Institute of Animal Health and Cattle Development (INDEGSAL), University of León, 24071 León, Spain.
Department of Animal Medicine, Surgery and Anatomy (Animal Medicine and Surgery), University of León, 24071 León, Spain.
Int J Mol Sci. 2020 Apr 13;21(8):2701. doi: 10.3390/ijms21082701.
Bull spermatozoa physiology may be modulated by melatonin. We washed ejaculated spermatozoa free of melatonin and incubated them (4 h, 38 °C) with 0-pM, 1-pM, 100-pM, 10-nM and 1-µM melatonin in TALP-HEPES (non-capacitating) and TALP-HEPES-heparin (capacitating). This range of concentrations encompassed the effects mediated by melatonin receptors (pM), intracellular targets (nM-µM) or antioxidant activity (µM). Treatment effects were assessed as motility changes by computer-assisted sperm analysis (CASA) of motility and physiological changes by flow cytometry. Melatonin effects were more evident in capacitating conditions, with 100 pM reducing motility and velocity (VCL) while increasing a "slow" subpopulation. All concentrations decreased apoptotic spermatozoa and stimulated mitochondrial activity in viable spermatozoa, with 100 pM-1 µM increasing acrosomal damage, 10 nM-1 µM increasing intracellular calcium and 1 pM reducing the response to a calcium-ionophore challenge. In non-capacitating media, 1 µM increased hyperactivation-related variables and decreased apoptotic spermatozoa; 100 pM-1 µM increased membrane disorders (related to capacitation); all concentrations decreased mitochondrial ROS production. Melatonin concentrations had a modal effect on bull spermatozoa, suggesting a capacitation-modulating role and protective effect at physiological concentrations (pM). Some effects may be of practical use, considering artificial reproductive techniques.
褪黑素可能调节公牛精子的生理学特性。我们将射出的精子用 TALP-HEPES（非诱导顶体反应）和 TALP-HEPES-肝素（诱导顶体反应）缓冲液冲洗以去除其中的褪黑素，并在其中孵育（4 小时，38°C）0-pM、1-pM、100-pM、10-nM 和 1-µM 浓度的褪黑素。这个浓度范围涵盖了褪黑素受体（pM）、细胞内靶点（nM-µM）或抗氧化活性（µM）介导的作用。通过计算机辅助精子分析（CASA）评估处理效果，用流式细胞术评估精子运动和生理变化。褪黑素在诱导顶体反应的条件下作用更为明显，100 pM 降低了精子的运动和速度（VCL），同时增加了“慢”亚群。所有浓度均降低了凋亡精子的数量，刺激了活精子中的线粒体活性，100 pM-1 µM 增加了顶体反应的损伤，10 nM-1 µM 增加了细胞内钙的含量，1 pM 降低了对钙离子载体的反应。在非诱导顶体反应的培养基中，1 µM 增加了与超激活相关的变量并降低了凋亡精子的数量；100 pM-1 µM 增加了膜紊乱（与诱导顶体反应有关）；所有浓度均降低了线粒体 ROS 的产生。褪黑素浓度对公牛精子有模态效应，表明其在生理浓度（pM）下具有调节诱导顶体反应和保护作用。考虑到人工生殖技术，一些作用可能具有实际用途。
