We have used Thomas-type recessed-tip pH-sensitive microelectrodes to measure the intracellular pH (pHi) in Xenopus eggs during both fertilization and ionophore activation . The average pH l in unfertilized eggs is 7.33 ± 0.11 (SD ; n = 21) with a resting membrane potential of -10.1 ± 3.5 (SD; n = 38) mV . Within 2 min after the onset of the fertilization potential, there is a slight, transient pH i decrease of 0.03 ± 0.02 (SD, n = 8), followed by a distinct, permanent pHl increase of 0.31 ± 0.11 (SD; n = 7) beginning -10 min after the start of the fertilization potential and becoming complete -1 h later. The pHi remains near this level of 7.67 ± 0.13 (SD, n = 10) through at least 10 cleavage cycles, but it is possible to discern pHl oscillations with a mean amplitude of 0.03 ± 0.02 (SD, n = 38) . Eggs perfused for at least 2 h in Na'-free solution with 1 mM amiloride exhibited all of these pH i changes, so these changes do not require extracellular Na' .Similar cytoplasmic alkalinizations that accompany the activation of metabolism and the cell cycle in a wide variety of cell types are discussed .The activation of development at fertilization results in a dynamic change in cellular activity including an increase in the metabolic rate and the initiation of cell cleavage . Recent investigations have revealed that among the earliest events triggered by sperm-egg fusion are changes in intracellular free Ca" and H+ concentrations. A large, transient increase in the intracellular free Cat`concentration occurs within a minute after fertilization in both invertebrate (43) and vertebrate (15, 34) eggs, and a permanent increase in intracellular pH (pHi) of -0,4 occurs after fertilization in marine invertebrates (23,24,38). This pHi rise requires a small amount of extracellular Na' (24, 39), and has been implicated in the dramatic increase in the rate of protein synthesis that occurs at fertilization in sea urchin eggs (18,(48)(49)(50) . We now report the first direct measurement of the intracellular pH during fertilization in the egg of the frog Xenopus laevis, a freshwater vertebrate . Here we also find a significant pHi rise accompanying the activation of development. In addition, we have detected an initial, small transient pHi fall preceding the larger permanent pHi rise, and a small, cyclical pHi fall associated with cleavage . None of these pHi changes appears to require extracellular Na'. MATERIALS AND METHODSMature eggs were obtained from Xenopus laevis females injected with 800 IU of human chorionic gonadotropin (Sigma Chemical Co., St . Louis, Mo .) . The unfertilized eggs with their jelly coat intact (as required for fertilization) were placed in the dry perfusion chamber to which they adhered. The chamber was then flooded with modified F, solution (21) and perfused either continuously or intermittently. Modified F, solution (referred to as F, throughout) contained (in 562 mM): NaCl, 31.25; KCI, 1.75; CaCl2, 0.25; MgC12, 0.06; Na2HP04, 0.5 ; NaOH, 1 .9 ; NaHCOa, 2.0; Tricine, 10.0 ; pH 7.8 .Intracellu...
31P NMR spectra ofmature eggs ofthe frog (Xenopus laevis) were taken prior to and after both fertilization and activation by a Ca2+/H+ ionophore (A23187). The eggs were constantly perfused with fresh well-buffered solution during the experiments, and the intracellular pH (pH1) was determined from the pH-dependent chemical shift of the internal Pi peak. The detection of this Pi peak in the presence of overlapping yolk phosphoprotein signals was accomplished by a T2 experiment which discriminated against the broader yolk phosphoprotein peak. The average pH; of the unfertilized, fertilized, and activated eggs was 7.42, 7.66, and 7.64, respectively. Thus, a cytoplasmic alkalinization of 0.24 pH unit occurs within 90 min after fertilization. These values are practically identical to pH, measurements made in this laboratory on Xenopus eggs by using pH-sensitive glass microelectrodes. These 31p NMR studies also indicate that extracellular pH changes as large as 3 pH units had no effect on pHi.We also found that phosphocreatine levels are very sensitive to metabolic perturbations such as oxygen depletion or metabolic inhibitor application. These treatments resulted in a rapid decrease in the phosphocreatine concentration; the ATP concentration declined only slowly after the phosphocreatine peak had disappeared.It has long been suspected that changes in the egg's intracellular ion concentrations must be important steps in the activation of development because simple manipulations of the ionic environment are effective in stimulating artificial parthenogenesis (1). Although most ofthe earlier data pointed to the involvement ofintracellular Ca2" (1-4), the most successful parthenogenetic treatments often included NH3 which was known to change intracellular pH (pH5) (1,(5)(6)(7)(8). Subsequent attempts to measure pHi during fertilization by using antimony electrodes (9) or injected indicator dyes (10,11) were inconclusive due to technical problems. Recently, the involvement of pH, changes in activation has been postulated for the sea urchin egg (12-14), and advances in pH-sensitive microelectrodes have allowed reliable direct measurements ofthe pH, increase that is triggered at fertilization (15). Application of this same technique in this laboratory has revealed a similar pHi increase after fertilization in the egg of the freshwater vertebrate Xenopus laevis (16). However, because microelectrode impalement may cause some injury to the eggs, it seemed desirable to confirm this finding with the noninvasive technique of 31P NMR.Here .25-cm Petri dish. Each dish was then scored for rotation before the eggs were placed in the NMR tube. The data presented here were taken from batches exhibiting >80% rotation. These eggs proceeded to cleave normally in the NMR tube throughout our experiments and, when removed, looked identical to control embryos growing in a shallow Petri dish. Ionophore activation was accomplished in a similar manner by adding a small amount ofA23187 (Calbiochem; 1 mg/ml in ethanol) to the Petri dish so...
SUMMARYIonic coupling was found in a!! investigated fibroblastoid cells of 7 permanent cell lines in culture, whereas in 7 epithelioid cell lines no couulina could be detected. These established lines consisted of cells of normal or malignant origin as we!! as cells that were able to, or failed to, produce tumors, but the only relation with ionic coupling appeared to be morphology. The ionic coupling between fibroblastoid cells was unaffected by the presence of fetal calf serum instead of calf serum; culturing in media conditioned by non-coupled cells; variation of the potential difference and phase of the cell cycle. Coupled cells could be depolarized by decreasing the bicarbonate concentration in the media; non-coupled cells were unaffected.
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