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

Abstract

The intracellular ionic distribution in uncleaved and cleaving Ambystoma eggs was investigated by analysing the influx of 3H2O, 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 (aNai, aKi and aCli) using intracellular ion-selective microelectrodes. The electrical membrane potential (Em) and membrane resistance (Rm) 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 Em 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.

3H2O influx revealed the existence of a single intracellular water compartment (3.06 μl/egg) and a low water permeability (5.35 × 10−5 cm sec−1). Na+, K+ and Cl− concentrations were constant at 54.1, 72.1 and 73.1 mM respectively, while aNai, aKi and aCli 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 Em of ca 40 mV and a specific membrane resistance of 39 kOhm cm2. Em could be described by a constant field equation with a permeability ratio PK/PNa= 0.073. Shortly after the onset of first cleavage, Em rapidly decreased concomitant with a rise in Rm (68.5 kOhm cm2). This was interpreted as a drop in Na+ permeability. During the cleavage process Em progressively hyperpolarized and Rm 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 cm2). Both in normally cleaving eggs and in eggs cleaving in the absence of the vitelline membrane Em behaved according to the constant field equation, PNa/PK being 0.69 and 0.39, respectively. The differences with other amphibian eggs were discussed.