精子运动是精子从子宫向输卵管迁移的重要因素。在体外保存精子期间,精子产生过量的ROS,损害其功能。本研究旨在探讨在稀释培养基中加入吡咯并喹啉醌(PQQ)是否能提高冷冻公羊精子质量,然后阐明机制。用含有不同剂量的PQQ(0nM,10nM,100nM,1000nM,10,000nM),并储存在4℃。精子运动模式,质膜完整性,顶体完整性,线粒体膜电位,活性氧(ROS)水平,丙二醛(MDA)水平,超氧化物歧化酶(SOD)活性,保存后测量ATP水平。此外,免疫印迹法检测精子中NADH脱氢酶1(MT-ND1)和NADH脱氢酶6(MT-ND6)的表达。此外,还评估了精子获能和精子与透明带结合的能力。观察到PQQ的添加显着(p<0.05)改善了公羊精子运动,膜完整性,和顶体在保存过程中的完整性。PQQ处理组中具有高线粒体膜电位的精子百分比远高于对照组。此外,补充PQQ也降低了精子MDA和ROS水平,同时增加ATP水平。有趣的是,用PQQ处理的精子中MT-ND1和MT-ND6蛋白的水平也高于对照组。此外,向培养基中添加100nMPQQ可降低MT-ND1和MT-ND6蛋白中的ROS损伤。100nMPQQ的添加显着(p<0.05)增加了诱导获能后公羊精子中的蛋白质酪氨酸磷酸化。此外,100nMPQQ治疗组中的精子-透明带结合能力的值也远高于对照组。总的来说,在冷冻精子保存期间,PQQ通过猝灭ROS水平以减少ROS损伤并维持精子线粒体功能来保护公羊精子质量。并保留了精子的高受精能力。
Sperm motility is an important factor in the migration of sperm from the uterus to the oviduct. During sperm preservation in vitro, sperm generates excessive ROS that damages its function. This study aims to investigate whether the addition of pyrroloquinoline quinone (
PQQ) to the diluted medium could improve chilled ram sperm quality, and then elucidates the mechanism. Ram semen was diluted with Tris-citric acid-glucose (TCG) medium containing different doses of
PQQ (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM), and stored at 4 °C. Sperm motility patterns, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, reactive oxygen species (ROS) levels, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and ATP levels were measured after preservation. Furthermore, the expressions of NADH dehydrogenase 1 (MT-ND1) and NADH dehydrogenase 6 (MT-ND6) in sperm were also detected by western blotting. In addition, sperm capacitation and the ability of sperm to bind to the zona pellucina were also evaluated. It was observed that the addition of PQQ significantly (p < 0.05) improved ram sperm motility, membrane integrity, and acrosome integrity during preservation. The percentage of sperm with high mitochondrial membrane potential in the
PQQ treatment group was much higher than that in the control. In addition, supplementation of
PQQ also decreased the sperm MDA and ROS levels, while increasing ATP levels. Interestingly, the levels of MT-ND1 and MT-ND6 protein in sperm treated with PQQ were also higher than that of the control. Furthermore, the addition of 100 nM PQQ to the medium decreased ROS damage in MT-ND1 and MT-ND6 proteins. The addition of 100 nM
PQQ significantly (p < 0.05) increased protein tyrosine phosphorylation in ram sperm after induced capacitation. Furthermore, the value of the sperm-zona pellucida binding capacity in the 100 nM PQQ treatment group was also much higher than that of the control. Overall, during chilled ram- sperm preservation, PQQ protected ram sperm quality by quenching the ROS levels to reduce ROS damage and maintain sperm mitochondrial function, and preserved the sperm\'s high ability of fertilization.