超低温冷冻过程引起猪精子产生“似凋亡”变化

doi:10.11843/j.issn.0366-6964.2013.11.010

摘要/Abstract

摘要：

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。

关键词: 低温保存, 似凋亡, 线粒体膜电位, 精子, 猪

Abstract:

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 fluorescence antibody Kit with flow cytometry (FCM) was used to determine the changes of phosphatidylserine (PS) location and mitochondrial membrane potential (Δψm) of boar sperm before and after 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 Δψm of equilibrated and cryopreserved sperm (P<0.05).Compared with fresh sperm (19.6%), the percentage of sperm with low Δψm 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 activities 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 obviously. This might be another important reason for low pregnancy rate of frozen-thawed sperm.

Key words: cryopreservation, apoptosis-like, mitochondrial membrane potential, sperm, boar

中图分类号:

S828
S814.8

赵娜，甄林青，胡启蒙，王亮亮，李新红. 超低温冷冻过程引起猪精子产生“似凋亡”变化[J]. 畜牧兽医学报, 2013, 44(11): 1766-1774.

ZHAO Na, ZHEN Lin-qing, HU Qi-meng, WANG Liang-liang, LI Xin-hong. Cryopreservation Procedure Induces the Alterations of “Apoptosis-Like” of Boar Sperm[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(11): 1766-1774.

参考文献

［1］ANZAR M, KROETSCH T, BOSWALL L, et al. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes［J］.Anim Reprod Sci, 2011,126(1-2):23-31.

［2］HASEGAWA A, YONEZAWA K, OHTA A, et al. Optimization of a protocol for cryopreservation of mouse spermatozoa using cryotubes［J］. J Reprod Dev, 2012, 58(1):156-161.

［3］BENSON J D, WOODS E J, WALTERS E M, et al. The cryobiology of spermatozoa［J］.Theriogenology, 2012,78(8):1682-1699.

［4］JOHNSON L A, WEITZE K F, FISER P, et al. Storage of boar semen［J］. Anim Reprod Sci,2000, 62: 143-172.

［5］CHANAPIWAT P, KAEOKET K , TUMMARUK P. Cryopreservation of boar semen by egg yolk-based extenders containing lactose or fructose is better than sorbitol［J］. J Vet Med Sci, 2012,74(3): 351-354.

［6］MORRELL J M,WALLGREN M C.Centrifugation of boar semen［J］. Reprod Domest Anim, 2011, 46(2):18-22.

［7］MALO C, GIL L, CANO R, et al. Fennel (Foeniculum vulgare) provides antioxidant protection for boar semen cryopreservation［J］. Andrologia, 2012,44:710-715.

［8］SATORRE M M, BREININGER E, BECONI M T. Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm［J］. Theriogenology, 2012,78(7):1548-1556.

［9］CASA I, ALTHOUSE G C. The protective effect of a 17 ℃ holding time on boar sperm plasma membrane fluidity after exposure to 5 ℃［J］. Cryobiology, 2013, 66(1):69-75.

［10］DIDION B A, BRAUN G D, DUGGAN M V. Field fertility of frozen boar semen: A retrospective report comprising over 2600 AI services spanning a four year period［J］. Anim Reprod Sci, 2013, 135(1-2):112-118.

［11］CHAVEIRO A, SANTOS P, SILVADA F M. Assessment of sperm apoptosis in cryopreserved bull semen after swim-up treatment: A flow cyto reproductive health care center/planned parenthood study［J］. Reprod Dom Anim, 2007, 42:17-21.

［12］PEÑA F J, JOHANNISSON A, WALLGREN M, et al. Assessment of fresh and frozen-thawed boar semen using an annexin-V assay: a new method of evaluating sperm membrane integrity［J］. Theriogenology, 2003b, 60:677-689.

［13］WÜNDRICH K, PAASCH U, MONIKA L, et al. Activation of caspases in human spermatozoa during cryopreservation-an immunoblot study［J］. Cell Tissue Banking, 2006,7:81-90.

［14］GARCÍA B M, FERNÁNDEZ L G, FERRUSOLA C O, et al. Membrane lipids of the stallion spermatozoon in relation to sperm quality and susceptibility to lipid peroxidation［J］. Reprod Domest Anim, 2011,46(1):141-148.

［15］MARTIN G, CAGNON N, SABIDO O, et al. Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa［J］. Hum Reprod, 2007, 22:380-388.

［16］PAASH U, RAKESH K, SHARM A, et al. Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa［J］. Biol Repreod, 2004, 71:1828-1837.

［17］WATERHOUSE K E, GJELDNES A, TVERDAL A, et al. Alterations of sperm DNA integrity during cryopreservation procedure and in vitro incubation of bull semen［J］.Anim Reprod Sci,2010, 117(1-2):34-42.

［18］SILVA P F N, GADELLA B M, Detection of damage in mammalian sperm cells［J］. Theriogenology, 2006,65: 958-978.

［19］GREED D R, REED J C. Mitochondria and apoptosis［J］. Science, 1998, 281:1309-1312.

［20］TAMER M S, AGARWAL A, ZBOROWSKL M, et al. Andrology lab corner utility of magnetic cell separationas a molecular sperm preparation technique［J］. JAndrol, 2008, 29(2):134-142.

［21］BRATTON D L, FADOK V A, RICHTER D A, et al. Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase［J］. J Biol Chem, 1997, 272:26159-26165.

［22］MARTIN G, SABIDO O, DURAND P, et al. Cryopreservation induces an apoptosis like mechanism in bull sperm［J］. Biol Reprod, 2004,71:28-37.

［23］ORTEGA-FERRUSOLA C, SOTILLO-GALÁN Y, VARELA-FERNÁNDEZ E, et al. Detection of “Apoptosis-Like” changes during the cryopreservation process in equine sperm［J］. J Androl, 2008, 29(2):213-221.

［24］JEONG Y J, KIM M K, SONG H J, et al. Effect of α-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis related genes［J］. Cryobiology, 2009, 58: 181-189.

［25］KHAN D R, AHMAD N M, ANZAR A A. Apoptosis in fresh and cryopreserved buffalo sperm［J］. Theriogenology, 2009, 71 : 872-876.

［26］BAILEY J L, BILODEAU J F, CORMIER N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon［J］. J Androl,2000, 1:1-7.

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