One of the leading causes of male infertility is defective sperm function, a pathology that commonly arises from oxidative stress in the male germline. Oxidative stress is induced through excessive reactive oxygen species (ROS) production leading to lipid peroxidation of the plasma membrane and apoptosis. Lipid peroxidation, in turn, results in the generation of cytotoxic aldehydes such as 4-hydroxynoneal (4HNE), further elevating oxidative stress cascades. To investigate the specific mechanisms by which 4HNE is produced in developing germ cells, comparative proteomics was performed on isolated round spermatids exposed to 4HNE and their untreated counterparts. This study revealed a highly significant, 28-fold increase in the enzyme 15-arachidonate-lipoxygenase (ALOX15) following exposure to 4HNE. The ALOX15 protein belongs to a family of non-heme iron containing enzymes implicated in lipid oxygenation. This role has the potential to exacerbate ROS production and hence accentuate the levels of oxidative stress experienced by the cell. Given this, we sought to characterise the functional role of ALOX15 in human spermatozoa.
To assess ALOX15 activity within human spermatozoa, the selective inhibitor 6,11-dihydro[1]benzothiopyrano[4,3-b]indole (PD146176) was utilized. Simultaneous treatment of spermatozoa with PD146176 alongside an oxidative stress insult (H2O2) yielded a significant reduction in mitochondrial and cytosolic ROS production as well as a concomitant suppression of lipid peroxidation. Moreover, our functional analysis of oxidatively stressed human spermatozoa revealed that ALOX15 inhibition led to significant improvements in sperm motility parameters, acrosome reaction rates and the competence of these cells to bind to homologous zona pellucidae compared to the non-inhibited control population. Such results support the hypothesised involvement of ALOX15 in accentuating the oxidative stress burden within spermatozoa and present a possible therapeutic strategy for combating ROS-mediated male fertility issues.