THE STUDY OF SPERM ENERGY METABOLISM MEASURED USING 13C MAGNETIC RESONANCE SPECTROSCOPY

Studying energy metabolism in sperm may be helpful in understanding the relationship between poor sperm motility and infertility. Sperm motility is fuelled by Adenosine triphosphate (ATP). Main ATP related production pathways are anaerobic glycolysis and oxidative phosphorylation (OXPHOS). These pathways yield different metabolic products. By using a labelled substrate, 13C, metabolic pathways for ATP production can be tracked by Magnetic Resonance Spectroscopy (MRS). In a previous 13C MRS sperm study, only lactate and bicarbonate were detected. Lactate may represent anaerobic glycolysis but the origin of bicarbonate is unclear as it could be from OXPHOS associated with pyruvate dehydrogenase, pyruvate carboxylase, or the Krebs cycle. Thus, the main aim of this study was attempt to detect Krebs cycle intermediates using 13C MRS. Several variables were investigated including Krebs cycle inhibitors, incubation temperature, U-13C glucose and 2-13C pyruvate labelled substrates, hyperpolarised 2-13C pyruvate and manipulation of oxygen availability. Inhibitor, succinate, significantly increased sperm vitality and decreased the MRS lactate integral from U-13C glucose after 18 hours incubation. It suggested that inhibition did occur but the Krebs cycle intermediate concentration was below the detection level of 13C MRS. By incubating sperm with 2-13C pyruvate at 37oC and 39oC, lactate and bicarbonate were consistently detected and their concentrations were significantly higher than those derived from U-13C glucose. Yet, no Krebs cycle intermediates were detected. However, disappearance of the bicarbonate signal from 2-13C pyruvate metabolism by succinate suggests that bicarbonate was indeed produced in the Krebs cycle. Even though dissolution- DNP could overcome the sensitivity issue faced by MRS, presently it has limitations in detecting metabolites of interest in boar sperm due to the short half-life of the hyperpolarisation substrate. Consequently, the origin of the bicarbonate cannot yet be confirmed. The effect of oxygen on sperm metabolism showed that the energy producing pathway for boar sperm was dominated by anaerobic glycolysis rather than mitochondrial metabolism regardless of the oxygen concentration in the environment that they were incubated. Hence Krebs cycle intermediates were not observed even in an oxygen rich atmosphere