The present study was conducted to investigate the effect of cold storage time on apoptosis of cumulus cells (CCs) from porcine ovaries, and to compare the sensitivity of four apoptosis‐detection methods. Porcine ovaries were stored in physiological saline solution at 4°C for 0, 7, 24 and 48 hr, and then cumulus cells or granulosa cells (GCs) in antral follicles were retrieved to detect cell apoptosis. Cumulus cells isolated from stored ovaries for 24 hr presented obvious apoptosis using terminal deoxynucleotidyl transferase (TdT)‐mediated d‐UTP nick end‐labeling (TUNEL) assay. A typical DNA ladder pattern of apoptosis was observed in GCs 24 hr post storage treatment. The mean Olive Tail Moment of CCs was significantly increased after 24 hr using comet assay; however, the mean tail migration and mean tail DNA increased gradually after 7 hr of storage. In addition, annexin V/PI staining assay showed an obvious increase in apoptotic CCs (Annexin V positive, PI negative) 7 hr after treatment, and the apoptotic rate reached to a peak at 24 hr followed by a decline after 48 hr of storage to the level at 7 hr. In conclusion, cold storage of porcine ovary in physiological saline solution induced a time‐dependent increase in apoptosis of cumulus cells, and annexin V/PI staining combined with comet assay provided a sensitive and reliable method to detect early damages in cumulus cells induced by cold storage of ovary.

中文翻译：

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4℃卵巢保存时间对猪窦卵泡卵丘细胞凋亡的影响

本研究旨在研究冷藏时间对猪卵巢卵丘细胞（CCs）凋亡的影响，并比较四种凋亡检测方法的敏感性。将猪卵巢在4°C的生理盐水中保存0、7、24和48小时，然后取出窦卵泡中的卵丘细胞或颗粒细胞（GCs）检测细胞凋亡。使用末端脱氧核苷酸转移酶 (TdT) 介导的 d-UTP 缺口末端标记 (TUNEL) 测定，从储存的卵巢中分离 24 小时的卵丘细胞呈现出明显的细胞凋亡。在储存处理后 24 小时的 GC 中观察到典型的细胞凋亡 DNA 阶梯模式。使用彗星试验 24 小时后，CC 的平均 Olive Tail Moment 显着增加；然而，平均尾部迁移和平均尾部 DNA 在储存 7 小时后逐渐增加。此外，annexin V/PI 染色显示处理后 7 小时凋亡 CCs（Annexin V 阳性，PI 阴性）明显增加，凋亡率在 24 小时达到峰值，随后在储存 48 小时后下降至7小时的水平。综上所述，猪卵巢在生理盐水溶液中冷藏诱导卵丘细胞凋亡呈时间依赖性增加，膜联蛋白V/PI染色结合彗星试验为检测低温诱导的卵丘细胞早期损伤提供了一种灵敏可靠的方法卵巢的储存。细胞凋亡率在 24 小时达到峰值，随后在储存 48 小时后下降至 7 小时的水平。综上所述，猪卵巢在生理盐水溶液中冷藏诱导卵丘细胞凋亡呈时间依赖性增加，膜联蛋白V/PI染色结合彗星试验为检测低温诱导的卵丘细胞早期损伤提供了一种灵敏可靠的方法卵巢的储存。细胞凋亡率在 24 小时达到峰值，随后在储存 48 小时后下降至 7 小时的水平。综上所述，猪卵巢在生理盐水溶液中冷藏诱导卵丘细胞凋亡呈时间依赖性增加，膜联蛋白V/PI染色结合彗星试验为检测低温诱导的卵丘细胞早期损伤提供了一种灵敏可靠的方法卵巢的储存。