Intracellular pH of the preimplantation mouse embryo: Effects of extracellular pH and weak acids

Abstract

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.

Measurement of the Intracellular pH in Human Stomach Cells: A Novel Approach To Evaluate the Gastric Acid Secretory Potential of Coffee Beverages

“…When cultivated cells are treated with potassium calibration buffers of varying pH values ranging from 5.5 to 8.4 in the presence of the ionophor nigericin, the intracellular pH (pH i ) and the extracellular pH (pH ex ) equilibrate. This allows the determination of pH i by analyzing the pH-characteristic fluorescence of SNARF-AM (18,25,26). In this study, treatment of HGT-1 cells with potassium calibration buffers adjusted to various pH values between 6.8 and 8.2 accordingly resulted in characteristic changes of SNARF-AM fluorescence.…”

Altering Intracellular pH Disrupts Development and Cellular Organization in Preimplantation Hamster Embryos1

“…The studies presented here showed that the weak acid DMO and the weak base TMA can be used to alter the pH i of hamster 2-cell embryos. Alteration of pH i with weak acids and bases has been used by a number of researchers to determine the role of pH i in a variety of cellular functions [8,16,30,[43][44][45][46]. In the work presented here both TMA and DMO, at 20 mM, inhibited development, with many embryos in both treatments unable to progress be1852 yond the 2-cell stage.…”

Intracellular pH Plays a Critical Role in Glucose-Induced Time-Dependent Potentiation of Insulin Release in Rat Islets

“…We demonstrated a similar pH-lowering effect of HEPES in rat islets by measuring intracellular pH in the presence and absence of HEPES. Intracellular pH measurements were performed in whole islets using the fluorescent dye carboxy SNARF-AM, an agent that has been used successfully to measure intracellular pH in multicellular systems (23)(24)(25)(26). Islets loaded with 5 mol/l SNARF-AM were maintained in KRBH (or other media as indicated), and the intracellular pH was recorded at 37°C during a 20-min period.…”