Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra 50, 40064, Ozzano dell'Emilia, BO, Italy.
INFA-AUB, University of Bologna, Via Gandolfi 16, Cadriano, BO, Italy.
Theriogenology. 2023 Sep 15;208:8-14. doi: 10.1016/j.theriogenology.2023.05.021. Epub 2023 Jun 1.
Bull spermatozoa depend equally on glycolysis and oxidative phosphorylation for the maintenance of the energy necessary for their proper functioning. The aim of the present work was to delineate the mitochondrial activity of bull spermatozoa after incubation with specific inhibitors of the different mitochondrial complexes and evaluate their ROS production. Thawed bull sperm cells (30 × 10 mL in Tyrode's extender) were incubated 1 and 3h at 37 °C with rotenone 5 μM (ROT), complex I inhibitor; dimethyl-malonate 10 mM (DMM), complex II inhibitor; carbonyl cyanide m-chlorophenyl hydrazine 5 μM (CCCP), uncoupling agent; antimycin A 1 μg/mL (ANTI), complex III inhibitor; oligomycin 5 μM (OLIGO), ATP synthase inhibitor, and 0.5% DMSO, vehicle (CTR). Sperm motility and kinematics were assessed by Hamilton Thorn IVOS 12.0. Mitochondrial membrane potential, mitochondrial O production and HO intracellular content were evaluated by BD FACSCalibur flow cytometer, and sperm viability (SYBR-14/PI) and mitochondrial activity (JC-1/SYBR-14/PI) were assessed by epifluorescence microscopy. A multivariate analysis was performed on the results. In addition, sperm kinematic features, registered for each motile spermatozoon, were studied by cluster analysis. The incubation during 1 or 3 h in presence of the inhibitors of mitochondrial functionality only had a minor effect on motility parameters, decreasing the proportion of the SP1 (fast progressive) subpopulation after 3 h of incubation with ROT, ANTI or OLIGO. The percentage of live spermatozoa with active mitochondria was reduced under the effect of ANTI and CCCP both at 1 and 3 h. In conclusion, mitochondrial function is somehow impaired in frozen thawed bull sperm as not all live cells showed active mitochondria. These results support the findings that bull spermatozoa can alternatively rely on oxidative phosphorylation or glycolysis for energy obtainment and that their mitochondria are less affected by ETC inhibitors.
牛精子依赖糖酵解和氧化磷酸化来维持其正常功能所需的能量。本研究的目的是描述在不同线粒体复合物的特异性抑制剂孵育后牛精子的线粒体活性，并评估其 ROS 产生。解冻的牛精子细胞（30×10 个 mL 在 Tyrode 稀释液中）在 37°C 下用鱼藤酮 5 μM（ROT）、复合物 I 抑制剂；二甲马来酸 10 mM（DMM）、复合物 II 抑制剂；羰基氰化物 m-氯苯腙 5 μM（CCCP）、解偶联剂；抗霉素 A 1 μg/mL（ANTI）、复合物 III 抑制剂；寡霉素 5 μM（OLIGO）、ATP 合酶抑制剂和 0.5%DMSO（CTR）孵育 1 小时和 3 小时。通过 Hamilton Thorn IVOS 12.0 评估精子运动和运动学。通过 BD FACSCalibur 流式细胞仪评估线粒体膜电位、线粒体 O 产生和 HO 细胞内含量，通过荧光显微镜评估精子活力（SYBR-14/PI）和线粒体活性（JC-1/SYBR-14/PI）。对结果进行了多变量分析。此外，通过聚类分析研究了每个运动精子的精子运动学特征。在存在线粒体功能抑制剂的情况下孵育 1 小时或 3 小时仅对运动参数有轻微影响，在用 ROT、ANTI 或 OLIGO 孵育 3 小时后，降低了 SP1（快速进展）亚群的比例。在 1 小时和 3 小时时，ANTI 和 CCCP 的作用下，具有活性线粒体的活精子的百分比减少。总之，冷冻解冻后的公牛精子的线粒体功能受到一定程度的损害，因为并非所有活细胞都显示出活跃的线粒体。这些结果支持以下发现：公牛精子可以通过氧化磷酸化或糖酵解来获取能量，并且它们的线粒体受电子传递链抑制剂的影响较小。
