Metabolic changes in Japanese medaka (Oryzias latipes) during embryogenesis and hypoxia as determined by in vivo 31P NMR

In vivo 31P nuclear magnetic resonance spectroscopy (NMR) was used to determine phosphometabolite changes in medaka (Oryzias latipes) during embryogenesis and hypoxia. NMR data were acquired using a flow-through NMR tube perfusion system designed to both deliver oxygenated water to embryos and accommodate a hypoxic challenge. Measurements of embryogenesis at 12- and 24-h intervals throughout 8 days of development (n=3 per time point, 900 embryos per replicate) and during acute hypoxia (n=6, 900 embryos at Iwamatsu stage 37 per replicate) were performed via NMR, and replicate samples (n=4, 250 embryos each) were flash frozen for HPLC analysis. The hypoxic challenge experiment consisted of data acquisition with recirculating water (pre-hypoxic control period; 1 h), without recirculating water (hypoxic challenge; 1 h), then again with recirculating water (recovery period; 1.3 h). Concentrations of ATP, phosphocreatine (PCr), orthophosphate (Pi), phosphomonoesters (PME), phosphodiesters (PDE), and intracellular pH (pHi) were determined by NMR, and ATP, ADP, AMP, GTP, GDP, and PCr were also determined via HPLC. During embryogenesis, [ATP] and [PCr] as determined by HPLC increased from 1-day post fertilization (DPF) levels of 0.93±0.08 and 2.48±0.21 μmol/mg (dry tissue), respectively, to 7.24±0.77 and 15.66±1.08 μmol/mg, respectively, by day 8. [ATP] and [PCr] measured by both NMR and HPLC fluctuated over 1-3 DPF, then increased significantly (p<0.05) over 3-8 DPF, while [PME] and [PDE] decreased (p<0.05) throughout embryogenesis. NMR and HPLC measurements revealed 1-3, 4-5, and 6-8 DPF as periods of embryogenesis significantly different from each other (p<0.05), and representing important transitions in metabolism and growth. During hypoxic challenge, [ATP] and [PCr] declined (p<0.05), [PME] and [PDE] decreased slightly, and [Pi] increased (p<0.05). All phosphometabolites returned to pre-hypoxia concentrations during recovery. The pHi decreased (p<0.05) from 7.10±0.03 to 6.94±0.03 as a result of hypoxia, and failed to return to pre-hypoxic levels within the 1.3-h recovery phase. Results demonstrate the utility of in vivo 31P NMR to detect significant alterations in phosphorylated nucleotides and phosphometabolites at specific developmental stages during medaka development and that late-stage medaka utilize PCr to generate ATP under hypoxic conditions.