Probing human sperm metabolism using 13C-magnetic resonance spectroscopy.

study questionCan 13C-Magnetic Resonance Spectroscopy (MRS) of selected metabolites provide useful information about human sperm metabolism and how glycolysis or oxidative phosphorylation are used by different sperm populations? SUMMARY ANSWER: Sperm populations, prepared by density gradient centrifugation (DGC) and incubated with either 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate, showed consistent evidence of metabolism generating principally lactate and more intermittently bicarbonate, and significantly more lactate was produced from 13Cu-glucose by vital or motile sperm recovered from the 40/80% interface compared to those from the pellet, which could not be accounted for by differences in the non-sperm cells present. WHAT IS KNOWN ALREADY: Previous studies have focused on CO2 or other specific metabolite production by human sperm and there remains considerable debate about whether glycolysis and/or oxidative phosphorylation is the more important pathway for ATP production in sperm. STUDY DESIGN, SIZE, DURATION: Sperm populations were prepared by DGC and subjected to 13C-MRS to answer the following questions. (i) Is it possible to detect human sperm metabolism of 13C substrates implicated in energy generation? (ii) What are the kinetics of such reactions? (iii) Do different sperm populations (e.g. '80%' pellet sperm and '40%' interface sperm) utilise substrates in the same way? Semen samples from 97 men were used in these experiments; 52 were used in parallel for aims (i) and (ii) and 45 were used for aim (iii). PARTICIPANTS/MATERIALS, SETTING,

methodsSperm populations were prepared from ejaculates of healthy men using a Percoll/Phosphate Buffered Saline (PBS) DGC and then incubated with a range of 13C-labelled substrates (13Cu-glucose, 13Cu-fructose, 13C1-pyruvate, 13C1-butyrate, 13C3-lactate, 13C2,4-D-3-hydroxybutyrate, 13C5-l-glutamate, 13C1,2-glycine or 13Cu-galactose) along with penicillin/streptomycin antibiotic at 37°C for 4 h, 24 h or over 48 h for an estimated rate constant. Sperm concentration, vitality and motility were measured and, for a subset of experiments, non-sperm cell concentration was determined. A 9.4 T magnetic resonance spectrometer was used to acquire 1D 13C, inverse gated 1H decoupled, MRS spectra. Spectrum processing was carried out using spectrometer software and Matlab scripts to determine peak integrals for each spectrum. MAIN RESULTS AND THE ROLE OF CHANCE: 13Cu-glucose, 13Cu-fructose and 13C1-pyruvate were consistently converted into lactate and, to a lesser extent, bicarbonate. There was a significant correlation between sperm concentration and lactate peak size for 13Cu-glucose and 13Cu-fructose, which was not observed for 13C1-pyruvate. The lactate peak did not correlate with the non-sperm cell concentration up to 6.9 × 106/ml. The concentration of 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate (1.8, 3.6, 7.2 or 14.4 mM) had no influence on the size of the observed lactate peak over a 4 h incubation. The rate of conversion of 13C1-pyruvate to lactate was approximately three times faster than for 13Cu-glucose or 13Cu-fructose which were not significantly different from each other. After incubating for 4 h, the utilisation of 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate by sperm from the '40%' interface of the DGC was no different from those from the pellet when normalised to total sperm concentration. However, after normalising by either the vital or motile sperm concentration, there was a significant increase in conversion of 13Cu-glucose to lactate by '40%' interface sperm compared to pellet sperm (Vital = 3.3 ± 0.30 × 106 vs 2.0 ± 0.21 × 106; P = 0.0049; Motile = 7.0 ± 0.75 × 106 vs 4.8 ± 0.13 × 106; P = 0.0032. Mann-Whitney test P < 0.0055 taken as statistically significant). No significant differences were observed for 13Cu-fructose or 13C1-pyruvate. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Only 13C labelled metabolites that accumulate to a sufficiently high concentration can be observed by 13C MRS. For this reason, intermediary molecules in the metabolic chain are difficult to observe without trapping the molecule at a particular step using inhibitors. Non-sperm cell concentration was typical of the general population and no link was found between these cells and the magnitude of the 13C-lactate peak. However, it is possible that higher concentrations than the maximum observed (6.9 × 106/ml) may contribute to exogenous substrate metabolism in other experiments. WIDER IMPLICATIONS OF THE

findings13C-MRS can provide information on the underlying metabolism of multiple pathways in live sperm. Dysfunction in sperm metabolism, as a result of either impaired enzymes of lack of metabolisable substrate, could be detected in sperm by a non-destructive assay, potentially offering new treatment options to improve overall sperm quality and outcomes for reproduction. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.