Carrageta David F, Freire-Brito Laís, Guerra-Carvalho Bárbara, Ribeiro João C, Monteiro Bruno S, Barros Alberto, Oliveira Pedro F, Monteiro Mariana P, Alves Marco G
Endocrine and Metabolic Research, UMIB-Unit for Multidisciplinary Research in Biomedicine, ICBAS-School of Medicine and Biomedical Sciences, University of Porto, 4050-313 Porto, Portugal.
Laboratory of Physiology, Department of Immuno-Physiology and Pharmacology, ICBAS-School of Medicine and Biomedical Sciences, University of Porto, 4050-313 Porto, Portugal.
Antioxidants (Basel). 2023 Feb 8;12(2):409. doi: 10.3390/antiox12020409.
Mitochondrial uncoupling proteins (UCPs) are central in the regulation of mitochondrial activity and reactive oxygen species (ROS) production. High oxidative stress is a major cause of male infertility; however, UCPs expression and function in human spermatozoa are still unknown. Herein, we aimed to assess the expression and function of the different homologs (UCP1-6) in human spermatozoa. For this purpose, we screened for the mRNA expression of all UCP homologs. Protein expression and immunolocalization of UCP1, UCP2, and UCP3 were also assessed. Highly motile spermatozoa were isolated from human normozoospermic seminal samples (n = 16) and incubated with genipin, an inhibitor of UCPs (0, 0.5, 5, and 50 µM) for 3 h at 37 °C. Viability and total motility were assessed. Mitochondrial membrane potential and ROS production were evaluated. Media were collected and the metabolic profile and antioxidant potential were analyzed by H-NMR and FRAP, respectively. The expression of all UCP homologs () mRNA by human spermatozoa is herein reported for the first time. UCP1-3 are predominant at the head equatorial segment, whereas UCP1 and UCP2 are also expressed at the spermatozoa midpiece, where mitochondria are located. The inhibition of UCPs by 50 µM genipin, resulting in the UCP3 inhibition, did not compromise sperm cell viability but resulted in irreversible total motility loss that persisted despite washing or incubation with theophylline, a cAMP activator. These effects were associated with decreased mitochondrial membrane potential and lactate production. No differences concerning UCP3 expression, however, were observed in spermatozoa from normozoospermic versus asthenozoospermic men (n = 6). The inhibition of UCPs did not increase ROS production, possibly due to the decreased mitochondrial activity and genipin antioxidant properties. In sum, UCPs are major regulators of human spermatozoa motility and metabolism. The discovery and characterization of UCPs' role in human spermatozoa can shed new light on spermatozoa ROS-related pathways and bioenergetics physiology.
线粒体解偶联蛋白（UCPs）在调节线粒体活性和活性氧（ROS）生成中起核心作用。高氧化应激是男性不育的主要原因；然而，UCPs在人类精子中的表达和功能仍不清楚。在此，我们旨在评估不同同源物（UCP1 - 6）在人类精子中的表达和功能。为此，我们筛选了所有UCP同源物的mRNA表达。还评估了UCP1、UCP2和UCP3的蛋白表达及免疫定位。从人类正常精液样本（n = 16）中分离出高活力精子，并与UCPs抑制剂京尼平（0、0.5、5和50 μM）在37°C下孵育3小时。评估了活力和总运动能力。评估了线粒体膜电位和ROS生成。收集培养基，分别通过¹H - NMR和FRAP分析代谢谱和抗氧化潜力。本文首次报道了人类精子中所有UCP同源物（）mRNA的表达。UCP1 - 3主要位于头部赤道段，而UCP1和UCP2也在线粒体所在的精子中段表达。50 μM京尼平对UCPs的抑制作用导致UCP3被抑制，这并未损害精子细胞活力，但导致不可逆的总运动能力丧失，尽管经过洗涤或与环磷酸腺苷（cAMP）激活剂茶碱孵育，这种丧失仍持续存在。这些效应与线粒体膜电位降低和乳酸生成减少有关。然而，在正常精子症男性与弱精子症男性（n = 6）的精子中，未观察到UCP3表达的差异。UCPs的抑制并未增加ROS生成，这可能是由于线粒体活性降低和京尼平的抗氧化特性所致。总之，UCPs是人类精子运动能力和代谢的主要调节因子。UCPs在人类精子中作用的发现和表征可为与精子ROS相关的途径和生物能量生理学提供新的线索。
