Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos

Methods are described for preparing and analyzing single preimplantation mouse embryos fora variety of metabolites and cofactors (glucose-6-P, fructose-6-P, fructose-1, 6-bisphosphate, ATP, AMP, P i , citrate, isocitrate, α-ketoglutarate, and malate). Oil-well and enzymatic cycling techniques are combined to provide the sensitivity needed to measure the amounts present (10 −12 to 10 −15 moles). After experimental treatment, embryos are collected on glass slides and freeze-dried. They can then be stored indefinitely under vacuum at −25 °C without deterioration. With these procedures, the embryos were collected at successive stages of development and subjected to starvation and refeeding with glucose, pyruvate or both. The results confirm the existence of a block at early stages at the P-fructokinase step. This may be due to inhibition by the very high citrate levels present. The data suggest that glycolysis is turned on late in preimplantation development by the rise in fructose-6-P, a deinhibitor of P-fructokinase. In the citrate cycle, no step between citrate and α-ketoglutarate is rate-limiting, but a step between α-ketoglutarate and malate appears to impede the flux at early embryonic stages.