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

STUDY QUESTION: Can 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, METHODS: Sperm 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 FINDINGS: 13C-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.

1H Magnetic Resonance Spectroscopy of live human sperm.

STUDY QUESTION: Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? SUMMARY ANSWER: Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface. WHAT IS KNOWN ALREADY: Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80%' sperm. MAIN RESULTS AND THE ROLE OF CHANCE: DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87. LIMITATIONS, REASONS FOR CAUTION: Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm. WIDER IMPLICATIONS OF THE FINDINGS: 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.

Studies of the dynamics of nuclear clustering in human syncytiotrophoblast.

Syncytial nuclear aggregates (SNAs), clusters of nuclei in the syncytiotrophoblast of the human placenta, are increased as gestation advances and in pregnancy pathologies. The origins of increased SNAs are unclear; however, a better appreciation of the mechanism may give insight into placental ageing and factors underpinning dysfunction. We developed three models to investigate whether SNA formation results from a dynamic process of nuclear movement and to generate alternative hypotheses. SNA count and size were measured in placental explants cultured over 16 days and particles released into culture medium were quantified. Primary trophoblasts were cultured for 6 days. Explants and trophoblasts were cultured with and without cytoskeletal inhibitors. An in silico model was developed to examine the effects of modulating nuclear behaviour on clustering. In explants, neither median SNA number (108 SNA/mm(2) villous area) nor size (283 µm(2)) changed over time. Subcellular particles from conditioned culture medium showed a wide range of sizes that overlapped with those of SNAs. Nuclei in primary trophoblasts did not change position relative to other nuclei; apparent movement was associated with positional changes of the syncytial cell membrane. In both models, SNAs and nuclear clusters were stable despite pharmacological disruption of cytoskeletal activity. In silico, increased nuclear movement, adhesiveness and sites of cytotrophoblast fusion were related to nuclear clustering. The prominence of SNAs in pregnancy disorders may not result from an active process involving cytoskeleton-mediated rearrangement of syncytial nuclei. Further insights into the mechanism(s) of SNA formation will aid understanding of their increased presence in pregnancy pathologies.

Analysis of syncytial nuclear aggregates in preeclampsia shows increased sectioning artefacts and decreased inter-villous bridges compared to healthy placentas.

Syncytial nuclear aggregates (SNAs) are increased in pregnancy complications and include 'true' syncytial knots and inter-villous bridges. Apparent nuclear overlay caused by sectioning artefacts are frequently counted from single sections. Haematoxylin and eosin stained serial sections were assessed for frequency of SNA subtypes in placentas from normal, preeclamptic and fetal growth restricted (FGR) pregnancies. There were more sectioning artefacts and syncytial knots and fewer bridges in samples from preeclampsia compared to controls. There were no significant differences between FGR and control samples. This suggests the villous tree in preeclampsia has less inherent structural support and trophoblast cell dynamics are different.

Oxygen transport by rabbit embryonic blood: high cooperativity of hemoglobin-oxygen binding.

Oxygen equilibrium curves (OECs) have been determined in blood of embryonic and adult rabbits (Oryctolagus cuniculus) using a thin film method (modified Hemoscan). The gestational age of the rabbits was from 12 days to 14 days, the end of the embryonic period. Measurements were made at 37 degrees C and a PCO2 of 21, 42, or 71 mmHg. The most striking finding was an embryonic OEC which was steep above 50% saturation. The Hill plot in this upper region gave a mean nH value of 5.3 in the embryos, the first finding of nH significantly greater than 4 in any normal eutherian mammal. On hemolysis, nH dropped below 4. Oxygen affinity of 14 day embryonic blood was higher than that of adult blood: respective P50 values at PCO2 = 42 mmHg were 29.6 mmHg and 32.5 mmHg. The P50s were somewhat lower at earlier stages. The Bohr effect was measured (as delta log P50/delta log PCO2) in 14 day embryos. Its value was 0.26, about 10% lower than in adults. The results show an apparent aggregation of Hb tetramers, as found in marsupials, but no right shifting of the embryonic OEC compared to the adult OEC.

Blood O2 transport in newborn and adult of a very small marsupial (Sminthopsis crassicaudata).

Blood O2 transport and hemoglobin types have been studied in a Dasyurid marsupial (Sminthopsis crassicaudata) at the neonatal (10-20 mg) and adult (16 g) stages, and in part of the transition period. In neonates the blood was embryonic in type with erythrocytes nucleated and containing two Hb types both different from adult Hb. The oxygen equilibrium curves (OECs) at day 1 had a P50 of 38 mmHg at 36 degrees C and PCO2 = 43 mmHg. This is lower than in other neonatal marsupials, but higher than in fetal or neonatal eutherian mammals. Adult P50 under the same conditions were higher (59 mmHg), the normal relationship in viviparous animals. Hill plots of neonatal OECs showed a sharp upward bend at about 50% saturation. As in other embryonic and neonatal marsupials, in the upper part of the plot nH was greater than 4. This indicates aggregation of Hb tetramers. The Bohr effect of neonatal blood at higher PCO2 values (43-71 mmHg PCO2) was zero. The special features of neonatal blood had largely disappeared by day 6.

High cooperativity of hemoglobin-oxygen binding in embryonic rabbit blood.

In rabbit embryos, 13 or 14 days after conception, the oxygen equilibrium curve climbs steeply above about 50% saturation. The value of the Hill coefficient (nH) in the upper part of the curve is significantly greater than 4, indicating aggregation of tetramers or interaction between adjacent tetramers. This is the first time this has been described in blood of a normal eutherian (placental) mammal.

Blood O2 transport and Hb types in the embryonic Tammar Wallaby (Marsupialia, Macropus eugenii).

Blood oxygen transport and hemoglobin type have been studied in the developing Tammar Wallaby from 20 days gestation, just after the circulation first forms, until 28 days gestation, 2 days after birth. The oxygen equilibrium curves (OECs) of the embryonic whole blood had high P50 values, mean being 44.6 mmHg at 36 degrees C, PCO2 = 40 mmHg. In contrast to other viviparous species studied, the embryonic OECs were well to the right of the maternal OEC. There was no significant change in P50 throughout the age range studied. The curves had high Hill coefficients above about 50% saturation (mean = 5.65), indicating cooperativity between hemoglobin tetramers. The Bohr effect (measured as delta log P50/delta log PCO2) was low, about half that of the adult. The early embryos (days 20 and 21) had only 2 or 3 Hb types, with the other embryonic Hbs appearing by 22 days of gestation. The proportion of the four embryonic Hbs changed during development although one type was always predominant (> 45%). This is the first serial study of blood O2 carriage and Hb type in developing marsupial embryos. The finding of a right-shifted embryonic OEC throughout intrauterine development suggests that, contrary to current belief, a right-shifted curve may be physiologically advantageous to a developing embryo.