DEVELOPMENTAL

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141,330-343 (1990)

Membrane Conductance Patterns during Fertilization Are Sperm Dependent in Two Sea Urchin Species R. E. KANE Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 96822 Accepted May 29, 1990 The influence of the egg and sperm on the conductance changes at fertilization in the sea urchin were investigated through cross-fertilization of two Hawaiian species, Ttipne-ustes gratilla and PseudoboZetia indiana. The current-voltage (I-V) relation, measured in voltage-clamped eggs at intervals over the period 2-16 min following the rise to a positive membrane potential that signals sperm attachment, differs significantly in the two species. The magnitude of the conductance change depends on the species of the fertilizing sperm in both homologous and heterologous crosses. This supports the hypothesis that currents during this period arise from sperm membrane channels incorporated into the egg at sperm-egg fusion. Measurements of conductance during the first 90 set, which includes the period of the major inward current correlated with cortical granule breakdown and elevation of the fertilization envelope, showed that the magnitude and timing of the maximum current also differed in the two species. This conductance change presumably involves an activation of egg membrane channels initiated by the sperm and would be expected to be characteristic of the egg species. However, in cross-fertilized eggs the magnitude and timing of the conductance change over this period also depends on the species of the sperm with little identifiable egg contribution, indicating that the fertilizing sperm can modulate the egg response to influence these events. o lsso Academic PWSS, IIIC. INTRODUCTION

Changes in membrane conductance associated with fertilization of the sea urchin egg give rise to a shift in membrane potential to more positive values (Steinhardt et al, 1971). The resting potential of the unfertilized urchin egg has been calculated to be -70 to -80 mV on the basis of independent flux data (Jaffe and Robinson, 1978; Chambers and de Armendi, 1979), but the potentials measured with microelectrodes are frequently more positive and this has led to some dispute on this point (Dale and de Santis, 1981). Failure to reach the calculated potential is generally attributed to electrodeinduced leaks in the high-resistance unfertilized egg (Hagiwara and Jaffe, 1979; Nuccitelli and Grey, 1984; Whitaker and Steinhardt, 1985). On fertilization the membrane potential rises to +lO to +20 mV; this change in potential is responsible for the fast block to polyspermy in the sea urchin, as sperm do not penetrate eggs at these potentials (Jaffe, 1976; Shen and Steinhardt, 1984). A permissive range of membrane potential for fertilization of the eggs of Lytechinus variegatus has been defined using voltage clamp procedures (Lynn and Chambers, 1984). Eggs clamped at more positive potentials show no apparent electrical response to sperm, while at more negative potentials the percentage of eggs undergoing activation (cortical granule breakdown and elevation of the fertilization envelope) without sperm entry increases. 0012-1606/90 $3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form resewed.

The currents associated with the conductance changes at fertilization in the sea urchin have been investigated with voltage clamp procedures and similar current patterns were observed during sperm attachment and fertilization in L. variegatus (Lynn et cd, 1988) and L. $ctu.s (David et al, 1988). The current profile associated with fertilization in L. variegatus has been divided into three phases (Lynn et al, 1988): a sharp current onset with a shoulder current continuing for approximately 10 set, followed by an increase to a peak inward current which cuts off at approximately 30 set, and then a slow decline of the remaining current over the same period during which eggs not voltage clamped return to a membrane potential of approximately -70 mV. There is good evidence that sperm attachment and membrane fusion give rise to the conductance changes of phase 1 (McCulloh and Chambers, 1985,1986), while the later conductance changes associated with cortical granule exocytosis and egg activation presumably involve the activation of egg channels by the sperm. The first stage of fusion may be transient and reversible (Whitaker et aZ., 1989), which could explain why, in attempts to relate the time course of the electrical and ultrastructural events during fertilization, the earliest point at which fusion of sperm and egg membranes could be detected was 5 set after the onset of the conductance increase (Long0 et al., 1986). Two species of Hawaiian sea urchins, Tripneustes gratilla and Pseudoboletia indiana, undergo membrane con330

R. E.

KANE

Conductance during Sea Urchin Fertilization

ductance changes in three phases, but have significantly different patterns of membrane conductance change when current-voltage (I-V) relations are measured in voltage clamped eggs during the period 2-16 min after fertilization (Kane and McCulloh, 1988). In hybrids of the two species both the measured currents and the pattern of current change during the period 2-8 min are characteristic of the fertilizing sperm (Kane, 1989). These observations support the hypothesis that the currents during this period result from the persisting activity of sperm channels introduced at fertilization (Chambers, 1989). A rapid, ramp-based I-V determination was used in the experiments reported here to extend these measurements of conductance change to the first 10 to 90 set following sperm-egg interaction, which includes the period of the peak inward current believed to arise from sperm-activated egg channels (Chambers, 1989). Membrane conductance changes during this period also differ significantly in the two species but, unexpectedly, both the magnitude and timing of the maximum current in hybrid crosses between them depended on the species of the fertilizing sperm. The precise interactions responsible are unknown, but the sperm determines the size and temporal profile of the membrane conductance changes throughout this period in these two urchin species. MATERIALS

AND

METHODS

Gametes. Gametes of the Hawaiian sea urchins T. gratilla and P. indiana were obtained by injecting 0.56 M

KC1 into the body cavity. Sperm were collected directly from the gonopore and stored “dry” at 0°C. Eggs were collected and washed 2~ in natural sea water (SW) and stored at room temperature (23-24°C). Eggs were dejellied before use by washing 2~ in 0.55 M NaCl, 0.01 M KCl, 5 mMTris, pH 8.3, returned to SW, and transferred to 35-mm plastic petri dishes previously treated with 0.0005-0.001% protamine sulfate to cause adherence (Steinhardt et al., 1971). For fertilization, dry sperm were diluted in the ratio of 2 ~1/40 ml of sea water and 10 ~1 of this dilute suspension added near the impaled egg. All experiments were done at 23-24°C. For cross-fertilizations the vitelline envelope of the eggs was removed with trypsin. A stock solution of 1 mg/ml of 2~ crystallized trypsin (ICN Biochemicals, Cleveland, OH) in sea water was prepared and stored at 0°C (maximum 1 day). The procedure of Epel(l970) was used for membrane removal, with a final trypsin concentration of 2.5 pg/ml in sea water used for treatment of the eggs of T. gratilla and 1 pg/ml for P. indiana. Samples of eggs were tested with heterologous sperm at intervals; although fertilization envelopes are not formed, the surface change at fertilization evident in phase contrast is adequate for scoring. When the per-

331

centage fertilization reached 90% or better, the trypsin solution was aspirated off and the eggs were washed 3~ in SW. With these trypsin concentrations, membrane removal required 20-30 min in both species. Trypsintreated eggs were impaled and fertilized immediately as spontaneous activation, indicated by surface changes and an increase in outward current, can occur after extended storage of treated eggs. Removal of the vitelline envelope had no detectable effect on the magnitude or rate of the conductance changes at fertilization when control and trypsin-treated eggs of each species were compared after fertilization with homologous sperm; all homologous fertilizations reported in the text used eggs which had not been trypsin-treated. Electrophysiological methods. Electrodes were pulled from l-mm glass tubing containing a glass fiber to aid filling (F. Haer, Brunswick, ME), using a vertical puller (Model 720, David Kopf Inst., Tujunga, CA). When filled with 0.5 M K,SO,, 20 mM NaCl and 0.5 mM K citrate (Lynn and Chambers, 1984) the electrodes used had resistances of 25-45 MQ. Painting these electrodes with clear nail polish to within approximately 100 pm of their tips reduced the transmural capacitance sufficiently to allow single electrode voltage clamping. The bath electrode was Ag-AgCl with a SW agar bridge. Eggs were impaled by means of a short (5-20 msec) oscillation induced by an increase in the negative capacitance. Electrical measurements and voltage clamping utilized an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) with membrane potential and current displayed on a Nicolet 301 digital oscilloscope (Nicolet Instruments, Madison WI). Single electrode voltage clamping (Wilson and Goldner, 1975; Finkel and Redman, 1984) was done at switching frequency of 3 kHz and filtered at 300 Hz. The input of the sample-and-hold amplifier (monitor output) was continuously observed on a Tektronix 2205 oscilloscope (Tektronix Instruments, Beaverton, OR) while adjusting the gain and phase of the clamp. Membrane potential and current were recorded on a Gould 220 recorder (Gould, Inc., Cleveland, OH) and these outputs were also digitized (Model VR-10, Instrutech Corp., Mineola, NY) and recorded on VCR tape (Sony Corp., Tokyo, Japan). Experimental protocols were set up and analyzed with the pClamp program (Version 5.0) of Axon Instruments. In the first program for I-V measurements current was measured at lo-mV intervals between +30 and -110 mV, each voltage step having a duration of 450 msec and a 25msec ramp to and from the holding potential of -20 mV. Steady-state currents averaged over 275 to 425 msec were plotted vs the clamped membrane potential; only the inward rectification in the eggs of P. indiana continued to increase over this period. Ramp I-V measurements used a negative-going ramp between

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+30 and -110 mV, with a total protocol duration of 1 sec. The protocol begins with a step from the holding potential (-10 or +lO mV) to +30 mV, followed by a 40-msec hold to allow the current to settle before beginning the measurement ramp with a duration of 840 msec, utilizing 420 data points between +30 and -110 mV (6 msec and three data points/mV). The voltage protocol used is illustrated on each figure. Currents are compared at -75 mV in all experiments and the calculated average and standard error is given for the number of cases in parentheses. The I-V plots illustrated in the figures are examples in which the currents at -75 mV approximated the average for all runs of that protocol. RESULTS

The Conductance Pattern at 2-16 min Following Attachment in T gratilla and P. indiana

Sperm

As in other sea urchin species, few of the eggs of !l! gratilla or P. indiana spontaneously attain a resting membrane potential in the range of -70 mV after impalement with a microelectrode. Current injection can be used to hyperpolarize the membrane which often results in a resting potential closer to -70 mV; this response presumably involves sealing of the electrode-induced leak (Chambers and de Armendi, (1979). A maximum of -0.5 nA was injected and reduced as the resistance increased, so as to maintain V, at approximately -80 mV; in most cases the current required to maintain -80 mV decreases to less than -0.1 nA within 10 min and then declines more slowly. Eggs which required more than -0.15 nA of current to maintain -80 mV after 20 min of impalement were not used. Eggs which had a resting potential near -75 mV responded to the fertilizing sperm with a typical action potential followed by an activation potential, which reaches +lO to +20 mV (Fig. 1A). When membrane potential is voltage clamped during fertilization, the current pattern in these eggs (Fig. 1B) has the same three phases as described by Lynn et al (1988) in L. variegatus, and which are indicated in the figure: phase 1, from the onset of the sperm-induced current to a shoulder maximum; phase 2, the period of major conductance increase ending at the point of maximum current, and phase 3, a rapid and then more gradual decline toward the holding current, defined as ending when the current has reached 10% of the maximum value (beyond the limit of the figure). When the membrane potential was clamped at -10 mV, in the permissive range for sperm entry, the current pattern was similar in the eggs of both Hawaiian species, but a shift of the voltage clamp to more negative potentials during phase 3 caused a much larger increase

VOLUME

141.1990

in current in T. gratilla than in P. indiana eggs. Currents were measured at a membrane potential of -75 mV, as this is close to the potassium reversal potential in the sea urchin egg (Jaffe and Robinson, 1978; Chambers and de Armendi, 1979) and currents at this potential should be insensitive to changes in potassium conductance. At 2 min after the rise to a positive membrane potential which signals sperm attachment, the current at -75 mV in T. gratilla eggs was more than -1 nA, while that in P. indiana eggs was approximately -0.2 nA. This conductance difference in the two species was further investigated by determining the current-voltage (I-V) relations during the period 2-16 min following sperm attachment. The I-V relation in unfertilized eggs of the two species was first measured at membrane potentials between +30 and -110 mV at intervals of 10 mV. The major difference in the resulting plots (Fig. 2) is the presence of inward rectification below -75 mV in the eggs of P. indiana, similar to that described in starfish eggs by Hagiwara and Takahashi (1974), which is much less evident in T. gratilla eggs. The voltage clamp was released before insemination to allow the eggs to shift to the positive fertilization potential following a successful sperm attachment, as this provides an unambiguous indication of fertilization and minimizes polyspermy. Eggs which did not rest at a membrane potential in the range of -75 mV were injected with sufficient current to maintain V, at -50 to -60 mV; the potential of these eggs rose rapidly to the same positive range following fertilization, but without the overshoot seen during the action potential. No difference was observed in the subsequent development of these eggs. Zero time in all experiments was the initial rise to the positive fertilization potential. At 1 min 50 set membrane potential was clamped at -20 mV and the I-V relationship measured at 2 min, using the same protocol as with the unfertilized egg. A clamped membrane potential of -20 mV was maintained and the I-V relation measured at 4, 8,12, and 16 min following sperm attachment. After the final I-V determination, the electrode was removed and the subsequent development of the zygote observed to insure that normal division followed. In each case the current measured in the unfertilized egg was subtracted from the 2- to 16-min current, so the I-V plots represent the steady-state conductance change in relation to the unfertilized egg. Successive I-V curves at 2,4, and 8 min for the two species are illustrated in Figs. 3A and 33. Inward current increases markedly at V, below -10 mV in T. gratilla at 2 min, while in P. indiana the increase in current at negative potentials is much smaller. Two-min currents at -75 mV in the two species are compared in Table 1. The inward currents in T. gratilla decline to = -0.20 nA by 8 min, while in P. indiana inward currents remain in the same range over the period 2 to 8

R. E. KANE 50

-

.5

PHASE 25

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Conductance during Sea Urchin Fertilization 1

2

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FIG. 1. (A) Membrane potential changes during fertilization of an egg of Z! gratiUa An action potential to +3 mV is followed approximately 40 set later by the activation potential, which rises to +16 mV. (B) Currents during fertilization of an egg of !l! gratilla, membrane potential voltage clamped at -10 mV. The three phases of current defined by Lynn et al. (1988) are identified.

min, often with a small increase in inward current below -40 mV and evidence of increased inward rectification at 4-8 min. Outward current at positive membrane potentials increases in the eggs of both species over the period 8-16 min (data not shown). This current increases with time and the reversal potential moves toward -70 mV; it presumably represents the developing K+ conductance dependent on cytoplasmic alkalinization @hen and Steinhardt, 1980).

The Conductance Pattern at 2-16 min following Sperm Attachment in H&rids of T gratilla and P. indiana The differing current patterns during fertilization in these two species suggested that the influence of sperm and egg on the conductance changes which follow fertilization might be distinguished in hybrids. P. indiana and T. gratilla can be cross-fertilized after removal of the vitelline envelope with trypsin. This procedure has

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FIG. 2. Steady-state current-voltage (I-V) relations in unfertilized eggs of Z! gratilla and P. indiana over the range of membrane potential from +30 to -110 mV. The 15-step voltage protocol used to generate the I-V curve is shown in the inset (holding potential -20 mV, step duration 500 msec).

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1.0 1

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FIG. 3. (A) Current-voltage relations in a Z! gratillu egg at 2, 4, and 8 min after sperm attachment. The 15-step voltage protocol used to generate these I-V curves is shown in the inset (holding potential -20 mV, step duration 500 msec). Currents in the unfertilized egg are subtracted in this and all following plots. The 2-min current at -75 mV in this example is -1.11 nA. (B) Current-voltage relations in a P. indiana egg at 2,4, and 8 min after sperm attachment. The 2-min current at -75 mV in this example is -0.16 nA.

no detectable effect on the conductance changes at fertilization as trypsin-treated eggs of either species fertilized with homologous sperm yielded current values indistinguishable from those measured in untreated eggs. Electrophysiological procedures and protocols used with the hybrids were the same as in control fertiliza-

tions. In both hybrid crosses the pattern of current change is very similar to those obtained in homologous fertilizations with the same sperm (Figs. 4A and 4B). The characteristic shape of the current vs voltage curve at 2 min in T. gratilla eggs fertilized by T. gratdla sperm is duplicated in P. indiana eggs fertilized by T. gratillu

R.E. TABLE

KANE

during Sea Urchin Fertilization

Conductance

1

CURRENTIN~AZ MIN AFTERSPERMATTACHMENT, MEASUREDAT-75 mV T. gratillu 0 T. gratilla 6 P. indiana 8 Note. Currents brane

potential T gratillu and ward current.

-1.22 -0.13

f 0.11 (n = 32) f 0.03 (72 = 14)

P. indiana P -1.18 -0.19

k 0.16 (n = 14) f 0.02 (n = l.4)

measured 2 min after sperm attachment at a memof -75 mV in homologous and heterologous crosses of P. indiam Values in nA + SE; negative indicates in-

sperm and the small increase in inward current often seen at 2-8 min in homologously fertilized P. indiana eggs also occurs in T gratilla eggs fertilized by P. indiana sperm. The measured currents at 2 min at a membrane potential of -75 mV are indistinguishable from those measured in homologous fertilizations with the same sperm, as illustrated in Table 1. The conductance at 2 min following fertilization in the hybrids thus depends on the fertilizing sperm, with no apparent influence exerted by the egg. By 8 min inward currents are small in both crosses. The small increase in inward rectification seen at 4-8 min in homologous crosses of P. indiana is also present when P. indiana eggs are fertilized by T. gratilla sperm and is the only egg-dependent conductance. Between 8- and 16-min outward currents increase in both hybrids, as in the homologous crosses. Ramp Measurement of Conductance Changes during Period lo-90 see following Sperm Attachment

the

The relative influence of sperm and egg during the earlier period of the major conductance increase, concurrent with cortical granule breakdown and elevation of the fertilization envelope, is of interest as it involves the activation of egg membrane channels by the sperm. Baseline currents are changing too rapidly to allow the steady-state I-V protocol to be used to acquire data during this period and voltage ramps were explored as means of rapidly obtaining conductance data. The voltage-sensitive channels of the egg, which are responsible for the action potential (Chambers and de Armendi, 1979; Hagiwara and Jaffe, 1979; David et al., 1988), are activated by positive-going ramps, but these channels are not activated by negative-going ramps. Conductance data for T gratilla and P. indiana eggs can be acquired rapidly by means of a single ramp from +30 to -110 mV. The ramp requires 840 msec and the total time for completion of the protocol is 1 sec. Current differences between 1 set ramps and slower ramps are in the range of the standard deviation of the holding current at -80 or -10 mV (x0.02 nA); the response to these ramps does

335

not change significantly until the protocol time is reduced to 250 msec or less. The conductance patterns measured with a 1-set ramp during homologous fertilizations were similar to those obtained with the steadystate 15-step protocol at 2,4,8, and 16 min and the average currents at -75 mV using the ramp protocol were also comparable to those obtained with the step protocol: the 2-min current in T. gratilla was -1.48 + 0.17 nA (n = 14) and in P. indiana was -0.22 + 0.02 nA (n = 16). To determine the I-V relation during the first 90 sec. of fertilization with the ramp protocol, the membrane potential must be voltage clamped at approximately 5 set after the rise to a positive membrane potential which signals fertilization. The eggs were held in discontinuous current clamp during insemination, as this reduces the tendency to oscillation at initiation of the voltage clamp. The holding potential between the ramps was +lO mV, the approximate fertilization potential reached by the eggs. Ramps were run and recorded at lo-see intervals between 10 to 90 sec. Ten seconds is the approximate duration of the current shoulder which defines phase 1 (Lynn et aC, 1988) and a shoulder is only rarely identifiable in plots of current vs time. A typical series of plots of current vs voltage for T gratilla is illustrated in Fig. 5A. The I-V relations show very little current at membrane potentials between +30 and -10 mV, with a reversal potential near +lO mV. The curves inflect at approximately -10 mV and during the first 20 to 30 set after fertilization the current-voltage relation is close to linear at more negative potentials. The plots effectively rotate about this inflection as their slope increases with time, until the maximum current (= -4 nA at -75 mV) is reached at 30 or 40 sec. These are apparent maxima, as the true maximum may fall between the ramps. After the maximum inward current is reached the plots gradually assume a curved shape at potentials between -10 and -110 mV, with reduced current values, similar to the those previously seen at 2 min. In P. indiana the maximum inward current occurs more quickly after the initial events of fertilization and is considerably smaller (Fig. 5B). The I-V relation at 10 set is rarely linear at potentials more negative than -10 mV and usually displays some curvature; the apparent maximum current ( = -1 nA at -75 mV) is reached at 10 or 20 sec. Currents following the maximum rapidly decrease to the small values previously measured at 2 min. Trypsin treatment of the unfertilized eggs of either species to remove the vitelline envelope had no detectable effect on the conductance changes during fertilization, as the shape of the I-V relation and the magnitude of the currents were indistinguishable in comparisons of control and trypsin-treated eggs. The maximum inward currents measured in the two species at a membrane potential of -75 mV are com-

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1.o A

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FIG. 4. (A) Current-voltage relations in a P. indiana 0 X Z! gratiUa 6 hybrid at 24, and 8 min after sperm attachment. The E-step voltage protocol used to generate these I-V curves is shown in the inset (holding potential -20 mV, step duration 500 msec). The 2-min current at -75 mV in this example is -1.23 nA. (B) Current-voltage relations in a T. gruttila P x P. indiana 6 hybrid at 2,4, and 8 min after sperm attachment. The 2-min current at -75 mV in this example is -0.22 nA.

pared in Table 2. Plots of current at -75 mV vs time illustrate the difference in size and timing of the maximum current in the two species (Fig. 6). Averaging the data from the ramp I-V measurements at lo-set intervals on the two species, the maximum current in T. grat&z occurs at 32 3- 2 set (n = lo), while in P. indiana the maximum current occurs at 16 -t 2 set (n = 9).

Ramp Measurements of Conductance the Period lo-90 set in H&rids

Changes during

This rapid measurement of conductance during the period of maximum inward current can then be applied to hybrids of T gratilla and P. indiana The conductance changes in eggs of P. indiana fertilized with T. gratilla

337

Conductance during Sea Urchin Fertilization

R.E. KANE 1.0

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.,

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FIG. 5. (A) Current-voltage relations in a T gratillu egg at 20, 30, and 50 set after sperm attachment. The voltage ramp generate these I-V curves is shown in the inset (holding potential of +lO mV indicated by dotted line, ramp +30 mV to -110 total protocol duration 1 set). Apparent maximum at 30 set; current at -75 mV in this example is -4.22 nA. (B) Current-voltage indiana egg at 20,30, and 50 set after sperm attachment. Apparent maximum at 20 set; current at -75 mV in this example

sperm (Fig. ‘7A) have a pattern and magnitude similar to that in l’! gratilla eggs fertilized with T gratilla sperm. The I-V relation for these hybrids is close to linear at potentials more negative than -10 mV for 2030 set and the apparent maximum inward current is reached at 30 or 40 sec. After the maximum current has been reached the plots become increasingly nonlinear

protocol used to mV in 340 msec, relations in a P. is -1.36 nA.

and assume the characteristic shape previously seen in homologously fertilized T gratilla eggs at 2 min. Conversely, when eggs of T. gratilla are fertilized with P. indiana sperm, the conductance changes resemble those in homologously fertilized P. indiana eggs (Fig. ‘7B). Currents at -75 mV in the hybrid crosses are given in Table 2; the maximum inward current measured in the

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DEVELOPMENTAL BIOLOGY TABLE

2

CURRENTIN nA AT MAXIMUM, MEASURED AT -75 mV T grutillu T. gratilh

d

P. indiana 8

0

P, indiana

-4.19 + 0.35 (n = 14) -1.15 + 0.13 (n = 7)

P

-4.29 + 0.29 (n = 6) -1.54 + 0.07 (n = 16)

Note. Maximum currents measured during fertilization at a membrane potential of -75 mV in homologous and heterologous crosses of T. gratilla and P. indiana, Values in nA 2 SE; negative indicates inward current.

hybrids cannot be distinguished from that measured in homologous fertilizations with the corresponding sperm. The average time at which the maximum current is reached is also characteristic of the fertilizing sperm, being 32 + 3 set in P. indiana P X T gratilla $ crosses (n = 6) and 20 + 5 set in T gratilla 0 X P. indiana 8 crosses (n = 7). Conductance Changes in Polyspermic Eggs

If the larger values of maximum current measured in hybrids of P. indiana 0 x T gratilla $ involve an increase in egg conductance stimulated by the foreign sperm, egg conductance might also be expected to increase in the presence of additional P. indiana sperm in polyspermie eggs. Polyspermy is blocked when V, is allowed to rise to the positive range at fertilization, but eggs whose

VOLUME 141,199O

membrane potential is voltage clamped in the permissive range lack this protection and the frequency of polyspermy increases with increasing sperm density. In dispermic eggs of P. indiana (indicated by the direct division to four cells, as these eggs are not sufficiently transparent to allow fertilization cones to be reliably counted), the average maximum inward current was -2.60 f 0.09 (n = 5) and in two eggs which cleaved irregularly to more than four cells the average maximum current rose to -4.04 + 0.28. Attempts to force higher levels of polyspermy by the addition of sperm suspensions 10x the usual concentration increased the maximum current, with the highest reaching -5.57 nA, greater than that measured in P. indianu eggs fertilized with T gratilla sperm. This egg, and several others with maximum inward currents above 3 nA, divided normally to two cells at first cleavage. This suggests that several additional sperm attached to the egg, acted to stimulate the maximum current and then separated, analogous to the events that occur in eggs which are activated and not fertilized by a single sperm (Lynn and Chambers, 1984, Lynn et aL, 1988). DISCUSSION

Membrane Conductance at 2-8 min Is Sperm Determined in T gratilla and P. indiana

During the period 2 to 8 min following sperm attachment, T. gratilla and P, indiana display distinctive membrane conductance patterns. The currents mea-

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FIG. 6. Current at a membrane potential of -75 mV during the period 10 to 90 set in the Z! gratilh egg of Fig. 5A and the P. indiunu egg of Fig. 5B. The magnitude and time of the maximum current in these two examples is close to the averages for the respective species. The line is a cubic spline interpolation of the data points.

R.E. KANE

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-60

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-60

-40

MEMBRANE

POTENTIAL (mV)

Q X P. indiana

-20

0

I

I

20

40

d

-1.0

2 5

-2.0

s is 2

-3.0

-4.0

20sec 30 set 50sec

--

-5.0

-120

-100

-60

-60

-40

-20

MEMBRANE

POTENTIAL (mV)

0

20

40

FIG. 7. (A) Current-voltage relations in a P. indiana P X !l! gratilla 6 hybrid at 20,30, and 50 set during fertilization. The voltage ramp protocol used to generate these I-V curves is shown in the inset (holding potential of +lO mV indicated by dotted line, ramp +30 mV to -110 mV in 340 msec, total protocol duration 1 set). Apparent maximum at 20 set; current at -75 mV in this example is -4.29 nA. (B) Current-voltage relations in a Z! gratillu P x P. indiana 8 hybrid at 20,30, and 50 set during fertilization. Apparent maximum at 20 set; current at -75 mV in this example is -1.25 nA.

sured at a membrane potential of -75 mV are approximately six times larger in the eggs of T. gratilla than in P. idiana at 2 min; they then decline in the former species so that currents are small and approximately equal in both species by 8 min. These marked differences

in the membrane conductance changes following fertilization in these two species and the ability to make interspecies crosses provide an opportunity to verify the postulated origin of this conductance from introduced sperm channels.

340

DEVELOPMENTALBIOLOGY V0~~~~141.1990

Lynn et al. (1988) have divided the current profile during fertilization into three phases, which were illustrated in Fig. 1B. These profiles are viewed (Chambers, 1989) as consisting of two components: component A, generated by a sperm-mediated conductance increase, is responsible for the shoulder current of phase 1; component B, generated by an increase in the conductance of the egg membrane in response to the sperm, is responsible for the major inward current of phase 2. Component B cuts off after its maximum, leaving the continuing decline of component A as the current seen in phase 3. The origin of the component A current from sperm channels was suggested by experiments using a “loose” patch clamp during fertilization of the eggs of L. variegatus (McCulloh and Chambers, 1985, 1986), which allowed conductance changes to be monitored in a membrane patch 5-10 pm in diameter. When the patch was outside the region of sperm attachment its conductance did not change during the currents of phase 1, but did increase transiently during the major inward current of phase 2. This indicates that the channels responsible for the phase 1 currents were localized outside the patch, possibly in the vicinity of the sperm, while the channels responsible for the component B current opened in a wavelike manner over the surface of the egg. In the next experiment sperm were added to the patch pipet, so the region of sperm attachment was limited to the membrane patch. The potential inside the pipet was voltage clamped with a sine wave and capacitance measured with a lock-in amplifier. In this case the conductance change responsible for the currents of phase 1 occurred in the membrane patch and thus was associated with the site of sperm attachment. The conductance increase was accompanied by an increase in capacitance, indicating further that the conductance change occurs at the time of membrane fusion, when sperm channels are incorporated into the egg plasma membrane. The continuing activity of introduced sperm channels as the basis of the current during phase 3 was supported by observations on L. variegatus eggs in which activation, but not fertilization, occurred: in these cases the sperm detaches from the egg and the putative sperm-dependent current of phase 3 is absent (Lynn et aZ., 1988). The hybrid crosses of T. gratilla and P. indiana provide independent evidence that the currents during phase 3 represent the continued activity of sperm channels introduced at fertilization. The characteristic I-V profiles and current magnitudes at 2-8 min in hybrids of these two species clearly depend on the fertilizing sperm, which can be attributed to the persistence of functional sperm channels in the egg membrane during phase 3. The persistence of sperm membrane proteins in the surface of the sea urchin zygote after fertilization has been demonstrated by Gunderson et al (1986) and

by Nishioka et al., (1987), using labeled antibodies to sperm-specific proteins. The only identifiable egg influence on the currents during this period is a small increase in inward rectification at potentials below -75 mV in the eggs of P. indiana fertilized by either sperm. Activation without fertilization also occurs in T. gratilla and P. indiana; as in L. variegates, the current returns to a value close to baseline after the major inward current in these eggs, indicating the absence of introduced sperm channels during phase 3 (data not shown). Membrane Conductance Changes during the Maximum Inward Current in T gratilla and P. indiana In T gratilla eggs I-V plots during the first minute following sperm attachment show very little current at positive potentials, with a reversal potential in the range of the positive fertilization potentials measured in unclamped eggs. There is inward rectification below -10 mV, with an almost linear current-voltage relation at negative membrane potentials. Current increases with time until the maximum inward current is reached at 30-40 set and following the maximum the relation shifts from linear and the current declines, displaying the characteristic curvature previously seen 2 min after fertilization with T gratilla sperm. In P. indiana the maximum current is considerably smaller and the process proceeds more quickly. The early I-V plots are already curved and the maximum current is reached by 20 set, followed by a rapid decline. The differences in response to fertilization in the two species are evident in the plot of current vs time at -75 mV (Fig. 6), which illustrates the more rapid attainment of a smaller maximum current in P. indiana as compared to T. gratilla. The similarity of the I-V curves in the two species at more positive potentials (Figs. 5A and 5B) causes the difference in maximum inward current between the two species to decline as the current vs time plots are shifted to this range. At -10 mV, in the permissive range for sperm entry and utilized for the fertilization of the voltage clamped egg of Fig. lB, the maximum current is small and similar in the two species, but continues to be reached more rapidly in P. indiana than in T. gratilla. Current-voltage relations at fertilization with similar characteristics have been measured in the eggs of the starfish, Mediaster aequalis by Lansman (1983). These eggs have a diameter of 1 mm and the currents are larger by a factor of 100 than those in the eggs of the Hawaiian sea urchins, in proportion to their larger surface area. The currents also extend over a longer period, attributable at least in part to the lower temperature of the experiments (15-17’C). The maximum inward current was -320 nA at 8 min in M. aequalis eggs and increased linearly at more negative V,, as it does in the

R. E. KANE

Conductance during

urchin eggs studied here; the reversal potential was approximately +8 mV, also similar to that in the urchin. The current declined to -100 nA at 17 min, comparable to the decline between maximum current and 2 min in the urchin eggs. The decline of the fertilization current during this period was not due to the development of an outward counter-current and the author concluded that the time course of the current reflected a time-dependent change in membrane conductance. A similar conclusion was reached in investigations of the currents during fertilization in the urchin L. p&us (David et al, 1988). Origin

of these Currents and their

Control by the Sperm

The experiments of McCulloh and Chambers, (1985, 1986), which provided clear evidence that the component A current of phase 1 originates at the site and time of sperm and egg membrane fusion, also indicated that the conductance change responsible for the component B current of phase 2 swept as a wave over the entire egg surface from the point of sperm attachment. This conductance change, which is responsible for the major inward current, might be expected to be characteristic of the egg rather than the sperm as it presumably results from the activation of constituent egg channels over a large area of membrane, far removed from the site of sperm attachment. Independent support for the role of constituent egg channels in the generation of the conductance change of phase 2 is provided by experiments demonstrating that this conductance change occurs after activation of sea urchin eggs with calcium ionophore, in the absence of sperm (Steinhardt and Epel, 1974; Chambers et aL, 1974). However, the membrane current measurements in control and hybrid embryos of T gratilla and P. indiana reported here do not support the combination of a characteristic egg-based current of fixed value with a sperm-based current as the basis of the maximum inward current. The value of the egg contribution to the maximum current can be approximated by subtracting an estimate of the continuing sperm current from the maximum current. The measured current at 90 set was used as an estimate of the sperm current, as the (longer duration) maximum current in T. gratilla is completed by this time, while the smaller currents in P. indiana are almost level during this period and insensitive to the time chosen (Fig. 6). Subtracting the average values and rounding, this yields an egg contribution to the maximum of -2.8 nA in T. gratilla and -1.3 nA in P. indiana (these currents are equivalent to component B in the terminology of Chambers (1988). The contribution of the T. gratilla egg to the maximum inward current when fertilized with P. indiana sperm is reduced to -1.1 nA and the contribution

Sea Urchin Fertilization

341

of the P. indianu egg when fertilized with T gratilla sperm increases to -2.7 nA, both clearly dependent on the fertilizing sperm. In these two urchin species, the temporal characteristics of the I-V relation during fertilization and the magnitude of both the sperm-based and the egg-based contributions to the maximum current depend on the sperm, with only the minor inward rectification of P. indiana being a distinguishable egg contribution. Calculations for polyspermic P. indiana eggs give an average value for the egg contribution to the maximum inward current of -2.3 nA in dispermic eggs and -3.6 nA in irregularly cleaving eggs; division by the estimated egg contribution of -1.3 nA in monspermic fertilization suggests an egg response to two and three sperm, respectively. In the case of the highest maximum current recorded in polyspermic eggs of P. indiana, dividing the calculated egg contribution of -5.2 nA by -1.3 nA suggests that four sperm were attached, but the subsequent division of this egg to two cells indicates that only one sperm was functional. These events may be a variant of those in eggs which are activated and not fertilized by a single sperm at more negative V, (Lynn et al, 1988); the attached sperm undergo a temporary fusion and are able to stimulate an increase in egg membrane conductance but are not incorporated in the zygote. The large value of inward current shows that the eggs of P. indiana are capable of conductance increases considerably larger than those measured after fertilization by !l! gratilla sperm. An increase in sodium conductance is believed to play a major role in the generation of the activation potential in the echinoderm egg (Steinhardt et ah, 1971; Ito and Yoshioka, 1973; Chambers and De Armendi, 1979) and more recent studies suggest that changes in Na+ and K+ conductance are involved, both in several species of sea urchin eggs (Lynn and Chambers, 19&1; Obata and Kuroda, 1987; David et al., 1988) and in a starfish egg (Lansman, 1983). Several of these authors have suggested that the conductance changes might be due to a cation channel nonselective to sodium and potassium, similar to that described in cardiac cells by Colquhoun et aZ., (1981). If the currents measured in homologous and heterologous crosses of T. gratilta and P. indiana result from the activation of similar channels in the egg membrane, then the difference in conductance induced by the sperm of the two species and its increase in polyspermic eggs imply that these eggs are capable of a graded response in the number of channels activated by the sperm. A direct relation between the number of channels opened and the degree of polyspermy has been demonstrated in eggs of the echiuroid worm, Urechis caupo (Jaffe et al, 1979). In these eggs the fertilizing sperm opens existing sodium channels in the egg mem-

342

DEVELOPMENTAL BIOLOGY

brane and in polyspermic eggs the number of channels opened, as measured by sodium uptake, increased linearly with the number of sperm entering. The channel described by Colquhoun et ab, (1981) is also calcium-activated and it-or separate calcium-activated Na+ and K+ channels-would provide a link between the membrane conductance changes and the calcium transient at fertilization in the urchin egg, whose timing is associated with the conductance change (Eisen et al, 1984). A calcium-activated sodium conductance has been shown to contribute to the long duration fertilization potential in the eggs of the nemertean worm CerebratuZus Zacteus (Kline et ah, 1986). If calcium-sensitive channels are involved in the conductance changes at fertilization in the urchin, then some difference in the calcium transient induced by these two species of sperm at fertilization would be necessary for the difference in the conductance response. The microinjection of inositol 1,4,5trisphosphate (IP,) into sea urchin eggs initiates a calcium transient in the cytoplasm and activates the egg (Whitaker and Irvine, 1984; Turner et aZ., 1986) and it has recently been reported that sea urchin sperm contain enough IP, to activate eggs (Iwasa et ah, 1989). A simple (or perhaps simplistic) explanation of the differing egg conductance responses initiated by the sperm of the two Hawaiian species might be based on differing contributions of IP, by the sperm at fertilization and a similar mechanism used to explain the increased conductance of polyspermic eggs. However, unlike the situation in Urechis caupo where Na+ channels are opened only the vicinity of the fertilizing sperm (Gould-Somero, 1981), the modulation by a sperm-contributed activator of a wave of conductance change passing over the entire surface of the sea urchin egg is more difficult to visualize, particularly if the increase in free calcium concentration stimulated by IP, proceeds by means of a positive feedback loop involving the calcium-stimulated production of IP, (Swann and Whitaker, 1986). The apparently complete control of the conductance response of the egg by the fertilizing sperm in T. gratilla and P. indiana provides a challenge to any proposed mechanism of the activation process. The author expresses his appreciation to Dr. E. L. Chambers for the opportunity of working in his laboratory and to Dr. D. M. McCulloh for many enlightening discussions. Neither should be held responsible for any errors herein, which are solely those of the author. This research was supported by National Institutes of Health RCMI Grant RR03061 to the University of Hawaii and by National Institutes of Health Grant GM14363. REFERENCES CHAMBERS, E. L. (1989). Fertilization in voltage-clamped sea urchin eggs. In “Mechanisms of egg activation” (R. Nuccitelli, G. N. Cherr, and W. H. Clark, Eds.), pp. l-18. Plenum, New York. CHAMBERS, E. L., and DE ARMENDI, J. (1979). Membrane potential of eggs of the sea urchin, Lytechinus variegatus. Exp. Cell Res. 122, 203-218.

VOLUME 141,199O

CHAMBERS, E. L., PRESSMAN, B. C., and ROSE, B. (1974). The activation of sea urchin eggs by the divalent ionophores A 23187 and X-537 A. Biochem Biophys. Res. &mm. 60,126-132. COLQUHOUN, D., NEHER, E., REUTER, H., and STEVENS, C. F. (1981). Ionic current channels activated by intracellular Cain cultured cardiac cells. Nature (London) 294,752-754. DALE, B., and DE SANTIS, A. (1981). Maturation and fertilization of the sea urchin oocyte: An electrophysiological study. Den BioL 85,474484. DAVID, C., HALLIWELL, J., and WHITAKER, M. (1988). Some properties of the membrane currents underlying the fertilization potential in sea urchin eggs. J. PhysioL (London) 402,139-154. EISEN, A., KIEHART, D. P., WIELAND, S. J., and REYNOLDS,G. T. (1984). Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs. J. Cell BioL 99,1647-1654. EPEL, D. (1970). Methods for the removal of the vitelline membrane of sea urchin eggs. II. Controlled exposure to trypsin to eliminate postfertilization clumping of embryos. E3cp. Cell Res. 61,69-70. FINKEL, A. S., and REDMAN, S. J. (1984). Theory and operation of a single microelectrode voltage clamp. J. Neurosci. Methods 11, lOl127. GOULD-SOMERO, M. (1981). Localized gating of egg Na+ channels by sperm. Nature (Landon) 291,254-256. GLJNDERSON,G. G., MEDILL, L., and SHAPIRO, B. M. (1986). Sperm surface proteins are incorporated into the egg membrane and cytoplasm after fertilization. Dev. BioL 113,207-217. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg membranes. Anna Rev. Biqhys. Bioeng. 8.385-416. HAGIWARA, S., and TAKAHASHI, K. (1974). The anomalous rectification and cation selectivity of the membrane of a starfish egg cell. J. Mew&r. BioL 18,61-80. ITO, S, and YOSHIOKA, K. (1973). Effect of various ionic compositions upon the membrane potentials during activation of sea urchin eggs. Ezp. Cell Res. 78,191-200. IWASA, K. H., EHRENSTEIN, G., DEFELICE, L. J., and RUSSELL, J. T. (1989). Sperm contain enough inositol 1,4,5-trisphosphate to activate eggs. J. Cell BioL 109, 128a. JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature (London) 261, 68-71. JAFFE, L. A., GOULD-SOMERO, M., and HOLLAND, L. (1979). Ionic mechanism of the fertilization potential of the marine worm, Urechis caupo (Echiura). J. Gen. PhysioL 73,469-492. JAFFE, L. A., and ROBINSON, K. R. (1978). Membrane potential of the unfertilized sea urchin egg. Dev. BioL 62,215-228. KANE, R. E. (1989). The postfertilization conductance pattern is sperm dependent in two sea urchin species. J. Cell Bid 109,126a. KANE, R. E., and MCCULLOH, D. M. (1988). Different channels appear after fertilization in two Hawaiian sea urchin species. J. CeU Bid 107,172a.

KLINE, D., JAFFE, L. A., and KADO, T. (1986). A calcium-activated sodium conductance contributes to the fertilization potential in the egg of the nemertean worm Cerebratulus la&us. Dev. BioL 117,184193. LANSMAN, J. B. (1983). Voltage-clamp study of the conductance activated at fertilization in the starfish egg. J. PhysioL (Lcmdon) 345, 353-372.

LONGO, F. J., LYNN, J. W., MCCULLOH, D. M., and CHAMBERS, E. L. (1986). Correlative ultrastructural and electrophysiological studies of sperm-egg interaction of the sea urchin Lytechinus wuriegatus. Dew. BioL 118,155-166. LYNN, J. W., and CHAMBERS, E. L. (1984). Voltage clamp studies of fertilization in sea urchin eggs. I. Effect of clamped membrane potential on sperm entry, activation, and development. Dew. BioL 102, 98-109.

LYNN, J. W., MCCULLOH, D. H., and CHAMBERS, E. L. (1988). Voltage

R. E. KANE

Conductance during Sea Urchin Fertilization

clamp studies of fertilization in sea urchin eggs. II. Current patterns in relation to sperm entry, nonentry, and activation. Dev. Biol 128,305-323. MCCULLOH, D. H., and CHAMBERS, E. L. (1985). Localization and propagation of membrane conductance changes during fertilization of the sea urchin Lytechinus variegates. J. CeU Biol. 101,230a. MCCULLOH, D. M., and CHAMBERS, E. L. (1986). When does the sperm fuse with the egg? J. Gen. Phvsiol 88,38a. NISHIOKA, D., PORTER, D. C., TRIMMER, J. S., and VACQUIER, V. D. (1987). Dispersal of sperm surface antigens in the plasma membranes of polyspermically fertilized sea urchin eggs. Exp. Cell Res. 173,628-632. NUCCITELLI, R., and GREY, R. D. (1984). Controversy over the fast, partial, temporary block to polyspermy in sea urchins: A reevaluation. Dev. Biol. 103, l-17. OBATA, S., and KURODA, H. (1987). The second component of the fertilization potential in sea urchin (Pseudocentrotus depressus) eggs involves both Na+ and K+ permeability. Dev. Biol. 122,432-438. SHEN, S. S., and STEINHARDT, R. A. (1980). Intracellular pH controls the development of new potassium conductance after fertilization of the sea urchin egg. Exp. Cell Res. 125,55-61. SHEN, S. S., and STEZNHARDT, R. A. (1984). Time and voltage windows

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for reversing the electrical block to fertilization. Proc Nat. Acad Sci USA 81,1436-1439. 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. SWANN, K., and WHITAKER, M. (1986). The part played by inositol trisphosphate in the propagation of the fertilization wave in sea urchin eggs. J. Cell Biol 103,2333-2342. TURNER, P. R., JAFFE, L. A., and FEIN, A. (1986). Regulation of cortical granule exocytosis in sea urchin eggs by inositol1,4,5-trisphosphate and GTP-binding protein. J. Cell Biol. 102,70-76. WHITAKER, M., and IRVINE, R. F. (1984). Inositol l,l,&trisphosphate microinjection activates sea urchin eggs. Nature (London) 312.636639. WHITAKER, M., SWANN, K., and CROSSLEY, I. (1989). What happens during the latent period at fertilization. In “Mechanisms of Egg Activation” (R. Nuccitelli, G. N. Cherr, and W. H. Clark, Eds.), pp. 157-171. Plenum, New York. WILSON, W. A., and GOLDNER, M. M. (1975). Voltage clamping with a single microelectrode. J. Neurobioiol. 6,411-422.