Paul R K, Kumar D, Naqvi Smk
ICAR-Central Sheep and Wool Research Institute, Rajasthan, India.
Reprod Domest Anim. 2017 Dec;52(6):1052-1060. doi: 10.1111/rda.13023. Epub 2017 Jul 25.
Antioxidants are known to prevent the reactive oxygen species (ROS)-mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid-protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll . Sperm pellet was resuspended in soya lecithin-Tris-fructose diluent (400 × 10 cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3-5°C for 72 hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo-osmotic swelling test (HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X-100 were resolved by SDS-PAGE and quantified using Quantity One software (Bio-Rad, USA). The rapid motility, linearity and straight-line velocity (VSL) were found significantly (p < .05) higher in the antioxidant-treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant-treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower (p < .05) in the sperm extract of antioxidant-treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase (p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant-treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage.
已知抗氧化剂可防止哺乳动物精子在低温保存过程中活性氧（ROS）介导的膜脂过氧化损伤。我们在此假设，ROS还会影响脂质-蛋白质相互作用，从而降低膜的完整性以及蛋白质与双层膜的锚定。抗氧化剂通过清除ROS来防止这些损伤。对帕坦瓦迪公羊的射精样本进行主观评估后合并，并使用Percoll进行离心。将精子沉淀重悬于含有抗氧化剂（100 IU/ml过氧化氢酶 + 10 mM还原型谷胱甘肽）或不含抗氧化剂的大豆卵磷脂-三羟甲基氨基甲烷-果糖稀释液（400×10⁶细胞/ml）中。将等分试样在橱柜中冷却至5°C，然后在3-5°C的冰箱中保存72小时。在0、24、48和72小时进行精子活力、生存力、脂质过氧化（LPO）和低渗肿胀试验（HOST）。用0.5% Triton X-100提取的精子蛋白通过SDS-PAGE分离，并用Quantity One软件（美国伯乐公司）进行定量。在储存48小时时，发现抗氧化剂处理组的快速运动性、线性度和直线速度（VSL）显著高于对照组（p < 0.05）。发现两组之间精子生存力相当。在48小时和72小时时，与对照组相比，抗氧化剂处理的样本中HOST反应更高，LPO更低。总体而言，与对照组相比，抗氧化剂处理组的精子提取物中蛋白质P1（106.09 kDa）、P2（87.00 kDa）和P4（51.14 kDa）含量更低（p < 0.05）。与抗氧化剂处理组相比，对照组精子提取物中P4（51.14 kDa）的含量在更早时间（48小时与72小时）增加（p < 0.05）。总之，结果表明抗氧化剂可降低精子中的LPO，从而在储存48小时和72小时时提高精子活力、质膜完整性并保护蛋白质与质膜的锚定。
