Energy metabolism of the early mouse embryo

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Effect of inhibiting nitric oxide production on mouse preimplantation embryo development and metabolism

Nitric oxide (NO) is a free radical that functions as a cell signaling molecule but at high concentrations can be toxic. It is formed from arginine, which is consumed by the mouse blastocyst, but its effect on early embryo development has been little studied. In this study, the role of NO in mouse preimplantation development has been examined in terms of developmental rate and oxidative metabolism. Zygotes were cultured in one of four media; potassium simplex optimization medium (KSOM), KSOM with amino acids (KSOMaa), KSOM without glutamine (KSOM-glut), or KSOM with 0.5 mM arginine (KSOMarg) ± L-NAME (a specific inhibitor of NO production). End points were Day 4 blastocyst rates, cell counts determined using bisbenzimide and oxygen consumption. In KSOM and KSOM-glut, the blastocyst rate was decreased by 1 mM L-NAME from 50.2% ± 3.1% and 37.4% ± 4.5% to 6% ± 3% and 0%, respectively. In KSOMaa, cavitation rates were unaltered but the blastocysts contained fewer cells (P < 0.001). Blastocysts cultured in KSOM and KSOM-glut consumed significantly more oxygen than those cultured in KSOMaa (P < 0.001 and P < 0.05, respectively). However, the addition of 0.1 mM or 1 mM L-NAME to KSOMaa significantly increased the amount of oxygen consumed (P < 0.05 and P < 0.001, respectively). The data suggest a physiological role for NO in mouse preimplantation metabolism and development. One possibility is that NO may limit oxygen consumption at the blastocyst stage at the level of mitochondrial cytochrome c oxidase

Oxygen consumption and energy metabolism of the early mouse embryo

Oxygen consumption of preimplantation and early postimplantation mouse embryos has been measured using a novel noninvasive ultramicrofluorescence technique, based on an oil-soluble, nontoxic quaternary benzoid compound pyrene, whose fluorescence is quenched in the presence of oxygen. Pyruvate and glucose consumption, lactate production, and glycogen formation from glucose were also measured. Preimplantation mouse embryos of the strain CBA/Ca × C57BL/6 were cultured in groups of 10–30 in 2 μl of modified M2 medium containing 1 mmol l−1 glucose, 0 mmol l−1 lactate, and 0.33 mmol l−1 pyruvate, for between 4–6 hr. Day 6.5 and 7.5 embryos were cultured singly in 40 μl M2 medium for between 2–3 hr. Oxygen consumption was detected at all stages of development, including, for the first time, in the early postimplantation embryo. Consumption remained relatively constant from zygote to morula stages before increasing in the blastocyst and day 6.5–7.5 stages. When expressed as QO2 (μl/mg dry weight/hr), oxygen consumption was relatively constant from the one-cell to morula stages before increasing sharply at the blastocyst stage and declining to preblastocyst levels on days 6.5 and 7.5. Pyruvate was consumed during preimplantation stages, with glucose uptake undetectable until the blastocyst stage. Glucose was the main substrate consumed by the 6.5 and 7.5 day embryo. The proportions of glucose accounted for by lactate appearance were 81%, 86%, and 119% at blastocyst, day 6.5, and day 7.5 stages, respectively. The equivalent figures for glucose incorporated into glycogen were 10.36%, 0.21%, and 0.19%, respectively. The data are consistent with a switch from a metabolism dependent on aerobic respiration during early preimplantation stages to one dependent on both oxidative phosphorylation and aerobic glycolysis at the blastocyst stage, a pattern which is maintained on days 6.5 and 7.5. Our technique for measuring oxygen consumption may have diagnostic potential for selecting viable embryos for transfer following assisted conception techniques in man and domestic animals

Metabolism of the early embryo: energy production and utilization

The vast majority of studies on early mammalian embryo metabolism have been concerned with the generation, rather than the fate, of ATP. Thus, research has focused on the uptake or metabolism of energy sources added to embryo culture media. There have been an even greater number of studies in which metabolism as such has not been measured, but rather where blastocyst formation has been used as an endpoint to assess the effect of different energy sourees. It is generally agreed that the early phases of mammalian preimplantation development are relatively quiescent metabolically, relying on substrates such as pyruvate, lactate, or amino acids, which are metabolized aerobically (1–5)

The delivery of PEBBLE nanosensors to measure the intracellular environment

Cellular introduction of PEBBLEs (photonic explorers for bioanalysis with biologically localized embedding) has been investigated by a wide variety of methods in a range of cell types. These methods include surface functionalization with CPPs (cell-penetrating peptides), pinocytosis, commercial lipid transfection agents, cytochalasin D, picoinjection, and Gene gun bombardment. This paper will overview several of the most popular methods used for the introduction of PEBBLE nanosensors to the cellular environment and discuss the efficacy of the techniques

Substrate utilization and maturation of cumulus cell-enclosed mouse oocytes: evidence that pyruvate oxidation does not mediate meiotic induction

This study was performed to address the possible role of pyruvate in meiotic induction in mouse oocytes. Cumulus cell-enclosed oocytes from primed, immature mice were cultured in 7.5 μl microdrops under oil for 9 or 18 h in medium containing 4 mmol hypoxanthine l−1 plus 0.23 mmol pyruvate l−1, l mmol pyruvate l−1, or 1 mmol pyruvate l−1 plus 5.5 mmol glucose l−1. When compared with cultures containing 0.23 mmol pyruvate l−1, 1 mmol pyruvate l−1 induced germinal vesicle breakdown, and this was preceded by an increase in pyruvate utilization. Addition of glucose prevented both the increase in pyruvate consumption and the meiotic induction. When different combinations of pyruvate and glucose were tested on oocyte maturation in microdrop cultures, a high concentration of pyruvate or glucose alone was stimulatory to maturation. Addition of the complementary energy substrate prevented the induction of germinal vesicle breakdown and reduced the amount of substrate consumption. During spontaneous maturation in vitro, oocyte–cumulus cell complexes consumed glucose for the first 3 h; however, during the second 3 h period, which followed germinal vesicle breakdown, glucose consumption decreased and net pyruvate utilization was initiated. Treatment of hypoxanthine-arrested oocytes with dichloroacetate, an activator of pyruvate dehydrogenase, stimulated pyruvate consumption but had no effect on germinal vesicle breakdown. Although FSH stimulates meiotic resumption, no changes in pyruvate consumption were observed in response to this gonadotrophin. Measurement of oxygen consumption by hypoxanthine-treated complexes revealed no effect of high concentrations of pyruvate on respiration, and FSH treatment resulted in a suppression of oxygen utilization. These data indicate that, in mouse oocyte–cumulus cell complexes, pyruvate and glucose can each modulate metabolism of the other substrate, and this can significantly influence meiotic maturation of the oocyte. In addition, augmentation of pyruvate oxidation does not appear to play a mediating role in meiotic induction triggered by energy substrate manipulation or gonadotrophin treatment

Two tales of one city: data, inference and Carthaginian infant sacrifice

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Expression and activity of hexokinase in the early mouse embryo

The maximal activity and Michaelis constant, KM, of hexokinase have been measured in the peri-implantation mouse embryo using an ultramicrofluorescence technique. In addition, transcript detection of the predominant isoenzyme hexokinase I has been determined in single preimplantation mouse embryos at successive stages of development using reverse transcriptase-mediated cDNA amplification. Maximal hexokinase activity decreased dramatically peri-implantation, from 0.97 ± 0.19 nmol/μg protein/h at the blastocyst stage to 0.31 ± 0.05 nmol/μg protein/h on day 6.5. The KM remained relatively low and constant over this period (0.23–0.39 mM), indicating the absence of the hexokinase type IV isoenzyme. The pattern of hexokinase activity resembled that of glucose consumption suggesting a possible regulatory role for the enzyme during this period of development. Hexokinase I mRNA was detected in the oocyte and all preimplantation stages of development. The blastocyst polymerase chain reaction (PCR) product, when cloned and sequenced was found to be 98% homologous with mouse tumour hexokinase I. Taken together, these data suggest that the hexokinase gene is not under transcriptional control during early mouse embryo development but plays a significant role in the regulation of glucose consumption. A role for hexokinase in the phosphate-induced inhibition of early embryo development is also proposed

Oxygen uptake and carbohydrate metabolism by in vitro derived bovine embryos

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 0–6 of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medium supplemented with 1.0 mmol pyruvate l−1, 10 mmol d,l-lactate l−1 and 1.5 mmol glucose l−1 and also 3 mg BSA ml−1 and, 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 0–4 of culture (0.24–0.27 nl per embryo h−1), but increased with the initiation of compaction (0.39 nl per embryo h−1) and continued to increase with the formation and expansion of the blastocoel (0.9 nl per embryo h−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 (60–85 pmol per embryo h−1 from day 0 to day 4) increasing with compaction (124 pmol per embryo h−1) and blastulation (221 pmol per embryo h−1). For precompaction stages, 93–96% 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

Oxygen consumption by day 7 bovine blastocysts: determination of ATP production

Using a fluorescence technique which measures changes in oxygen concentration in embryo incubation medium, we have measured the uptake of oxygen by small groups of Day 7 bovine embryos recovered from superovulated and inseminated heifers (n = 8). The amount of glucose consumed by the embryos was also determined by analysing the spent medium following the oxygen assay. Oxygen consumption was successfully recorded from 17 out of 22 attempted assays and measured at 0.66 ± 0.08 nl embryo−1 h−1. There was a significant difference in oxygen consumption between blastocyst stage (0.84 ± 0.09) and expanded blastocyst stage (0.51 ± 0.09) embryos (P < 0.05). Mean ±(SEM) glucose uptake was 14.7 ± 1.9 pmol e−1 h−1 for all blastocysts; there was no significant difference in glucose consumption between blastocyst and expanded blastocyst stage embryos. From these values and assuming virtually all the glucose is metabolised via the Embden-Meyerhoff pathway, the rate of ATP production can be calculated at 205 ± 23 pmol e−1 h−1 (range 45–390 pmol e−1 h−1), of which approximately 85% is derived from oxidative phosphorylation. This rate represents a rapid turnover of ATP and demonstrates that metabolism in Day 7 bovine embryos is comparable to that of the more active tissues of the body