Sphingolipids modulate redox signalling during human sperm capacitation.

study questionWhat role do sphingolipids have in mediating human sperm capacitation? SUMMARY ANSWER: Sphingosine 1-phosphate (S1P) mediates the acquisition of fertilizing competency in human spermatozoa by engaging with its Gi-coupled receptor S1PR1 and promoting production of reactive oxygen species such as nitric oxide and superoxide anion. WHAT IS KNOWN ALREADY: Bioactive sphingolipids, such as S1P, are fundamental for regulating numerous physiological domains and processes, such as cell membranes and signalling, cell death and proliferation, cell migration and invasiveness, inflammation, and central nervous system development. STUDY DESIGN, SIZE, DURATION: Semen samples were obtained from a cohort of 10 healthy non-smoking volunteers (18-30 years old) to investigate the role of S1P in sperm. PARTICIPANTS/MATERIALS, SETTING,

methodsPercoll-selected human spermatozoa were incubated at 37°C for 3.5 h in BWW media with or without foetal cord serum ultrafiltrate (FCSu), sphingosine (Sph), or ceramide (Cer). Spermatozoa were also incubated with or without pharmacological inhibitors of sphingolipid metabolism. Protein tyrosine phosphorylation was determined by immunoblotting. The acrosome reaction was determined by PSA-FTIC labelling of the acrosome and analysed using fluorescence microscopy. Intracellular nitric oxide (NO•) production was determined using a DAF-2DA probe. Immunocytochemistry was performed to localize and assess the functional relationship of key components of lipid signalling in spermatozoa. Sperm viability and motility of the samples were evaluated by the hypo-osmotic swelling (HOS) test and computer-aided sperm analysis (CASA). Statistical differences between groups were determined using ANOVA and Tukey's test. Normal distribution of the data and variance homogeneity were assessed using Shapiro-Wilk and Levene's test, respectively. A difference was considered significant when the P-value was ≤0.05. MAIN RESULTS AND THE ROLE OF CHANCE: S1P mediates the acquisition of fertilizing competency in human spermatozoa by engaging with its Gi-coupled receptor S1PR1. We found that S1PR1 redistributes to the post-acrosomal region upon induction of capacitation. S1P signalling promotes the activation of the PI3K-AKT pathway, leading to NO• production during sperm capacitation. L-NAME, an nitric oxide synthase inhibitor, impaired the Sph- and Cer-dependent capacitation. Additionally, Sph and Cer promote superoxide anion (O2•-) production, and the extracellular addition of superoxide dismutase (SOD) prevented Sph- and Cer-dependent capacitation, suggesting that Sph and Cer stimulate O2•- production during sperm capacitation. Protein kinase type R (PKR), ceramide kinase (CERK), and protein kinase C (PKC) are responsible for translocating and activating sphingosine kinase 1 (SphK1), which is necessary to promote S1P production for sperm capacitation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The utilization and actions of sphingolipids may differ in spermatozoa of different species. WIDER IMPLICATIONS OF THE

findingsSphingolipid metabolites such as Sph, Cer, S1P, and ceramide 1-phosphate (C1P) play a crucial role in inducing human sperm capacitation. Our research has provided new insights into fundamental sphingolipid processes in human sperm, including the importance of C1P in translocating and activating SphK1 as well as the S1P signalling to regulate the PI3K/AKT/NOS pathway to generate NO• for sperm capacitation. We are the first to identify the presence of PKR in human spermatozoa and its role in the phosphorylation activities of SphK1 with the subsequent activation of S1P signalling. Furthermore, our study has identified that S1PR1 and S1PR3 are involved in capacitation and the acrosome reaction, respectively. These findings shed light on a novel mechanism by which sphingolipids drive capacitation in human sperm and pave the way for further exploration of the role of bioactive sphingolipid metabolites in this process. Lastly, our studies lay the foundation for examining the lipid profile of infertile males, as potential discrepancies can affect the functional capacity of spermatozoa to reach fertilizing potential. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by the Canadian Institutes of Health Research (CIHR), grant number PJT-165962 to C.O.F. S.S. was awarded a Research Institute-MUHC Desjardins Studentship. There are no competing interests to report.