Membrane potential of the unfertilized sea urchin egg

DEVELOPMENTAL

BIOLOGY

Membrane

62, 215-228 (1978)

Potential

LAURINDA *Department

of the Unfertilized

A. JAFFE*J’

AND

7,1976;

Egg

R. RosINsoNt~*

KENNETH

of Physiology, University of California, Los Angeles, Biology, Purdue University, West Lafayette, Received September

Sea Urchin

California 90024 and TDepartment Zndiana 47907

of

accepted in revised form September 13,1977

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 t 0.2 pmole/cm2.sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential ( - 70 mV) and specific resistance (420 kn . cmz). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were -75 -C 3 mV and 380 k M .cmz.

simply exposed to radioactive tracer ions in a gently stirred suspension. Therefore, The resting potential of the unfertilized echinoid egg has been a matter of dispute. it is unlikely that the measurement damReported microelectrode measurements ages the eggs. Tracer flux measurements of resting potential, as well as of specific range from -5 to -75 mV (Hiramoto, resistance and potassium selectivity, were 1959; Steinhardt et al., 1971; Higashi and then compared with microelectrode measKaneko, 1971; Ito and Yoshioka, 1972; urements of these characteristics. We also Uehara and Katou, 1972; Tupper, 1973; determined the potassium selectivity of Chambers et al., 1974; J&e, 1976; Macthe membrane by measuring passive soKenzie and Chambers, 1977; Okamoto et dium influx as a function of external poal., 1977). Because the resting potential tassium concentration. might be involved in controlling metabolic activities and also as a basis for further MATERIALS AND METHODS electrical studies, it is important to know All experiments were done with Stronwhich resting potential is natural. To find supplied by Pagylocentrotus purpuratus, out, we determined the resting potential cific Biomarine Co., Venice, Calif. Eggs by an independent method: We measured and sperm were obtained by injection of fluxes of the pertinent ions with radioac0.5 M KC1 into the coelomic cavity. Most tive tracers. The flux data can be used to experiments were done in artificial seacalculate resting potential by means of water of the following composition: 483 the constant-field equations. This method mM Na+, 55 mM Mg2+, 10 m&f Ca2+, 10 does not involve the use of microelecmM K+, 563 mM Cl-, 28 mM Sod*-, 2.5 trodes, nor does it necessitate holding the m&f HC03-, 10 mM Tris, pH 8.0. When eggs down to a surface. The eggs are the potassium concentration was varied, 1 Present address: Marine Biological Laboratory, K+ was substituted for Na+ (or Na+ for Woods Hole, Massachusetts 02543. K+). Some of the microelectrode experi2 To whom reprint requests should be sent. Presments were done in natural seawater, and ent address: Division of Molecular and Cellular some of the tracer experiments were done Biology, National Jewish Hospital, 3800 E. Colfax Avenue, Denver, Colorado 80206. in Tris-free artificial seawater. The results INTRODUCTION

215 0012-1606/78/0621-0215$02,00/O Copyright 0 1978 by Academic Press, Inc. All rights

of reproduction

in any form reserved.

216

DEVELOPMENTALBIOLOGY

of these experiments were identical to the ones done in the presence of Tris buffer. All experiments were done at 15°C. Microelectrode methods. Eggs were hand centrifuged and agitated with a jet of seawater from a spray bottle, alternately several times. This procedure removed the jelly coats from some of the eggs. The suspension of eggs was poured into a plastic petri dish (Falcon 1007); it was found that the dejellied eggs stuck to the bottom of the dish, so that they could be penetrated by microelectrodes (Chambers et al., 1974; Jaffe, 1976). Unstuck eggs were rinsed away. The basis of the adhesion between the egg surface and the plastic may be electrostatic attraction between charges on the cell surface and charges on the plastic surface, in which case this method is similar to the method of attaching eggs to protamine-coated surfaces (Steinhardt et al., 1971). Damage to the eggs by the dejellying and attachment procedure is relatively minor, if it occurs at all. The attached eggs look normal and can be fertilized. They cleave, form blastulas, and hatch off the surface. The dish containing the eggs was supported in a Plexiglas frame and was observed under a Wild stereoscope at 50x. Solution exchange was accomplished by introducing new solution with a syringe, while withdrawing old solution with an aspirator. The solution could be changed while the microelectrode was in the cell. Microelectrodes were prepared by filling the glass capillary with fibers of glass wool and then injecting the micropipet with 3 M KC1 from a syringe. Resistances of the electrodes, before penetration, varied from 30 to 60 hizR. A cell was penetrated by bringing the electrode to the surface so that it produced a slight depression and then applying an oscillating current through the electrode by transiently turning up the “negative capacitance.” The current may cause penetration by a direct electrical effect, or it may be that

VOLUME 62, 1978

the electrical oscillation sets up a mechanical oscillation. It has been suggested that this application of current might activate the eggs. Sufficiently large currents have been shown to cause fertilization membrane elevation (Ito and Yoshioka, 1973). However, we rarely saw fertilization membrane elevation when the electrode was inserted. In those occasional cases, the egg was discarded. (We don’t know whether the membrane elevation was caused mechanically or electrically.) Possible microelectrode artifacts of this sort were the reason we decided to do the tracer flux measurements. One electrode was used for both recording voltage and passing current; the ir drop across the electrode was electronically balanced with a WPI amplifier (Model M4-A). The amplifier was slightly modified because of the necessity to use very small currents: The input voltage to current conversion factor was changed to 1 mV = lo-‘* A. The zero level and leakage current of the amplifier were adjusted before each electrode penetration; this procedure was necessary because the nominal leakage current of the amplifier (5 x lo-‘* A) is large enough that it could affect potential measurements on these very high input resistance cells (3-mV shift for 5 x lo-l2 A). Membrane potential and applied current were recorded on a strip chart recorder. In most cases, the penetrated egg was fertilized before removing the electrode. When sperm were added, a fertilization membrane always formed. For later observation, the cell which had been penetrated was marked by drawing a box on the bottom of the petri dish with a felt-tip pen. The eggs cleaved and developed normally except in cases of polyspermy. Tracer flux methods. The methods used in these experiments were similar to those described elsewhere (Robinson, 1976). Eggs from a single female were suspended

JAFFE

AND ROBINSON

Membrane Potential of Urchin Egg

in the appropriate artificial seawater (ASW) and the radiotracer was added (either 42K, 22Na, 36C1, or 45Ca). The eggASW mixture was gently stirred and samples were removed at various times. The extracellular tracer was removed by several (four or five) washes in a hand centrifuge, and the eggs were evenly distributed in ringed planchets, dried, and counted in a low-background, gas-flow planchet counter. Standards were prepared by adding small volumes of radioactive ASW to planchets containing the same number of eggs as the samples. In order to remove a small (0.03 pmolel egg) nonzero intercept in the sodium uptake data, which was presumably due to extracellular binding, it was necessary to extend the washing time to 15 min. A consideration of the rate of exchange calculated from our influx data (K, - 10m5,’ set) makes it clear that an insignificant amount of tracer sodium would be lost from the eggs in this time. The number of eggs in the suspensions was determined by drawing eggs into 5~1 capillary tubes and counting the eggs in several (10-20) such tubes. This method was compared to that of Robinson (1976) and found to give identical results. The eggs were tested for fertilizability before and after each experiment and were always >95% fertilizable. RESULTS

Microelectrode

AND ANALYSIS

Measurements

Upon penetration with a microelectrode, the unfertilized S. purpuratus egg initially has a small resting potential and a small input resistance. Within a few minutes, however, the potential and resistance of the membrane usually begin to increase. Figure 1 shows an example of this process. The increase in membrane potential from -4 to -77 mV is accompanied by an increase in membrane resistance from 140 to 670 m. The change in membrane potential is 90% complete in 3

217

FIG. 1. Membrane potential and input resistance as a function of time after penetration of an S. purpuratus egg with a microelectrode. (O-0-0) Membrane potential, (O-O-01 input resistance. Arrows indicate time of microelectrode penetration. Input resistance was determined by passing a small current pulse and measuring the resulting voltage deflection. Bars on input resistance points are estimates of the possible error due to reading data off the chart record.

min, and after that the properties remain stable for the duration of the record (30 min) . In different eggs, the time between initial penetration and attainment of a -7OmV resting potential varies from 1 to 30 min. In some cases, -70 mV is never reached. Commonly, just before reaching the steady level of -70 mV, there is a transient positive shift in potential lasting less than a minute (small shift is shown in Fig. 1). The shift is an action potential, probably initiated by injury current, or sometimes as an off response to an applied inward current pulse. The action potential will be described in detail below. Once the membrane potential reaches -70 mV, it stays approximately constant for an hour or longer, or until the electrode comes out, or the egg is fertilized. This time-dependent change in resting potential of the unfertilized sea urchin egg has been reported by several other investigators (Higashi and Kaneko, 1971; Ito and Yoshioka, 1972; Uehara and Katou, 1972; Chambers et al., 1974). The concomitant increase in membrane resistance has also been reported (Chambers et al., 1974, Fig. 2, Lytechinus uariegatus). It has been suggested that the change in membrane potential represents recovery from pene-

218

DEVELOPMENTAL BIOLOGY

tration damage (Ito and Yoshioka, 1972; Uehara and Katou, 1972); alternatively, however, it might be due to the appearance of potassium channels characteristic of the activated egg (Higashi and Kaneko, 1971; Steinhardt and Mazia, 1973). To distinguish between these possibilities, we used the following reasoning: A recovery process would be accompanied by an increase in membrane resistance, while activation of potassium permeability would be accompanied by a decrease in TABLE

VOLUME 62, 1978

membrane resistance toward the small resistance characteristic of the fertilized state. Table 1 shows a comparison of the membrane resistance of the unfertilized -7O-mV egg with that of the fertilized egg. The input resistance of the unfertilized egg is approximately 500 Tu2R,compared to 80 MQ for the fertilized egg. This sixfold difference in membrane resistance is not consistent with the idea that the -70-mV unfertilized egg has the potassium permeability characteristic of the ac1

MEMBRANE PROPERTIES OF Stronmlocentrotus numuratus EGGS Resti;fapotenInput ;e&tancea Specific resistanceb (Kfi. cm*) (mV)

K+ selectivity (YE/W&d (mV)

Microelectrode measurements Unfertilized eggd Types A and B

GV-Stage Fertilized

oocytef eg@

Tracer flux calculationsn Unfertilized egg

-75 (n -72 (n -65 (n -73 (n -64 (n

k = -+ = 2 = k = ” =

3’ 27) 3 33) 419) 6 2) 2 6)

640 (‘2 430 (n 420 (n 20 (n 80 (n

-70,

-

77

700

2 = + = + =

90 8) 40 4) 200 21)

= 2) k 20 = 6)

380 (n 260 (n 250 (n

2 = + = + = -

60 8) 30 4) 120 211

56 (n 57 (n

+ = 2 = -

1 3) 2 2)

-

110 f 30 (n = 6)

-

420

-

“For unfertilized eggs, Types A, B, and C, input resistance was measured from the tangent to the IV curve at the resting potential (see Fig. 3). All cells used for these measurements had resting potentials of -75 f 2 mV. For unfertilized egges, type D, and for GV-stage oocytes and fertilized eggs, input resistance is only approximate, being calculated in most cases from the voltage response of the membrane to one or two applied current pulses. * Specificresistances were calculated from input resistances, using 6.0 x 10m4cm2 for the membrane area of the unfertilized egg and 13.5 x 10m4cm2 for the membrane area of the fertilized egg (measured from electron micrographs, Eddy and Shapiro, 1976). Specific resistance of the GV-stage oocyte was not calculated, because of the uncertainty about membrane area. c K+ selectivity was measured as in Fig. 4. All cells for these measurements had resting potentials in standard artificial seawater of -75 + 2 mV. E was measured at K, + = 300, 100, 30, and 10 mM. In three of the five cells, E was also measured at 3 mM. Over this range of K,+ concentrations, E was a linear function of log K,+. K+ selectivity was not measured for unfertilized eggs, type D, or for GV-stage oocytes or fertilized eggs. d Microelectrode measurements of unfertilized eggs are classified according to action potential amplitude and duration: types A, B, C, and D, as described in the text. e All data are expressed as mean -C SE; n = number of eggs studied. f Fully grown oocyte @O-pm diameter) with intact germinal vesicle. 0 Ten minutes after fertilization. At this time, the development of increased K+ permeability is probably not quite complete (Steinhardt et al., 1972). h Calculations are explained in the text. Two resting potential values are listed: The first is obtained from Eq. (1); the second is the potassium equilibrium potential (see text explaining Fig. 7).

JAFFE AND ROBINSON

tivated egg. The observation of an increase in membrane resistance following penetration (fivefold increase in Fig. 1) is consistent with the idea that initially there is a leak around the microelectrode, and that this leak gradually heals, allowing one to record the true -70-mV resting potential. Having concluded from the above argument that eggs with small resting potentials were damaged, we limited all further microelectrode studies to those cells with a resting potential larger than -60 mV. Within this group, there is variation in the ability of the egg membrane to produce an action potential in response to an outward current pulse. This action potential is similar in shape to action potentials in other cells, but it has a very slow time course (Jaffe, 1976). The response shows two inflections in its rise, one near -50 mV and a second near -10 mV. Similar action potentials have been observed in other sea urchin eggs (Okamoto et al., 1977) and in starfish oocytes and eggs (Hagiwara et al., 1975; Miyazaki et al., 1975a,b; Shen and Steinhardt, 1976). Figure 2 shows some of the variability of the action potentials in different S. purpuratus egg cells. Type A response outlasts the stimulus. Type B response does not outlast the stimulus, but does maintain an oscillating positive potential for the duration of a 5-set pulse, with an amplitude of 3 x lo-” A or less. Like types A and B, type C

D-.,_.,

-

--.q

219

Membrane Potential of Urchin Egg

~0552110-%,,

urL

FIG. 2. Action potentials of the unfertilized S. purpuratus egg membrane. The top trace is voltage against time; the bottom trace is current against time. The dotted line indicates zero millivolts. A, B, and C are progressively weaker action potentials. D is a case where, even when the stimulus current was increased, no action potential occurred.

123456 1(x10%mps)

FIG. 3. Resistance of the unfertilized S. pq~uratus egg membrane. The abscissa is applied current; the ordinate is steady-state voltage deflection from the resting potential (-74 mV). A smooth curve was drawn connecting the points; the slope of the tangent to this curve at the resting potential is the resistance.

response has inflections in its rise (often only the first inflection), but after a brief peak the response falls off. Type D response is nonregenerative, or almost nonregenerative. Table 1 lists membrane properties of the unfertilized egg: resting potential, resistance, and potassium selectivity. The eggs are classified on the basis of the amplitude and duration of their action potential, as defined above. The characteristics of type A and B cells are about the same: the resting potential is -75 * 3 mV, the specific resistance is 380 * 60 kn .cm2, and the membrane is potassium selective, showing a 55-mV change in membrane potential per lo-fold change in external potassium concentration. Figures 3 and 4 show data used to obtain these values. Type C cells have a slightly smaller resting potential and a slightly smaller specific resistance, but show the same potassium selectivity. Type D cells have a distinctly smaller resting potential and show a large range in specific resistance. Potassium selectivity of these cells was not investigated. For comparison, Table 1 lists a few electrical measurements made on immature oocytes [fully grown, germinal vesicle (GV) stage] and fertilized eggs (10 min after fertilization). Like the unfertilized egg, the oocyte and fertilized egg have

DEVELOPMENTAL BIOLOGY VOLUME62, 1978

220

membrane potentials near -70 mV; their input resistances, however, are 5 to 30 times smaller than that of the unfertilized egg.

brane, 6 x lop4 cm2, the fluxes of potassium and sodium (Jx and &) can be calculated and are 0.60 and 0.10 pmol/ cm2*set, respectively, for the experiments shown in Fig. 5. [Electron micrographs Tracer Flux Measurements indicate that the surface of the S. purpurThe time course of uptake of 42K and atus egg is convoluted. Eddy and Shapiro 22Naby a single batch of unfertilized eggs (1976) estimate that the total surface area is shown in Fig. 5. The curves are linear is on the order of 6 x lop4 cm2. This is over the period of the experiments, so threefold larger than the surface area calback-flux can be neglected and the amount culated from egg radius: 2.0 x 10e4cm2.1 of ions entering the eggs can be deter- Table 2 lists the results of one other such mined from their specific activities in the measurement of JK and JNa on a single seawater. Using the area of the egg mem- batch of eggs. Also shown there are several measurements of JK, JNa, Jcl, and JCa, each done on different batches of eggs but by the same methods. It is clear from Table 2 that the potassium flux is at least four times as great as the sodium flux; in the two cases where both fluxes were measured on the same batch of eggs, JK was five- and sixfold greater than JNa, respectively. Chloride flux is even smaller, being only 15% of the average potassium flux. Calcium flux is an order of magnitude smaller than the other fluxes, so that its direct effect on the FIG. 4. Potassium selectivity of the S. purpurumembrane potential can be disregarded. tus egg membrane. The abscissa is the concentration As discussed below, these data indicate of potassium outside the cell; the ordinate is membrane potential. A straight line was drawn connectthat the plasma membrane of the unfertiling the points. The slope of this line is a measure of ized sea urchin egg is primarily permeable the membrane’s potassium selectivity: A slope of 57 to potassium and that the membrane pomV/lO-fold change in external potassium would tential is near the potassium equilibrium indicate that the membrane was exclusively potaspotential. sium permeable. The observed slope was 55 mV/lOSince the concentrations of these ions fold change.

Counts 5mm

P~corrdes K(Nal egg

7

5

IO

15

20

25 30 35 40 45 Exposure Time ( man)

FIG. 5. The uptake of 42K and **Na into unfertilized made simultaneously, with eggs from a single female.

50

55

60

eggs of S. purpurutus.

The measurements

were

JAFFE

AND ROBINSON

Membrane TABLE

TRACER

FLUXES

IN UNFERTILIZED

Potential

of Urchin

221

Egg

2

Strongylocentrotus

purpuratus

EGGS

Experiment 1” 2” 3 4 5 6’ 7” Sd 9d 10d

0.16 0.10 0.087

0.80b 0.60

0.073 0.073 0.0083 0.0053 0.50 0.43 0.23 0.50 f 0.2e

0.12 ? 0.04

0.073

0.0068 ? 0.002

a J, and J,, were measured simultaneously, with eggs from a single female. * Values are expressed as picomoles per square centimeter. seconds. c Measurements of J,, were done by measuring uptake at a single time point, rather than at several time points as for the other ions. This method is subject to error if there is insufficient washing. However, the error would result in an overestimate of the flux, so for the purpose of demonstrating that K+ flux is much larger than Ca*+ flux, these measurements are meaningful. d From Robinson (1976), corrected for surface area determined from electron microscopy. Calculations in the 1976 paper were based on surface area determined from cell radius.

(K+, Na+, and Cl-) in the sea urchin egg are known, it is possible to use our flux measurements to calculate analytically the membrane potential using the Hodgkin and Katz (1949) formulation of the Goldman equations (1943). These equations were derived with the assumptions that each ion moves independently of each other ion, that all ion pumps are electrically neutral, and that the field across the membrane is constant. The Goldman voltage equation is: E

=“zlnK,+ m

F

aNa,+

&+

aN%+

@Cl,

@Cl,,’

than the potassium equilibrium potential and more negative than the sodium equilibrium potential; thus the outward movement of potassium and the inward movement of sodium will be passive and ,=p?=

Ki

exp(F%IRT)

PK

where Em = membrane potential; R, T, and F have their usual meanings; subscript o designates external ion activities (concentrations); subscript Y’ designates internal ion activities (assumed equal to concentrations; see discussion below); (Y = PNaIPK; and /3 = PclIPK. These permeabilities (P,, PNa, and PO) can be expressed as functions of the membrane potential and fluxes (e.g., Robinson and Jaffe, 1973). One many assume that the membrane potential is more positive

K

However, the membrane potential may be either smaller or larger than the choride equilibrium potential (EC, ), so

pI = PA = K ew W,IRT) pK

(‘I

. J?.

Na,

if Em is more negative

Cli

Jc, JK

’

than EC,, or

if E,,, is more positive than Ea. If these quantities are substituted into Eq. (l), the resulting equations are quadratic in exp (FE,,,/RT) and can be solved exactly for E,,,: -b + (b’ - ~uc)“~ 9 (2) 2a ( ) where (for Em more negative than EC,) E, = Rg In

222

DEVELOPMENTAL BIOLOGY

CJoK Ja u-N3K,.JNa+ __. Na,

JK

Clj

VOLUME 62, 1978

3. Of course, one should use activities rather than concentrations, but, with one

JK ’

a+&.J+q.Jf,

TABLE K

K

c = -K,, Ion

or (for E,,, more positive than l&) Ki Nai a=---

JNa

Na,

External concentration= 10 mM

K+

JK JK

K,+----c;” I$

c=-

Jc,

cl,

(

3

INTRACELLULAR AND EXTRACELLULAR ION CONCENTRATIONS IN SEA URCHIN EGGS

JK 1 *

In applying these equations, we have used our influx measurements. It has been shown that the potassium influx and efflux in S. purpurutus eggs are equal to within 2% (Robinson, 1976). It is assumed that chloride is also in the steady state. In Fig. 6 are shown plots of E,,, as a function of JNa/JK from Eq. (21, with Jcl/ JK as a parameter. The appropriate expressions for a, b, and c were used, as explained above. In making these plots, we used published values for the internal ion concentrations summarized in Table

483 mM

Na+

cl-

563 mM

L2The external

Internal concentrationb 220 mM 210 + 12 mM (SD) 200-240 mM (activity) 45 pmole/egg = 210 mM 40 mM 52 k 17 mM (SD) 0.16 Ki, = 35 mM 80 ma4 80 k 33 mM (SD) concentrations

Reference

Rothschild and Barnes, 1953 Steinhardt et al., 1971 Robinson, 1976

Rothschild and Barnes, 1953 Hori, 1965

Rothschild and Barnes, 1953 are those in sea-

water.

* The internal concentrations are composites of the values reported in the literature. The individual literature values are listed below each composite value. Except as indicated, the measurements are concentrations, rather than activities.

o-io-2o-

-3oEm (mV) -40

-80

-

I 0.1

I 0.2

, 0.3

I 0.4

I 0.5 JN.

I 0.6

I 0.7

I 0.8

I 0.9

I 1.0

JK FIG. 6. Calculated membrane potential as a function of the sodium to potassium flux ratio (&,A&). The plot is made for four different values of J,-,/JK . The curves were generated from Eq. (2). The marked point corresponds to the average value of JN,/& for the two experiments where both fluxes were measured on the same batch of eggs and the average value of Jc,/& (Table 2), and indicates a membrane potential of -70 mV.

JAFFE AND ROBINSON

Membrane

exception (Steinhardt et al., 19711, these are not available. The marked point in Fig. 6 corresponds to our average values of J&J, (for experiments done on eggs from the same female) and Jcl/JK from Table 2 and indicates a membrane potential of -70 mV. Figure 6 demonstrates that uncertainties in the flux ratio measurements could have only a small effect on our conclusion that the resting potential is near -70 mV. Even if both JN,/JK and J,-,/JK are doubled from the average values in Table 2, the predicted membrane potential from Fig. 6 is -62 mV. Since the membrane is primarily permeable to potassium, and the membrane potential is near the potassium equilibrium potential, the specific conductance can easily be estimated [Hodgkin, 1951, Eq. (1911: g = g, = RG JK = 2.0 x lo-” Rp’/cm2. This does not take into account the contribution to the conductance from sodium and chloride, so the actual conductance would be expected to be somewhat larger. If these ions are taken into account [Hodgkin and Katz, 1949, Eq. (S.O)], the calculated conductance is 2.4 x lo-” mho/cm”, usingP, = 1.3 x 10-scm/sec;P,,/P, = 5.1 x 1O-3 (calculated from Experiments 1 and 2 in Table 2, in which JNa and JK were measured simultaneously with eggs from a single female); P,,/P, = 2.4 x lop2 (calculated from average values of Jcl and KK in Table 2); and E,,, = -70 mV. In terms of resistance, this corresponds to 420 kn - cmS. Because of possible uncertainties in the above approach (discussed in some detail below) an alternate method for determining the state of the egg membrane was used. We varied the external potassium concentration between 3 and 30 mM and measured the effect on sodium influx. If the membrane were a pure potassium electrode, the potential would obey the Nernst equation:

of Urchin

Potential

223

Egg

Em=El

!!!?

F

nKi’

The passive sodium influx is related the membrane potential by:

F& Jm = PN, RT __ 1-

to

Na, exp (FE,/RT)

(Hodgkin and Katz, 1949). Although an action potential can be produced by the sea urchin egg cell, it is purely calcium dependent (Okamoto et al., 1977); therefore, sodium influx is, by definition, passive. The relationship between sodium influx and external potassium concentration can then be obtained by substitution:

JNa= PNa(In%)

(1 -F:iK,)

.

A plot of this equation (normalized to the influx at 10 u-&f K+) is shown in Fig. 7. Also shown in Fig. 7 are plots of similar equations derived under the assumptions that the sodium and chloride permeabilities were each one-tenth and one-hundredth of the potassium permeability. Clearly, the data best fit the assumption that the membrane potential is a potassium diffusion potential, with negligible contributions from either sodium or chloride. The potassium diffusion potential, calculated from the Nernst equation and

JN. JN.(lOmM

K’) ILo

II/,

,

3

~=pcl~o.olpK

IO 20 [K*]o (rnfd,

30

FIG. 7. Sodium influx as a function of external potassium concentration. Data were obtained by measuring Z2Na uptake during ZO-min pulses. Closed and open circles represent two different batches of eggs. The three curves were drawn with the indicated assumptions concerning the permeabilities of potassium, sodium, and chloride.

224 the potassium is -77 mV.

DEVELOPMENTALBIOLOGY

activities

listed in Table 3,

VOLUME 62, 1978

Horowitz et al., 1958). (ii) The activity of potassium in the sea urchin egg is known (Steinhardt et al., 1971). Intracellular seDISCUSSION questration of sodium may occur; however, We have determined the membrane po- even if the cytoplasmic sodium activity tential of the unfertilized sea urchin egg were zero, the predicted membrane potential from Eq. (2) would be -69 mV. (iii) by measuring influxes of sodium, potassium, and chloride. Using these fluxes and Only the existence of K+-K+ exchange difpublished ion concentrations, we have calfusion, then, could introduce an error culated the egg’s membrane potential by which would lead to a calculation of -70 means of the constant-field equations. The mV when the real potential was more posivalue of membrane potential determined tive. In human red blood cells, 20% of the from fluxes was observed with microelecpotassium efflux is exchange diffusion (Glynn and Karlish, 1975). As we have trodes. Membrane resistance and potaspointed out, a 20% error (or even a 50% sium selectivity determined by the two methods are also in agreement (Table 1). error) in our estimate of the passive potasWe have also measured the response of sium flux would not affect our conclusion the passive sodium influx to changes in that the membrane potential is about -70 mV. The possibility, however remote, the external potassium concentration and of an unprecedentedly large fraction of we found that it varied in a way that would be expected if the membrane potenpotassium exchange diffusion cannot be ruled out; it is for this reason that experitial were a potassium diffusion potential. ments relating sodium influx to external Our conclusion is that the membrane potential is approximately -70 mV, as potassium concentration were done. [It the result of a relatively small, but highly might seem that a more direct test would selective permeability to potassium. Be- be to measure potassium efflux as a funccause we disagree with several published tion of external potassium (e.g., Powers and Tupper, 1974). This is not so because reports, we discuss below various possible objections to our conclusion. We then pres- both of the possibilities between which we are trying to distinguish would give ent a possible explanation of the variation between different batches of eggs. the same result, qualitatively.] These results clearly show that the membrane Possible Sources of Error in Using Ion potential is dominated by potassium and Fluxes to Calculate Membrane Potential must be near its equilibrium potential of There are several possible sources of - 77 mV. If exchange diffusion of potassium error in using fluxes to calculate memor sodium exists at all, it must be a small brane potentials as in Eq. (2) and Fig. 6: fraction of the total. Both the microelectrode results (Fig. 4) an electrogenic sodium pump, exchange in sodium influx as a diffusion, single-file effects, ignorance of and the variation function of external potassium concentrainternal ionic activities. On careful examtion (Fig. 7) indicate that the membrane ination, however, only one of these, K+K+ exchange diffusion, remains as even a behaves as a potassium electrode even at possible difficulty in this particular case. 3 mM K,+ and thus the membrane potential would be -107 mV. However, the (i) An electrogenic sodium pump, Na+coefficients calculated from Na+ exchange diffusion, or Cl--Clex- permeability change diffusion would all cause us to set the data of Table 2 (PN,lPK = 5.1 x 10e3; membrane potential at too positive a P,,/P, = 2.4 x 10-Y predict [from Eq. (111 a membrane potential of -87 mV at 3 value, likewise for a “single-file” or “longpore” effect (Hodgkin and Keynes, 1955; mM KO+. The reason for this discrepancy is

JAFFE

AND ROBINSON

Membrane

Potential

of Urchin Egg

225

tion. Since the egg cell has a very high input resistance (about 640 m, Table l), even a very small leak around the microelectrode would greatly reduce the membrane potential. In the fertilized egg, due to the lower input resistance (about 80 MQ, Table 11, the leak around the microelectrode would have less effect on the membrane potential. This explains why, while the membrane potential of the unfertilized egg has been a matter of dispute, the membrane potential of the fertilized or activated egg has generally been reported to be close to -70 mV (Hiramoto, 1959; Steinhardt et al., 1971, 1972; Higashi and Kaneko, 1971; Ito and Yoshioka, 1972; Uehara and Katou, 1972; Steinhardt and Mazia, 1973; Tupper, 1973; Chambers et al., 1974; Steinhardt and Epel, 1974; Mazia et al., 1975). There have been several studies on the ion selectivity properties of sea urchin eggs with -lO-mV membrane potentials, all of which concluded that the membrane is primarily chloride permeable (Steinhardt et aZ., 1971; Higashi and Kaneko, 1971; Ito and Yoshioka, 1973). However, the shifts in membrane potential caused by varying chloride were very small, approximately 10 mV/lO-fold change, nowhere near the 57 mV/lO-fold change expected for a chloride-selective membrane. Such small potential shifts might result from an artifact such as a change in the liquid junction potential between the test The -IO-mV Egg Is Damaged solution and the cytoplasm exposed to the The gradual hyperpolarization from - 10 test solution at a leakage site. (See for mV toward the final membrane potential example, Hagiwara et al., 1960.) In any of -70 mV has been reported in other case, with such small effects of chloride chloride cannot be the only microelectrode studies of unfertilized sea substitution, urchin eggs, and it has been suggested (or even the principle) per-meant ion. Subthat it represents a recovery from penetrastitutions of sodium by choline or Tris, or tion damage (Higashi and Kaneko, 1971; of potassium by sodium, sometimes afIto and Yoshioka, 1972; Uehara and Ka- fected the membrane potential slightly, tou, 1972; Chambers et al., 1974; Mac- sometimes not. In summary, the ionic subKenzie and Chambers, 1977; Okamoto et stitution experiments do not explain the al., 1977). The concomitant increase in ionic basis of the -lO-mV membrane pomembrane resistance (Fig. 1; Chambers et tential. The simplest explanation of the al., 1974, Fig. 2) supports this interpretageneral lack of ion selectivity is that there

unclear; however, as pointed out earlier, the existence of a single-file effect for K+K+ or exchange diffusion of Cl would both cause an underestimate of PK relative to the other permeabilities and lead to the calculation of too positive a membrane potential, especially at low K,,+. The analysis used in this paper can be applied to other cells if the relevant fluxes and concentrations are known. For example, if the flux data of Robinson and Jaffe (1973) are substituted into Eq. (21, a membrane potential of -31 mV is predicted for the unfertilized Fucus egg. Originally, direct measurements of this membrane potential by microelectrodes yielded values of -19 (Bentrup, 1970) and -22 mV (Weisenseel and Jaffe, 1972). Later measurements indicated that the “true membrane potential of the unfertilized F. serratus egg is somewhat more negative than -33 mV” (Weisenseel and Jaffe, 1974). The case of the Fucus egg, however, is different in one important respect from the present case. The Na+-K+ and Cl--K+ flux ratios are much larger for Fucus than for S. purpuratus. An examination of Fig. 6 shows that at these higher flux ratios, the predicted membrane potential is far more sensitive to uncertainties in the flux ratios and also to uncertainties in intracellular ion activities. Therefore, we feel less certain that the tracer fluxes correctly predict the membrane potential of the Fucus egg.

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is a leak around the microelectrode. One further reason for believing that the -lO-mV egg is damaged is the observation by Higashi and Kaneko (1971) that eggs with -lO-mV membrane potentials were “not always fertilizable,” while eggs with larger steady potentials were “always fertilized.” ‘. The -70-mV Potential quence of Activation

Is Not a Conse-

Since the fertilized or activated egg has a membrane potential close to -70 mV, it has been suggested that the -70 mV potential of the “unfertilized” egg might be observed because the egg is activated. There is some evidence that activation of potassium permeability can occur independently of the cortical reaction (Steinhardt et al., 1973; Mazia et al., 1975), so the absence of fertilization membrane elevation does not prove that potassium permeability has not been activated. Our arguments against this suggestion are the following: (i) The resistances (measured with microelectrodes) and the potassium permeabilities (measured with tracers) of the unfertilized and fertilized eggs differ, by factors ranging from 2.5 to 18fold in different reports (Table 1; Chambers, 1949; Chambers and Chambers, 1949; Tyler and Monroy, 1959; Tupper, 1973). The only characteristic in which the unfertilized state resembles the activated state is membrane potential; otherwise the membranes are quite different. (ii) The tracer flux calculation of a -70 mV resting potential argues against the possibility of activation, since the methods of measuring fluxes are very gentle and very unlikely to activate the eggs. (iii) Sodium influx determinations in different external potassium concentrations, indicating a membrane potential near -70 mV, were also obtained with gentle methods. We do not deny that in some cases the eggs may have been overly mature or “slightly activated” when they were removed from the female. This possibility

VOLUME 62, 1978

will be discussed more in the next section. In some cases slight activation could have occurred as a result of the microelectrode technique. This possibility is suggested by the fact that, with prolonged penetrations, we occasionally have seen a gradual loss of the action potential and a decrease in resistance, while the potential remained at about -70 mV. We suspect that in some cases in which it took up to 30 min after penetration to reach the -70-mV potential, such an activation process may have been occurring, gradually overcoming the depolarizing effect of a leak around the microelectrode. However, with brief penetrations, the effect of the microelectrode is negligible, since we have noted that the penetrated and surrounding eggs show the same susceptibility to polyspermy, a direct reflection of the state of the membrane (Jaffe, 1976). In any case, these phenomena suggestive of activation never occurred in eggs which we classified types A and B in Table 1. If we limit our analysis to these eggs, our conclusion is still the same. For the three reasons stated above, we conclude that the unactivated, undamaged egg has a membrane potential of about -70 mV. Variability Pattern

among Batches of Eggs: of Membrane Maturation?

A

Between batches of eggs, we observed considerable variation in potassium influx, more than would be expected due to experimental error. This variability in potassium permeability was also evident in the variability of resistances measured with microelectrodes. Perhaps this variability in laboratory eggs could be related to the fact that the animals are induced to spawn at a time different from their natural cycle. Possibly, some of the eggs, although meiotically mature, have an immature membrane or an overly mature or “slightly activated” membrane. Maturing starfish eggs exhibit a minimum potassium permeability at the time of natural fertilization, and larger perme-

JAFFE AND ROBINSON

Membrane

abilities either before or after this time (Miyazaki et al., 1975b; Shen and Steinhard& 1976; Runnstrom, 1949). The mechanism of these changes in potassium permeability is unclear; there could be changes in the amount of membrane, in the number of potassium channels, or in the permeability of individual potassium channels. It appears that a similar pattern of membrane maturation occurs in the sea urchin egg. The germinal vesicle-stage oocyte and the fertilized or activated egg both have high potassium permeability relative to the unfertilized egg. (See Table 1; also Chambers, 1949; Chambers and Chambers, 1949; Tyler and Monroy, 1959; Steinhardt et al., 1972; Tupper, 1973. The unfertilized membrane is potassium selective, but the total potassium permeability is smaller.) Thus, the mature unfertilized egg is located in time between two states of higher potassium permeability. An egg removed from the adult slightly too early or slightly too late might have higher than normal potassium permeability. The results described in this paper represent work on approximately 100 females studied over a period of 12 months. Without observing the eggs for a period of several years, we cannot make any correlation of membrane properties with seasonality. It is clear, however, that eggs from any one female have similar membrane properties. Between batches of eggs, there are differences in the ability of the membrane to produce an action potential. Because the action potential serves a function, that of preventing polyspermy (Jaffe, 19761, we believe that the eggs which can produce an action potential are closest to the natural state. The tracer flux and microelectrode experiments were done in the laboratories of L. F. Jaffe and S. Hagiwara, respectively. We would like to thank them for their advice in conducting the experiments and preparing the manuscript. Financial support was provided by the NIH and the NSF. REFERENCES BENTRLJP, F. W. (1970). Elektrophysiologische

Un-

Potential

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tersuchungen am Ei von Fucus serratus: Das Membranpotential. Planta 94, 319-332. CHAMBERS, E. L. (1949). The uptake and loss of K4’ in the unfertilized and fertilized eggs of Strongylocentrotus purpuratus and Arbacia punctulata. Biol. Bull. 97, 251-252. CHAMBERS, E. L., and CHAMBERS, R. (1949). Ion exchanges and fertilization in echinoderm eggs. Amer. Natur. 83, 269-284. CHAMBERS, E. L., PRESSMAN, B. C., and ROSE, B. (1974). The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A. Biochem. Biophys. Res. Commun. 60, 126-132. EDDY, E. M., and SHAPIRO, B. M. (1976). Changes in the topography of the sea urchin egg after fertilization. J. Cell Biol. 71, 35-48. GLYNN, I. M., and KARLISH, S. J. D. (1975). The sodium pump. Annu. Reu. Physiol. 37, 13-55. GOLDMAN, D. E. (1943). Potential, impedance and rectification in membranes. J. Gen. Physiol. 27, 37-60. HAGIWARA, S., KUSANO, K., and SAITO, S. (1960). Membrane changes in crayfish stretch receptor neuron during synaptic inhibition and under action of gamma-aminobutyric acid. J. Neurophysiol. 23, 505-515. HAGIWARA, S., OZAWA, S., and SAND, 0. (1975). Voltage clamp analysis of two inward current mechanisms in the egg cell membrane of a starfish. J. Gen. Physiol. 65, 617-644. HIGASHI, A., and KANEKO, H. (1971). Membrane potential of sea urchin eggs and effect of external ions. Annot. Zool. Japon. 44, 65-75. HIRAMOTO, Y. (1959). Electric properties of echinoderm eggs. Embryologia 4, 219-235. HODGKIN, A. L. (1951). The ionic basis of electrical activity in nerve and muscle. Biol. Rev. Cambridge Phil. Sot. 26, 339-409. HODGKIN, A. L., and KATZ, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 108, 37-77. HODGKIN, A. L., and KEYNES, R. D. (1955). The potassium permeability of a giant nerve fibre. J. Physiol. 128, 61-88. HORI, R. (1965). The sodium and potassium content in unfertilized and fertilized eggs of the sea urchin. Embryologia 9, 34-39. HOROWICZ, P., GAGE, P. W., and EISENBERG, R. S. (1958). The role of the electrochemical gradient in determining potassium fluxes in frog striated muscle. J. Gen. Physiol. 51, 193s-203s. ITO, S., and YOSHIOKA, K. (1972). Real activation potential observed in sea urchin egg during fertilization. Exp. Cell Res. 72, 547-551. ITO, S., and YOSHIOKA, K. (1973). Effect of various ionic compositions upon the membrane potentials during activations of sea urchin eggs. Exp. Cell Res. 78, 191-200. JAFFE, L. A. (1976). Fast block to polyspermy in sea

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urchin eggs is electrically mediated. Nature (kmdon) 261, 68-71. MACKENZIE, D. O., and CHAMBERS, E. L. (1977). Fertilization of voltage clamped sea urchin eggs. Clin. Res. 25, 643a. MAZIA, D., SCHATTEN, G., and STEINHARDT, R. A. (1975). Turning on of activities in unfertilized sea urchin eggs: Correlation with changes of the surface. Proc. Nat. Acad. Sci. USA 72, 4469-4473. MIYAZAKI, S., OHMORI, H., and SASAKI, S. (1975a). Action potential and non-linear current-voltage relation in starfish oocytes. J. Physiol. 246,37-54. MIYAZAKI, S., OHMORI, H., and SASAKI, S. (197513). Potassium rectifications of the starfish oocyte membrane and their changes during oocyte maturation. J. Physiol. 246, 55-78. OKAMOTO, H., TAKAHASHI, K., and YAMASHITA, N. (1977). Ionic currents through the membrane of the mammalian oocyte and their comparison with those in the tunicate and sea urchin. J. Physiol. 267, 465-495. POWERS, R. D., and TUPPER, J. T. (1974). Some electrophysiological and permeability properties of the mouse egg. Develop. Biol. 38, 320-331. ROBINSON, K. R. (1976). Potassium is not compartmentalized within the unfertilized sea urchin egg. Develop. Biol. 48, 466-472. ROBINSON, K. R., and JAFFE, L. F. (1973). Ion movements in a developing fucoid egg. Develop. Biol. 35, 349-361. ROTHSCHILD, L., and BARNES, H. (1953). The inorganic constituents of the sea urchin egg. J. Exp. Biol. 30, 534-544. RUNNSTR~M, J. (1949). The mechanism of fertilization in metazoa. Advan. Enzymol. 9, 241-327. SHEN, S., and STEINHARDT, R. A. (1976). An electrophysiological study of the membrane properties of the immature and mature oocyte of the batstar,

VOLUME 62, 1978 Patiria miniata. Develop. Biol. 48, 148-162. STEINHARDT, R. A., and EPEL, D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. USA 71, 1915-1919. STEINHARDT, R. A., LUNDIN, L., and MAZIA, D. (1971). Bioelectric responses of the echinoderm egg to fertilization. Proc. Nat. Acad. Sci. USA 68, 2426-2430. STEINHARDT, R. A., and MAZIA, D. (1973). Development of K+ conductance and membrane potentials in unfertilized sea urchin eggs after exposure to NH,OH. Nature (London) 241, 400-401. STEINHARDT, R. A., SHEN, S., and MAZIA, D. (1972). Membrane potential, membrane resistance, and an energy requirement for the development of potassium conductance in the fertilization reaction of echinoderm eggs. Exp. Cell Res. 72, 195203. TUPPER, J. T. (1973). Potassium exchangeability, potassium permeability, and membrane potential: Some observations in relation to protein synthesis in the early echinoderm embryo. Develop. Biol. 32, 140-154. TYLER, A., and MONROY, A. (1959). Changes in rate of transfer of potassium across the membrane upon fertilization of eggs of Arbacia punctulata. J. Ezp. 2001. 142, 675-690. UEHARA, T., and KATOU, K. (1972). Changes of the membrane potential at the time of fertilization in the sea urchin egg with special reference to the fertilization wave. Develop. Growth Difi. 14, 175184. WEISENSEEL, M. H., and Jaffe, L. F. (1972). Membrane potential and impedance of developing fucoid eggs. Develop. Biol. 27, 555-574. WEISENSEEL, M. H., and JAFFE, L. F. (1974). Return to normal of Fucus egg membrane after microelectrode impalement. Exp. Cell Res. 89, 55-62.