that can produce action potentials. (ap's). Young oocytes (sometime during metaphase. I to first polar body stage) can generate trains of spontaneous.
DEVELOPMENTAL
BIOLOGY
98,
47-59
(1983)
Spontaneous Action Potentials Produced by Na and Cl Channels in Maturing Rana pipiens Oocytes L. C. Department
of Botany, Received
June
University
SCHLI~HTER
of Townto,
4, 1982; accepted
Toronto,
in revised
form
Ontario, February
M5S
1Al
Canada
7, 1983
The electrical excitability of maturing Rana pipiens oocytes was studied using intracellular recording and voltageclamp techniques. Naturally ovulated oocytes, removed from the body cavity within a few hours after ovulation, possess voltage-sensitive Na and Cl channels that can produce action potentials (ap’s). Young oocytes (sometime during metaphase I to first polar body stage) can generate trains of spontaneous action potentials: no chemical treatment or current injection is required. This is the first report of spontaneous repetitive firing in an egg cell membrane. As the oocyte matures, the duration of each ap increases because the outward Cl- current decreases. Middle-aged oocytes (about first polar body stage to metaphase II) have continuously positive membrane potentials (V,,,‘s). Mature, activatable (metaphase II) oocytes have negative V,‘s when impaled but can produce a long-lived ap when depolarizing current is injected. The ap’s differ fundamentally from ap’s in other excitable cells, including eggs: the Na+ current develops slowly and does not inactivate; most of the outward current is carried by Cl-, not by K+; the Cl channel is lost or is rendered insensitive to voltage as the oocyte matures.
sitive Ca and Na channels in eggs is also unclear. In some species the Ca action potential may be a part of the electrical response to fertilization and may help establish the fast block to polyspermy (see Hagiwara and Jaffe, 1979). In the absence of sperm or another activating stimulus, current must be injected to elicit the Ca action potential. The existence of Cl (or anion-selective channels) in amphibian oocytes has long been recognized (Maeno, 1959). One type of Cl channel generates the activation or fertilization potential of mature Runa oocytes (Ito, 1972). Xenopus Levis oocytes at the germinal vesicle (GV) stage spontaneously generate a Cl-dependent current that disappears shortly after hormonal stimulation (Robinson, 1979). By applying acetylcholine, a selective increase in Cl- permeability can be evoked in GV stage Xenopue oocytes (Kusano et aL, 1977). In contrast to the ion channels that generate action potentials, the Cl channels in Rana oocytes are not thought to be voltage dependent. An activation potential is not evoked by injecting current (Cross and Elinson, 1980) but can be elicited by injecting calcium (Cross, 1981). The present study comprises two parts. This paper shows for the first time that immature Rana pipiens oocytes have voltage-sensitive Na and Cl channels that can be opened simply by changing the membrane potential. When maturing oocytes are exIjosed to normal amphibian Ringer’s solution these ion channels can produce spontaneous action potentials: no current injection or chemical is required. This is the first account of spontaneous, repetitive firing in an oocyte. Changes in the
INTRODUCTION
Following the discovery of electrical excitability in tunicate eggs (Miyazaki et ab, 1972), action potentials have been found in immature and mature oocytes (eggs) of several classes of animals (see review by Hagiwara and Jaffe, 1979). In almost all the oocytes examined, the rising phase of the action results from Ca2+or Na+ and Ca2+influx through voltage-sensitive channels. The action potentials end because the channels carrying inward current inactivate with time and because there is a small outward K+ current. Until recently, Na-selective, voltage-sensitive channels in egg membranes had only been found in tunicate eggs (Miyazaki et al, 1972,1974). These Na channels are like those of adult excitable cells such as squid axon, except that they open and close more slowly (Okamoto et aL, 1976). Voltage-dependent Na channels have recently been found in immature Xenopus Levis oocytes at the germinal vesicle stage (Kado et ah, 1979; Baud et aL, 1982). The Xenopus Na channels differ substantially from others in that they require a depolarizing current pulse lasting many seconds to induce the regenerative response. After firing, the action potential can last many minutes because the Na channels do not inactivate and because there is little or no outward K+ current. No physiological role for the long-lived action potential was postulated because the conditions required to open the Na channels are unlikely to be met naturally (Baud et UL, 1982). The biological significance of the other voltage-sen47
0012-1606/83 $3.00 Copyright All rights
0 1983 by Academic Press. Inc. of reproduction in any form reserved.
48
DEVELOPMENTAL
BIOLOGY
form of the action potentials between metaphase I and metaphase II are described and analyzed by investigating the underlying ionic currents using the voltageclamp technique. The second paper demonstrates a possible role for the action potentials in oocyte maturation. MATERIALS
AND
METHODS
Oocytes. Sexually mature northern leopard frogs, Rana pipiens, were~obtained commercially and stored at 4°C until used. Female frogs were injected intraperitoneally with one or two homologous, macerated pituitaries and intramuscularly with 0.5-3 mg progesterone, the lower doses being used as the natural breeding season approached. After 16-20 hr at 18”C, ovulated, jelly-free oocytes were removed from the body cavity and allowed to continue maturing in vitro in normal amphibian Ringer’s at 20-22°C. Solutions. Normal amphibian Ringer’s solution contained 111 mM NaCl, 1.9 mM KCl, 1.1 mM CaClz, 0.8 mMMgS04, and 2.5 mMHepes’ buffer and was adjusted to pH 7.8 with about 4 mM NaOH. For some experiments Cl, Na, K, or Ca concentrations were altered. Chloride-free Ringer’s solution was made by substituting equimolar amounts of KNOB for KCI, Ca(NO& for CaClz, and Na-methanesulfonate for NaCl. For Na-free solution choline chloride or tetraethylammonium chloride replaced NaCl and KOH replaced NaOH. KC1 was simply omitted for K-free solution. Solutions with elevated ion concentrations were made by adding Namethanesulfonate (high Na) or Ca(NO& (high Ca) or KNOa (high K). All solutions were adjusted to pH 7.8. Experiments were conducted at 19-21°C. Electroph@ologg. A single oocyte was placed in a small petri dish or in a Plexiglas perfusion chamber. Solutions were changing by washing five times using inlet and outlet syringes (petri dish) or by continuous flowthrough driven by a peristaltic pump (perfusion chamber). Complete solution exchange took l-3 min. For recording membrane potential (V,) oocytes were impaled with a glass microelectrode containing either 3 M KC1 or a 2:l mixture of 3 M KC1 and 0.6 M KzS04 and V, was recorded between an intracellular and a similar extracellular microelectrode. The differential output of the amplifier (input impedance, 2 X 10” Q; leakage current 50 Hz, full scale). For injecting current to evoke action potentials, to monitor membrane resistance or to volti Abbreviations used: a, ionic activity; ap, action potential; c, ionic concentration; Es,, equilibrium potential for Na; GV, germinal vesicle; Hepes, Z-(N-2-hydroxyethylpiperazine-iV’-yl)ethane sulfonic acid; PPT, postprogesterone treatment; R,, membrane resistance; V,,,, membrane potential.
VOLUME
98, 1983
age clamp the membrane, a second KCl-filled microelectrode was implanted. The microelectrodes had resistances of 50-60 MO (normal glass) or 2-10 MQ (thinwalled glass). With the same tip diameter, microelectrodes pulled from thin-walled glass have much lower resistances than those made from normal glass (Jacobson and Mealing, 1980). For voltage clamping, the two microelectrodes were positioned at an angle of about 60” with respect to each other. This reduces the spatial nonuniformity of membrane potential during a voltageclamp pulse (Eisenberg and Engel, 1970). Membrane current was monitored by a current-to-voltage converter connected to a Ag/AgCl wire in the bath. Stage of maturity. Freshly ovulated oocytes from several females were boiled, cut open, and examined for the presence of the germinal vesicle. The germinal vesicle had completely broken down in all those examined. The meiotic stage of some oocytes from which electrical recordings were made was determined either in fixed, sectioned material or in the living oocyte. Some oocytes were fixed overnight in Smith’s fixative, embedded in paraffin, sectioned at 9 pm, stained with Feulgen’s reagent and light green, and observed at 250X magnification. The presence of a metaphase spindle with dense, cross-like chromosomes (tetrad chromatids) indicated an oocyte at metaphase I. Metaphase II was indicated by the presence of a spindle and a polar body. In living oocytes, the stage of maturity was determined by observing their external appearance under a dissecting microscope. The stages were assigned according to the description by Subtelny and Bradt (1961), as follows. On recently ovulated oocytes, a distinct black pit near the animal pole marks the position of the metaphase I spindle. After several hours, the pit disappears and the first polar body emerges. About 2 hr later a second pitmarks the position of the metaphase II spindle. Oocytes become activatable 2 to 3 hr later. In the present study oocytes at metaphase I had a distinct black pit encircled by one or two concentric white rings. Subsequently, the white rings faded to a white or gray spot and the black pit disappeared as the first polar body emerged from it. Finally, at metaphase II a second black pit was observed and on fixed, whole oocytes a polar body was found. RESULTS
Spontaneous
Action Potentials
Oocytes removed from the body cavity within a few hours after ovulation and transferred to normal Ringer’s solution often fired trains of spontaneous action potentials (ap’s). In three experiments spontaneous firing was seen in 11 out of 20, 15 out of 22, and 9 out of 14 young oocytes (18 to 25 hr postprogesterone treatment, PPT). These oocytes were impaled with one fine-
L. C. SCHLICHTER
met
Oocyte
Action
49
Potentials
I, Ringer’s
a
b
JI”“““““““““““““““““““L
50
25
-25
C
d
40 7.5 50 25 0 -25
I-
FIG. 1. Voltage records of spontaneous and evoked action potentials in young (metaphase I) oocytes recorded at 18 to 20 hr postprogesterone treatment (PPT). In this and all subsequent figures the vertical scale bars are in millivolts for membrane potential and nanoamperes for current traces. The horizontal calibration bars are in seconds. (a and b) Oocyte ICl, 18-19 hr PPT. (a) Oocyte was impaled with two microelectrodes. (b) Spontaneous ap’s began 3 minutes after withdrawing one microelectrode. This oocyte was still producing ap’s 32 min later when recording ended. (c) Oocyte IC2,20 hr PPT. Spontaneous ap’s began immediately after one fine-tipped microelectrode was implanted. This trace shows the first 10 ap’s. Firing continued until the microelectrode was withdrawn about 30 min later. (d) Oocyte VCI, 18 hr PPT. With two microelectrodes in the oocyte, ap’s were not spontaneous but could be evoked by injecting depolarizing current. The membrane partly repolarized during each current pulse.
tipped microelectrode (50-60 MQ resistance). In contrast, when oocytes were impaled with two coarser microelectrodes only 6 out of more than 200 oocytes fired spontaneously. This suggests that damage reduces the ability of oocytes to fire action potentials. Figures 1 and 2 show data to support this hypothesis. Figure 1 (parts a and b) shows spontaneous ap’s in a young oocyte removed from the body cavity at 17 hr PPT and impaled 1 hr later. The membrane potential (V,) was -8 mV and the membrane resistance (R,) was 8 ma when the oocyte was first impaled with two microelectrodes. The membrane resistance gradually increased to 12 MQ, where this voltage record begins (Fig. la). The V,,, spontaneously began to oscillate with increasing amplitude around an average of -7 to -8 mV. When V,,, reached about 0 mV an ap fired, reaching a peak at +60 mV. The membrane spontaneously repolarized, undershooting the resting potential to reach a trough at -27 mV. The ap lasted 8 to 10 sec. Subthreshold oscillations of this kind were seen in five oocytes all of which were impaled with two microelectrodes. When one microelectrode was withdrawn from this oocyte (Fig.
lb), after a 3-min lag the ap’s were spontaneous and lacked subthreshold oscillations. Otherwise, the form of the ap was the same as that in Fig. la: the peak V, was +61 mV initially, the ap lasted 8 to 10 set, and the most negative potential reached was about -28 mV. Occasionally, young oocytes began to fire spontaneously as soon as they were impaled with one fine-tipped microelectrode (Fig. lc). Experiments in which the same oocyte fired spontaneously with one microelectrode but not with two show that damage can prevent firing. For those young oocytes that did not fire spontaneously with one microelectrode, ap’s could be evoked by injecting current (Figs. Id and 5a). For these oocytes, the possibility cannot be excluded that a large resting conductance for an ion with a negative equilibrium potential (e.g., K+) prevents the membrane from depolarizing to the threshold for firing. For the oocyte illustrated in Fig. Id, while current was injected V, reached a peak of +75 to 80 mV but quickly fell toward 0 mV. The undershoot of V, at the end of the current pulse was characteristic of oocytes at early metaphase I (48-19 hr PPT).
50
DEVELOPMENTAL
Ringer’s a 25 hr
b
BIOLOGY
VOLUME
98, 1983
24hr C
2%
1
SL
I (nA)
b
-4oFIG. 2. Effect of leakage conductance on the action potentials. Each oocyte Oocyte VC2,25 hr PPT. Voltage record of a spontaneous action potential. (b) a depolarizing current pulse. This oocyte did not fire spontaneously at any relations for the two oocytes, obtained by clamping the membrane to different Figs. 5a’-d’ for examples of membrane currents under voltage clamp.) Lines
Figure 2 compares the resting (leakage) conductance of a spontaneously firing oocyte with an oocyte of the same stage that did not fire spontaneously. Both oocytes were 24 to 25 hr PPT and had ap’s that reached a peak of +50 to +55 mV and lasted ‘70 to 80 set (Figs. 2a and b). The main difference between these oocytes is that the spontaneously firing oocyte had a smaller leakage conductance. Leakage conductance is measured as the slope of the current versus voltage curve constructed by briefly stepping V, to various command potentials using a voltage-clamp circuit. At voltages more negative than the resting (zero-current) potential the ionic channels causing the action potential did not open: the current versus voltage curves for these oocytes were linear (Fig. 2~). The slope conductance of the spontaneously firing oocytes was 0.075 pmho compared with 0.21 pmho for the oocyte that fired only in response to injected current. This supports the hypothesis that damage by impalement does not cause spontaneous firing but is more likely to prevent firing. A larger leakage conductance would be more effective in holding V, near the reversal potential for the leak (-12 to -14 mV in these two oocytes) thus inhibiting firing.
was impaled with two microelectrodes for voltage clamping. (a) Oocyte VC3,24 hr PPT. An action potential evoked by injecting time. Note the different time scale. (c) Current versus voltage voltages for 10 set and measuring the current at 2 sec. (See were fitted by eye.
Changes in the Wavefwm during Maturation
of the Action Potential
As the oocytes matured there were characteristic changes in the form of the action potentials. The most obvious changes were a decrease in the peak potential and an increase in the duration. Voltage records typical of the various stages are shown in Fig. 3 and examples in which the spike waveform was correlated with the stage of maturity are shown in Table 1. Figure 3a shows a voltage record from a young, metaphase I oocyte that fired spontaneously. The peak voltage of each ap was about +58 mV and the most negative potential was about -28 mV. Each ap lasted 8 to 10 set (measured at 0 mV). Figure 3b shows spontaneous ap’s that began immediately after the oocyte was impaled with one microelectrode. About 30 min out of the entire 70-min recording are shown, taken from the middle of the record. Unlike the ap’s in part a, these changed considerably in form during the recording period. The peak V, was initially +57 mV, drifting downward to reach f53 mV after 40 min. During this time the most negative potential (trough) changed from -21 to -12
L.
C. SCHLICHTER
Oocyte TABLE
FORM
Form
OF THE ACTION
of ap”
(br
POTENTIALS
Time PPT)
Action
51
Potentials
1 DURING
OOCYTE
MATURATION
Stage
Morphology External
and activatability
morphology
Brief, evoked by current (e.g., Fig. Id)
15 16 17.8
met I met I pb just forming
Two white Two white pb present,
rings + black pit rings + black pit no spot or pit
Spontaneous, (e.g., Figs.
18.5 21
pb given off pb just forming
pb present pb present,
+ gray spot no spot or pit
25
met
pb present
+ black
repetitive lb, c, 3a, b, 4)
V, always positive (e.g., Figs. 3c, d)
II
Internal Spontaneous, then (e.g., Fig. 3b)
V,,, positive
pb given met met met
19.5 24 24.5 26.3 27.5
V, always positive (e.g., Figs. 3c, d)
off I I II
met II
pit
morphology
pb complete, no spindle long slender spindle, no pb dense X-shaped chromosomes, pb + spindle barrel-shaped
spindle,
spindle
no pb
Activatability V,,, negative when impaled, ap evoked by current, V, stayed positive (e.g., Figs. 3% 6)
” Abbreviations
used: ap, action
potential;
met met met met met met met met met
44 45 48 49 51 52 48 48.5 49 met
I, met
II, first
and second
mV. The duration increased from 20 set for the first spike to 100 set for the last spike. Ultimately, V, remained positive until the recording ended 30 min later. The presence of both a metaphase spindle and a polar body showed that this oocyte was at metaphase II when it was fixed immediately at the end of the electrical recording. Figures 3c and d show voltage records from two more mature oocytes. In part c, V, was +40 mV when the voltage electrode entered, it rose to t45 mV then fell to about +41 mV where it remained throughout the recording. In part d the oocyte had a positive V, when impaled. In this segment of the record V,,, had stabilized at about +40 mV when a 25-nA hyperpolarizing current pulse was injected to repolarize the membrane. When the current pulse was turned off the membrane spontaneously depolarized and another ap fired within 12 sec. V, reached a peak of +55 mV, decreased and reached a plateau of about +40 mV. This shows that not only is V, positive when oocytes at this stage of maturity are impaled, but I’,,, cannot be made neg-
II II II II II II II II II metaphase;
Siblings Siblings Siblings Siblings Siblings Siblings Siblings Siblings Siblings pb, polar
body;
fully mature, shock activatable fully mature, shock activatable fully mature, shock activatable fully mature, shock activatable fully mature, shock activatable fully mature, shock activatable mature, developed when fertilized mature, developed when fertilized mature, developed when fertilized PPT,
postprogesterone
treatment.
ative except during a pulse of strongly hyperpolarizing current. Figure 3e shows a recording from a mature (metaphase II) oocyte impaled with two microelectrodes at 49 hr PPT. The resting potential was -21 mV and R, was about 9 MO near the resting potential. When a sufficiently long current pulse was injected an ap fired in an all-or-none manner similar to that of other excitable cells. The peak V, was +46 mV, leveling off at +40 mV where it remained throughout the approximately 7-min recording period. Table 1 shows that the form of the ap was approximately correlated with the meiotic stage of the oocyte. From sometime during metaphase I until about the time the first polar body was given off, oocytes produced brief ap’s that were often spontaneous and repetitive (e.g., Figs. la, b, 3a, 4). At about the time the second black pit appeared (metaphase II) the ap’s lengthened then remained at a positive potential (e.g., Fig. 3b). The transition from spontaneous, repetitive firing to a continuously positive V,,, was seen in about 10 oocytes that
52
DEVELOPMENTALBIOLOGY
VOLUME 98,1983
Ringer’s met
a
I
40
50 25 0 -25 i
met II
b 5
so
0 P/mm
- -
_ - -
- -
_ --
.-
--
-
-
-
-
-
--
--
---
----
----
1
-----------_-------_---
-5d
met II
?YE
!Ir ------------y!! \L -50
1
FIG. 3. Changes in the form of the action potentials during maturation. The bathing solution was normal Ringer’s throughout. Oocytes were impaled with one microelectrode for parts a and b and with two microelectrodes for parts c-e. (a) Oocyte IC3, 18-19 hr PPT. Young, metaphase I oocyte with spontaneous, recurrent action potentials. (b) Oocyte IC4, 24-25.5 hr PPT. Spontaneous action potentials. V, was still about f35 mV when the microelectrodes were withdrawn about 20 minutes later. Ooeyte was at metaphase II at the end of the recording (metaphase spindle and polar body found). (c) Oocyte IC5, 32 hr PPT. V, was positive when the oocyte was impaled and remained at about +41 mV during the approximately 5-min recording period. (Stage not determined.) (d) Oocyte IC6, 26-27.5 hr PPT. V, was positive when the oocyte was impaled. A large hyperpolarizing current ended the ap but when the current pulse ended, another ap fired spontaneously. V, remained at about +40 mV until the recording ended about 11 min later. Oocyte was at metaphase II at the end of the recording. (e) Oocyte VC4, 49 hr PPT. Mature, metaphase II oocyte showing characteristic all-or-none response to injected current. The notch at the arrow corresponds to the end of the current pulse and is at a voltage above the threshold for firing. V, remained at about +40 mV until the recording ended about 7 min later.
were impaled with one microelectrode. For the next several hours oocytes had positive Vm’swhen impaled (e.g., Figs. 3c, d). In two experiments with middle-aged oocytes (27 to 35 hr PPT) 6 out of 8, and 24 out of 33 oocytes had positive membrane potentials when they were impaled with a single microelectrode. Older metaphase II oocytes whose siblings were activatable by an electric shock or by fertilization had ap’s of the form shown in Figs. 3e and 6. V, was negative on impalement
but a long-lived depolarization could be elicited by injecting current. The longest recording made of a positive V,,, during an ap was about 30 min, hence I do not know how long the ap can last. If sufficient hyperpolarizing current was injected during an ap, V,,, became negative. The V, returned to its negative resting level when the current was turned off and remained there indefinitely if no more current was injected. This form of ap was seen in about 45 out of 50 fully mature oocytes.
L.
young,
C. SCHLICHTER
Oocyte Action Potentials
53
20 hr
q ~)/qrf$l~~>: JIJjrzz -25 R
Na
170
Cl 0
FIG. 4. Spontaneous ap’s: effects of changing &, and ~$1. Oocyte IC7, 20 hr PPT. In normal Ringer’s (R) action potentials were spontaneous. For Na-free Ringer’s (Na 0), TEACI replaced NaCl; for high Na Ringer’s (Na 170), 55 mM Na-methanesulfonate was added; for Cl-free Ringer’s (Cl 0), Na-methanesulfonate replaced NaCl, KNOs replaced KCI, and Ca(NO& replaced CaC12.
The remaining oocytes had small resting potentials and low resistances and ap’s did not fire when current was injected. This is characteristic of oocytes damaged before or during impalement. Ionic Basis of the Action Potentials Figures 4-6 show the effects of changing external concentrations of ions on the form of the ap’s and on the ion currents measured under voltage clamp. Where ionic activities are discussed they were calculated from the Debye-Huckel equation. Figure 4 shows a recording of membrane potential from a young oocyte impaled at 20 hr PPT. In normal Ringer’s the peak V,,, was +45 to 46 mV but when all the Na was replaced by tetraethylammonium, the ap’s stopped. When normal Ringer’s was restored the ap’s were again spontaneous, reaching a peak of +46 to 47 mV. Increasing the external Na concentration (c&J by one-half (Na 170) caused the peak V,,, to become 9 mV more positive. If the change in external Na+ activity (a”,,) is considered and if Na+ permeability dominates, then the Nernst equation predicts a $8.6 mV shift in the peak V,. All five spontaneously firing oocytes, for which at, was changed from ‘74 to 104 mM (concentration changed from 111 to 170 m&I), responded with a shift of +8.5 to +9 mV in peak V,. This is evidence that the Na conductance dominates at the peak of the ap. External Cl strongly affects the spike waveform but does not affect the peak V,. The peak of the ap’s in normal Ringer’s was +45 mV and their duration (measured at 0 mV) was 54 to 58 sec. When all the external Cl was replaced by methanesulfonate the peak V, was unchanged but the ap did not repolarize. This experi-
ment was repeated on six other spontaneously firing oocytes with the same results: when c& was sufficiently low the membrane did not repolarize during the recording period. This is evidence that Cl- entry helps to repolarize the membrane to end each action potential. Data in Fig. 5 give further support for the hypothesis that the rising phase of each ap results from Na+ influx and the falling phase depends mainly on Cl- efflux. With this oocyte action potentials were elicited by injecting current. In normal Ringer’s (part a) the resting potential between ap’s was -32 mV. Each ap reached a peak of about +80 mV then fell to +5 to +lO mV within 8 sec. When &, was reduced by 90%, injecting current no longer caused the regenerative changes in V, that are characteristic of ap’s. Reducing c& by 90% lengthened the ap’s from 8 set to about 18 set but had little effect on the peak potential (+80 mV in part a, compared with +78 mV in part c). When c& was further reduced ten fold the ap’s broadened to 50 to 60 set but the peak amplitude (+75 mV) was not much affected. The effects of changing c{, and c& on the spike waveform can be explained by changes in ionic currents measured during voltage clamp pulses. Part d’ shows that with low external Cl the initial current during a voltage-clamp pulse was outward and time dependent but soon became inwardly directed (see traces at +22 and +47 mV). At a characteristic potential (+75 mV in this example) the current was constant with time for 1 to 2 set (arrow in Fig. 5d’) before increasing rapidly in the outward direction. This is the reversal potential for the inward current and is at the same potential as the peak of the ap. This is evidence that the peak of each ap is at or near the equilibrium potential for the ion carrying the inward current (Na). When more Cl was present in
a’
0
50
1
-33
26
46
b’
_------
/ r--24
Na
-36
44
--
-
-_I_
C’
c ‘1 10 Cl
-
-
-
--t
47
SO---
effects on voltage and current of changes in 8. and et,. Oocyte VC5,18-19 hr PPT. Upper traces (a-d) show membrane potential (mV) and some ap’s indicate the peak potential reached. Lower traces (al-d’) membrane current (nA) during lo-set-long voltage clamp pulses of each current trace. Outward current is upward. Arrow in d’ at +75 mV shows the reversal potential for the inward current. Solutions: Na with Na substituted by choline; c and c’, l/10 Cl with NaCl substituted by Na-methanesulfonate and the remaining Cl substituted substitutions as above. Note that in the lower current traces the scale is different for a’.
------
10
b ‘ilO
injected current to the potentials a and a’, normal by nitrate; d and
~~]~/~~~ til--i”--- --r8--- --/75 ---
-40
-20
O-
20.
40.
r
--------
60-
0.
1
r
20
FIG. 5. Evoked ap’s: (nA). Numbers above indicated at the right Ringer’s; b and b’, l/10 d’, l/100 Cl with same
-50
0
50
100
young, 18-19 hr a Ringer’s
L.
C. SCHLICHTER
Oocyte
the bathing medium (part c’) the outwardly directed current developed at a less positive potential (see trace at f48 mV) and was larger; however, the reversal potential for the inward current was not much affected (+78 mV). Again, the peak of the ap was at the reversal potential. When c’& was increased further (part a’) the inwardly directed current was obscured by the large outward current; however, the reversal potential and peak of the ap (+80 mV) were about the same as before. The increase in outward current with increasing c& is easiest to see at the reversal potential for the inward current: at other potentials the inward current is superimposed on the outward current. At the reversal potential for the inward current in each solution the outward current (measured at 10 set) was 65 nA in 1.1 mM c&, 120 nA in 11 mM c&, and 270 nA in 111 mM c&. This is evidence that Cl- influx contributes most of the outward current that repolarizes the membrane at the end of each ap. If external Na is reduced, Na+ efflux can contribute to the total outward current. Reducing CL, lo-fold (part b’) greatly increased the outward current at all potentials above about +24 mV. If the equilibrium potential for Na (ENa) is +80 mV in normal Ringer’s then it is about +20 mV in l/10-Na Ringer’s. Above this potential the Na+ current is outward and adds to the outward Cl- current; therefore, in this medium there can be no regenerative action potential (see part b). Note that reducing c&, or c& had little effect on the resting (zero-current) potential: V,,, slightly hyperpolarized (2 to 7 mV). The peak of the ap in mature (metaphase II) oocytes depends on ai, as it does in immature oocytes. Figure 6 shows a voltage record of a mature (metaphase II) oocyte impaled with two microelectrodes at 44 hr PPT. V,,, was -25 mV when the oocyte was first impaled, then it stabilized at -33 mV. .R, at the resting potential was 7 MQ. The ap was evoked by injecting a pulse of depolarizing current. At the positive plateau potential R, (1 MO) was seven times smaller than it was at the negative resting potential, indicating a greater ionic conductance during the ap than at rest. In normal Ringer’s the peak V, was +33 mV. Ten-fold or one-hundred-fold changes in c& caused at most a 2-mV shift in V,. Raising or lowering cg lo-fold also shifted V, by 2 mV at most. In contrast, a l.&fold increase in cia caused a +9 mV shift, exactly as predicted by the Nernst equation. Reducing cia (l/4 Na) caused a -15 mV shift in V, from that in normal Ringer’s. This is less than the predicted change of -35 mV, suggesting that the permeability to some other ion(s) is significant at this potential (+15 mV). Some Na channels close at this potential, as judged by the increase in membrane resistance. Therefore, the ion conductances that determine the resting potential contribute more to the membrane potential than they
Action
55
Potentials
do at the peak of the ap. Further reducing &, (l/l0 Na) brought V,,, below threshold and a large increase in R, accompanied the closing of Na channels. This figure shows that the long-lived ap of mature oocytes resulted from an increase in Na+ conductance and that during the ap the conductances for K+ and Cl- were insignificant. Two lines of evidence suggested that the progressive increase in ap duration with age resulted from a gradual loss of the Cl- current. First, the ap’s of young oocytes ended because a large outward Cl- current repolarized the membrane. By reducing c& this outward current was decreased and each ap lasted longer. Second, the ap of mature oocytes lasted many minutes and was insensitive to changes in c&. To further test the hypothesis regarding the ap duration, effects of changing c& were compared with normal changes during maturation in the same oocyte. Figure 7 shows that the effect of reducing c& is similar to the natural changes during maturation. At 23.5 hr PPT (parts a and a’) the ap lasted about 22 see and the peak potential (+61 mV) was close to the reversal potential for the inward current (+62 mV). As V,,, was stepped more positive than +34 mV the outward Cl- current appeared and increased with time and with voltage. Reducing c& lo-fold greatly reduced this outward current (part b’) and lengthened the ap (part b). V, was still +35 mV when the recording ended after about 7 min. The peak of the ap was at the reversal potential for the inward current (+55 mV). The same oocyte was allowed to mature for 2 more hr in normal Ringer’s then retested (parts c and c’). The ap lasted 3.5 min and the peak (+46 mV) was near the reversal potential (+48 mV). The membrane currents were similar to those in l/lo-Cl Ringer’s at an earlier stage: the outward current was greatly reduced compared with that at an earlier stage in normal Ringer’s (part a’). Oocytes younger than this have still larger outward Cl- currents and briefer ap’s (see Fig. 5). Therefore, the normal change in the length of the ap as the oocyte matures can be accounted for by a decrease in outward Cl- current. DISCUSSION
Early in the first meiotic division, oocytes of Rana piNens are able to fire trains of action potentials. As the oocyte matures, the repolarizing phase of the action potential lengthens and eventually the membrane potential remains positive. The form of the ap was correlated with the meiotic stage but there was not always an exact correspondence of stage with spike waveform. Therefore, for the following discussion maturing oocytes will be classed according to the form of the ap. Young oocytes (metaphase I to first polar body stage)
L.
C.
Oocyte
SCHLICHTER
Action
57
Potentials
a
b
C
R , 23.5 hr
l/10 Cl, 24 hr
R, 26 hr
a'
4o lo
57
20
34
0
--
-
---
L
-20 -40
---
43
38
100 50
48
i/G---
- J
----
62
-_-
0 I/ -
---
-
c
i
50
40
-50 0 A- ----
-e
-;
-2
22 -
---
i
FIG. 7. Comparison of the effects of age with those of changing c&. Oocyte VC6. a and a’ in normal Ringer’s at 23.5 l/l0 Cl-Ringer’s at 24 hr PPT: c and c’ in normal Ringer’ at 26 hr PPT. In a-c the upper trace is membrane potential trace is injected current (nA). The peak potential of each ap is indicated. Horizontal time scale is 20 set (a) or 40 set show membrane currents (nA) recorded during a series of lo-set long voltage clamp pulses to the potential indicated trace. Note that the current scale changes during each series.
had brief ap’s that were repetitive if damage was not too severe. Middle-aged oocytes (first polar body to early metaphase II) had a continuously positive membrane potential (V,). Fully mature (always metaphase II) oocytes had negative resting potentials but could be forced to produce a long-lasting ap by injecting current. There are several lines of evidence that ap’s fire spontaneously in young and middle-aged oocytes. (1) In young oocytes, it frequently happened that spontaneous repetitive firing occurred when one fine-tipped microelectrode was implanted (62% of oocytes from 3 females)
hr PPT, b and b’ in (mV) and the lower (b and c). Parts a’-c’ above each current
but not when two microelectrodes were used (3% of oocytes from more than 40 females). Excessive damage seems to prevent firing. (2) Voltage-clamp measurements showed directly that spontaneous firing seemed to be characteristic of oocytes with a lower leakage conductance. Oocytes that did not fire spontaneously always had larger leakage conductances than the spontaneously firing oocyte in Fig. 2a. (3) Middle-aged oocytes had positive membrane potentials (ap’s) when impaled (74% of oocytes from two experiments). They could not be prevented from firing an ap by transiently
58
DEVELOPMENTALBIOLOGY
injecting current to make V, negative. (4) Mature oocytes had a negative V, when impaled which shows that the Na channels did not open spontaneously. An ap could be evoked by injecting depolarizing current and ended by injecting hyperpolarizing current (about 90% of oocytes from >lO experiments). This shows that damage by impalement did not open the Na channels or keep them open after a current pulse was injected to evoke the ap. (5) The following paper shows that the peak potential of the ap (ENa) decreases throughout maturation as Na enters during the trains of ap’s and that preventing the ap’s also prevents the decrease in ENa, The decrease in ENa occurs without a microelectrode in the oocyte, further evidence that spontaneous ap’s occur in the absence of impalement. The possibility cannot be excluded that slight damage stimulates repetitive firing and more damage prevents firing. If, in future, a noninvasive technique can be used to measure rapid changes in membrane potential in Rana oocytes, this would provide more information about the effect of impalement on repetitive firing. There exist several reports on the membrane potential of amphibian oocytes at different stages of maturity. Why are there no reports of spontaneous or evoked ap’s? There are at least four reasons why ap’s have not previously been found. (1) There are very few reports for oocytes at the right stage of maturity (from metaphase I to metaphase II). For Rana pipiens oocytes, most electrical measurements have been made at or before ovulation, or on mature oocytes (e.g., Morrill and Watson, 1966; Morrill et ak, 1966; Ito, 1972; Cross and Elinson, 1980; Cross, 1981). In three studies, oocytes were examined after germinal vesicle breakdown (GVBD) and before metaphase II. In one (Schlichter and Elinson, 1981), the bathing solution was always 10% Ringer’s. Action potentials do not fire in 10% Ringer’s because there is no inward Na+ current. In the other two studies (Ziegler and Morrill, 197’7; Weinstein et aL, 1982), the membrane potential was zero or slightly positive after GVBD and no action potentials were seen. In the latter two studies the oocytes were dissected from the follicles and matured in vitro. This treatment may have prevented action potentials but the possibility of damage has not been ruled out. (2) The ap’s are only spontaneous if the membrane is not much damaged. Spontaneous firing was associated with a large R, (e.g., 13 m0 or 1000 KQ. cm2 in Fig. 2). Previous reports on V, from amphibian oocytes at the appropriate stages show much lower resistances. For example, reported values of R, for immature Bufo oocytes are from 25 KG. cm’ (Maeno, 1959) to 200 K0. cm2 (Iwao et al, 1981). These values probably in-
VOLUME 98,1983
dicate damage. Furthermore, studies on Rana oocytes at the correct stage have used large microelectrodes (up to 6 pm tip diameter) inserted as deeply as 100 to 300 pm into the oocyte (Morrill et al, 1971; Ziegler and Morrill, 1977). I found that Rana oocytes are very sensitive to damage: for ap’s to reliably occur one fine-tipped microelectrode was inserted less than 10 pm into the oocyte. (3) Large depolarizing currents must be injected to evoke ap’s in oocytes that fail to fire spontaneously. Most previous studies have not done this (for example, Morrill et aZ., 1971; Ziegler and Morrill, 1977). However, when Maeno (1959) injected current into Bufo oocytes he saw a response that had some features of an ap but it is impossible to know from his data what the change in V, signified. (4) Action potentials may not exist in oocytes of all species. Immature Xenopus oocytes can produce a long ap but only under unnatural conditions (Baud et ah, 1982). I have found that long-lived action potentials can be evoked in mature oocytes of Rana clamitans and Xenopus laevis when sufficient current is injected (unpublished results). For Rana clamitans no chemical treatment is necessary; whereas, Xenopus oocytes require pretreatment with a disulfide-reducing agent such as thioglycolate. The ionic basis of the ap’s in Rana pipiens oocytes is unique in several respects. The rising phase of each ap results from an inward Na+ current through voltagesensitive Na channels. The Na channels do not inactivate; therefore, the ap can last many minutes provided there is insufficient outward current to repolarize the membrane. When the membrane does repolarize, the necessary outward current is carried by Cll influx through voltage-sensitive Cl channels. Voltage-sensitive Na channels have been found in tunicate oocytes and in GV-stage Xenopus laevis oocytes, but they differ fundamentally from those in Rana oocytes after germinal vesicle breakdown. The Na+ current in tunicate oocytes develops rapidly (time constants of the order of 10 msec) and rapidly inactivates (within 100 msec) thus ending the ap (Okamoto et al., 1976). In Xenopus oocytes the Na channels must be induced by holding V, more positive than about +30 mV for several seconds (Baud et al, 1982). The current then develops slowly (time constants of the order of seconds) and does not inactivate. The Na channels in Rana oocytes do not require induction: they begin to open as soon as the membrane potential is stepped above threshold. The current develops slowly (time constants of the order of 1 set) and does not inactivate. Of the Na channels in oocytes, only those in Rana oocytes open spontaneously. The mechanism for repolarizing the membrane dur-
L. C. SCHLICHTER
ing repetitive firing differs from other oocytes and from other excitable cells. In most excitable cells one or more type of K-selective channel produces an outward K+ current that repolarizes the membrane to help end each action potential. In addition, the Na channels and usually the Ca channels inactivate rapidly. (For recent reviews see: Thompson and Aldrich (1980) for K channels; Cahalan (1980) for Na channels; Hagiwara and Byerly (1981) for Ca channels.) A detailed study of the properties of the Na and Cl channels in Rana oocytes is in progress. The form of the action potential changed dramatically during the period from metaphase I to metaphase II. The most pronounced changes were an increase in ap duration and a decrease in the peak potential of each ap (ENa). The changes in ENa are described and discussed in detail in the following paper. The increasing duration of the action potentials can be accounted for by a gradual decrease in Cl- permeability. Results of experiments in which external ion concentrations were changed showed that Cl- entry was necessary for repolarizing young oocytes. Reducing c& reduced the outward current and lengthened the ap’s. This mimicked the normal changes in Cl- current during maturation. Older oocytes had a smaller outward Cl- current and longer ap’s. The ap of fully mature oocytes did not depend on external Cl and could last more than 30 min. Therefore, during normal maturation the Cl channel appears to be lost or altered in such a way that it is no longer sensitive to voltage. A question for further study is whether these Cl channels are the same as those responsible for activation and fertilization potentials in mature oocytes but existing in a form that can respond to activating stimuli, not to voltage. It is a pleasure to thank Drs. J. Dainty and R. P. Elinson for stimulating discussions and Drs. Dainty and L. A. Jaffe and Mr. J. W. Tanner for helpful criticisms of the manuscript. Thanks also to Drs. R. T. Kado and C. Baud for help in building the voltage clamp. This work was supported by a grant from the Natural Sciences and Engineering Research Council (Canada) to J. Dainty. REFERENCES BAUD, C., KADO, R. T., and MARCHER, K. (1982). Sodium channels induced by depolarization of the Xenopus laevis ooeyte. Proc. Nat. Acud.
Sci. USA 79,3188-3192
CAHALAN, M. (1980). Molecular properties of sodium channels in excitable membranes. In “The Cell Surface and Neuronal Function” (C. W. Cotman, G. Poste, and G. L. Nicholson, eds.), Vol. 6, pp. l47. Elsevier/North-Holland, Amsterdam. CROSS,N. L. (1981). Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog, Rana pipiens. Dew. BioL
85, 380-384.
CROSS,N. L., and ELINSON, R. P. (1980). A fast block to polyspermy in frogs mediated by changes in the membrane potential. Dev. BioL 75.187198.
Oocyte
Action
Potentials
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EISENBERG,R. S., and ENGEL, E. (1970). The spatial variation of membrane potential near a small source of current in a spherical cell. J. Gen
Physiol
55, ‘736-757.
HAGIWARA, S., and BYERLY, L. (1981). Calcium channel. Annu Rev. Neurosci. 4, 69-125. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Ann Rev. Biophys. Bioeng. 8385-416. ITO, S. (1972). Effects of media of different ionic composition on the activation potential of anuran egg cells. Dev. Grwwth Difleren 14, 217-227. IWAO, Y., ITO, S., and KATAGIRI, CH. (1981). Electrical properties of toad oocytes during maturation and activation. Dev. Growth otfferen. 23,89-100. JACOBSON,S. L., and MEALING, G. A. R. (1980). A method for producing very low resistance micropipettes for intracellular measurements. Electroenceph. Clin Neurophys. 48, 106-108. KADO, R. T., MARCHER, K., and OZON,R. (1979). Mise en evidence d’une depolarisation de longue duree dans l’ovocyte de Xenopus laevis. C. R. Acad. SC. Paris Serie D 288, 1187-1189. KUSANO, K., MILEDI, R., and STINNAKRE, J. (1977). Acetylcholine receptors in the oocyte membrane. Nature (London) 270, 739-741. MAENO, T. (1959). Electrical characteristics and activation potential of Bufo eggs. J. Gen, Physiol. 43, 139-157. MIYAZAKI, S., TAKAHASHI, K., and TSUDA, K. (1972). Calcium and sodium contributions to regenerative responses in the embryonic excitable cell membrane. Science 176, 1441-1443. MIYAZAKI, S., TAKAHASHI, K., and TSUDA, K. (1974). Electrical excitability in the egg cell membrane of the tunicate. J. PhysioL 238, 37-54. MORRILL, G. A., KOSTELLOW, A. B., and MURPHY, J. B. (1971). Sequential forms of ATP-ase activity correlated with changes in cation binding and membrane potential from meiosis to first cleavage in R. p-ipiens. Exp. Cell Res. 66, 289-298. MORRILL, G. A., ROSENTHAL, J., and WATSON, D. E. (1966). Membrane permeability changes in amphibian eggs at ovulation. J. Cell Physiol. 67, 375-382. MORRILL, G. A., and WATSON, D. E. (1966). Transmembrane electropotential changes in amphibian eggs at ovulation, activation and first cleavage. J. Cell PhysioL 67, 85-92. OKAMOTO, H., TAKAHASHI, K., and YOSHII, M. (1976). Membrane currents of the tunicate egg under the voltage-clamp condition. J. PhysioL 254, 607-638.
K. R. (1979). Electrical currents through full-grown and maturing Xewpus oocytes. Proc. Nat. Acad Sci. USA 76, 837-841. SCHLICHTER, L. C., and ELINSON, R. P. (1981). Electrical responses of immature and mature Rana pipiens oocytes to sperm and other activating stimuli. Dev. Bid 83, 33-41. SUBTELNY,S., and BRADT, C. (1961). Transplantations of blastula nuclei into activated eggs from the body cavity and from the uterus of Rana pip&s. II. Development of the recipient body cavity eggs.
ROBINSON,
De-v. BioL
3,96-114.
THOMPSON, S. H., and ALDRICH, R. W. (1980). Membrane potassium channels. In “The Cell Surface and Neuronal Function” (C. W. Cotman, G. Poste, and G. L. Nicholson, eds.), Vol. 6, pp. 49-85. Elsevier/ North-Holland, Amsterdam. WEINSTEIN, S. P., KOSTELLOW, A. B., ZIEGLER, D. H., and MORRILL, G. A. (1982). Progesterone-induced down-regulation of an electrogenie Na+,K+-ATPase during the first meiotic division in amphibian oocytes. J. Membr. BioL 69.41-48. ZIEGLER, D., and MORRILL, G. A., (1977). Regulation of the amphibian oocyte plasma membrane ion permeability by cytoplasmic factors during the first meiotic division. Deu. BioL 60, 318-325.
