目的:精子冷冻被认为是辅助生殖技术(ART)计划中的有效方法,它对精子功能有有害影响,由于活性氧(ROS)的产生。本研究旨在探讨线粒体醌(MitoQ)在精子冷冻过程中抑制线粒体ROS产生的潜力。
方法:本研究共收集了20个人类正常精子样本。样本分为四组,每个含有不同浓度的MitoQ(0、0.2、2和20nM),然后进行冻结。解冻后,评估精子悬浮液的参数,包括运动性,形态学,顶体完整性,三磷酸腺苷(ATP)水平,细胞内ROS,生存能力,染色质包装,DNA变性,DNA片段化,以及抗氧化剂的表达(GPX,SOD)和凋亡(Bax,Bcl2)基因。
结果:结果表明,在2nM组中,精子的总和进行性迁移率显着增加,而在20nM组中显著降低(p≤0.05)。在所有测试浓度下,精子形态均未明显改善(p≥0.05)。细胞内ROS水平在2和20nM的浓度下显示出显著的降低和增加,分别为(p≤0.05)。此外,观察到生存能力显着增加,ATP,顶体完整性,染色质包装,用2nM的MitoQ处理后的非变性和非片段化的DNA,与对照组相比(p≤0.05)。关于基因表达,氧化应激基因的相对表达在2nM组中增加,在20nM组中减少(p≤0.05),与对照组相比,凋亡基因的表达没有显着差异(p≥0.05)。所有比较均相对于对照组进行。
结论:向精子冷冻培养基中添加最佳浓度的MitoQ(2nM)不仅可以改善精子功能参数并减少DNA损伤,而且还刺激抗氧化基因的表达,为精子冷冻保存带来更大的好处。
OBJECTIVE: Sperm freezing is considered as an effective way in assisted reproductive technology (ART) programs, it has detrimental effects on sperm function, due to the production of reactive oxygen species (ROS). This study aimed to investigate the potential of Mitoquinone (MitoQ) in inhibiting the production of mitochondrial ROS during sperm freezing.
METHODS: A total of 20 human normozoosperm samples were collected for this study. The samples were divided into four groups, each containing different concentrations of MitoQ (0, 0.2, 2, and 20 nM), and then subjected to the freezing process. After thawing, the sperm suspensions were evaluated for parameters including motility, morphology, acrosome integrity, adenosine triphosphate (ATP) level, intracellular ROS, viability, chromatin packaging, DNA denaturation, DNA fragmentation, as well as the expression of antioxidants (GPX, SOD) and apoptotic (Bax, Bcl2) genes.
RESULTS: The results showed that total and progressive mobility of sperms significantly increased in the 2 nM group, while significantly decreased in the 20 nM group (p ≤ 0.05). Sperm morphology did not significantly improve across all the tested concentrations (p ≥ 0.05). Intracellular ROS levels showed a significant decrease and increase in the concentrations of 2 and 20 nM, respectively (p ≤ 0.05). Furthermore, a significant increase was observed in viability, ATP, acrosome integrity, chromatin packaging, and non-denatured and non-fragmented DNA after treatment with 2 nM of MitoQ, compared with the control group (p ≤ 0.05). Regarding gene expressions, the relative expressions of oxidative stress genes were increased in the 2 nM group and decreased in the 20 nM group (p ≤ 0.05), while no significant difference was observed in the expressions of apoptotic genes compared with the control group (p ≥ 0.05). All the comparisons were made with respect to the control group.
CONCLUSIONS: Adding the optimal concentration of MitoQ (2 nM) to the sperm freezing medium not only improves sperm functional parameters and reduces DNA damages, but also stimulates the expression of antioxidant genes, leading to even greater benefits for sperm cryopreservation.