Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos

Abstract

The consumption of oxygen, uptake of pyruvate and glucose and production of lactate were determined for groups of bovine embryos produced in vitro from the one-cell to the blastocyst stage (day 0\p=n-\6of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medium supplemented with 1.0 mmol pyruvate l \ m=-\ 1, 10 mmol d,l-lactate l \m=-\1 and 1.5 mmol glucose l \m=-\1and also 3 mg BSA ml \m=-\1and, from day 5 of development, 10% (v/v) fetal calf serum. The amount of ATP production was determined from oxygen consumption and the proportion of glucose taken up that could be accounted for by lactate production. The data revealed that oxygen consumption was relatively constant from days 0\p=n-\4of culture (0.24\p=n-\0.27 nl per embryo h \ m=-\ 1) , but increased with the initiation of compaction (0.39 nl per embryo h\m=-\1) and continued to increase with the formation and expansion of the blastocoel (0.9 nl per embryo h \m=-\1). Both pyruvate and glucose uptake followed similar patterns. Furthermore, when plotted against oxygen consumption, both pyruvate and glucose uptake increased significantly (P < 0.001) in a linear relationship (R2 = 0.61 and 0.49, respectively). Lactate production also increased with development and accounted for 40% of glucose uptake at day 0 of culture (putative zygotes), increasing to 70% by day 2 (eight-cell stage) and 100% of glucose uptake from day 4 of culture onwards. ATP production followed a similar pattern to that of oxygen consumption (60\p=n-\85 pmol per embryo h \ m=-\ 1 from day 0 to day 4) increasing with compaction (124 pmol per embryo h \ m=-\ 1) and blastulation (221 pmol per embryo h\m=-\1). For precompaction stages, 93\p=n-\96%of ATP production was derived from oxidative phosphorylation, decreasing to 82% with compaction. ATP produced by oxidative phosphorylation could be accounted for by the uptake of pyruvate, suggesting that bovine embryos produced in vitro utilize little endogenous substrates when appropriate exogenous substrates are present in the culture medium. The data revealed that bovine embryos were dependent on oxidative phosphorylation for energy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with compaction. It follows that oxidizable substrates, such as pyruvate and certain amino acids, are preferred in embryo culture medium during development in vitro.

Actions of thermal stress in two-cell bovine embryos: oxygen metabolism, glutathione and ATP content, and the time-course of development

“…The rate of oxygen consumption in embryos at 38.5 8C was , 2.8 £ 10 213 moles/s. This value is closer to the value of 0.14 £ 10 213 moles/s for bovine morula reported by Shiku et al (2001) than for values reported by Thompson et al (1996) of , 9 £ 10 211 moles/s for bovine embryos from day 0 -4 of culture. Reported values for bovine blastocysts range from 0.4 -1.5 £ 10 29 moles/s , Kaidi et al 2001, Donnay et al 2002.…”

Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation

“…The increase in oxidative activity observed in sperm-activated oocytes corresponds to the initiation of pronuclear formation and first mitotic division in putative zygotes, suggesting increased demands for energy for these events. It has been observed that one-and two-cell bovine embryos are dependent on mitochondrial oxidative phosphorylation for energy supply, consuming oxidative substrates to produce ATP (Kim et al 1993, Thompson et al 1996. Coincidently, a higher oxygen consumption rate was detected before cleavage in bovine zygotes (Lopes et al 2010).…”

Mitochondrial Malate-Aspartate Shuttle Regulates Mouse Embryo Nutrient Consumption

“…Oxygen Consumption-Rates of oxygen consumption by embryos were assessed by an non-invasive pyrene method (10,11). Embryos were incubated in 2 l of media in a 5-l polymerase chain reaction micropipette loaded with 1 l of pyrene (Sigma) dissolved in paraffin oil (BDH) that had been pregassed overnight in a gas phase of 20% oxygen, and each end of the tube was then sealed (10).…”

“…For each experiment a control of 1 mg/ml yeast that had been incubated overnight in 60 mM glucose for zero oxygen and a 20% oxygen control where the PCR tube was loaded with medium and no embryos were included. The levels of oxygen consumption were then calculated by using a program that relates the linear change in pyrene fluorescence to changes in oxygen levels and models the diffusion of oxygen along the length of medium and pyrene (10,11). Oxygen consumption was ex- Enzyme Extraction-For whole embryo extraction, individual embryos were washed in saline and placed in 500 nl of enzyme extraction buffer containing 100 mM K 2 HPO 4 , 30 mM KF, 1 mM EDTA, 5 mM mercaptoethanol, 2 g/liter bovine serum albumin, and 0.5 g/liter phenylmethylsulfonyl fluoride at pH 7.5 (12).…”