In this study we used imaging techniques with the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to investigate the control of cytosolic pH (pH(i)) in two-cell mouse embryos in nominally HCO3--free conditions. We found that the resting pH(i) of two-cell embryos (40-50 h after human chorionic gonadotropin) in HCO3--free M2 was 7.31 +/- 0.01 (n = 172 embryos), which is significantly above the level predicted if H+ is at electrochemical equilibrium. We showed that two-cell embryos contain a H+-monocarboxylate cotransport system with apparent Michaelis constants for D-lactate, L-lactate, and pyruvate of 11.5, 3.7, and 3.5 mM, respectively. It is inhibited by p-chloromercuribenzoic acid (300 mu M), p-chloromercuriphenylsulfonic acid (300 mu M), and alpha-cyano-4-hydroxycinnamate (1 mM) and is insensitive to 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid (500 mu M). We also showed that the pH(i) response to the acid load produced by an NH4Cl pulse has two components, one due to H+-monocarboxylate cotransport and the other due to Na+/H+ exchange. We found no evidence that a H+ conductance was responsible in these cells for the recovery in pH(i) after an acid load.
