In vitro produced bovine zygotes show substantial variation in the time required to complete the first cell cycle and in their in vitro development potential. A
Individual Day-7 embryos (morulae to expanded blastocysts) were incubated with radiolabelled substrates and karyotyped to determine the sex. In Exp. 1, embryos were incubated for 3 h with D-[1-14C]glucose, as a measure of the activity of the pentose-phosphate pathway (PPP) and D-[5-3H]glucose, as a measure of total glucose metabolism. The labelled products 14CO2 and 3H2O were collected throughout the measurement period. Total glucose metabolism in male embryos was twice that in female embryos and increased between the morula and expanded-blastocyst stages. Relative to total glucose metabolism, PPP activity was four times greater in female than in male embryos. In Exp. 2, embryos were cultured with D-[1-14C]glucose, and L-[3,4-3H(N)]glutamine (a measure of Krebs cycle activity) in the presence of brilliant cresyl blue, a stimulator of the PPP. Glutamine metabolism increased from the morula to expanded-blastocyst stages. Relative to the metabolism of glutamine, the activity of the PPP was one-third greater in female than in male embryos.
After maturation in vitro for 0, 6, 12, 18 or 24 h, the metabolism of radiolabelled glucose, glutamine, pyruvate and glycine by individual cattle oocytes was measured for 3 h. The metabolism of glucose through the Embden-Meyerhof (1.77-2.66 pmol per oocyte per 3 h) and pentose-phosphate (0.39-0.75 pmol per oocyte per 3 h) pathways was low and did not change over time. The oxidative metabolism of glucose carbon through the Krebs cycle was low throughout maturation, but increased significantly (P < or = 0.05) at 6 h (0.41 pmol per oocyte per 3 h) and 18 h (0.69 pmol per oocyte per 3 h). Pyruvate, glutamine and glycine metabolism in the Krebs cycle increased during culture. Pyruvate metabolism increased significantly from 0 h (17.3 pmol per oocyte per 3 h) to 6 h (23.3 pmol per oocyte per 3 h) and reached a maximum at 12 h (30.8 pmol per oocyte per 3 h). Glutamine metabolism was unchanged from 0 to 12 h (0.89 pmol per oocyte per 3 h), and then increased significantly at 18 h (2.25 pmol per oocyte per 3 h). Glycine metabolism increased significantly from 6 h (0.21 pmol per oocyte per 3 h) to 12 h (0.46 pmol per oocyte per 3 h) and reached a maximum at 18 h (0.68 pmol per oocyte per 3 h). The results suggest that oxidative metabolism increases, and is the major site of cellular energy production, during maturation of the cattle oocyte in vitro.
The metabolism of, and retention of radioactivity from, radiolabelled glucose, glutamine and pyruvate were measured in individual cattle embryos produced in vitro from the 2-cell to hatched blastocyst stage. Uptake was defined as the numeric sum of metabolism and retention of radiolabel. Glucose metabolism increased significantly between the 8- and 16-cell stages, but was accompanied by a much larger increase in glucose uptake. Consequently, the proportion of glucose uptake that was metabolized through the pentose-phosphate and Embden-Meyerhof pathways reached a minimum at those stages. From the compacted morula stage onward, the calculated uptake of [14C]glucose was only 25 to 33% of that calculated for [5-3H]glucose. This suggests that 66 to 75% of glucose carbon leaves the embryo, after metabolism to phosphoenolpyruvate, in some form other than CO2. Little or no glucose metabolism by the Krebs cycle could be detected at any stage. Both glutamine and pyruvate metabolism were relatively high at the 2- and 4-cell stages, declined to a minimum at the compacted morula stage and then increased with blastulation. Glutamine metabolism continued to increase with expansion and hatching of the blastocyst, but pyruvate metabolism did not. This suggest that, relative to the activity of the pathway from pyruvate to 2-oxoglutarate, the activity of the 2-oxoglutarate-to-oxaloacetate segment of the Krebs cycle is of increasing significance during expansion and hatching of the cattle blastocyst.
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