Spermatozoa rely on kinase/phosphatase mediated protein phosphorylation to modulate cellular processes in the absence of de novo transcription and translation. In congruence, recent studies have detailed extensive remodelling of the sperm protein phosphorylation signature during post-testicular maturation in the epididymis and subsequent capacitation. However, cellular functions in spermatozoa, including phosphorylation signalling, are intrinsically under threat of disruption by oxidative stress due to the cell’s paucity of cytoplasmic antioxidants and enrichment in polyunsaturated fatty acids. This renders the intracellular environment of spermatozoa uniquely susceptible to lipid peroxidation leading to the production of cytotoxic reactive carbonyl species such as 4-hydroxynonenal (4HNE). In turn, increased intracellular 4HNE is associated with a loss of sperm-egg recognition impacting fertilization capacity.
For the first time in human spermatozoa, we have quantified a distinct dysregulation of protein phosphorylation by the exogenous application of low levels of 4HNE (50 µM) using high-resolution tandem mass spectrometry. Specifically, we observed a 4HNE-induced dysregulation of 30.3% of the total phosphopeptides detected in spermatozoa (375 of 1,239), including those within A-kinase anchoring protein 4 (AKAP4), sperm acrosome-associated protein 9 (SPACA9) and sperm flagellar protein 2 (SPEF2). Moreover, application of a lipid-based strategy to protect human sperm function during 4HNE treatment (through the pharmacological inhibition of arachidonate 15-lipoxygenase (ALOX15)), reduced phosphopeptide dysregulation (12.5% of total phosphopeptides) during 4HNE exposure. Proteins protected from 4HNE-induced dysregulation included SPACA9 and SPEF2. However, aberrant phosphorylation of AKAP4 proved refractory to lipoxygenase inhibition. We are now poised to further investigate oxidative stress induced dysregulation of protein phosphorylation signatures caused by environmental stressors with our in silico analyses able to reveal kinases/phosphatases responsible for this dysregulation. Moreover, we have demonstrated a protective but also selective effect of ALOX15 inhibition on phosphorylation signalling in human sperm with potential applications extending to the use of ALOX15 inhibition in medically assisted reproduction.