超低温冷冻过程引起猪精子产生“似凋亡”变化

Cryopreservation Procedure Induces the Alterations of “Apoptosis-Like” of Boar Sperm

测定超低温冷冻保存过程能否诱导猪冻融精子产生"似凋亡"变化,探讨猪冻融精子活力降低的分子机理。利用Annexin-V/PI及JC-1荧光抗体试剂盒与流式细胞术相结合的方法,检测猪精子冷冻前后及不同培养时间冻融精子质膜中磷脂酰丝氨酸(PS)位置及线粒体膜电位(Δψm)变化;蛋白免疫印迹分析方法测定猪精子Bcl-2、Caspase-9及Caspase-3等"凋亡"因子含量变化。猪精子超低温冷冻保存后活性精子的比例(54.4%)显著低于鲜精组(87.9%)(P<0.05),其中PS移位的精子"似凋亡"比例约为26.8%,同鲜精组(5.6%)相比增加21.2个百分点(P<0.05);降温平衡及冷冻复苏后猪精子线粒体膜电位变化显著,其中低Δψm精子比例显著增加(P<0.05);同新鲜精子(19.6%)相比,冷冻复苏后(79.4%)低Δψm精子比例提高59.8个百分点(P<0.05);冻融精子中Bcl-2、Caspase-9及Caspase-3等凋亡因子活性显著高于鲜精子及降温平衡后的精子(P<0.05),由此证明冻融过程能诱导猪精子进入凋亡状态。超低温冷冻保存能导致猪冻融精子质膜磷脂酰丝氨酸(PS)移位及线粒体膜电位降低,同时激发精子Bcl-2、Caspase-9及Caspase-3等凋亡因子活性,诱导成活冻融精子提前进入"似凋亡"状态,明显缩短冻融精子存活时间,这可能是猪冻精繁殖力降低的又一诱因。

The objective of the present work was to analyze whether cryopreservation could induce the ＂apoptosis-like＂ alteration of frozen-thawed boar sperm, and study the molecular mechanism of the fertility reduction of frozen-thawed sperm. Annexin-V/PI combined with the JC 1 fluores cence antibody Kit with flow cytometry （FCM） was used to determine the changes of phosphati dylserine （PS） location and mitochondrial membrane potential （△ψm） of boar sperm before and af- ter cryopreservation with different incubation times . Content changes of ＂apoptosis＂ factors（Bcl-2, Caspase-9 and Caspase-3）in boar sperm were validated by immunoblotting analysis. The results showed that the motility of the cryopreserved sperm （54. 4%） was lower than fresh sperm （87. 9%） （P 0.05）, in which the percentage of ＂apoptosis-like＂ of sperm with PS dislocation （26.8%） was 21.2% higher（P〈0.05） than fresh sperm （5.6%）. The cryopreservation caused an significant increase in the percentage of low Aq）m of equilibrated and cryopreserved sperm （P〈0.05）. Com- pared with fresh sperm （19.6%）, the percentage of sperm with low △ψm increased 59.8% after cryopreservation （P 〈0. 05）. The activating levels of apoptotic factors （Bcl-2, Caspase-9 and Caspase-3）in cryopreserved sperm were remarkably higher than the fresh and equilibrated sperm （P〈0.05）. Present evidences indicated that cryopreservation could induce spermatozoa into the ＂apoptosis-like＂ alteration. This study indicated that cryopreservation could not only lead to the dislocation of PS and reduced the mitochondrial membrane potential, but also stimulate the activi- ties of apoptotic factors （Bcl-2, Caspase-9 and Caspase-3）, induce the frozen-thawed sperm into ＂apoptosis-like＂ state in advance, and shorten the survival time of frozen-thawed spermatozoa ob- viously. This might be another important reason for low pregnancy rate of frozen-thawed sperm.