Spermatozoa are known to be amongst the most environmentally responsive cells in the body, which grants them the capacity to sense critical cues in the female reproductive tract that signal readiness for fertilisation. However, this sensitivity also culminates in a mature sperm cell that is highly vulnerable to exogenous oxidative stress. While a notable lack of cytoplasmic antioxidant content underpins a degree of this stress sensitivity, mammalian spermatozoa have been historically charted to possess a phospholipidome replete with an abundance of polyunsaturated fatty acids (PUFA-PLs) that are key substrates for oxidative attack. Despite this long-held view, we have no clear information regarding which stages of germ cell maturation are specifically enriched in PUFA-PLs and which enzymes contribute to these membrane characteristics.
In this study, we examined the phospholipidome and lipid modulating-proteome of three distinct mouse germ cell stages, pachytene spermatocytes (PSc), round spermatids (RSt) and spermatozoa, through high-resolution tandem mass spectrometry approaches. These approaches demonstrated that spermiogenesis results in an enrichment of ester and ether-linked phosphatidylethanolamines (PE), the latter of which may indicate an intrinsic sensitivity to the cell death modality ferroptosis (P<0.05). While spermiogenesis gave rise to widespread and significant (P<0.05, 1.5-fold change) changes in phospholipid abundance, synthesis/remodelling of lipids was more profound during the maturation of PSc to RSt. Examining lipid structural characteristics revealed a significant increase (P<0.01) in PUFA-PLs containing six or more double bonds in RSt compared to PSc, indicating a profound increase in oxidation potential in RSt. In accounting for this enrichment in PUFA-PLs, our proteomic data revealed the presence of several PL remodelling enzymes, including lysophosphatidylcholine acyltransferase 3 (LPCAT3), in RSt that were absent or lowly expressed in PSc. Further examination of these data will permit the design of novel stage-specific strategies to fortify germ cell membranes during periods of environmental stress.