Intracellular ionic compartmentation, electrical membrane properties, and cell membrane permeability before and during first cleavage in the Ambystoma egg

Abstract

SUMMARYThe intracellular ionic distribution in uncleaved and cleaving Ambystoma eggs was investigated by analysing the influx of 3H,0, by determining the total content of Na+, K+ and Cl-in extracts of eggs at different stages by both flame spectrophotometry and ion-selective microelectrodes, and by the continuous measurement of the Na+, K+ and Cl-activities (uh, uk and a&) using intracellular ion-selective microelectrodes. The electrical membrane potential (E,) and membrane resistance (R,) were measured continuously in uncleaved and normally cleaving eggs as well as in eggs cleaving after removal of the vitelline membrane. The latter eggs expose their newly formed cleavage membrane to the external medium. Ionic permeability of the cell membrane before and during cleavage was analysed by a statistical comparison of the experimentally determined relationship between E,,, and the ionic gradients across the cell membrane with those predicted theoretically from a constant field equation in dependence on the relative permeability, through insertion of the measured intracellular ion activities.3H,0 influx revealed the existence of a single intracellular water compartment (3.06 @l/egg) and a low water permeability (5.35 x 10M5 cm set-I). Na +, K+ and Cl-concentrations were constant at 54.1, 72.1 and 73.1 mM respectively, while nk,, uk and u&r were constant at 5.8, 51.8 and 59.7 mM respectively. It was concluded that all Cl-ions are in solution, while 12.5 % of all K+ and 86 % of all Na+ is bound. The uncleaved egg showed a positive E,,, of ca 40 mV and a specific membrane resistance of 39 kOhm cm'. E,,, could be described by a constant field equation with a permeability ratio P K /P N,==O.O73. Shortly after the onset of first cleavage, E,,, rapidly decreased concomitant with a rise in R, (68.5 kOhm cm2). This was interpreted as a drop in Nat permeability. During the cleavage process E, progressively hyperpolarized and R, decreased due to the insertion of a small fraction (3.3 %) of the newly formed intercellular membrane into the cleavage furrow. This new membrane had a low specific resistance (0.69 kOhm cm"). Both in normally cleaving eggs and in eggs cleaving in the absence of the vitelline membrane E,,, behaved according to the constant field equation, PNa/PK being 0.69 and 0.39, respectively. The differences with other amphibian eggs were discussed.In a series of previous reports [IA] we have small electrical membrane potential-E,-presented a detailed description of the changes (-10 mV) and a high specific membrane rein electrical membrane properties, cell mem-sistance (74 kOhm cm2), while any permbrane permeability, and intracellular distriselectivity was absent. Starting at 6-8 min bution of Na+, Kf and Cl-ions during the after the onset of first cleavage, E, hyperfirst cleavage of eggs of the amphibian polarized from -10 to -20 mV and the Xenopus laevis. The uncleaved egg showed a electrical membrane resistance-R,,-de- creased from 850 to 500 kOhm. The intracellular Na+, K+ and Cl-concentrations 59, 87, 63 mM/l ...

Neutral carrier sodium ion-selective microelectrode for intracellular studies

“…In contrast to the monensin based electrode, where the detection limit is slightly above the physiological Na+ activity (10~2 M, vertical bar in Figure 3), the neutral carrier electrode does make intracellular Na+ activities accessible. This is all the more true if the intracellular K+ concentration becomes smaller, as is observed in several living cells (19)(20)(21).…”

Chapter 5 An Old Enigma: the Gray Crescent of Amphibian Eggs

“…Tbe gênerai conclusion is that the newly formed membrane bas différent pbysiological properties from the "old" one; it probably arises from tbe large reserve of membrane material that is présent in tbe cytoplasm. In particular, tbe permeability to K* ions of tbe new membrane that forms tbe furrow is much bigher than that of tbe preexisting membrane (de Laat et al, 1975); tbe new membrane contains fewer intramembrane particles than tbe old one (Bluemink et al, 1976).…”

“…L0vtrup (1962) bas suggested that the permeability to water and even to oxygen might be higher in the gray crescent than in other parts of the fertilized egg. This remains, for the time being, purely hypothetical and new investigations of the question, with the élégant microelectrode techniques applied to amphibian eggs by de Laat et al (1974de Laat et al ( , 1975, would be extremely welcome: they should show whether permeability to water and ions is indeed higher in the gray crescent area than in the ventral marginal zone. L0vtrup and Pigon (1958) and more recently Landstrom and L0vtrup (1975) have tested the idea that dorsoventrai polarity might be linked to différences in permeability to oxygen; the basic principle of the two sets of experiments is the same: to place an amphibian egg, at the 2-or 4-cell stage, in a tightly fitting tube, in order to decrease the oxygen content of the surrounding water by the respiration of the egg itself.…”

Hydrocolloid Coating of Xenopus laevis Embryos

“…Mineral concentration within the embryo (stage 2) before coating was 73.1 ( 3.6, 72.5 ( 3.6, and 12.7 ( 0.6 mM for Na, K, and Ca, respectively (Table 2). De Laat et al (1975) found similar Na and K concentrations (58.6 and 87.3 mM, respectively) in X. laevis embryos at the same stage of development.…”