Adenosine triphosphate content of preimplantation mouse embryos.

The utilization of energy substrates by preimplantation mouse embryos undergoes several changes during development in vitro (Brinster, 1965a, b) but very little is known about the level of high-energy containing compounds, such as ATP at these stages. In the present study, the content ofATP in freshly collected preimplantation mouse embryos has been measured. For each estimation, fifty to 150 embryos were collected by flushing the reproductive tracts of superovulated female albino mice with modified KrebsRinger bicarbonate medium (Brinster, 1965b). Unfertilized and fertilized ova were removed from the Fallopian tubes 20 to 24 hr after the injection of hcg and the surrounding cumulus cells were removed by incubation in hyaluronidase solution (Brinster, 1965c). Later developmental stages of the embryos were flushed from the Fallopian tubes and uterus at specific times after the injection of hcg. After washing by transfer through 2 ml of fresh medium, the embryos were extracted with 100 μ of cold 2-5% perchloric acid for 2 hr and the perchlorate removed by precipitation as the potassium salt. In a preliminary experiment, embryos were lysed by freezing and thawing in water but this treatment gave inadequate extraction of the ATP. Assay of ATP was carried out on aliquots of the neutralized perchloric acid extract of the embryos by the procedure of Stanley & Williams (1969) using a Packard Model 3002 Tri-Carb liquid scintillation spectrometer run at 5° Cwith the detector gate switched out of coincidence. A series of reaction mixtures containing ATP standards or embryo extract were prepared and allowed to equilibrate at room temperature for 20 min. Ten seconds after adding a suitable aliquot of firefly luciferase extract, the reaction vessel was lowered into the counting well and the counts accumulating in the first 6 sec of counting re¬ corded. An aliquot of neutralized perchlorate, equivalent to that of the embryo extracts, was included in the ATP standards. Internal standards of ATP were also added to the embryo extracts to correct for quenching. The levels of ATP in mouse embryos at various stages of preimplantation development are given in Table 1, together with the summary of the analyses of variance. There was a significant (P<0-01) linear decrease in ATP with time over the range of stages studied but all other comparisons between stages were not significant. The gradual decline in the amount of ATP present in the mouse embryo during preimplantation development is similar to that recorded in the