enables nonfrozen storage, an alternative approach for preserving the maternal genome at cool or ambient temperature.
将猫的生发泡（GV）压缩并注入受体细胞质的能力，使得探索一种避免整个卵母细胞冷冻的新的生育力保存策略成为可能。本研究的目的是了解水分流失和储存时间对GV DNA完整性的影响。未成熟的猫卵母细胞在微波辅助脱水0、5、10、15、20、25、30或40分钟之前，先暴露于1.5M海藻糖中10分钟。然后使卵母细胞再水化，以评估染色质构型和DNA片段化发生率（TUNEL检测）。在最初30分钟内，水分含量从1.7逐渐降至0.1gH2O/gDW（p<0.05），但40分钟后不再下降（p>0.05）。随着时间推移，染色质构型未受影响（p>0.05）。处理0至30分钟期间，发生DNA片段化的GV百分比未改变（p>0.05）（范围为6.1%-12%），但40分钟后增加（p<0.05）至32.5%。接下来，研究了干燥30分钟后在两个不同的零上温度下储存的影响。将加载有卵母细胞的微波处理过的滤膜分别密封在防潮袋中，并在4°C或室温下储存0至8周。在4°C或室温下储存8周后，水分含量从0.12逐渐降至0.10gH2O/gDW（p<0.05）。发生DNA片段化的GV百分比从0天（14.3%）到2天（30.0%-33.0%）增加了一倍多（p<0.05），但此后保持稳定（p>0.05）（1至4周，25.0%-35.0%）。总体结果表明，使用微波处理将哺乳动物GV脱水至非致死水分含量并实现非冷冻储存是可行的，这是在凉爽或室温下保存母源基因组的一种替代方法。
