The production of radiolabelled CO2 from [U-14C]glucose, [1-14C]lactate, and [U-14C]acetate was used to study the oxidative metabolism of embryos recovered from sheep, mice and cattle. Sheep embryos showed an increasing capacity to oxidize glucose after the 4- to 8-cell stage and oxidative turnover of this substrate at the blastocyst stage was four times that at the early stages. Decarboxylation of carbon-1 of lactate followed a pattern similar to that seen for glucose oxidation, but acetate oxidation was low and did not follow the trends with development seen for the other substrates. Furthermore, estimates of incorporation of acetate into the macromolecules of sheep embryos were low compared with similar estimates for glucose and, unlike glucose, did not increase with development. Oxidation of all three substrates by mouse embryos increased with development but the rate of CO2 production from acetate was low compared with that from the other substrates. A combination of lactate or glucose with acetate had no influence on the utilization of acetate by mouse morulae/early blastocysts, nor did acetate influence utilization of the other substrates. Cattle morulae/early blastocysts also produced more CO2 from glucose and lactate than from acetate and the incorporation of carbon from acetate into the macromolecules of these embryos was less than from glucose. Overall, cattle embryos showed the greatest ability to metabolize acetate but were not as effective in using the other substrates as either sheep or mouse embryos at the same stage.
The production of carbon dioxide and lactate from glucose by sheep embryos and samples of extraembryonic membranes was measured during a 2.5 h incubation period. Both embryos and their membranes were active in the glycolytic and oxidative utilization of glucose and, in general, the utilization of glucose per unit weight fell as development progressed from Day 13 to Day 19 of pregnancy. Both oxidation of glucose and glycolysis by the extraembryonic tissues, expressed as activity per microgram dried tissue, fell progressively with development. The rate of decline in CO2 production was greater than the rate for glycolysis and, as a consequence, the contribution of glycolysis to the estimated energy yield from the catabolism of glucose rose with time. In the embryo, both glucose oxidation and glycolysis peaked on Day 15 with estimates of adenosine triphosphate (ATP) production from glucose per microgram dried tissue on this day being 50% above those on Day 13 and 100% above those on Day 17. In general, the estimated yields of ATP from glucose were similar for structures of the same developmental age except that, at Day 19, it was calculated that the rate of ATP production by embryos was double that by the extraembryonic membranes. In incubations using 5.56 mM glucose as sole exogenous energy source, glucose turnover by embryos and embryonic membranes tended to be higher in a bicarbonate-buffered medium than in HEPES (4-(2-hydroxyethyl)-1-piperazincethane sulfonic acid) and phosphate-buffered media. As a result, the estimate of ATP yield plus the contribution of oxidative pathways to this yield were significantly higher in this medium than in the others. Glucose turnover by the embryo and its membranes in bicarbonate-buffered medium containing 0.56 mM glucose plus the alternate substrates, lactate and pyruvate, was severely depressed. Further experiments using samples of trophoblast and yolk sac indicated that both reduction in glucose concentration and the presence of the other substrates contributed to this suppression. Furthermore, an interaction between these factors was evident with the effects of alternative substrates being exaggerated when glucose concentration was low.
Incorporation of glucose into the internal biochemical pools of the sheep embryo and samples of extraembryonic membranes was measured during a 2.5 h incubation in the presence of radiolabelled glucose. Very little glucose was incorporated into the glycogen pools by either the embryo or its membranes and never represented more than 5% of total incorporation. Approximately 65% of label was isolated in the non-glycogen acid-soluble fraction of samples and the remainder was incorporated into non-glycogen macromolecules. The embryonic disc of the day-13 conceptus had the highest rate of incorporation per mg dried weight of any structure studied. Synthesis of non-glycogen macromolecules by the day-13 disc was five to six times that of either day-15 or day-17 embryos. On day 19 very low rates of incorporation into the isolated embryo were found during culture. Evidence suggests that this was a result of limitations on the diffusion of substrate into the embryo because incubation of fragmented embryos produced rates similar to those found on days 15 and 17. Incorporation of glucose into the intracellular pools of extraembryonic membranes per mg dried weight remained relatively low and stable over the period studied and there were only minor differences in the rate of incorporation between membranes. Incorporation of glucose by embryos and extraembryonic membranes was equally as good in phosphate-buffered media as in bicarbonate-buffered solutions and was unaffected by changes in the concentration of lactate and pyruvate in the culture medium.
Acetate metabolism by the sheep conceptus was assessed by measuring CO2 production during a 2.5-h incubation of embryos and samples of the extraembryonic membranes in HEPES-buffered media containing 1.12 mM [U-14C]acetate. The rate of oxidation of acetate by embryonic tissue showed little change between Days 13 and 15 of pregnancy but greatly decreased by Days 17 and 19. By contrast, oxidation of the substrate by the trophoblast increased substantially with development and was five times the early rate by Day 19. Oxidation of acetate by the yolk sac also increased 4-fold between Days 17 and 19. The addition of glucose to incubations of extraembryonic membranes resulted in some reduction in the oxidation of acetate by the yolk sac and allantois but had little effect on the trophoblast. At Days 13 and 15, the rate of oxidation of acetate by the embryonic disc was 6-7 times that by the trophoblast. As development progressed, this situation was reversed and by Day 19 the trophoblast metabolized more than five times the amount of acetate per microgram than did the Day-19 embryo. Although acetate metabolism by yolk sac and allantois on Day 17 was low, its metabolism by the yolk sac increased to values similar to those for the trophoblast at Day 19 but its utilization by the allantoic membrane remained low. Comparison of the estimates of ATP generated from acetate by these tissue with those published for glucose demonstrates that acetate is much less effective than glucose for the provision of metabolic energy.
Of the substrate carbon incorporated from 1.12 mM [U-14C]acetate as sole energy substrate during a 2.5-h incubation, a large proportion was isolated in the acid-soluble fraction. Although there was no significant change over time in the rate of entry into this pool for embryos, the rate of accumulation by the trophoblast and yolk sac increased as development progressed. At Days 13 and 15 of pregnancy, incorporation of acetate into the acid-insoluble fraction of embryos accounted for almost half the total label accumulated. The rate of this incorporation fell rapidly over time and by Day 19 less than 30% of carbon accumulated was in this fraction. By contrast, the rate of incorporation into this fraction by trophoblastic tissue was low at Day 13 but rose dramatically as development progressed. Incorporation by the yolk sac into acid-insoluble components also rose with time. At the early stages of pregnancy studied, lipid synthesis accounted for the majority of acetate carbon accumulated by the conceptus in the acid-insoluble pool. At later stages of development, incorporation into lipids constituted a minor pool of acetate carbon. Some acetate carbon was found in the glycogen fraction of the conceptus. The rate of incorporation into the acid-soluble glycogen fraction by embryos was constant throughout the period studied. By contrast, trophoblast increased its rate of incorporation markedly into both acid-soluble and acid-insoluble glycogen pools as did the yolk sac from Day 17.(ABSTRACT TRUNCATED AT 250 WORDS)
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