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目的 探讨精子体外获能进程与细胞凋亡之间的联系 ,以及获能过程中精子凋亡对体外受精的影响。方法 猪精子经钙离子载体A2 3187(IA)诱导处理后 ,在TALP以及添加 10 %胎牛血清 (TALP +FCS组 )、2 5 μmol/L核酶抑制剂 (ATA)、TALP +ATA组 3种培养液中孵育 ,以考马斯亮蓝和TUNEL检测顶体反应 (AR)率和凋亡率 ;同时行体外受精 ,检查卵母细胞穿透率和卵裂率。结果 鲜精液的精子凋亡率为 5 .7% ,经洗涤、上游处理后凋亡率下降为 3% ;获能培养过程中 ,尽管IA浓度影响精子寿命 ,胎牛血清推迟获能进程 ,但凋亡仅与AR直接相关 ,获能完成后 4小时左右出现明显的凋亡增长峰 ,ATA可阻止获能后的凋亡效应 ;3种获能处理的精子体外受精后 ,TALP和TALP +FCS组有超过 30 %以上的受精卵不发生卵裂 ,而TALP +ATA组仅为 2 0 % ,与阴性对照组相当。结论 精子在顶体反应发生的同时可能启动和 (或 )激活凋亡机制 ,4小时后出现凋亡效应 ;精子DNA的异常断裂影响受精卵的发育能力。
Objective To investigate the relationship between sperm capacitation and apoptosis in vitro and the effect of sperm apoptosis on in vitro fertilization during capacitation. Methods Pig spermatozoa were induced by calcium ionophore A2 3187 (IA), then cultured in TALP medium supplemented with 10% fetal bovine serum (TALP + FCS), 25 μmol / L ribozyme inhibitor (ATA) The culture medium was incubated with Coomassie brilliant blue and TUNEL to detect the rate of acrosome reaction (AR) and the rate of apoptosis. At the same time, in vitro fertilization was performed to check the rate of oocyte penetration and cleavage. Results The apoptosis rate of spermatozoa in fresh semen was 5.7%. After washing, the apoptosis rate of spermatozoa was decreased to 3% after upstream treatment. In the process of capacitation culture, although the IA concentration affected the life span of sperm, Apoptosis was only related to AR directly. After 4 hours, the obvious apoptotic peak appeared, and ATA could prevent the apoptotic effect after capacitation. After in vitro fertilization, TALP and TALP + FCS In the group with more than 30% fertilized eggs, cleavage did not occur, but only 20% in the TALP + ATA group, which was comparable to the negative control group. Conclusion Sperm may activate and / or activate the apoptosis mechanism at the same time of acrosome reaction, and the apoptotic effect occurs after 4 hours. The abnormal rupture of sperm DNA affects the developmental ability of fertilized eggs.