calcium action potentials in the developing oocytes ... - Semantic Scholar

J. exp. Biol. 119, 287-300 (1985) Printed in Great Britain © The Company ofBiobgists Limited 1985

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CALCIUM ACTION POTENTIALS IN THE DEVELOPING OOCYTES OF AN INSECT, RHODNIUS PROLIXUS BY M. J. O'DONNELL Department of Biology, York University, Dotxmsview, Ontario, Canada, M3J1P3 Accepted 21 May 1985

SUMMARY

1. Action potentials (APs) have been recorded for the first time from the oocytes of an arthropod, the haematophagous insect Rhodnius prolixus. In saline containing Zmmoll" 1 calcium, APs could be evoked by depolarizing the oocyte membrane from its resting potential of — 50 mV to a threshold between —35 and — 40 mV. The mean duration and overshoot of the APs were 2-6 s and +16mV, respectively. 2. APs could not be evoked by depolarization of follicular epithelia that had been separated from the oocyte. It is concluded that the APs are generated at the oocyte cell membrane. 3. The overshoot of the APs was unaffected by the addition of tetrodotoxin (3 jrniol I"1) or the removal of Na + (choline replacement) from the bathing saline. 4. APs could be reversibly blocked by lmmoll" 1 La3+ or lOmmolT 1 Co 2+ . Verapamil at SO^moll"1 and lOOjzmoll"1 reduced AP duration by 30% and 50%, respectively. The overshoot increased by 30 mV when bathing saline Ca2+ concentration was increased from 2 to 20mmoll~1. These results suggested the occurrence of a Ca2+ influx during the rising phase of the AP. 5. Addition of Ba2+ increased the overshoot and duration of APs. In Caz+-free saline, addition of 2mmoll~1 Ba2+ resulted in the spontaneous production of a series of action potentials. The duration of APs was as long as 120 s in 20mmoll~1 Ba2+ saline. It is suggested that Ba2+ may block a voltage-sensitive potassium conductance. 6. Possible functions of action potentials during fertilization and early development are discussed. INTRODUCTION

Action potentials have been recorded in unfertilized eggs of mice and hamsters (Georgiou, Bountra, Bland & House, 1984) and a variety of marine invertebrates (reviewed by Hagiwara & Jaffe, 1979). In general, the rising phase of the action potential results from an influx of Ca2+ and/or Na + through voltage-sensitive channels. These channels in the oocyte membrane may be present in preparation for Key words: Action potentials, calcium, oocytea, verapamil.

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development into excitable cells. Alternatively, they may amplify the potential changes at fertilization that are responsible in some species for preventing polyspermy (Jaffe, 1976). However, action potentials have not been reported for the oocytes of arthropods. A recent study of the oocytes of two species of decapod crustaceans has demonstrated a sustained hyperpolarization at fertili2ation (Goudeau, Pascale & Goudeau, 1984). Depolarization of the oocyte by perfusion with potassium-rich saline did not result in action potentials. In previous studies of insects, action potentials could not be elicited from the mature eggs of Drosophila (Miyazaki & Hagiwara, 1976) or the developing oocytes of the locust (Wollberg, Cohen & Kalina, 1976). This paper provides the first description of action potentials in the oocytes of an arthropod, the haematophagous insect, Rhodnius pmlixus. These action potentials are calcium-dependent, and can be evoked by depolarization of the oocyte cell membrane.

METHODS

Ovaries were dissected under control saline (Table 1) from mated adult Rhodnius prolixus obtained from a laboratory culture. Each of the two telotrophic ovaries consists of seven ovarioles, each of which may contain a number of ellipsoidal follicles in different stages of development. Each oocyte is surrounded by a layer of follicle cells. Only one follicle is vitellogenic; all earlier stages are arrested in previtellogenesis (Huebner & Anderson, 1972). Vitellogenic follicles range in size from 400 to 600 /im; previtellogenic follicles are less than 400 /im in length. Each ovariole was separated from the surrounding sheaths and allowed to equilibrate in control saline at room temperature for IS—60min. In some experiments the follicular epithelium was separated from the oocyte by tearing the follicle with a sharp needle under saline and flushing out the contents of the oocyte. An oocyte or follicular epithelium was then transferred to a chamber (volume 2 ml) constructed from polyvinyl chloride tubing, and perfused at 8—10 ml min"1. The oocyte or epithelium was held in place by two Ushaped weights constructed from glass or steel rods about 200 (tin in diameter. One weight was placed across the interfollicular tissue between the vitellogenic and Table 1. The composition of the experimental solutions (concentrations in mmol l~') Control NaCl

KC1 MgCl2

CaCfe

Glucose BIS-TRIS* BaCl2 Choline Cl

129 8-6 8-5 2 34 15

Na+-free Ca2+-rich 8-6 8-5 2 34 IS

129

104 8-6 8-5 20 34 15 —

Ca2+-rich, 2 Ba 2+ , 20 Ba2+, Na+-free Ca2+-free 20Ba 2+ Ca2+-frcc Ca2+-free Mg*+-free 8-6 8-5 20 34 15 — 104

132 8-6 8-5 — 34 15

101 8-6 8-5 2 34 15 20

129 8-6 8-5 — 34 15 2

104 8-6 8-5 — 34 15

20

141 8-6 — 2 34 15 —

Salines containing cobalt (10 mmol 1 l ) and lanthanum (1 mmol 1 ') were made by direct addition to control Baline. All salines were buffered with BIS-TRIS and adjusted to pH 6-8 by addition of 5 mmol T 1 HCI. • Bis (2-hydroxyethyl)imino tris (hydroxymethyl)methane.

Action potentials in insect oocytes

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pre-vitellogenic follicle; the other was placed proximal to the vitellogenic follicle across the corpus luteum, which is the empty and degenerating follicular epithelium of the previously ovulated oocyte. Intracellular recording Electrical potential was measured by penetrating the follicular epithelium and oocyte membrane with a glass microelectode filled with 3 m o i r 1 KC1 (20-40 MQ resistance). For vitellogenic oocytes it was not feasible to inject current and measure potential with a single electrode and bridge balance techniques because of the large currents (10~7A) required. Current was injected, therefore, through a second microelectrode which was fabricated from thin-walled glass tubing (lmm o.d., 0-78mm i.d.) to facilitate passage of large currents. The resistance of this microelectrode when filled with 3moll" 1 KC1 was 4-8MQ. The microelectrodes were connected through Ag/AgCl half-cells to high impedance (> 1011 Q) preamplifiers (WPI model 707 or FD 223). During current injection, the current injection amplifier was switched to breakaway mode and current pulses were supplied directly to the microelectrode through a pulse generator and stimulus isolation unit. Current intensity was monitored by a current-to-voltage converter which functioned as a virtual ground. The bath was connected to the virtual ground through 3 mol I"1 KCl-agar bridges and Ag/AgCl half-cells. To impale an oocyte the microelectrode was advanced slowly until a rise in resistance indicated that the tip had contacted the follicle surface. The microelectrode was then advanced a further 40-50 //m and the baseplate of the micromanipulator was tapped gently with the forefinger. Alternatively, the cell could be impaled by transiently increasing the negative capacitance to apply an oscillating current through the microelectrode. The further advancement after contact was necessary for the microelectrode to penetrate the 30-40 [im thickness of the follicular epithelium surrounding the oocyte. Potentials and current were displayed on a dual beam recording oscilloscope and recorded on film or a two-channel strip chart recorder. RESULTS

Action potentials in Rhodnius oocytes For 49 follicles in control saline, the resting potential was —50 ± 1 mV (X ± S.E.). I nput resistances varied with the size of the follicle, and were in the range o f l - 2 x 105 Q. In response to depolarizing current injection, a regenerative depolarization lasting 2-6 ± 0-2s was produced (Fig. 1). The potential at the peak (Ep) was 16 ± 1 mV and the threshold for initiation of the regenerative event was about —35 to —45 mV. Generally, a stimulus lasting 50—70 ms was the minimum necessary to produce an action potential. Action potentials (APs) could also be produced if the oocyte was depolarized by potassium-rich (SOmmoll"1) saline, or, in some cases, as an anodebreak response at the end of a sustained hyperpolarization. In many cases, the transient depolarization produced by the introduction of a second microelectrode was sufficient to produce an AP. Insertion of a second electrode did not significantly affect the resting potential for more than a few seconds, indicating that impalement of the oocyte by microelectrodes is probably not associated with significant leak artefacts.

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20 mV

L 0-2 s

500 nA

Fig. 1. Action potentials produced by depolarizing current injection. In this and subsequent figures the dotted line indicates the potential recorded with the microelectrode in the bathing saline (0 mV). The lower trace is the output from a current-to-voltage converter, and indicates the current injected into the oocyte through the microelectrode. The upper trace represents the oocyte membrane potential measured by a second microelectrode.

500 nA

20 mV

Fig. 2. Effects of repeated stimulation upon action potential duration. Upper trace, current injected. Lower trace, superimposed recordings of oocyte membrane potential recorded during 10 injections of depolarizing current at 30-s intervals. The action potentials became successively shorter.

Fig. 2 shows the response of an oocyte to repeated stimulation at intervals of 15-20 s. Ep and resting potential decreased slightly and the duration of the action potential decreased to about 60% of the initial value. In other oocytes, repeated stimulation did not change the resting potential, but Ep decreased about 4mV after the first one or two APs and then did not change by more than 1—2mV during subsequent APs in the train. Repeated stimulation at intervals of less than about 60 s always shortened the APs, and it was not possible to evoke action potentials at a rate exceeding about once every 2—3 s, suggesting a refractory period of about 2 s. It is important to note that the oocyte in Rhodnius is surrounded by a layer of follicle


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20 mV

500 nA

Fig. 3. Effects of sodium-free saline upon action potentials. Lower trace: current injection. Upper traces: oocyte membrane potential. The longer action potential was recorded in sodium-free saline, and the shorter one was recorded in control saline.

cells which are well coupled to the oocyte (Huebner, 1981). Control experiments were conducted, therefore, to test if the follicle cell membranes contributed to the generation of action potentials in whole follicles. Isolated follicular epithelia were perfused with control saline and one or more follicle cells were impaled with microelectrodes. If the membrane potential was more negative than the threshold for the generation of an action potential in whole follicles, the perfusion solution was switched to saline containing lZOmmoir 1 K (sodium substitution) to depolarize the epithelium. For five epithelia, the membrane potential was —48±l-2mV in control saline and —27 ± 4 mV in the potassium enriched saline. Although the latter value is well above the —35 to —40 mV threshold for generation of action potentials in whole follicles, no action potentials were observed. Intact follicles always produced APs when exposed to saline containing 120mmoir' K + . It appears, therefore, that the generation of action potentials is a property of the oocyte cell membrane alone. The ionic basis of the resting potential in Rhodnius follicles will be described in a separate publication (M. J. O'Donnell, in preparation), but it is worth noting that the depolarization of follicle cells produced by exposure to saline containing 120 mmoll" 1 K + is much less than the 66 mV predicted for a potassium-selective membrane. Similarly, the resting potential of intact follicles changes by 30-35 mV for a ten-fold change in external potassium concentration (M. J. O'Donnell, unpublished results), much less than the 58mV predicted for a potassium-selective membrane. These data suggest that either other ions and/or electrogenic processes contribute significantly to the resting potential of the follicles. Ionic basis of action potentials Effects of sodium removal Replacement of sodium by choline neither prevented action potentials nor significantly changed the peak potential. Ep was 18 ± 3 mV in control saline and

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19 ± 3 mV in Na-free saline (N = 8). However, the duration of the action potential increased significantly from 3-1 ± 0-4 s in control saline to 5-4 ± 0-9 s in Na-free saline (Fig. 3). The resting potential in Na-free saline became about 8mV more positive, possibly because of inhibition of an electrogenic sodium pump contribution to resting potential. The experiments with sodium-free saline suggested that the rising phase of the action potential was not due to a sodium influx. Further support for this conclusion was obtained by application of the sodium-channel blocker tetrodotoxin (TTX). Even high concentrations (3/anoll" 1 ) of the TTX had no effect upon the Ep or duration of evoked action potentials (N = 4). Experiments involving alteration of bathing fluid calcium concentration or the application of calcium channel blockers were conducted, therefore, to test if the opening of calcium channels produces the action potential. Effects of divalent cations upon action potentials A ten-fold increase in the calcium concentration of the bathing saline increased spike height by 31 ± 1 mV (N= 11). In Na-free saline, a change from 2 to ZOmmoll"1 calcium produced an increase in Ep of 30 ± 2mV (N— 6; Fig. 4). These values are not significantly different from the 29-5 mV expected for a calcium-selective electrode at 22-23°C. In other systems, magnesium ions cannot substitute for calcium as charge carriers in voltage-sensitive calcium channels (Hagiwara & Byerly, 1981), and the maximum slope of the rising phase of the action potential (d V/dtmax) is decreased in the presence of magnesium (Hagiwara.& Takahashi, 1967). In Ca-f ree saline containing the normal concentration of magnesium (S-SmmolP 1 ), no action potentials were observed in response to larger depolarizing pulses than were necessary to elicit APs in control saline in the same follicles (TV = 4). In magnesium-free saline (sodium substitution),

20 mV

500 nA

Fig. 4. Effects of a ten-fold increase in external calcium concentration upon action potential overshoot (Ep) in sodium-free salines. The larger action potential was recorded in calcium-rich ( 2 0 m m o i r ' ) saline. The smalleT action potential was recorded in saline containing 2 m m o l P ' calcium. Lower trace, current injection. Upper traces, membrane potential.


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20 mV

500 nA

Fig. 5. Effects of magnesium-free saline on the initiation of action potentials. The action potential with the more rapid depolarization and more negative threshold wag measured in magnesium-free saline. The other trace was recorded in control saline. Upper trace, membrane potential. Lower trace, current injection.

20 mV

500 nA Fig. 6. Effects of barium upon action potential overshoot and duration. The shorter action potential was recorded in control saline. The larger and longer action potential was recorded in saline containing 2 0 m m o i r ' Ba and 2 m m o i r ' Ca. Lower trace, current injection. Upper traces, egg cell membrane potential.

the threshold for excitation was several millivolts more negative, and dV/dtmm increased from 4 ± 1 Vs~' to 7 ± 1 Vs~' (N —5). The peak potential, therefore, was reached 100-200ms sooner than in control saline (Fig. 5). Effects of barium It is well known that barium can substitute for calcium in producing calcium action potentials in other systems (Reuter, 1973). In saline containing Zmmoll" 1 calcium and 20 mmol I"1 Ba, Ep increased by 18 ± 5 mV (N = 7) and the duration of the action

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potential increased nearly four-fold (Fig. 6), to 11-4± Is, relative to the value in control saline (2-9 ± 0-3 s). In calcium-free saline containing 2 mmol I"1 Ba, the duration of the action potential increased approximately 16-fold relative to the value in control saline, and Ep increased from 13 ± 2mV (N = 10) to 22 ± 4mV. Moreover, in 9 of 10 follicles, spontaneous action potentials were observed when the bathing saline was changed from control saline to saline containing 2 mmol I"1 barium. When follicles were exposed to the 20mmol" 1 Ba in Ca-free saline, a series of as many as 25 spontaneous action potentials was observed (Fig. 7); individual action potentials early in the series lasted as long as 120 seconds. The oocyte was not refractory for most of the interval between spontaneous APs because depolarizing current injected during periods of spontaneous electrical activity evoked APs in Caz+-free saline containing 2 mmol I"1 (N = 3) or 20 mmol I"1 (N = 3) Ba2+. APs could also be evoked by current injection after cessation of spontaneous electrical activity, provided that the resting potential had not declined to values less negative than the threshold for the APs. For the first of each series of spontaneous APs, Ep increased 16 + 4 mV for a ten-fold increase in barium concentration. For evoked APs, the corresponding increase in Ep was 17 ± 5 mV (N= 6). Because the height of successive spontaneous action potentials declined (Fig. 7) it was difficult to measure precisely the effects of changes in bathing fluid barium concentration upon Ep, and these measurements, therefore, may be underestimates. In a separate series of experiments, lower concentrations of barium, 0-5 and 1-0 mmol I"1, were applied in calcium-free saline. The change in Ep for evoked action potentials in these salines was 10-5±0-6mV (N = 5), which exceeds the 8-7mV expected for a barium electrode in response to a two-fold concentration change. As

5min

Fig. 7. Spontaneous action potentials in calcium-free saline containing 20 mmol 1 ' barium. For the period between the arrows the perfusing solution was switched from control saline to calcium-free barium-rich saline.


295

500 nA

20 mV 0-2s Fig. 8. Reversible blockage of action potentials by cobalt. The longest action potential was produced in control saline. During exposure to cobalt (10 mmol ' in control saline), only electrotonic responses were recorded after the same or higher levels of depolarizing current. The shorter action potential was recorded at the lower stimulus intensity within 2 min of the removal of cobalt from the bathing saline. Upper trace, current injection. Lower trace, membrane potential.

discussed below, the increase in the duration and overshoot of the action potentials in barium-rich salines, and the production of spontaneous APs in Ca-free salines containing barium may reflect a second effect of barium, namely the inhibition of a voltage-sensitive potassium conductance. Calcium channel blockers The role of calcium in supporting action potentials in Rhodnius oocytes was also tested by examining the effects of agents known to block calcium channels in other systems. These include cations such as cobalt, manganese and lanthanum, and organic compounds such as verapamil (Hagiwara & Byerly, 1981; Reuter, 1983). Cobalt (10 mmol I"1) reversibly suppressed the action potential (N— 5), even if the intensity of the depolarizing stimulus was increased (Fig. 8). Lanthanum also blocked the action potential completely and reversibly when it was present in sodium-free saline at 1 mmol T 1 (N =3). These concentrations of cobalt and lanthanum also block calcium action potentials in other cells, such as mouse and hamster eggs (Georgiou etal. 1984). Verapamil blocked action potentials completely at 1 mmol I"1 (N=2), but the inhibition was only partly reversible. At lower concentrations (lOO/imoll"1), APs were blocked at the stimulus intensity used to elicit APs in control saline (N = 6). At higher current intensities, APs were elicited in five out of six oocytes, but were reduced in duration by 50 % relative to control saline (Fig. 9). Ep declined significantly from 12 ± 3 mV to 7 ± 3 mV (N= 5). In response to 50/imolI"1 verapamil, there was a reduction in action potential duration of about 30%, to 2-0 ± 0-3 s, relative

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to the value in control saline (2-8 ± 0-4 s; N - 4) and there was little or no reduction in Ep. Action potentials in pre-vitellogenic follicles and chorionating follicles Action potentials could also be recorded in pre-vitellogenic follicles (Fig. 10). Input resistances of previtellogenic follicles were 3-5 X lC^Q. The APs were of shorter duration than those of vitellogenic follicles, however, and in some cases could be elicited only by increasing the calcium concentration of the saline (20 mmol I"1), or by the addition of barium (20 mmol I"1). In some instances, depolarizing current 200 nA

20 mV

Fig. 9. Blockage of action potentials by verapamil. The action potential was recorded in control saline in response to the lower level of depolaraing current. The small electrotonic response was recorded at the same level of current injection in control saline containing 0 - 1 mmol P 1 verapamil. The smaller, shorter action potential was recorded at the higher level of current injection. Upper trace, current injection. Lower trace, membrane potential.

200 nA

20 mV

Fig. 10. Action potential recorded in a pre-vitellogenic follicle bathed in control saline. Current injection and potential measurement were performed with a single microelectrode using a bridgebalance technique. Upper traces: current injection. Lower traces, membrane potential.


297

500 nA

20 mV 0-5 s Fig. 11. Anode-break response of a pre-vitellogenic follicle. A series of three action potentials was recorded after removal of hyperpolariiing current. Upper trace, current injection. Lower trace, membrane potential.

pulses were ineffective in producing action potentials, but APs could be recorded at the end of a hyperpolarizing current pulse (Fig. 11). These anode break responses have been found in other egg cells in which the resting potential is more positive than the threshold for the action potential. Action potentials could also be recorded from follicles in which formation of the chorion had been initiated. It was not possible to record from follicles in which the chorionation was complete because the chorion could not be penetrated by microelectrodes. Effects of depolarizing current on oocytes of other insects In contrast, action potentials in response to depolarizing currents were not found in another reduviid, Triatoma sp. Nor was there evidence of action potentials in the oocytes from an orthopteran (Locusta migratoria) or a lepidopteran {Hyalophora columbiae).

DISCUSSION

The results of this study provide the first positive evidence for action potentials in the oocytes of an arthropod. The effects of either calcium channel blockers or changes in the external calcium concentration suggest the presence of a calcium influx during the rising phase of the action potential. In common with the calcium action potentials of other cells, the potential at the peak of the action potential is much less than the value expected from the equilibrium potential for calcium (Ec»; Reuter, 1973). Intracellular calcium activities are generally less than 5 X 10~7moll~1 (Reuter, 1983), and a value for Ep in excess of +100 mV would be expected, therefore, if the bathing saline contains 2 mmol I"1 calcium . One likely explanation is that the peak of the action potential is more negative than

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Ec» because an outward positive current is present along with an inward calcium current. It has been suggested, first by Fatt & Ginsborg (1958) for crustacean muscle and by other authors since (Reuter, 1973), that this outward current is carried by potassium. Although the ionic movements during the action potential of Rhodnius oocytes have not been examined in detail, several pieces of evidence are consistent with the presence of an outward potassium current during the action potential. It has been suggested that Ba2+ depresses K+ permeability changes during action potentials in barnacle muscle fibres, with the result that the duration of the action potential is prolonged (Hagiwara & Naka, 1964). In Rhodnius oocytes, addition of 20mmoir 1 barium to saline containing 2 m m o i r ' calcium resulted in a decreased slope in the falling phase of the action potential, and a nearly four-fold increase in overall duration. The increase in AP duration was more dramatic in the absence of external calcium. It is probable that Ba2+ blocks passive K + channels, resulting in a slow depolarization towards threshold and a spontaneous action potential. The increased duration of the action potential suggests that Ba2+ may also block an active outward K + current that may be involved in restoring the membrane potential to the resting level. In squid axon, internal Baz+ acts as a simple competitive inhibitor of K + with the K + conductance (Eaton & Brodwick, 1980). Barium might also increase AP duration if it is less effective at inactivating the Ca2+ channel. In Aplysia neurones and Paramecium, inward current decays more slowly with Ba2+ than with Ca2+ outside the cell membrane (Brehm & Eckert, 1978; Tillotson, 1979). It has been suggested that inactivation of Ca2+ channels in these cells involves a direct reaction between Caz+ that has entered the channel and the channel itself, and that Ba2+ is less effective than Ca2+ in this reaction (Brehm & Eckert, 1978; Tillotson, 1979). The decline in Ep of successive spontaneous APs might then be caused by a build-up of Ba2+ on the inside of the membrane. However, recent studies suggest that calcium channel inactivation is not dependent upon Ca2+ entry (Lux & Brown, 1984). The effects of barium, therefore, may be mediated primarily by its influences upon potassium conductances. An electrogenic pump may play a subordinate role in restoring the resting potential at the end of an action potential. Sodium-free salines depress membrane potential, consistent with the inhibition of an electrogenic Na:K exchange. Ouabain has similar effects (M. J. O'Donnell, unpublished results). The increase in duration of action potentials in sodium-free salines may reflect the absence of an electrogenic component to the restoration of the resting potential. Function of calcium action potential Because action potentials are found in chorionating oocytes, it appears likely that the capacity to produce action potentials is retained after chorionation. Sperm entry into the egg may, therefore, evoke an action potential, as has been observed in the eggs of a sea urchin (Jaffe, 1976). In the oocytes of other species, action potentials are involved in the rapid block of polyspermy (Jaffe, 1976). Many insects' eggs are polyspermic (Wigglesworth, 1972), although monospermy is a common occurrence in Drosophila (Hildreth & Lucchesi, 1963). It is not known if Rhodnius eggs are monospermic. However, even in the absence of an electrical block to polyspermy, the repeated penetration of the egg cell


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membrane by sperms could give rise to a prolonged series of action potentials that could either directly raise cytosolic calcium concentration or cause secondary release from intracellular stores (Gilkey, Jaffe, Ridgway & Reynolds, 1978). Changes in intracellular calcium concentration can serve as signals for the initiation of development (reviewed by Whitaker & Steinhardt, 1982). Alternatively, the channels may not be involved in developmental events around the time of fertilization, but may be present in preparation for future development into excitable cells. The absence of a significant sodium component in the action potential suggests that calcium spikes may be required first during embryogenesis. Barish (1984) has suggested that ion channels may segregate into identifiable cells as development progresses. This segregation, which might be involved in specifying determination, would cause differential excitability in the early embryo, as observed in the mollusc Dentalium (Jaffe & Guerrier, 1981). I am grateful to Dr Laurinda A. Jaffe for her helpful comments on an earlier version of the manuscript. Financial support was provided by a Natural Sciences and Engineering Research Council (Canada) operating grant. REFERENCES BARISH, M. E. (1984). Calcium-senBitive action potential of long duration in the fertilized egg of the ctenophore Mnemiopsis meidyi. Devi Biol. 105, 29-40. BREHM, P. & ECKERT, R. (1978). Calcium entry leads to inactivation of calcium channel in Paramecium. Science, NY. 202, 1203-1206. EATON, D. C. & BRODWICK, M. S. (1980). Effect of barium on the potassium conductance of the squid axon. J. gen. Physiol. 75, 727-750. FATT, P. & GINSBORG, B. L. (1958). The ionic requirements for the production of action potentials in crustacean muscle fibres. J. Physiol., Land. 142, 516-543. GEORGIOU, P., BOUNTRA, C , BLAND, K. P. & HOUSE, C. R. (1984). Calcium action potentials in unfertilized eggs of mice and hamsters. Q. Jl exp. Physiol. 69, 365—380. GILKEY, J. C , JAFFE, L. A., RIDGWAY, E. B. & REYNOLDS, G. T. (1978). A free calcium wave traverses the activating egg of the medaka, Oryzias latipes. J. Cell Biol. 76, 448-466. GOUDEAU, H., PASCALE, K. & GOUDEAU, M. (1984). Mise en evidence du potentiel de fecondation chez les Crustacea Decapodes Brachyoures Cardnus maenas et Mcaa squinado. C. R. hebd. Seanc. Acad. Set. Paris 299, 167-172. HAGIWARA, S. & BYERLY, L. (1981). Calcium channel. A. Rev. Neumsd. 4, 69-125. HAGIWARA, S. & JAFFE, L. A. (1979). Electrical properties of egg cell membranes. A Rev. Biophys. Bioeng. 8, 385-416. HAGIWARA, S. & NAKA, K. (1964). The initiation of spike potential in barnacle muscle fibers under low intracellular Ca 2+ . J. gen. Pkysiol. 48, 141-162. HAGIWARA, S. & TAKAHASHI, K. (1967). Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. 7- go- Physiol. 50, 583-601. HILDRETH, P. E. &LUCCHESI, J. C. (1963). Fertilization inDmsophila. 1. Evidence for the regular occurrence of monospermy. Devi Biol. 6, 262—278. HUEBNER, E. (1981). Oocyte-follicle cell interaction during normal oogenesis and atresia in an insect. J. ultrastruct. Res. 74, 95-104. HUEBNER, E. & ANDERSON, E. (1972). A cytological study of the ovary of Rhodmuspmlixus. III. Cytoarchitecture and development of the trophic chamber. J. Morph. 138, 1—40. JAFFE, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature, bond. 261, 68-71. JAFFE, L. A. & GUERRIER, P. (1981). Localization of electrical excitability in the early embryo of Dentalium. Devi Biol. 83, 370-373. Lux, H. D. & BROWN, A. M. (1984). Single channel studies on inactivation of calcium currents. Science, N.Y. 225, 432-434. MIYAZAKI, S. & HAGIWARA, S. (1976). Electrical properties of the Dmsophila egg membrane. Devi Biol. 53, 91-100.

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REUTER, H. (1973). Divalent cations as charge carriers in excitable membranes. Prog. Biopkys. molec. Biol. 26, 1-43. REUTER, H. (1983). Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature, Lond. 301, 569-574. TILLOTSON, D. (1979). Inactivation of Ca conductance dependent on entry of Ca ions in molluscan neurons. Proc. natn. Acad. Set. U.SA. 76, 1497-1500. WHITAKER, M. J. & STEINHARDT, R. A. (1982). Ionic regulation of egg activation. Q. Rev. Btophys. 15, 593-666. WIGGLESWORTH, V. B. (1972). The Principles ofInsect Physiology. Seventh edition. London: Chapman & Hall. WOLLBERG, Z., COHEN, E. & KALINA, M. (1976). Electrical properties of developing oocytes of the migratory locust, Locusta migratoria.J. Cell Physiol. 88, 145-158.