Controversy over the fast, partial, temporary block to polyspermy in sea urchins: A reevaluation

DEVELOPMENTAL

BIOLOGY

103, l-17 (198-i)

REVIEW Controversy over the Fast, Partial, Temporary Block to Polyspermy in Sea Urchins: A Reevaluation RICHARD NUCCITELLI AND ROBERT D. GREY Department of Zoology, Univeraity of California,Davis, California95616 Rec&ved October 11, 1989; amepted in revised form January 11, 1984

I. INTRODUCTION

species (reviewed by Gould-Somero and Jaffe, 1983, Jaffe and Gould, 1984, Whitaker and Steinhardt, 1982).2 Questions have recently been raised concerning the validity of the hypothesis that the fertilization potential is causally related to the FPT block at the plasma membrane in sea urchin eggs (Dale and Monroy, 1981). The challenge stems principally from reports that the magnitudes and temporal characteristics of the fertilization potentials in three Mediterranean species of sea urchins appear to differ strikingly from the comparable properties of the fertilization potential in species studied earlier by Jaffe (1976) and others. Using Strongyhxm trotu.s puywuratus, Jaffe found that unfertilized eggs with resting membrane potentials in the range of -60 to -70 mV responded to sperm by a rapid, action potentiallike depolarization to about +8 mV. Evidence that this “fertilization potential” was causally related to the fast block was as follows:

During the past decade considerable attention has focused on the hypothesis that the fertilizing spermatozoon initiates a block to polyspermy at the plasma membrane of the egg that is fast, partial, and temporary. This hypothesis was first proposed in 1952 by Rothschild and Swann who suggested that the wave of cortical granule exocytosis (cortical reaction) in sea urchin eggs was too slow to account for the predominance of monospermy. They postulated that the cortical reaction was preceded by a faster block, established within l-2 set after attachment of the fertilizing sperm. In contrast to the block established at the fertilization envelope by the cortical reaction, which is essentially absolute and permanent, the putative fast block was suggested to be partial and temporary’ (reviewed by Rothschild, 1956). In 1976, Laurinda Jaffe presented evidence that sea urchin eggs display a large, positive-going depolarization of the plasma membrane (termed the fertilization potential) within a few seconds after insemination, and that this change in potential alters the receptivity of the egg to sperm. The rapidity of the fertilization potential, and its occurrence well before the onset of the cortical reaction, suggested that the fast, partial and temporary (FPT) block was electrically mediated. Following publication of Jaffe’s first report, similar evidence for an ionic or electrically mediated block to polyspermy has been obtained from a variety of animal and plant

(1) Eggs that failed to depolarize to positive values became polyspermic and exhibited a small “second-step” depolarization. (2) Unfertilized eggs whose membrane potential was shifted to +5 mV or higher by injection of current could not be fertilized by the concentrations of sperm employed (106/ml), whereas most other eggs in the same dish were fertilized by this concentration. (3) When current was applied during fertilization in order to maintain the membrane potential of the egg at negative values, the egg became polyspermic.

i In much of the literature dealing with this hypothesis, the “relative speed” characteristic has achieved the most prominence, with the result that the mechanism proposed by Rothschild and Swann is often referred to as the “‘fast block” hypothesis. This abbreviated designation has apparently caused confusion in that some studies have overlooked the partial and temporary characteristics that were also key features of the Rothschild and Swann proposal. For the remainder of this review, therefore, we shall designate the block at the plasma membrane as the fast, partial, temporary (FPT) block.

z Not all species exhibit a sharply depolarizing fertilization potential. Large depolarizations do not occur at fertilization in medaka (fish) (Nuccitelli, 198Va,b), Pleurodelea (urodele amphibian) (Charbonneau et al, 1X43), or in hamster (Miyazaki and Igusa, 1981; Igusa and Miyazaki, 1983, Igusa et d, 1983), mouse (Jaffe et al, 1983; Iguaa et al, 1983), or rabbit (McCulloh et al, 1983). Sperm-egg fusion in some of these cases is insensitive to experimental alterations of membrane voltage. 1

Vcl12-16cW84 $3.00 Copyright All rights

0 19E4 by Academic Press, Inc. of repmduction in any form reserved.

2

DEVELOPMENTAL

BIOLOGY

In Paracentrotus lividus, Psammechinus microtuberculatus, and Sphaerechinua granularis, the species studied by Dale and his collaborators (DeFelice and Dale, 1974; Dale and DeSantis, 1981), the fertilization potentials appear to be much slower and their amplitude of depolarization far less than has been reported for St. purpuratw (JaRe, 1976) or L@echinus pictus (Chambers and DeArmehdi, 1979). The slower timing and lesser amplitude are related to the differences in resting membrane potentials of the unfertilized egg. In all the sea urchin species studied using microelectrode techniques, the rapid fertilization potentials are observed only in eggs with resting potentials of about -70 mV (see Section IV), Dale and DeSantis (1981) argue that such resting potentials are atypical, and that the majority of physiologically mature eggs of Pa, lividus and Sp. grand&s have resting membrane potentials of -10 to -30 mV, as measured by intracellular electrodes. If this claim is correct for these and other species, then the rapid, large amplitude fertilization potential proposed by Jaffe (1976) as the mechanism for the FPT block may be so rare in normal eggs as to be physiologically unimportant. The validity of the notion that the fertilization potential serves as an FPT block is further challenged by the report that in Sp. granularis and Pa. l&idus the fertilization potentials coincide with, rather than precede, the cortical reaction (Dale and DeSantis, 1981). If this is the typical relationship between these two events, then, in these species at least, the fertilization potential is probably too slow to meet the temporal characteristic required for an FPT block. Dale and DeSantis (1981) have suggested that the coincidence of the fertilization potential and the cortical reaction occurs in other species of sea urchins as well. The data from Pa liwidus and Sp. granularis suggest either that previous data obtained from St. purpuratus and L variegates may have been taken from atypical populations of eggs, or that the electrical block hypothesis is valid only for some species of sea urchins. Differences in procedure and interpretation are other obvious sources of discrepancy between data from different species reported by different investigators. In light of these discrepancies, and in view of the important issues raised by them, it is useful to reexamine the evidence for the electrical block hypothesis in sea urchin eggs. In comparison to other groups of animals, the literature on the electrical properties of sea urchin eggs is relatively abundant. It is therefore possible to begin to assess and compare the validity of various methods employed to measure these properties and, in some cases, to differentiate procedural artifacts from genuine differences between species. After -presenting a brief summary of the characteristics of polyspermy blocks (Section II) and of the ev-

VOLUME

103,13&1

idence that an FPT block is established at the plasma membrane in sea urchin eggs (Section III), our review will focus on three major questions that are central to the hypothesis that the fertilization potential provides the mechanism for the FPT block: (1) Are there truly major differences in the electrical properties of eggs from different species of sea urchins, or are the reported differences due to artifacts of procedure? (2) Which of the two reported patterns of fertilization potential (fast, high-amplitude, or slow, low-amplitude) is typical of the majority of healthy, physiologically mature eggs in a given species? (3) Does the typical fertilization potential meet the criteria for an FPT block, i.e., does it precede the cortical reaction, is it only a partial block, and is it only temporary in nature? II.

MAJOR

CHARACTERISTICS

OF

POLYSPER$iY

BLOCKS

In order to provide a perspective for the mechanism upon which the present review is focused it is useful to identify the four characteristics that distinguish the presently known mechanisms to prevent polyspermy. Elaboration of these characteristics can be obtained from the excellent review by Jaffe and Gould (1983); our purpose in the list that follows is primarily to provide definitions, with only brief reference to the best-studied species that display the characteristic. (1) Site. The best described sites of the block to polyspermy are the fertilization envelope (zona pellucida in mammals) and the plasma membrane. Other sites may include the jelly or cumulus that surrounds the fertilization envelope (see Schmell et a& 1983) and the perivitelline space that contains, at least for a time, the exudate from cortical granules. (2) Em. Some blocks that have been described are absolute, i.e., once established, the block excludes all sperm regardless of the concentration, age or vitality of the sperm in the medium surrounding the egg. The best known absolute blocks occur at the fertilization envelope in sea urchins, anurans, and some mammals. Absolute blocks have also been reported to.occur at the plasma membrane in rabbits (Braden et a& 1954), and Urechis (a marine worm) (Paul and Gould-Semero, 1976). Blocks that are not absolute in nature have been given the term “partial”; they are, by definition, mechanisms that reduce the probability of polyspermy, In contrast to absolute blocks, partial blocks can be experimentally overridden by high concentrations of viablesperm. Partial blocks have been described in the extrhcellular investments that lie peripheral to the fertilization envelope or zona and at the plasma membrane. The partial block at the plasma membrane, which is the subject of this review, has been most extensively studied in sea urchins and in Urechis.

NUCXXTELLI

1

'0

AND

Contw

GREY

I

I

I

I

20

40

60

80 Time

3

over the Fast Block to Polyapermy

I 100 (set9

I 120

L 140

I 160

1 180

200

FIG. 1. Time course of the establishment of the absolute block to polyspermy in Psammechinus miliaris. Eggs were inseminated at 0 time with high concentrations of sperm (9.11 X lO’/ml) to insure near-instantaneous fertilization. Adapted from Rothschild and Swann (1952, Fig. 3).

(3) Duration Blocks may be either permanent or temporary. In general, blocks of the absolute type are permanent. The partial block at the plasma membrane is temporary in all cases studied to date; the effective duration of partial blocks at other sites has not been described. (4) Relative speed When both partial, temporary blocks and absolute permanent blocks occur in the same egg, the terms “fast block” and “slow block” are often used to indicate the relative timing of the two.

the percentage of polyspermic eggs at each interval (Fig. 1). The maximum level of polyspermy occurred at about 60 set (range: 17-94 set at IS-lS”C!), after which time the percentage of polyspermic eggs remained constant. Similar measurements have been made for St. drobachiensis (Ginzberg, 1964) and St. purpuratus (Byrd and Collins, 19’75), yielding times of ,about 180 set (at 8°C) and 30 set (at 16’C), respectively. The absolute, permanent block is therefore not established instantaneously, and, in the absence of any other protective mechanism, the egg appears to remain vulnerable to

III. EVIDENCE FOR THE EXISTENCE OF A FAST, PARTIAL, AND TEMPORARY BLOCK TO POLYSPERMY IN SEA URCHIN EGGS

As early as 1919, E. E. Just noted that supernumerary sperm failed to enter eggs of Echinarachnius parma even prior to any visibly detectable initiation of the exocytotic cortical reaction or elevation of the fertilization envelope. Eggs of this species became polyspermic only with “heavy” insemination. The possibility that a polyspermy block precedes the cortical reaction was, however, first given serious consideration only following careful measurement of the time required to establish the absolute, permanent block that is mediated by the exocytosis of cortical granules. To measure the time at which the absolute, permanent block becomes functional, Rothschild and Swann (1952) fixed eggs of Ps. m&aris at intervals following insemination with high concentrations of sperm and calculated

TABLE

1

LAG TIME BETWEEN DEMONSTRATED ARRIVAL SPERM AND ONSET OF THE CORTICAL

Soeci& s

* Time between ‘Time between cTime between in this species is

OF THE FERTILIZING REACTION

Lag time (eec)

Reference

12 11.6 13.4” 18.8 18.4b” 30-40b 37.5 40

Dale and DeSantis, 1931 Allen and Griffin, 1963 Just, 1919 Epel et al, 197’7 Schatten and Huleer, 1933 Paul and Epel, 1971 J&e et a& 1978 Ginzburg, 1964

sperm engulfment and onset of cortical reaction. insemination and the onset of the cortical reaction. onset of fertilization potential and onset of cortical 20.6 eec.

reaction

4

DEVELOPMENTAL

BIOLOGY

supernumerary sperm for at least one-half minute in all three species. The interval between arrival of the Arst successful sperm and establishment of the absolute permanent block is composed of two distinct periods. First is an experimentally demonstrable “lag” period between attachment of the fertilizing sperm and onset of the cortical reaction (Table 1). This period, which appears to be relatively constant in any one species, ranges from 12 to 40 set among different species; a part of these differences may be attributable to the temperature of the region in which the species reside. The lag period is important because the egg is probably completely unprotected by the absolute permanent block during this time. The remainder of the time required for establishment of the absolute block is taken by the propagation of the wave of exocytosis that begins at the site of sperm entry (Table 2). Presumably, the egg is partially protected from polyspermy during this period, at least in those regions where the fertilization envelope has been elevated. Is the egg likely to be challenged by a second sperm during its period of vulnerability? The answer is dependent upon the ratio of motile, functional sperm to eggs in the fertilizing medium (see discussion of this point by Byrd and Collins, 1975). Since the actual concentration of sperm in natural mating conditions is unknown for sea urchins, investigators have studied the responsiveness of the eggs to a wide range of sperm concentrations. DeFelice and Dale (1979) examined the relationship between sperm concentration and successful sperm-egg collision’ by measuring sperm-triggered depolarizations of immature (germinal vesicle stage) oocytes. Oocytes at this stage do not fire a fertilization potential and do not prepagate a cortical reaction (Dale and DeSantis, 1981), thereby making it possible to measure the rate of successful sperm-egg collision without interference by these events. Since these measurements detect the rate qf successful fusion, and not simply the rate of collision of sperm with eggs, they should provide a reasonable estimate of the actual polyspermy threat posed by the various concentrations of sperm employed in experiments. As shown in Fig. 2, successful fusions increase monotonically over a wide range of sperm concentrations. It is important to note that at concentrations used for many studies of polyspermy in sea urchins (lO’/ml), sperm successfully fuse at a rate of slightly less than once per’second. Similar rates would generate *We use the term “collision” to indicate the sperm-egg interaction event(s) that result in depolarization of the egg’s plasma membrane. The exact physical relationship between the sperm and the egg at the time of the depolarization is unknown.

VOLUME

103,1984 TABLE

TIME

ELAPSED

Species Echinarachnius paw Strong¢rotus purpkratus St purpwatus Paracentrotus m&aria St drobachiensis

2

BETWEEN INITIATION AND COMPLETION OF THE EXOCYTOTIC WAVE

Time (se4

Temperature (“C)

Reference

16

?

Just, 1919

20-30 41 20

16 16 18

90

8

Paul and Epel, 1971 Jaffe et aL, 1978 Rothschild and Swann, 1949 Allen and Griffin, 1958 Ginzburg, 1964

lo-20 successful sperm-egg fusions during the lag period that precedes the cortical reaction in many species. The first attempt to test the hypothesis that a block of some sort is established prior to the cortical reaction was made by Rothschild and Swann (1952) who compared the egg’s “receptivity” to the first successful sperm with its receptivity to subsequent sperm during the period prior to establishment of the permanent block. They used as their measure of receptivity the rates of successful sperm-egg fusions: rates observed for initial fertilization were compared with rates at which polyspermy occurred. Rothschild and Swann expressed receptivity in terms of an abstract value “a” and provided appropriate statistical confirmation of its accuracy. A more intuitively obvious presentation of the differences in receptivity observed by Rothschild and Swann is shown in Fig. 3, in which we have compared the rate of the first successful fusion with the rate of fusion by supernumerary sperm.4 This comparison makes clear, as did Rothschild and Swann’s (Yvalues, that even during the first 20 set (i.e., prior to onset of the cortical reaction), eggs fuse with supernumerary sperm at a rate much slower than that for the first sperm. Rothschild and Swann’s data have recently been reanalyzed by Whitaker and Steinhardt (1982, Appendix B), who confirm the conclusion that receptivity of the egg is reduced by the fusion of the first sperm. The important consequence of this change is that the gtmbability of polyspermy is sig’ Rothschild and Swann measured rates over a wide range of sperm concentrations and were careful to adjust their data to take the obvious effect of sperm concentration into account. Most of the rate measurements used for the calculation of a values for unfertilized eggs were from dejellied eggs, while those used for fertilized eggs were from jellied eggs (Rothschild and Swann, 1952, Fig. 6). Since jelly increusca the probability of sperm-egg fusion (Rothschild and Swann, 1951), it is unlikely that the lower receptivity of already-fertilized eggs is due to this variable. The two rates replotted in Fig. 3 of this review are from experiments that used only jellied eggs.

NUCC~TELLI

AND

Controversy over the Fast Block to Polyspewny

GREY

.

l l l

.

.

.i

A

. .

10” 10’ Sperm Concentration

10’ (Number/ml)

FIG. 2. The relationship between sperm concentration and frequency of collision with the plasma membrane of immature oocytes of Psammechinw microt~ is or Pamcentrotzls lividus. “Collision” frequency was detected by step depolarizations of the oocyte’s plasma membrane. From DeFelice and Dale, 1979.

nificantly reduced (but not eliminated) during the interval between fusion of the first sperm and completion of the absolute block. The reduction in receptivity therefore fits the definition of a partial block to polyspermy; and since it occurs prior to the cortical reaction and vitelline envelope elevation, it also meets the criterion of a fast block. Byrd and Collins (1975) measured the kinetics of’ sperm-egg fusion in St. puvratus and concluded that no fast block-either absolute or partial-existed in this species (Fig. 4a). But our reexamination of their published data leads us to disagree with their conclusion that a partial fast block is absent. Indeed, when one compares the kinetics of fusion of the first sperm (which occurs within 3-5 set in their experiments) with the rates at which supernumerary sperm fuse (i.e., at times after 3-5 set), the latter rate is slower by a factor of 4 (Fig. 4). More recent results by Collins (cited by Epel and Vacquier, 1978) are also consistent with the concept of a rapid partial block in St. purpuratus. Collins, in effect, “titrated” the receptivity of the egg to sperm by first determining the concentration required to yield a synchronous cortical reaction in 100% of the eggs in the population. At this concentration, eggs were monospermic or slightly polyspermic. He found that the sperm concentration must be increased above this level by at least 20-fold before significant levels of polyspermy began to occur. This finding agrees with Rothschild and Swarm’s (1952) estimate of a 20-fold reduction in receptivity after the initial sperm-egg fusion. A similar observation was made with Pa m+otuber&at~

5

(DeFelice and Dale, 1979, Table 2): No significant increase in polyspermy was observed when the sperm con& centration was increased lo-fold from lo5 to lo6 sperm per milliliter, but a large increase in the percentage of polyspermy occurred when a concentration of lo7 sperm per milliliter was used. A somewhat different analysis of fertilization rates was undertaken by Presley and Baker (1970) using Echinus esculentus, a species in which elevation of the fertilization envelope is not completed until about 1.52 min after the fusion of the first sperm. Their results (Fig. 5) show clearly that the incidence of monospermy is far greater than predicted if receptivity of the egg surface were unchanged by the first sperm-egg fusion. Whitaker and Steinhardt (1982, Appendices A and B) have performed a further analysis of Presley and Baker’s data and corroborate the original authors’ conclusion that some mechanism operates prior to the cortical reaction to reduce the probability of polyspermy. Additional evidence for a polyspermy block preceding the cortical reaction comes from studies of the effect of nicotine, an agent long known to induce polyspermy (Hertwig and Hertwig, 1337). At high concentrations (Hagstrom and Allen, 1956), nicotine interferes with the cortical reaction and at least part of its polyspermyinducing capacity may be due to that effect. But nicotine also induces polyspermy at concentrations that do not 100

j

i l 80

0 Time

(set

after

insemination)

FIG. 3. An example of the contrast between the rate of initial fertilization (first) sperm-egg fusion and the rate of fusion of supernumerary sperm, as seen in data from two separate experiments by Rothschild and Swarm (1952, Figs. 2 and 3a) that employed jellied eggs and similar sperm concentrations (6.9 X lO’/ml and 9.1 X lo”/ ml, respectively). The difference in rates observed in these individual experiments illustrates the general trend observed in numerous experiments designed to measure the change in receptivity ((u) induced by the first sperm.

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DEVELOPMENTAL BIOLOGY

VOLUME 103, 1934

idence strongly supports the hypothesis that a fast, partial block to polyspermy occurs in sea urchin eggs. O-

IV. THE ELECTRICAL PROPERTIES OF THE SEA URCHIN EGG

.o -

,o -

Time

01”” 012345

’ Time

of additionof SDS(S) 10

bet

after

15

ineemination)

FIG. 4. Data (inset) from Byrd and Collins (1975, Fig. 2) replotted to expand the period of transition from monospermy to polyspermy (0, % fert.; 0, number of sperm nuclei per egg). Eggs of St. purpurutus were fertilized at 0 time by 8 X 10’ sperm/ml. Supernumerary sperm were killed with dilute SDS at intervals indicated by data points. Egga were subsequently fixed and scored for sperm nuclei.

The controversy over the hypothesis that the fertilization potential is an integral part of the mechanism of the FPT block centers on reported differences in the pattern and timing of the fertilization potential in fertilized eggs; these differences, in turn, are related to reported differences in the resting membrane potential in unfertilized eggs. At issue is the question of which of the reported patterns of fertilization potentials and which of the reported values for resting potentials best represent the physiological properties of mature eggs in the natural population. It is therefore essential to consider in some detail those fundamental electrical properties that are crucial for understanding the different patterns and values that have appeared in the literature. The electrical measurements published to date for sea urchin eggs are summarized in Table 3, which includes 26 papers on 14 species. Our survey of the literature shows significant variability in the values reported for the three parameters that are central to the electrical block hypothesis: the resting membrane potential, the action potential, and the fertilization potential. We shall therefore discuss the physiological significance of these parameters, in terms of the electrical block hypothesis, and assess the possible causes of the variability in their measurement. Resting Potential

block the cortical reaction. Further, it has the effect of increasing the rate of successful sperm-egg fusions even before the cortical reaction begins (Presley and Baker, 1970). This effect clearly argues for the existence of some polyspermy regulating mechanism preceding the cortical reaction. To summarize this brief survey, there is clear and compelling evidence that in most, if not all, species of sea urchins: (1) the egg remains unprotected by the absolute block during the interval between fusion of the first sperm and initiation of the cortical reaction, and may be only partially protected until the cortical reaction is completed; (2) at sperm concentrations 2 105/ ml, and in the absence of any:other protective mechanism, there is a high probability of successful fusion of at least one supernumerary sperm prior to the completion of the absolute block; and (3) the actual rate of successful sperm-egg fusion is substantially reduced after the arrival of the first sperm and before the completion of the absolute block. We conclude that this ev-

of the Un&rtilized

Egg

The value of the resting potential in the unfertilized egg is critically important because, as will be apparent, the shape of the curve that describes the fertilization potential depends on the level of the resting potential. As Table 3 shows, the most commonly reported membrane potential (in 13 papers) is around -10 mV, but there are also numerous reports (in 16 papers) that some eggs exhibit a more negative value of about -70 mV. It is interesting to note that the reported values of resting potentials do not vary continuously, but form a distinctly bimodal distribution (Fig. 6). Furthermore, in more than one-half of the papers the authors report that their data indeed fall into these two major populations of low and high resting potentials. What accounts for these contradictory sets of values for the resting potential? One technical problem that can affect the value reported by intracellular electrodes is the seal between the membrane and the electrode, and even a minor leak can cause spurious readings in the range of -10 mV (see Appendix). Measurements of

NUCCITELLI AND GREY

Cantmversyover the Fast Block to Polyspmmy

7

ioa

50

Number

of male

pronuclei

in egg

FIG. 5. Comparison of the actual levels of polyspermy (bars) and the levels predicted from a Poisson distribution (broken line) shown by Presley and Baker (1970, Text-Fig. 7B and C). Eggs of Echinue ~SW!Q&AS were fertilized by 5 X 10’ sperm/ml. Supernumerary sperm were 1killed by KC1 at 15 (left) or 80 set (right). Eggs were later fixed and scored for number of sperm nuclei.

the input resistance of the plasma membrane gives a good indication of the tightness of the membrane-electrode seal, since leaks would obviously result in low resistance values. Jaffe and Robinson (1978) pointed out the good correlation between resting potentials near -70 mV and high input resistance values; this correlation is documented in Table 4. Earlier, Ito, and Yoshidka (1972) had also suggested that the less negative values of the resting potential were the result of a poor electrode seal. They found that in normal seawater only 7% of the eggs from H. pulcherri~~ indicated a -40 mV resting potential, with the remainder averaging -10 mV. Because an elevated external Ca2+ concentration resulted in even more negative resting potentials (-56 mV), they speculated that an improved membrane-electrode seal had been induced by the calcium. Another observation which supports the idea that the membraneelectrode seal is critical is the fast sweep oscilloscope record of membrane voltage changes that accompany penetration by a microelectrode. Such records indicate that the membrane potential which is first measured upon impalement averages -66 mV and is followed within milliseconds by a depolarization initiated by the leak around the electrode (Chambers and deArmend& 1979). At least two reports, however, seem at variance with the idea that the -10 mV group of resting potentials are the result of leaks. Uehara and Katou (1972) found that when eggs were treated with detergent, which would

be expected to make them mare leaky, they often exhibited a rapid hyperpolarization to resting potentials in the -70 mV range. Somewhat similarly, Dale and DeSantis (1981) found that eggs from “suboptimal animals” or those “aged” in the laboratory also show a large incidence of highly negative resting potentials. Such “aged” eggs may not form tight membrane-electrode seals, but insufficient input resistance data were provided to check the correlation with membrane potential. These observations of more negative resting potentials in eggs which one might expect to be relatively leaky appear to contradict the above mentioned correlation between high resistance and more negative membrane potentials. We will speculate on a possible explanation for these contradictory observations in the Appendix, but present them here to convey the difficulty in determining the true resting potential using the microelectrode technique alone. Some investigators have argued that the -10 mV range of resting potentials does not result from a leak around the electrode since impalement by a second electrode does not significantly alter the resting potential recorded by the first electrode (see, for example, Dale and DeSantis, 1981). The “second impalement” test for leakage may be useful in nerve cells, but it is not a valid method for determining leakage around the electrode in unfertilized sea urchin eggs because the inherent resistivity of these cells is lo- to loo-fold higher than in most nerve cells. In cells with such high inherent re-

DEVELOPMENTAL BIOLOGY

8

VOLUME103,1984

a second leak will have only minor impact on inherently low resistance properties. Fortunately, the the resting potential if leakage is already occurring resting potential in unfertilized eggs has also been measured by a method that does not rely on impalement around the first electrode (see Appendix for elaboration sistivity,

of this point). It becomes apparent from these considerations that the controversy over whether values in the -10 mV range are leakage artifacts will be difficult to settle solely on the basis of microelectrode recording procedures. The issue is not decided by the correlation between high resistance and highly negative resting potentials since it can be argued that eggs with the -10 mV range of resting potentials simply have plasma membranes with

procedures. This alternative method involves simply the measurement of tracer ion fluxes across the plasma membrane. Since the voltage across that membrane is one of the forces that influences ion flux, it is possible to calculate the membrane potential from ion flux data. This technique does not damage the plasma membrane, as microelectrode impalement does, and it also has the advantage of measuring a population of eggs rather

TABLE3 FERTILIZATIONPOTENTIALMEASUREMENTS

Species

Year 1959 1971 1971 1972 1972

Hemicatrotua pl&mrrimzuc

1972 1972 1973 1973 1973

H. pu.lcMmua Temnopleuw Lpi&A Lgtechinw H. pukherrimus

1974 1976 1977 1978 1978 1919 1979 1979

L variepatue St~looentmtus

1979

1980 1981 1981 1982 1982 1982 1983

Lgtechinus pictua H. pu&herrimua L pi&e H. pukherrimwr

St nudus St. purparatw, Pameentmtus L u&ego&s L pi&la Psamntechinus Pa. lividus Pa lividw Pa. mtiiaris A. lixub St. purpumtu.3 Pa lividua Sphaerechinw A. punctulata

torewmatieun

prpumtua

lividus

macrotuberculo.tw

granularis

St purpuratus Pa 1ividu.a L variegatua Echinou, esculentup, Pa miliarb

Av. unfert. resting potential

Highest resting potential

-9 -8 -12 -6 to -14 -10 f l(20)

-15

-10 f 4 (82) -10 rt 4 (42) -10 -8 to -15 -12 f 5 (157) -56 f 25 (4)’ -33 + 32 (3) -70 + 6 (21) -7od -75 f 3 (27) -16 -75 + 1 (12) -75 -8 to -16

-70 -28 + 9 (18) -61 zk 9 (10)’ -42 -40 -47b -15 -58 -70 -78

IN SEA

Resistance (KC. cm-‘)

Fert. potential peak

0.35 4.6-19.8 -

+10 +5 +14 -23 f 3 (6)

-

URCHIN

EGGS

Fertilized membrane potential -65 -60 -80 to -70 -73

+1+ 4.4 (29) -24 + 14 (29) +7 + 7 (32) -14 + 5 (32) +10 -60 +12 -31 2 15 (62)

-80 -80

85 f 48 (3) 386 f 50 (8) 4-18 324 + 36 (16)’ -

+21 +12 + 7 (8) +12 +20 + 3.5 (7)

-8 to -15

-80

-751 -10 to -30

-90

-72 -70 to -8of -18 f 1 (35) -

Action potential threshold

-

Reference

-40

Hiram&o Steinhardt, et al Higashi; Higashi and Kaneko Steinhardt et o.l Uehara and Katou

-

Ito and Yoshioka Ito and Yoshioka Steinhardt and Maaia fipper Ito and Yoshioka

-

-76 -54 f 2 (6) -70 -

-80 -50 -40 -50 -80 -

-

-

-50

-

Tagiietti

10-50’

-10’ -

-

-

Jaffe Dale and DeSantis

-85

-

+20

-70

-

-90’ -80”

307

-67 5~ 2 (35) -

-40 -50

Jaffe and Tilney (reported by Schuel and Schuel) Jaffe et aL Dale et al. Hiilser and Schatten Whitaker and Steinhardt

23 * 0.5 (35) -

+17 f - 11 9 + 1 (35)

Chambers, et al. Jaffe Okamoto et al Jaffe and Robinson Dale et al Chambers and deArmendi Shen and Steinhardt DeFeiice and Dale

Note Values are in mV (*SD, N in parentheses). o 0.3% myristyl sulfate, laurylsulfate detergent added for 2-3 min. bObserved after 26 min in 1 mM NH,OH seawater at pH 9.1. ‘In 333 mM CaClr seawater. ‘In 30 mitf Ca artificial seawater. ‘This value assumes a surface area threefold larger than that of a smooth sphere 114 pm in diameter. This is the increase in surface area found using electron microscopy on other sea urchin species (Eddy and Shapiro, 1977; Schroeder, 1978). fin 1 m&f nicotine. g This value assumes a surface area threefold larger than that of a smooth sphere 90 pm in diameter. A While only a minority of eggs exhibit this high stable resting potential, they conclude from the initial E,,, within 0.5 msec of impalement that -80 mV is the true A’,.

NUCCITELLI

0

0

FIG. 6. Distribution potentials.

-20

-40 Em (mV)

GREY

AND

sea urchin egg resting

than a single egg. The calculation of resting potential requires only the values for the influx of Na+, K+, and Cl-, as well as the internal and external concentrations of these ions. These parameters have been measured in unfertilized eggs of St. purpuratus (Jaffe and Robinson, 1979) and in I, variegate (Chambers and deArmendi, 1979), yielding a calculated resting potential of -70 and -78 mV, respectively. This technique therefore corroborates the more negative resting potential values obtained by. microelectrode measurements in these same species.6 It is important to note that these influx measurements are made between 16 and 60 min after spawning (Jaffe and Robinson, 1978, Fig. 5). This is quite comparable to the “age” of the eggs used for electro6 It is interesting that the ion flux technique has not as yet revealed the bimodal distribution of potential values produced by the microelectrode technique in these and other species. We speculate on possible explanations of the bimodal distribution of values obtained by the microelectrode technique in the Appendix.

TABLE SPECIFIC

Species Less negative resting potential Hem~entrotua palhrrimm &techinus pictua Par-aentrocW, lividw, L

varifzgatua

More negative resting potential L variegates L uariwatua Strun&ocentmtua pwpuratua St pwquratus

RESISTANCE

4

MEASUREMENTS

Technique

Specific resistance (k&cm-r)

Microelectrode Microelectrode Microelectrode Microelectrode

0.35 5-26 10-50 23

Microelectrode Tracer flux Microelectrode Tracer flux

9

over the Fast Block to Polysmy

physiological studies, so one cannot argue that these tracer measurements use an “aged’ population of eggs. We therefore conclude that, in these two species at least, the true resting potential of the egg lies in the range of -70 to -80 mV, and we suggest that ion flux measurements will yield similar values for other species of sea urchins as well. In fact, the K+ and Cl- fluxes measured in Pa. Ziviclus (Christen et aL, 1979) are quite comparable to those values found in the other two species whose tracer fluxes have been determined, predicting a highly negative membrane potential. Therefore, unless the Na+ flux in Pa. lividus is found to be strikingly different from these other species, one would expect a similar highly negative resting potential in this species as well (in contrast to the values observed with electrode impalement). We can now begin to answer the question posed in the Introduction regarding species differences in electrical properties. In the two species in which the resting potential has been determined by two independent techniques, the resting potential falls in the highly negative range of approximately -70 mV. The less negative values (-10 mV range) reported for other species have, as yet, been determined only by the microelectrode impalement procedure which is subject to error because of leakage. Thus far there has been no clear proof that leakage artifacts are not responsible for the low resting potential values reported for these species, the “second impalement” test notwithstanding. In the absence of corroborating data from ion flux measurements, the -10 mV range of resting potentials in unfertilized sea urchin eggs must be viewed with skepticism, as must the conclusion that genuine species differences are responsible for the different values observed.

-80

-60

of published unfertilized

i%ntroversy

325” 386 429

ON SEA URCHIN

EGGS

Reference

E, (mv)

-15

-6 to -14 -10 to -36 -18 -75 -78 -75 -70

Hiramoto, 1959 Steinhardt et al, 1972 Dale and DeSantis, 1981 Hiilser and Schatten, 1982 Chambers Chambers Jaffe and Jaffe and

and deArmendi, and deArmend& Robinson, 1978 Robinson, 1978

1979 1979

*This value is calculated using a surface area threefold larger than that of a smooth sphere 114 pm in diameter. This is the increase in surface area found using electron microscopy on other sea urchin species (Eddy and Shapiro, 1977; Schroeder, 1978).

10

DEVELOPMENTAL BIOLOGY

Action Potential Action potentials have now been observed in eggs from six different species of sea urchins (Table 3), and a question of vital importance to the electrical block hypothesis is whether the capacity to fire an action potential is a universal characteristic of sea urchin eggs, or whether it is a property unique to certain species, or even perhaps an event confined to atypical eggs of certain species. By the term, “action potential” we refer to a large, rapid change in the voltage across the membrane which is triggered by a much smaller voltage change. This large voltage change results from the opening of a population of voltage-sensitive ion channels and exhibits two welldefined properties: it is very rapid, usually reaching its peak depolarization in less than 1 set; its amplitude is fixed and can be influenced only by the variations in the concentration of the specific ion species involved or by channel blockers. That unfertilized eggs from some species of sea urchins are capable of firing an action potential similar to that observed in nerve or muscle has been known since 1972 when Uehara and Katou detected “depolarizing spikes” in some, but not all, eggs of H. pulcherrimus. The spikes were triggered by a brief detergent treatment, but only in eggs with a relatively negative resting potential of -60 mV. Similarly, in unfertilized eggs of L variegatus, Chambers et al. (1974) observed “spike-like depolarizations” following addition of divalent cation ionophore, X537A, but, again, only in those few eggs that registered a large, negative resting potential upon impalement with an electrode. In the other sea urchin species thus far examined, action potentials are present only in eggs with resting potentials more negative than -40 mV (Jaffe, 1976; Okamoto et d, 1977; Chambers and DeArmendi, 1979; Hiilser and Schatten, 1982; Whitaker and Steinhardt, 1933). This voltage-sensitive characteristic is believed to be a property of the channels in the plasma membrane that regulate the flux of ions responsible for the rapid depolarization spike of the action potential. The ion involved in the action potential of sea urchin eggs is apparently Ca2+, since the action potential is nearly abolished when the concentration of calcium in the external medium is reduced to 10m4 M (Chambers and deArmendi, 1979). The absence of action potentials is unfertilized eggs with less negative resting potentials is explained by an important characteristic of these voltage-sensitive Ca2’ channels: when the membrane potential falls to a value less negative than -40 to -50 mV, they will no longer open and are said to be inactivated (Jaffe, 1976; Okamoto et uL, 1977; Chambers and deArmendi 1979; Whitaker and Steinhardt, 1933). Thus, in eggs with resting potenta values we positive than abut -50 m V, the f9rtilization

VOLUME 103,19&i

potential will lack a rapid clepolarizatti (action potent&d) component. As we will later show, the presence or absence of an action potential governs the relative speed of the fertilization potential. If eggs truly lack an action potential, it is doubtful that their fertilization potential will be rapid enough to serve as a mechanism for the FPT block. We can best address the issue of the action potential’s role in fertilization and the question of its universality in sea urchin eggs in the context of the fertilization potential. Fertilization Potential The fertilization potential in sea urchin eggs is a composite of several successive events involving rapid and discrete changes in the ionic permeability of the plasma membrane. The question of whether the fertilization potential acts as an FPT block hinges, in part, on the overall kinetics of this succession of membrane alterations. As previously indicated, the fertilization potential must precede the cortical reaction in order to qualify as a fast block to polyspermy. Our survey of the literature reveals at least two patterns of fertilization potential in sea urchin eggs, only one of which meets the temporal requirements of an FPT block. Before discussing these patterns and addressing the issue of which represents the physiological properties of a healthy population of eggs, we shall first discuss the several known ionic events that can contribute to the composite fertilization potential and thereby affect its kinetics. The relative order and contribution of each of these events are illustrated by a model of the fertilization potential that incorporates all ionic components thus far reported for sea urchin eggs (Fig. 7, solid line). This model assumes a highly negative resting potential as indicated by the ion tracer data discussed earlier, and include six phases. The first phase corresponds to a step depolarization triggered by a successful sperm-egg collision. This leads very quickly to the second phase, the rapid depolarization resulting from Ca2+-dependent action potential. There is then a slight repolarization after the action potential peak (Phase 3) which leads to a brief plateau (Phase 4). Next, there often follows a transient depolarizing phase which is due to an increase in Na+ permeability (Phase 5). Finally, there is a slow repolarization back to the resting potential (Phase 6). This model describes the main components of the fertilization potential in the form which we feel is most representative of the majority of eggs. We will now discuss the evidence supporting it. The first phase in the fertilization potential can be best observed on a millisecond time scale and is usually not apparent in the slower recordings made in eggs with

NUCCITELLI

25~

AND

GREY

*CD-l-

-

0

Controversy cnwr the Fast Block to Polyspermy

11

Q-03

I 20

I

I 40 Tlmr

1 60

‘4 ”

I 520

I 540

(a)

‘7. A model of the fertilization potential for the sea urchin egg (solid line). The model incorporates the six major phases (circled numbers) thus far observed upon fertilization of eggs with resting potentials in the -70 mV range. The dashed line represents the generalized pattern of Class II fertilization potentials (see text) which exhibits most of the same phases as the model, but is lacking Phases 2 and 3. FIG.

highly negative resting potentials in normal seawater because it is overshadowed by the rapid action potential (Phase 2) that follows it. In eggs with highly negative resting potentials this first phase is most easily seen in low Ca2+ seawater in which Phase 2 is markedly suppressed (Fig. 8). It is also obvious in eggs with less negative resting potentials since these eggs lack the rapidly rising depolarization phase, This “step depolarization” was first reported by Uehara and Katou (1972, replotted in Fig. 1OC here) and is also apparent in Figs. 1 and 3 of Ito and Yoshioka (1973) and in all of the fertilization potentials replotted in Fig. 10B of this review. Dale, and colleagues (1978) first showed that the step depolarization is accompanied by an increase in membrane noise and corresponds to the first electrical indication of sperm-egg interaction. The correspondence between the step depolarization and spermegg interaction was first reported by Dale and DeSantis (1981) and has recently been confirmed using video anal-

FIG. 8. Fertilization potential of L varifgatus egg in low Ca” (lo-’ M) seawater. Abscissa: time in units shown; ordinate: mV. Arrow marks the start of the depolarization in inseminated egg;, horizontal bracket: interval during which fertilization membrane (FM) elevation occurred. Dotted line near end of trace indicates a 5.5-min break during which the membrane potential steadily decreased. Temp. 25°C. Reproduced, with permission, from Chambers and deArmendi (19’79).

ysis in which the bioelectric recording of a microelectrode-impaled egg was electronically superimposed over the microscope image of the same egg at insemination (Schatten and Hiilser, 1983). The second phase is the very rapid depolarization, or action potential, which reaches a positive voltage in less than one second, and generally overshoots the ensuing plateau level. This was first reported by Higashi and Kaneko (1971, Fig. 7) in H. plcherrimus and was described in more detail by Jaffe in St. purpurat&i (1976) and by Chambers and deArmendi in L varkgatus (1979). When external Ca2+ is reduced loo-fold to 0.1 mM, the threshold for the action potential is not reached and the rapid depolarization fails to occur. Under this condition the Phase 1 step depolarization is still present, but there is a 12-set flat interval before the depolarization of Phase 5 (Fig. 8). This striking effect of low Ca2+ medium is strong evidence that Phases 2-4 involve the Ca2’ action potential. (Again, it is important to note that this action potential can be triggered only if the membrane potential is more negative than -40 mV. At less negative values the Ca2+ channels will no longer open and are said to be “inactivated.“) The return to the plateau level comprises Phase 3 of the fertilization potential and lasts 5-15 sec. The ionic mechanisms contributing to this step are not understood. The plateau itself (Phase 4), often exhibits a small, slower depolarization (Phase 5) due mainly to an increase in Na+ permeability, and its magnitude varies with changes in the external Na+ concentration (Steinhardt et al, 1971; Chambers and deArmendi, 1979). This relationship is illustrated by the experiment in Fig. 9 in which the plateau level is greatly reduced when the egg is in a low Na+ medium. In Phases 4 and 5, the membrane potential remains positive for 40-80 set after

12

DEVELOPMENTAL

BIOLOGY

+8mV @..--_____---_-_- ______- -___-----______----------. -2o-

-- Y

mV-40-

A

-6O1osec

-8Oj -20

,- - Y

mV-40 -60 11,

d

FM

B 1Osec -

VOLUME

103, 1934

fourth phase. The last two phases can be quite similar to the last phases of the Class I type in shape, time of onset, and duration (compare L. variegates in Figs. 10A and 10B). The third phase may occur at the same time as cortical granule exocytosis (Dale and DeSantis, 1981). The most slowly rising fertilization potential, recorded in H. pddwrrim~, is intermediate between the two major classes just described (Fig. 1OC). When the two major classes of fertilization potentials are compared, one striking difference is the absence of a rapidly rising depolarization in the Class II group, suggesting that the action potential, mediated by volt-

FIG. 9. Fertilization potentials of L variegut~ eggs in low Na+ (4.8 mM) seawater. Abscissa: time (see scale); ordinate: mV. Arrow and bracket as in Fig. 8. (A) Initial membrane potential more negative, and (B) initial membrane potential less negative than the threshold potential of action potential mechanism. Dotted lines are 50-set breaks in the traces during which the membrane potential gradually rose. Subsequent to peak at y, polarization occurred to -70 mV by 9.5 min (A) and 8.0 min (B). Temp., 22°C. Reproduced with permission, from Chambers and deArmendi (1979). Copyright, 19’79, Academic Press.

the initial depolarization. In the final phase (Phase 6) the membrane potential slowly returns to the highly negative, prefertilization level in about 8 min due to an increase in K+ permeability (Steinhardt et aL, 1972) which may be influenced by the increase in intracellular pH (Shen and Steinhardt, 1980). Fertilization potentials from six different sea urchin species are replotted on the same time scale in Fig. 10. Two common patterns emerge from these data (Class I and Class II) and exhibit a good correlation with the initial resting potential. The first class of fertilization potentials (Fig. lOA), recorded from eggs with highly negative resting potentials (-45 to -85 mV), has the six components described in our model, although, as mentioned, the first phase is normally not detected on this time scale (but can be seen in ion alteration experiments). The dominant feature of this class of fertilization potentials is the rapid Ca2+-dependent action potential that transforms the membrane potential of the egg to a positive value in less than one second. The depolarized level is maintained by an increased permeability of the plasma membrane to Na+, that corresponds to the fifth phase or component in our model. The second class of fertilization potentials is observed in eggs with less negative resting potentials (Fig. 10B). This class exhibits a four-component pattern. The first phase is again the small step depolarization of a few millivolts (Dale et uL, 1978); this phase is more easily seen in this class than in Class I. The step depolarization is often followed by a steady voltage level for about 10 set (Phase 2). A positive-going phase constitutes the third component, and the repolarization makes up the

-70

J

:

FIG. 10. Fertilization potentials measured in six species of sea urchin. (A) Those measured with highly negative initial resting potentials: solid line, L variegatua (replotted from Chambers and dedrmendi, 19’79, Fig. 5A); dashed line, St. purpuratua(replotted from Jaffe, 1980, Fig. 1A); dash-dot line, L pictus (replotted from Holland and Gould, unpublished data); dotted line, A. punctulato (replotted from Jaffe and Tilney, unpublished, but also found in Schuel and Schuel, 1981, Fig. 5). (B) Those measured fertilization potentials with less negative initial resting potentials: solid line, L variegatus (replotted from Hiilser and Schatten, 1982, Fig. 3h); dashed line, P. lividus (replotted from Taglietti, 1979, Fig. la); dash-dot line, also P. lividue (replotted from Dale and DeSantis, 1981, Fig. 5a). (Cl) The most slowly rising fertilization potential: H. pulcherrinrus; two examples (solid line replotted from Ito and Yoshioka, 1973, Fig. 1; dashed line replotted from Uehara and Kato, 1972, Fig. 3).

NUCCITELLI AND GREY

Contmersy

age-sensitive Ca 2+ channels, is not functioning in this group. The contrast is illustrated in Fig. ‘7, in which the dashed line represents the generalized Class II pattern. The lack of an action potential component in this class of records is not surprising, however, since the recorded resting potentials are clearly less negative than the threshold for the voltage-sensitive Ca2+ channels that are responsible for the action potential. Since the rapid action potential component is missing from Class II, the time required for depolarization of the plasma membrane is much greater than in Class I. In the Class II group, then, the positive-going phase of the fertilization potential is probably too slow to serve as an FPT block. But do these records, obtained solely from microelectrode impalement, accurately reflect the electrical properties of these species? We are led by this question back to the problem of the resting potential. If the true resting potential is the most commonly reported value from microelectrode data of -10 mV, then the action potential cannot be triggered at fertilization and the Class II pattern may be valid. If, however, the -10 mV resting potential is the result of leakage caused by impalement, then the Class II pattern may be an artifact. It would therefore be very helpful to know whether an egg which is not impaled with a microelectrode exhibits an action potential upon fertilization. A recent study by Whitaker and Steinhardt (1983) asked that exact question for E. esculentus and Ps. miliaris. By contacting the egg surface with a large microelectrode and applying a gentle suction, action potentials can be detected without actual impalement. They found that 91% of the population of eggs in these two species exhibited a large, rapid spike-like potential change upon fertilization. This would suggest that most eggs in the population must fall into the more negative resting potential group, i.e., Class I in our survey. In view of these results, what conclusions can be drawn about the Class II group of fertilization potentials? Do they truly represent the majority of the population in the species from which the samples were drawn, thereby reflecting a species-specific characteristic, or are they artifacts of procedure? Since the evidence for the Class II pattern rests solely on the impalement technique, we believe that corroboration of the low resting potential and absence of an action potential by noninvasive procedures is required before the pattern can be attributed to a genuine species difference. In the species to which they have been applied thus far, the noninvasive techniques have detected the highly negative resting potential and the rapid action potential, characteristic of the Class I pattern. We are led by this evidence, and by the previously discussed considerations, to the conclusion that this pattern probably more ac-

13

mmr the Fast Block to F’olyspermy

curately represents the electrical response of the sea urchin egg to the fertilizing sperm. V. THE

FERTILIZATION POTENTIAL AS A MECHANISM FOR THE FPT BLOCK TO POLYSPERMY

Does the Fertilization Potential Fit the Temporal Characteristics of the FPT Block? To be effective, a fast block mechanism must communicate the occurrence of the first successful spermegg collision (fusion?) to the entire egg surface within a second or so, based on the collision frequency data described in Section III. A rapid membrane potential change such as an action potential, could meet this requirement. In fact, an action potential-like depolarization is the only known cellular signal fast enough to do so since any chemical messenger would be temporally limited by diffusion across the loo-pm diameter of the cell. Even if the cytoplasm of the egg had no barriers, a diffusion-mediated signal would require about 4 set to cross the egg for a small ion such as Na+, and 8 set for a molecule the size of glucose. The rapid, positive-going depolarization feature of the Class I pattern clearly meets the “relative speed” or “fast” criterion for the FPT block, in that it changes the voltage across the entire plasma membrane by about 80 mV in loo-150 ms (Fig. 10A). The Class II pattern does not fit the temporal requirements of the FPT block, and should future evidence prove it not to be the artifact we presume it to be, the electrical block hypothesis would apparently not apply to species displaying this pattern.6 The second temporal requirement is that the fast block mechanism be temporary, since many species of sea urchin eggs can be refertilized if the permanent block (the fertilization envelope) is removed. Since the membrane voltage in the Class I pattern returns within about 90 set to a negative, nonblocking potential, this pattern is consistent with the temporal pattern of a transient block at the plasma membrane. So far as we are aware, there have as yet been no attempts to correlate the pattern of repolarization with receptivity of the plasma membrane to sperm. Can the Egg Plasma Membrane’s Receptivity Be Regulated lq Membrane Potential?

to Sperm

The evidence that the sperm receptivity of the egg’s plasma membrane is voltage-dependent has been exe In a recent study of early electrical events in Lytechinus variegatus eggs using intracellular microelectrodes, Schatten and Hiilser (1992) found resting potentials and fertilization potentials in most eggs to be similar to the pattern we describe here as Class II. That this pattern is an artifact produced by impalement was indicated by the higher incidence of polyspermy in impaled eggs. Whitaker and Steinhardt (1922, Table A4) have pointed out a similar incidence of impalement-induced polyspermy in the data of DeFelice and Dale (19’79).

DEVELOPMENTAL BIOLOGY

14

tensively reviewed elsewhere (Jaffe and Gould, 1984; Whitaker and Steinhardt, 1982). Briefly, the relationship between the potential across the plasma membrane of the egg and the receptivity of that membrane to sperm is evidenced by the following

experimental

results:

(1) When the membrane potential of the unfertilized egg is held positive by current injection, there is a reduction in the probability of fertilization (Jaffe, 1976; Dale et aL, 1978; Jaffe et d, 1982). (2) When the positive-going depolarization of the fertilization potential is reduced or eliminated by any of three different methods, the eggs become polyspermic. The first of these methods, current injection, was demonstrated by Jaffe in 1976. She found that 717 eggs were polyspermic if their fertilization potential was prevented from depolarizing above -30 mV by injection of current with a microelectrode. Since the plateau level of the fertilization potential is strongly dependent upon the external Na+ concentration (Fig. 9), reduction of the Na+ concentration in the extracellular medium also reduces the amplitude of the fertilization potential. Schuel and Schuel(l981) found that the incidence of polyspermy increases linearly up to 50% as the concentration of external Na+ is decreased from 400 to 100 mM. Eggs fertilized in Na-free seawater become highly polyspermic (Nishioka and Cross, 1978). Jaffe (1980) also reported 100% polyspermy in 120 or 50 mM Na+ using a sperm concentration which gave only 22% polyspermy in normal seawater. The third method involves treatment of eggs with nicotine which also reduces the amplitude of the fertilization potential. This drug acts to change the current-voltage relationship of the egg so that an outward current of a given magnitude causes a smaller membrane voltage change (Jaffe, 1980; Dale et aL, 1982). The fact that all three of these polyspermy-inducing perturbations reduce the amplitude of the fertilization potential argues that it is the fertilization potential that influences sperm-egg fusion, rather than a side effect of any one of the experimental conditions. What is not yet understood regarding sea urchins, or any other species, is the mechanism by which membrane potential affects the interaction of the plasma membrane with sperm. Recent cross-species fertilization studies suggest that the sperm may contribute the potentialsensitive component in the fertilization reaction (Jaffe et aL, 1982; Gould-Somero and Jaffe, 1983). There is little doubt, however, that in sea urchins the rate at which sperm can successfully enter an egg is voltage dependent. Is the Electrical

Block Partial

ar Absolute?

The block to sperm entry posed by positive membrane potentials in sea urchin eggs can be overcome by high sperm concentrations. This has been demonstrated with

VOLUME103,1934

two independent techniques for changing the membrane potential: In Jaffe’s (1976) experiments, when a concentration of lo6 sperm per milliliter was used, O/8 eggs fertilized when their potential was held between +l and +6 mV by the current injection technique. However, when a concentration of 4 X 10’ sperm per milliliter was used, 2/10 eggs fertilized even though their potential was held at the much higher value of +20 mV (more positive voltage levels were not tested (Jaffe et uL, 1982)). This result suggests that the block imposed by membrane potential is not absolute, although more cases are needed for a statistically significant confirmation of this idea. Further indication that the electrical block is only partial comes from experiments in which the concentration of extracellular Na was varied in order to modify the plateau voltage level (Phase 5) of the fertilization potential (Schuel and Schuel, 1981). Results of this experiment indicated a graded, rather than absolute, effect of membrane potential on polyspermy. The membrane potential appears to affect only the probability of successful sperm entry, rather than barring any concentration of sperm above a certain voltage level. This relationship between membrane potential and the probability of sperm entry therefore fits the definition of a partial block.

The normal electrical response of a sea urchin egg to fertilization is a fertilization potential that includes a very rapid depolarization to positive levels within O.l1 set of its initiation, and that returns the voltage across the plasma membrane to a nonblocking level within about 90 set and to the prefertilization level within about 10 min. This response is therefore fast enough to qualify for a fast block mechanism and is temporary in nature. It also behaves like a partial block since it can be overcome by very high sperm concentrations. Therefore, this fertilization potential pattern meets the essential criteria for the fast, partial, temporary block to polyspermy. The evidence to date suggests that the fast, high-magnitude fertilization potential plays an integral role in the fast block to polyspermy in at least five species of sea urchin eggs: St. pwpwatus, I; variegatus, L pi&~, E. emdentus, and Ps. miliaris. We predict that refined procedures will reveal the presence of an electrical block in other species as well. APPENDIX DIFFICULTIESINTHE MEASUREMENTOF MEMBRANE POTENTIALWITH MICROELECTRODES

The principal reason for the extensive variability in measurements of resting potential made by electrode

NUCCITELLI

AND

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Contmmy

impalement of unfertilized sea urchin eggs is that these eggs have an exceptionally high membrane resistance, considerably greater than is observed in most nerve cells. When a glass microelectrode punctures the plasma membrane, a membrane-electrode seal is made. If that seal does not have the same large resistivity as the membrane, ions will leak across the seal causing a kind of short circuit or depolarization, shifting the measured voltage away from the true resting potential. While most excitable cells such as nerve have resistivities of l-10 KQ. cm2, the resistivity of the unfertilized sea urchin egg is lo- to loo-fold higher. This means that the membrane-electrode seal in the unfertilized egg must be loto l&l-fold tighter to avoid ion leaks which would obscure the true potential. A list of the resistance measurements made in sea urchin eggs (Tables 3 and 4) indicates that eggs with more negative resting potentials also have greater specific resistances than those with less negative potentials. It is probable that the greater resistance reflects a better membrane-electrode seal and a more accurate measurement in the more negative resting potential cases. This idea is further supported by the results of the noninvasive tracer flux technique which also indicate the more negative resting potential and high specific resistivity in the two species to which it has been applied. If a leak introduced by the electrode is the cause of the lower resting potentials, why is there a bimodal distribution of measured potentials rather than a continuous distribution? The answer to this question was clearly presented by Miyazaki et al. (1975, Fig. 9b) and by Hagiwara and Jaffe (1979, Fig. l), and is based on the fact that these egg membranes do not behave like passive resistors. In certain voltage ranges a population of K+ channels closes to increase the membrane resistance. This means the membrane voltage can change abruptly when a small current crosses the membrane. In echinoderm eggs this “jump” region is often between -10 and -70 mV (Hagiwara et aZ., 19’76), explaining why these are often the most commonly measured voltages. This phenomenon will also be dependent on the overall K+ permeability of the cell. Cells with relatively few open K+ channels (such as the unfertilized eggs)7 are more sensitive to leakage currents because the K+ efflux cannot keep up as easily with the leak influx. If the K+ permeability increases (as in the fertilized egg), the same leakage current will cause less depolarization because ’ The fact that the unfertilized egg has fewer open K+ channels does not imply that the egg will exhibit a less negative membrane potential. Studies of membrane permeability using either ion tracer flux or microelectrode techniques indicate that K+ is still the most permeant ion (Jaffe and Robinson, 1978), so the membrane potential is expected to be close to the highly negative K+ equilibrium potential.

15

over the Fast Block to Polgqmmy

more K+ efflux can compensate for it. This is probably why the resting potential of the fertilized egg is always in the more negative potential range. The same leakage at the membrane-electrode seal has a much smaller effect on the observed potential due to the increased K+ efflux. It has been argued that if the measured membrane potential does not change much upon the insertion of a second electrode, then it probably did not change much when the first electrode entered, and the measured value can be assumed to be very close to the true potential (see for example, Dale and DeSantis, 1981). Reasoning similar to that in the preceding paragraph can be used to explain why the insertion of a second electrode cannot provide a more reliable determination of the membrane potential. This “second electrode” test is valid only when the leakage through the membrane-electrode seal is small compared to the K+ ion efflux through K+ channels. The leakage resistance (RL) will be in parallel with the K+ channel resistance (Rx), so these two will combine to give an overall resistance of RLRK/(RL + Rd. Suppose the leakage resistance is initially 10 times smaller than the K+ channel resistance (this is not unreasonable considering the range of resistivity values in Table 4): RK = 10 units RL = 1 unit R total= (RLRK)/(RL + RK) = lO/ll

= 0.91 units.

If another electrode is inserted, introducing more leakage and thereby reducing the leak resistance so that now RL = 0.05RK, the overall membrane resistance would be: RK = 10 units RL = 0.5 units R total = (RLRK/(RL + RK) = 5/10.5 = 0.48 units.

Thus, the apparent resistance changes by only 47% due to the second electrode while it fell from 10 units to 0.91 units (11-fold) when the first electrode was introduced. Therefore, the apparent membrane voltage could fall much more due to the first electrode impalement than as a result of the second impalement, depending upon the K+ permeability at impalement. If K+ permeability is low, the leakage current will more readily dominate the measured voltage. Based on this reasoning, we can speculate on a possible explanation for the contradictory observations described at the beginning of Section IV in which highly negative resting potentials were found in eggs with high input impedance as well as in eggs which were detergenttreated and therefore expected to be more leaky, or in “older,” more fragile eggs impaled several hours after spawning. If the detergent-treated eggs or the “older”

16

DEVELOPMENTAL BIOLOGY

VOLUME 103, 1984

eggs had increased K+ permeability compared to freshly spawned eggs, the leakage resistance would not dominate the overall membrane resistance as strongly and the measured voltage could better reflect the true resting potential. It is well documented that increased intracellular Ca” will increase K+ permeability (Meech, 1979) and both detergent-treated eggs as well as “older” eggs could well have increased intracellular Ca2+. Thus, the apparently contradictory observations might result from differences in the initial K+ permeability under these different circumstances.

GINZBURG, A. S. (1964). Mechanism of blockade of polyspermia in echinoderms. Doll Acad S& USSR Bid Sci 152, 1232-1235. GOULD-SOMERO, M., and JAFFE, L. A. (1984). Control of cell fusion at fertilization by membrane potential. In “Cell Fusion: Gene Transfer and Transformation” (R. F. Beers, Jr., and E. G. Bassett, eds.), pp. 27-38. Raven Press, New York. GRAY, J. (1922). A critical study of the facts of artificial fertilization and normal fertilization. Quo& J. Mzbrosc. Sci 66,419-437. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annzc Rev. Biophys Bioeng. 8,385-416. HAGIWARA, S., MIYAZAKI, S., and ROSENTHAL, N. P. (1976). Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell of a starfish. J. Gen Phl/sioL 67, 621-

We are indebted to Edward Chambers and Laurinda JatIe for helpful discussions, particularly with regard to the model of the fertilization potential and for the suggestion (from L.J.) for the data analysis in Fig. 4. This work was supported in part by NSF Grant PCM 81 18174 to R.N.

HAGSTROM, B. E., and ALLEN, R. D. (1956). The mechanism of nicotineinduced polyspermy. Exp. Cell Res. 10, 14-23. HERTWIG, O., and HERTWIG, R. (1887). Uber den Befruchtungs und Teilungsvargang des Tierischien Eies unter dem Einfluss ausserer Agentien. Jena 2. Naturwisa 20,120~241 and 455-510. HIGASHI, A. (1971). Correlation of the membrane potential of the sea urchin egg to its developmental activity. Zool. Mag 80,364-370. HIGASHI, A., and KANEKO, H. (1971). Membrane potential of sea urchin eggs and effect of external ions. Ann& ZooL Japan 44,65-75. HIRAMOTO, Y. (1959). Electric properties of echinoderm eggs. Em-

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NUCCITEUI AND GREY

Controversy over the Fast Block to Polyspermy

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