209
THE BIOPHYSICS OF THE EGG SURFACE OF
ECHINUS ESCULENTUS DURING FERTILIZATION AND CYTOLYSIS
BY LORD ROTHSCHILD
Trinity College, Cambridge
From the Sub-Department of Experimental Zoology, Cambridge
and the Marine Laboratory, Millport
(Received 21 June 1937)
(With One Text-figure)
an egg is fertilized, well-defined morphological changes occur. In spite of
this, physico-chemical differences between fertilized and unfertilized eggs have been
difficult to find. Nor have the increases in permeability and metabolic rate found
in certain eggs been shown to be of general incidence. Investigations of the
electrical properties of eggs are few, and the results have been inconclusive and
contradictory.
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.
WHEN
Table I. Main constituents of sea water (Wood's Hole) and of A. punctulata eggs
Molalities
Sea water (ao° C.)
Sodium
Potassium
Calcium
Magnesium
Chlorine
Sulphate
A. punctulata egg!
038
033
00105
0-0107
00534
0591a
0-0373
0-37
0-28
I 09
0-031
0-00003
1
Chemical asymmetry across an interphase is, of course, not necessarily associated with an
asymmetrical distribution of electrical charge.
JEB-Xvii
14
210
LORD ROTHSCHILD
The values have been computed from the data of Page (1927 a, b) 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 Pe'terfi (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 (Ej) varied in different experiments between
2 and 10 ix, while the other pipette (Et) which remained in the sea water round the
eggs varied between 8 and 30 /u.. The distal ends of the pipettes were connected
through sintered glass filters to saturated calomel half-cells, which were nonpolarizable 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,
1
The jelly wa» removed by washing with sea water.
Biophysics of Egg Surface of Echinus esculentus
211
7
which, owing to the narrow apertures of the pipettes, may reach io 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 x 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 x io9 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 x io~8 amp. div.^1, was used as an indicator, and a significant
deflexion could be obtained for an input voltage of o-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.
Switch
Thermionic bridge
Bndge galvanometer
Piprtie inrl electrode K,
Revenui£ key
Smarting switch
Fig. 1. Insulation: paraffin wax or air throughout. Switches: mercury/copper throughout.
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
1
This procedure indicate* whether any observed potential is due to an external E.M.F. or to »n
artifact caused by a high resistance in the preparation or electrodes (see p. 213).
14-3
LORD ROTHSCHILD
212
Table II. Unfertilized egg in sea water, 23. iii. 37
p.D. between electrodes £j and E, in mV.
Ei and E, in sea water
Ei in em
El and E, in sea water
Leads to bridge reversed
Leads to bridge reversed
Leads to bridge reversed
2-5 (Ei + )
2-5 (£1 + )
2-5 (£1 + )
2-S (£. + )
2-S (£1 + )
2-5 (£1 + )
Terminal diam. (int.) of Er: 3 p. Distance into cytoplasm: 10/i.
Terminal diam. (int.) of Et: 25 pElectrode system: Hg I Hg.Cl, | KC1 (sat.) |i KC1 (o-6 M) | KC1 (o-6 Min 1 % agar) | sea water |
cytoplasm | KC1 (o-6 M in 1 % agar) | KC1 (00 M) |i KC1 (sat.) | Hg,Cl, | Hg.
Remarks: Distance of £ , into cytoplasm is only approximate, e.g. ± 5 fi. Egg samples from
general culture produced normal membranes after insemination.
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.
Table III. Measurements of the P.D. across the cell membrane
No. of
P.D.
Remarks
Good fertilization membranes
Three successful fertilizations,
but poor membranes
No fertilization
(1) Unfertilized eggg with Ei within the egg:
(a) In normal sea water at pH 8-3
(b) In sea water at pH 7-0
12
10
O
O
(c) In sea water at pH 9-0
(2) Unfertilized eggs in normal sea water at
IO
O
44
0
(3) Fertilized eggs in normal sea water at
20
O
Fertilization membrane collapses
(4) Unfertilized eggs cytolysing in normal
sea water at pH 8-3
(5) Unfertilized eggs in sea water+isotonic
manitol, in ratio 10 : 40, at pH 7-0
(6) Unfertilized eggs in sea water + isotonic
dextrose, in ratio 1 : 19, at^H 7-4
5
0
7
O
Cytolysis induced by having too
flat a drop and by puncture
No cytolysis
(7) Unfertilized eggs in sea water + isotonic
dextrose, in ratio 1 : 19, at pH 8-4
10
IO
In seven cases, eggs cytolysed.
Faint indications of cytoplasm
becoming negative with respect
to outside, e.g. 0-25—0-5 mV.
p.D. insignificant. In three eggs
which did not cytolyse, no signs
of P.D.
c. 8 mV. Nine eggs cytolysed. P.D. 0 at
beginning, rose sharply during
(trancytolysis, and fell to zero at the
sient)
end of cytolysis (time 1-2 min.).
Inside of egg negative. One
egg did not cytolyse ->• no p.D.
o(?)
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,
Biophysics of Egg Surface of Echinus esculentus
213
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 and P.D. The D.C. resistance of the surface of the impaled egg
cannot be more than io* ohm cm.s 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.8, 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
214
LORD ROTHSCHILD
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
/>H 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/>H 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 />H 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 Valorua and other plant cells which have a
resistance of 10* to 2-5 x io* ohm cm.1
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 10* ohm cm.* For if complete impermeability were to
Biophysics of Egg Surface of Echinus esculentus
215
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 />H 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 ^>H 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
1
Sometimes thii process is not visible, but it presumably doe^ happen.
216
LORD ROTHSCHILD
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 HC1 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 CH+ 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 Runnstrom (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 -o 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 />H 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.
SUMMARY
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 10* ohm cm.1
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.
REFERENCES
BLINKS, L. R. (1930). J. gen. Phytiol. 13, 495.
COLE, K. S. (1938). J. gen. Pkyriol. 12, 37.
COLE, K. S. & COLE, R. H. (1936). J. gen. Phytiol. 19, 625.
EPHRUSSI, B. & RAPKINE, L. (1938). Arm. Physiol. Phyricochim. biol. 4, 386.
GELFAN, S. (1931). Proc. Soc. exp. Biol., N.Y., 29, 58.
KOPAC, M. J. (1936). Publ. Carneg. Instn, No. 452, p. 359.
KREBS, H. A. & JOHNSON, W. A. (1937). Biochem. J. 31, 772.
NEEDHAM, J. & NEBDHAM, D. M. (1926). Proc. roy. Soc. B, 99, 173.
PAGE, I. H. (1927a). Biol. Bull. Wood's Hole, 82, 164.
(1927A). Biol. Bull. Wood's Hole, 82, 168.
PETERFI, T. (1923). Handb. biol. ArbMeth. 5, 479.
RUNNSTROM, J. (1933). Biochem. Z. 288, 257.
(1935). Biol. Bull. Wood's Hole, 69, 345.