The inclusion of specific amino acids in conventional culture media has been shown to enhance mammalian embryo development in vitro. Amino acids have been shown to confer their benefits to the preimplantation embryo in a number of different ways. However, their ability to buffer intracellular pH (pHi) has not been investigated. Thus, the aim of this study was to determine if amino acids regulate pHi in the mouse preimplantation embryo. pHi was determined using carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. Incubation with 5,5-dimethyl-2,4-oxazol-idinedione (DMO), a non-metabolizable weak acid, resulted in a significant intracellular acidification in the zygote, 2-, 4- and 8-16-cell embryo. However, in the presence of groups of amino acids, the degree of acidification due to DMO was markedly reduced in the mouse embryo up to the 4-cell stage. Specifically, non-essential amino acids and glutamine had the greatest capacity to buffer pHi in the early embryo. The ability of amino acids to buffer pHi was not apparent from the 8-16-cell stage onwards. In contrast to the precompacted embryo, the morula did not undergo a significant decrease in pHi until exposed to DMO concentrations > or = 10 mM in the absence of amino acids. This may be due to the generation of a permeability seal during compaction, thus enabling the morula to regulate its own pHi. This regulatory ability could either be reversed by causing the morula to decompact, or created by inducing premature compaction in the 8-16-cell embryo. Data presented in this study indicate that amino acids act as buffers of pHi in the early embryo and play a key role in regulating cell physiology. Further evidence for this was provided by the result that only those embryos cultured in 30 mM DMO in the presence of non-essential amino acids and 1 mM glutamine did not block at the 2-cell stage, but grew on to develop into expanded blastocysts.

Although intracellular pH (pHi), is a regulator of numerous biological processes, it has received relatively little attention with regard to the physiology of the mammalian preimplantation embryo. Interestingly, there is some controversy as to whether the early embryo can recover from an acid load. The significance of this is that two constituents of mouse embryo culture media are pyruvate and lactate. These carboxylic acids are utilised by the early mouse embryo for energy production. However, as weak acids, pyruvate and lactate may induce perturbations in the pHi and thus alter the physiology of the embryo. The aims of this study were therefore to measure the pHi of the mouse preimplantation embryo and to determine the effect of lactate on pHi at different developmental stages. The pHi was measured using the ratio-metric fluorophore carboxy-seminaphthorhodafluor-1-acetoxymethylester (SNARF-1) in conjunction with confocal microscopy. The pHi increased significantly with development from the zygote to the morula stage. Furthermore, at concentrations greater than 5 mM, lactate caused the pHi of the zygote to become significantly more acidic. It was demonstrated that facilitative transport in association with a smaller passive component was responsible for the movement of lactate into the zygote. Metabolic studies revealed that, through their acidifying effect, weak acids caused a reduction in glycolytic activity in the early embryo. In contrast, the pHi of the compacted embryo remained unchanged by the presence of lactate in the external media. Furthermore, incubation with weak acids did not affect the rate of glycolysis in the morula. These data suggest that, by the generation of a transporting epithelium at compaction, the embryo develops the ability to regulate pHi against an acid load.

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