猪冷冻精子的细胞凋亡及凋亡途径初步研究

Preliminary Studies on Apoptosis and Apoptotic Pathways of Frozen Boar Spermatozoa

旨在研究低温冷冻解冻对精子细胞凋亡的影响,并探讨凋亡发生的可能相关途径。采用流式细胞仪检测解冻后精子内的ROS水平和TUNEL染色后的细胞凋亡情况,使用酶标仪检测精子内ATP含量,利用相对荧光定量PCR技术检测不同凋亡途径中相关基因的表达量变化。结果表明:冷冻后精子活力、顶体和质膜完整率显著下降(P<0.05);冷冻精子TUNEL染色后的凋亡率(80.4%)与新鲜组(9.7%)相比显著升高(P<0.05);冷冻组精子内ROS绿色荧光比率(58.2%)亦显著高于新鲜组(P<0.05);与新鲜组0.926μmol·L-1的ATP浓度相比,冷冻组精子的ATP浓度(0.247μmol·L-1)则显著下降(P<0.05);qRT-PCR检测结果显示,冷冻组中TNF-α、Fas、Caspase家族、P53和Bax基因相对表达量升高,其中Fas、P53、TNF-α、Caspase-8和Caspase-9等相对表达量显著升高(P<0.05);BCL-2、BIRC-5、MnSOD、CuZnSOD和SURVIVN基因相对表达量降低,其中BIRC-5、MnSOD、SURVIVN显著降低(P<0.05)。结果表明:低温冷冻解冻会使猪精子细胞发生严重的凋亡,死亡受体外源性凋亡途径和线粒体内源性凋亡途径均可能介导了冷冻后精子的凋亡。

In order to investigate the effects of cryopreservation on boar spermatozoa apoptosis and apoptotic pathway,the flow cytometry was used to detect the apoptosis state and ROS level of boar frozen spermatozoa.ATP content was measured by luminometer,and the mRNA expression level of genes involved in different apoptotic pathways were measured by real-time quantitative RT-PCR method.The results showed that the motility,normal acrosome rate and plasma membrane integrity rate of sperm after freezing were decreased greatly(P<0.05).The TUNEL-positive spermatozoa rate(80.4%)from freezing group were much higher than that of fresh group(9.7%)(P<0.05).The percentage of ROS-positive spermatozoa in freezing group(58.2%)was also obviously higher than that of fresh group(P<0.05).The cryopreservation caused a significant decrease of ATP concentration in freezing group(0.247μmol·L-1)comparing with fresh group(0.926μmol·L-1)(P<0.05).qRT-PCR results showed that the relative expression levelfrom promoting apoptosis genes(TNF-α,Fas,Caspase,P53 and Bax)were up-regulated,and in which the expression of Fas,P53,TNF-α,Caspase-8and Caspase-9increased greatly after cryopreservation(P<0.05).The relative expression level from inhibiting apoptosis genes(BCL-2,BIRC-5,MnSOD,CuZnSODand SURVIVN)were down-regulated,and in which the expression of BIRC-5,MnSODand SURVIVNdecreased greatly(P<0.05).The results indicated that cryopreservation could lead to boar spermatozoa apoptosis.Not only extrinsic death receptor but also intrinsic mitochondrial signaling pathway might play key roles in mediating spermatozoa apoptosis after cryopreservation.