The Biophysics of the Egg Surface of Echinus Esculentus During Fertilization and Cytolysis

The eggs of Echinus esculentus are somewhat peculiar in that they show a considerable change in metabolism and permeability after fertilization or parthenogenetic activation. These might well be expected to produce chemical changes within the eggs and concomitant changes in the electrical properties of the cell surface.¹ Two of the electrical properties of the surface of these eggs, their d.c. resistance and the potential difference, if any, across their surface, are closely related to the chemical constitution of the eggs and the external medium. Certain ions are found both in sea water and in sea-urchin eggs. Their respective molalities in sea water (Wood’s Hole) and in the unfertilized eggs of Arbacia punctulata are shown in Table I.

The values have been computed from the data of Page (1927 a, EXBIO_15_2_209C10b) and Ephrussi & Rapkine (1928). There are two possible explanations of the marked concentration differences: (1) that the cell is actively concentrating certain ions by the expenditure of energy, although these ions are free to diffuse through the cell surface; or (2) that these ions are not free to diffuse through the cell surface. Experiments on the volume changes in sea-urchin eggs when in contact with solutions of varying osmotic pressure (made by dilution or evaporation of sea water) indicate that the cell surface is very impermeable to ionizing solutes. This block to diffusion might be due to cationic or anionic impermeability, or both, and the data in Table I confirm this. The condition of the cell surface which prevents the diffusion of ionizing solutes across it may or may not be associated with a high electrical resistance. In fact, inferences concerning the mobilities of various ions across the cell surface deduced from a consideration of the volume changes of eggs in solutions of varying osmotic pressure, give no indication of the mobilities of various ions across the cell surface when under the influence of an electric field. For instance, if the cell surface is almost totally impermeable to anions, but permeable to cations, measurements of the volume changes in solutions of different osmotic pressure would indicate a general impermeability of the cell surface, as is the case in the eggs of E. esculentus. On the other hand, measurements of the electrical resistance indicate simply the force necessary to overcome the short-range restrictions on the diffusion of cations. Cole’s experiments on the impedance of A. punctulata eggs (1928, 1936) indicate that this impermeability is both cationic and anionic. This author has at times gone so far as to suggest that the d.c. impedance of the egg is infinite. It will be shown that this concept is inconsistent with these experiments.

In these experiments the static electrical properties across the cell membrane of the egg of E. esculentus have been investigated before and after fertilization, and during cytolysis. The external medium was sea water, modified in certain experiments by dilution and alteration in pH. The experiments show that there is no measurable static potential difference across the egg surface before or after activation, nor after cytolysis, but that transient electrical changes occur when the egg cytolyses in certain abnormal solutions.

About 20–30 ripe unfertilized eggs¹ were placed on a cover-slip in a moist chamber. Two micro-pipettes controlled by Péterfi (1923) micro-manipulators were inserted in the moist chamber. The pipettes were filled with NaCl or KC1 isotonic with sea water with or without 1 % agar-agar. The terminal internal diameter of the pipette which entered the egg (E₁) varied in different experiments between 2 and 10 μ, while the other pipette (E₂) which remained in the sea water round the eggs varied between 8 and 30 μ. The distal ends of the pipettes were connected through sintered glass filters to saturated calomel half-cells, which were non-polarizable at the current densities employed in the measurements. The electrodes were connected to a voltmeter of the thermionic bridge type. The bridge was designed so as to be relatively independent of the resistance of the electrode system, which, owing to the narrow apertures of the pipettes, may reach 10⁷ or more ohms. As the potential to be measured is applied between the grid and cathode of a valve, it is only necessary to use a valve which passes a very small grid current under normal working conditions to ensure that the measured potential is independent of the resistance of the electrode system. The grid current of the valve used (W.E.D. 96475) was 5 × 10 ⁻¹⁴ amp. at a grid bias of −3 V. and an anode voltage of + 8 with respect to ground. Thus for all ordinary measurements, the resistance of the electrode system may be ignored, as it has to reach a value of 2 × 10⁹ ohms before the bridge is appreciably affected. Precautions of the usual type were taken to ensure that the apparatus and preparation were electrostatically screened and were independent of the atmospheric conditions which sometimes make high electrical insulation difficult at marine stations.

As the voltmeter is stable, its sensitivity is a function of the sensitivity of the galvanometer in the bridge circuit. A C.I.C. moving coil galvanometer (C. 213790), with a sensitivity of 5·4 × 10⁻⁸ amp. div.⁻¹, was used as an indicator, and a significant deflexion could be obtained for an input voltage of 0·1 mV. It is doubtful, however, if steady potential differences of less than 1−2 mV. can be considered as very significant in ordinary biological systems, and readings are given only to the nearest 0·5 mV.

The experimental procedure was as follows: The two pipettes were immersed in the sea water, and any electrical asymmetry between the electrodes noted. The egg was then impaled on the micro-pipette E₁ and the galvanometer reading noted. The leads from the preparation to the voltmeter were then reversed and another reading taken.¹ While the pipette is inside the egg, the egg is examined microscopically for pathological symptoms. Galvanometer readings are noted. When the experiment is over, the pipettes are again placed in the sea water and any p.d. between them noted. A typical experiment is appended in Table II.

Unfertilized eggs can be fertilized with the micro-pipette inside the egg, and normal membrane formation occurs. Insemination is made with a third pipette by injecting a small drop of an active sperm suspension into the egg culture in the moist chamber. Thus puncture or any diffusion from the tip of the micro-pipette do not prohibit the activation process.

The results are summarized in Table III.

The absence of any steady p.d. across the cell membrane of fertilized and unfertilized eggs might be due to faulty apparatus. It has been established independently that this is not so, and the experiments on cytolysing eggs confirm this supposition. Alternatively, the act of puncture might injure or stimulate the cell, causing depolarization. As the primary purpose of these experiments was to investigate the effect of activation on the electrical properties of the cell surface, and, as fertilization takes place quite satisfactorily although there is no static p.d. across the cell surface, it may be concluded (1) that the absence of p.d. is not associated with any pathological state of the egg, and (2) that if puncture stimulates the egg, as in the case of the frog’s egg, this stimulation has no effect on the developmental potentialities of the egg up to the formation of the fertilization membrane. It might perhaps be thought that the pipette never enters the egg, but causes an invagination of the cell surface. This objection has been raised before in experiments on inserting pipettes into sea-urchin eggs, but, in the opinion of the writer, the entry of the pipette into the cytoplasm can be established quite definitely by visual means. Furthermore, pressure could be applied at the distal end of the electrode system used in these experiments, forcing some of the fluid out of the tip of the pipette. The fluid which exudes from the pipette diffuses through the cytoplasm. If Ca⁺⁺ ions are present in this fluid, no diffusion takes place owing to the formation of some form of precipitation membrane (calcium proteinate?) which usually occurs when naked protoplasm comes into contact with a fluid containing Ca⁺⁺ ions. If the pipette does not enter the egg, the Ca⁺⁺ fluid would diffuse through the sea water in the ordinary way and no interphase would be formed. This effect of Ca⁺⁺ ions indicates the necessity of using Ca⁺⁺-free solutions in the pipettes. If this were not done, p.d. measurements would be across two interphases: (1) that between the cytoplasm and the external sea water, and (2) that between the cytoplasm and the electrolyte in the micro-pipette.

There is, therefore, no appreciable p.d. across the cell membrane before or after fertilization. This somewhat unexpected condition is also found in the starfish egg (Gelfan, 1931). This does not preclude the possibility that fertilization is associated with transient electrical changes which would not be recorded on an instrument designed to measure steady potentials. In fact, the electrical changes which occur during cytolysis in special solutions are slow transients, the wave forms of which are not recorded accurately by the indicator used in these experiments.

d.c.resistance andp.d. The d.c. resistance of the surface of the impaled egg cannot be more than 10⁶ ohm cm.² A small but definite current flows from the grid to the cathode or ground in all thermionic valves. In these experiments the grid and cathode are shunted by the preparation, and a potential is developed between the grid and cathode of the valve; this potential is not due to an external e.m.f. and therefore its sense is not reversed by reversing the leads from the preparation to the valve. The potential is the product of the resistance of the preparation and the grid current of the valve (Ohm’s law). If the resistance of the preparation is more than 10⁶ ohm cm.², this potential becomes detectable through the deflexion of the galvanometer in the bridge circuit. As this does not happen, the resistance of the preparation is clearly less than the above value.

It is possible, and from the results of Blinks (1930), Kopac (1936) and others on the resistance of Valonia it is probable, that the manipulation involved in these experiments may cause a temporary decrease in resistance, but the resultant permeability increase, if it occurs, has no effect on the capacity of the egg to be fertilized. These experiments confirm the idea that the cell surface is relatively impermeable.

Let us consider the distribution of H+ between the egg and its medium. The pH of sea water is about 8·2, while according to Needham & Needham (1926) the pH of the cytoplasm is about 6·6. As this steady state is maintained, evidently the H+ ions inside and outside the cell are not absolutely free to diffuse according to their electro-chemical potentials in their respective environments. There are two probable alternative explanations: (1) that the cell membrane is not permeable to H⁺ ions, or (2) that the cell membrane is permeable to H⁺ ions, but that they are prevented from passing through the membrane by short-range forces restricting the anions and other cations. (This latter hypothesis is put forward to explain the p.d. across the nerve membrane; in this case the cation is K⁺.) If these forces prevent H⁺ ions from crossing the cell surface, and the ordinary principles of thermodynamics are applicable in this system, there should be a p.d. across the cell membrane when the egg is in normal sea water, and this p.d. should vary as the pH of the sea water is altered. As neither of these things happen, two possibilities exist: (1) that the cell membrane is impermeable to H⁺ ions, or (2) that the cell membrane is permeable to H⁺ ions but that the sum of all the possible other potentials across the cell surface depending on (a) the activities of all other ions inside and outside the cell, and (b) dipole or other structures in the cell surface, add up exactly to minus the p.d. produced by the difference in pH inside and outside the cell. And furthermore, that these conditions persist even if the external medium is varied by dilution, even though the pH ratios remain constant.

As a working hypothesis, the concept of the cell membrane being impermeable to H⁺ ions is simpler. A similar analysis could be made for all the other ions in sea water whose concentration differs markedly from that within the egg. The absence of any p.d. in diluted sea water indicates a similar conclusion; that the egg, when intact, is relatively impermeable to these ionizing solutes which could contribute to the production of a p.d. across the cell membrane. This condition persists after fertilization, though it must be reiterated that this does not preclude the possibility of transient electrical changes during activation.

An attempt has been made to measure the d.c. resistance of the cell membrane before and after activation. If two electrodes are placed inside the egg and two outside, one pair may be used to flow current across the cell surface, while the other pair measures the ohmic drop of potential across the cell surface. This experiment is technically very difficult, and the results were unsatisfactory, though there are indications that the d.c. resistance of the cell surface is not higher and may very well be lower than that of Valonia and other plant cells which have a resistance of 10⁴ to 2·5 × 10⁴ ohm cm.²

Having established that there is no p.d. across the normal cell surface before or after activation, it is still necessary to explain why the cell membrane has a d.c. impedance of less than 10⁶ ohm cm.² For if complete impermeability were to exist, the cell membrane should behave like a pure capacitance, as Cole has suggested. Such a condition is hardly likely to exist in a biological system, and the current which does flow across the cell surface must be carried by ions, there being no evidence for appreciable electron conduction in a system of this type. It is perhaps possible that current only flows through the seal round the point of entry of the pipette, but this still does not explain the absence of p.d. It is possible that the ions concerned in the flow of current are not those which would be concerned in the production of any potential, but this explanation is not satisfactory, as an intact egg in diluted sea water still has no p.d. across the cell surface. As an impaled egg in diluted sea water swells, the seal round the point of entrance of the pipette is not completely permeable to ionizing solutes. Further experiment is needed to solve this problem.

Cytolysis. When an egg is punctured in sea water at pH 8·3, 7·0, or 9·0, there is no cytolysis unless too flat a sea-water drop is used in the moist chamber, a condition which is well known to cause disintegration of the cell. The cytolysis is not associated with any potential changes. Beginning with a change in the morphological structure of the cytoplasm which makes it appear more granular, the cytolysis ends in the total disruption of the cell membrane. The reaction is over in about 1–2 min. The fertilization membrane which elevates during the process¹ becomes the only membrane round the cytoplasm, and the egg contents are homogeneously dispersed throughout the space inside it. This form of cytolysis has been called white cytolysis. As in some cases the fertilization membrane can be seen during this process, it appears that the fertilization membrane is not permeable to Ca⁺⁺ ions in this form of cytolysis. Were the fertilization membrane permeable to Ca⁺⁺ ions, the cytoplasm exuding from the cell surface would immediately reform a surface and the cytoplasmic contents would not be dispersed. Chloroform causes black cytolysis at the pH of normal sea water, in which the fertilization membrane, the perivitelline space and the cell surface remain as discrete structures. The cytoplasm becomes densely granular and blackish in colour and appeared almost to be “fixed”. These conditions probably indicate that during chloroform cytolysis the fertilization membrane is permeable to Ca⁺⁺ ions.

Neither an increase nor a decrease in the concentration of H⁺ ions in otherwise normal sea water appear to make the cell unstable as regards cytolysis. But at pH 9·0 fertilization does not occur when the pipette is in the cytoplasm. If, however, the sea water is diluted with isotonic sugar solution, serious instability occurs. At the pH of the normal Millport sea water (8·2) and at pH 8·4, cytolysis is almost certain and this is probably due to the decrease in the Ca⁺⁺ concentration of the external medium, a factor which decreases the stability of these eggs. At pH 8·4, a p.d. appears across the cell surface during cytolysis. As this is a transient, neither absolute size nor wave form can be determined accurately by a slow period indicator. The simplest working hypothesis to explain this condition, when we remember that a decrease in C_H+ in the sea water appears to increase the size of this p.d., is that an acid is produced inside the egg during cytolysis, and that the cell membrane becomes permeable to H⁺ ions and anions. Suppose the two micro-pipettes are placed in sea water and a small drop of HC1 (aqu) is placed near one of them. This electrode immediately becomes negative with respect to the other one. The acid tends to diffuse through the solution in the ordinary way. The narrow bore of the proximal pipette impedes the diffusion of the HCl up the pipette, a process which would make this electrode positive. The acid therefore diffuses through the solution, and, as the H⁺ ions have a higher mobility than the anions, the proximal electrode is left negative with respect to the other. Some such process may very well occur in the cytolysing egg. The transient nature of the p.d., its sense and its dependence on an abnormally low C_H+ tend to confirm this hypothesis. Other hypotheses could be invented to explain this transient p.d., but on external grounds there is additional evidence. The “acid of injury” is well known, and Runnström (1933, 1935) has reported the production of acid in cytolysing and in fertilized eggs.

Similarly, Gray and the author (unpublished) have shown that the respiratory quotient of sea-urchin eggs immediately after fertilization or cytolysis is very markedly more than 1·0 when the eggs are in sea water or its equivalent, provided that the external solution contains bicarbonate ions. We have also shown that after cytolysis there is a considerable decrease in the pH of the external medium. These facts would be consistent with the production by the eggs of an acid or pseudoacid such as aceto-pyruvic acid (Krebs & Johnson, 1937) after sperm activation or cytolysis.

1. Activation is not dependent on the presence of a steady P.D. across the cell membrane of the egg of E. esculentus.

2. The D.C. resistance of the impaled fertilizable egg is less than 10⁶ ohm cm.²

3. Cytolysis in diluted isosmotic sea water is associated with a transient P.D., which appears to be due to the production of acid within the egg.

I wish to record my thanks to the Director and Staff of the Marine Station, Millport, for the facilities I enjoyed while working there.