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Developmental change of a depolarization-induced sodium permeability in the oocyte of Xenopus laevis
DEVELOPMENTAL
BIOLOGY
(1983)
99,524-528
Developmental Change of a Depolarization-Induced Sodium Permeability in the Oocyte of Xenopus laevis CHRISTIANE Laboratoire
de Neurobiolcgie
Received March
Cellulaire
18, 1983;
BAUD’ CNRS G+sur- Yvette 91190, France
accepted in revised form May
18, 1983
In the full grown oocyte of Xenqpus luevti, a sodium permeability can be induced by depolarization potential with current injection. Voltage-clamp analysis has shown that depolarization causes a long-lasting of the membrane during which voltage-gated sodium channels become functional. In the present study, for the existence of these channels during cell growth. The channels appear during a very restricted growth, corresponding to Dumont stage V. A possible biological function is discussed. INTRODUCTION
has not been studied, but voltage alone is insufficient to cause them to open (Cross and Elinson, 1980; Grey et cd, 1982). The aim of the present study was to look for the existence of the sodium channels during development of the Xenopus oocyte, with the expectation that this could give a hint at their function. In the ovary, oocytes grow to a final diameter of 1.4 mm. Growth has been morphologically characterized and separated in six successive stages (Dumont, 1972). Channels have been induced in oocytes at different stages of development. Conductances (i.e., the ratio of current to driving force) have been compared. It is shown that sodium channels appear in the membrane during a very restricted period of cell growth, corresponding approximately to Dumont stage V.
Following the first finding by Miyazaki et al. (1972) of excitability in a tunicate egg, membrane potentialgated channels have been found in eggs and oocytes of many animal species (reviewed by Hagiwara and Jaffe, 1979); calcium-selective channels are the most widespread, sodium selective channels have been found only in tunicate and recently in Amphibians eggs (Schlichter, 1983a). In the oocyte of Xenopus luevis, a sodium-selective channel has been described, which displays some unusual features (Kado et aL, 1979; Baud et al, 1982; Baud and Kado, 1983). The channels have to be “induced” by a depolarization of several seconds to a positive potential; after induction they show activation as do other voltagegated channels; in addition, they do not inactivate; if the membrane potential is brought back to a negative value they close, displaying a “tail current”; if the membrane potential is maintained at a negative value, they slowly disappear, over several minutes. In spite of their particularities, these channels are probably similar to other membrane-gated sodium channels since they have a functional selectivity filter and a voltage-gating mechanism. The biological significance of membrane potentialgated channels in most egg cells is not known. In the egg of Urechis, Jaffe et aL (1979) have shown that an action potential due to calcium-selective voltage-gated channels amplifies the fertilization potential, the fertilization potential itself being due to sperm-gated sodium channels. However, in amphibians the fertilization potential is due to the activation of a chloride conductance only; the voltage dependence of these channels i Present address: Jerry Lewis Neuromuscular partment of Physiology UCLA, Los Angeles,
Research Center Calif. 90024.
MATERIAL
Copyright All rights
$3.00
0 1933 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
The animals were kept in laboratory holding tanks filled with tap water, and were lighted on a 12-hr daynight cycle. They had received no hormonal treatment of any kind. They were anesthetized in MS222 and a piece of ovary was surgically removed. Oocytes free of follicular cells were obtained by treatment in a 40 mg/ 100 ml Dispase (Neutral Protease Grade III, Boehringer) in saline solution for 2 hr. Oocytes were dissected free from their follicular cells with fine forceps. The vitelline layer, a net of fibers surrounding the oocyte (Dumont and Brummett, 1978), was not removed in the procedure. The oocytes were then kept in OR2 medium (Wallace et aL, 1973) having the ionic composition Na, 84.5 mM, K, 2.5 mM, Ca, 1 mM; Mg, 1 mM, PO1, 1 mM, Hepes, 5 mM, Cl, 86.5 mM, pH 7.4. The oocytes were generally used up to 3 or 4 days postdissection and kept at 5°C during the night. Electrophysiological measurements
De-
524 0012-1606/83
to positive modification I have looked period of cell
BRIEF
were done at 16-17°C. The bath was cooled by a Peltier cell placed under the chamber. Membrane potential control was achieved by a twomicroelectrode voltage-clamp amplifier. The electrodes were filled with 3 MKCl; their resistance ranged between 1 and 10 MOhms. Membrane potential and current were continuously recorded on a pressure ink pen recorder (Brush, Gould Inc.). The current was measured and the current-voltage curves were plotted by hand. The cell membrane surface area was quantified by the cell electrical capacitance. In each cell, a 1-nA current was injected in constant-current mode and the membrane potential shift was recorded and analyzed for resistance and capacitance measurements. The resistance was calculated as the ratio of the voltage shift to the applied current. The capacitance was calculated from the time constant of the exponential membrane potential change since this time constant is the product of the cell resistance and the cell capacitance. The cell capacitance was chosen as an index of the cell surface rather than the cell diameter, because the cell surface becomes covered with microvilli during cell growth (Dumont, 1972). Thus, cells of the same diameter can have very different membrane surface areas (Marcher and Kado, in preparation). RESULTS
Each cell was impaled with both electrodes and the membrane potential was allowed to recover for 10 to 20 min, until it reached a steady value. Typical membrane resting potentials were between -60 and -80 mV. A lnA pulse was applied and the membrane potential change was analyzed for resistance and capacitance measurements as described under Materials and Methods. Characteristics of the cell population studied are presented in Table 1.
CHARACTERISTICS
Capacitance W) O-50 50-100 loo-150 150-200 200-250 250-300
Dumont stage III IV IV V IV, v VI
TABLE 1 OFTHECELLPOPULATION Diameter (rm) (*SD) 460 + 650 f 880 + 1150 f 1200 f 1300
Resistance (Mohms) (*SD) 150 75 100 150 70
8.5 6.5 2.9 2.6 2.1
f 4.0 + 5.0 -t 0.5 + 0.7 f 0.6 3.05
STUDIED
ENa WV) (*SD) 79 80 84 88 88
+ + f + f 96
N 8.0 9.2 7.5 7.8 3.9
5 4 3 19 6 1
Note. Ceils were classified according to electrical capacitance, which is directly proportional to the membrane surface. Corresponding Dumont stages have been determined from diameter measurements, according to Dumont (1972). ENa is the potential at which the transient current through the induced channels reverses its direction. Cells have been obtained from two different animals.
NOTES
525
As described in the Introduction, the number of voltage-gated channels in a given cell depends on the duration (and possibly on the amplitude) of the inducing depolarization; furthermore, it also depends on the time during which the membrane is kept at a negative potential after induction, since the channels disappear. It was thus important to perform exactly the same sequence of depolarization on each cell. Figure 1A shows a chart recording of a standard experiment. During the first inducing depolarization, the current showed the typical sigmoidal time course corresponding to the induction. A series of 3-set pulses to different membrane potentials was then applied. Induced channels opened at the onset of the pulse and closed at the return to resting potential. The overall procedure lasted less than 3 min after the end of induction. In all traces leakage current was not subtracted. The current at the end of each pulse was measured and plotted against the membrane potential at which it was obtained (Fig. 1B). Extrapolation of the line obtained at negative potentials to the positive potential range gave an estimate of the current due to the passive properties of the cell membrane. The intersection of this line with the I-Vcurve gave the reversal potential. The reversal potential was also estimated by direct observation of the transient current during a test depolarization, as the potential at which this transient reverses its direction (see legend of Fig. 1). Reversal potentials estimated in both ways were in good agreement.
Variability of the Reversal Potential within D#erent Cells As shown in the second to last column of Table 1, the reversal potential showed a large variability within cells at similar stages. It is known that after defolliculation the Na-K ATPase is activated leading to a decrease of internal sodium activity (Vitto and Wallace, 1976; Wallace and Steinhardt, 1977), this extrusion taking place in 6 to 7 hr. Although the cells were allowed to sit for more than 6 hr at room temperature before impalement, a difference in sodium extrusion might account largely for the variability between cells at the same stage. Furthermore, there is a slight increase in the reversal potential during cell growth (a Student t test between the first and the second to last groups gave P < 0.05). This could also be due to a difference in the rate of extrusion, although other possibilities cannot be ruled out before a Na-sensitive electrode is used to clarify this point.
Variation of the Total Sodium Conductance When the Cell Size Increases Comparison of I- Vcurves from cells of different stages showed that the inward current was always smaller in
526
DEVELOPMENTAL BIOLOGY
7
nA
B
FIG. 1. (A) Standard test procedure for inducing and measuring I-Vcharacteristics of the channels in a given cell (upper trace, membrane potential; lower trace, current). A depolarization is applied to +80 mV for 30 sec. The current becomes slowly inward, following a sigmoidal time course (induction and opening). The membrane potential is then stepped to the resting level (-60 mV), the current goes to zero, following a large capacitive transient and an inward tail current. During the following test pulses, a rapid current component is observed, it corresponds to the opening of the channels which have been induced during the first long-lasting depolarization. The potential at which the direction of this current reverses is an estimate of the reversal potential for the sodium current. In this case it is between the potentials applied during the first two short pulses, at about +95 mV. Cell diameter: 1.04 mm, capacitance: 170 nF. (B) I-V curve obtained from the traces in A. Extrapolation of the curve obtained at negative potential to the positive potential (dotted line) allows an estimation of the sodium reversal potential, which is close to the potential estimated in A. However, in some cases such as the one presented here, a small discrepancy was observed, the potential estimated by the transient current being always larger than the crossing point potential. Since the former is more likely to reflect the true reversal potential, a slightly curved extrapolated line was drawn rather than a straight line. This curvature can he explained by a small amount of instantaneous rectification in the resting permeability.
smaller cells. In Fig. 2A, I-V curves obtained in three cells at different developmental stages are shown, corresponding to cell capacitance of 60, 170, and 220 nF. In these cells, the reversal potential was similar. In such a case, there was no indication of a shift of the curves along the voltage axis. Therefore it can be assumed that the channels have the same sensitivity to membrane
VOLUME 99,1983
potential at different stages. Thus, if the single channel conductance is unchanged, the total conductance measured at a given potential is proportional to the number of channels which can be induced in the membrane. The conductance at +50 mV was plotted against cell electrical capacitance. Data from 38 cells, with capacitances ranging from 30 to 280 nF, are shown in Fig. 2B. The numbers under the abscissa give the corresponding Dumont stage of development (Dumont, 19’72), based on diameter measurements. The plot shows that the sodium conductance is not a linear function of the membrane surface. In the early part of the plot (cell capacitance between 20 and 80 nF) the conductance slightly increases. It is followed by a period during which no more channels are added to the membrane although the cell surface undergoes a clear increase between 90 and 150 nF. This plateau period corresponds to the time during which the cell acquires its morphological differentiation between animal and vegetal poles, as observed by the concentration of the pigment at the animal pole (Dumont stage IV). There is a sharp increase of the total conductance when the cell capacitance changes from 150 to 180 nF corresponding to a diameter change between 900 and 1100 pm (end of stage IV and stage V). This is a period of very active yolk accumulation, after the morphological differentiation between animal and vegetal poles has taken place. These sodium channels were not observed in ovulated eggs from females stimulated with human chorionic gonadotropin, nor were they found in embryonic cells at early stages (data not shown). Thus they seem to be inducible only in the immatured oocyte. DISCUSSION
In the full grown oocyte of Xenopus laevis, electrically excitable sodium channels are present in the membrane, although they are not readily functional. They have to be induced by a long-lasting depolarization to a positive potential. This induction probably does not depend on de novo protein synthesis since cycloheximide has no effect on the induction rate (Baud and Kado, 1983). This suggests that the molecular components of the sodium channels do exist in the oocyte membrane or close to the membrane. In this report I have looked for their existence during the growth of the oocyte, by studing cells of different diameters, between 200 and 1300 pm. Assuming that channel sensitivity to membrane potential is the same during development of the cell, the number of channels that can be induced in the membrane is directly proportional to the conductance at a given potential. It is shown that the number of channels that can be induced increases drastically during a very short growth period, corresponding approximately to Dumont
BRIEF
NOTES
527
I-111 , I
FIG. 2. (A) to right. Dumont
I-V curves from three different cells, at different developmental stages, Notice that the current scale is different in each case. (B) Plot of the conductance stages are indicated below the cell capacitance.
stage V. However, from the data reported here, the underlying mechanism cannot be determined. An increased synthesis rate could occur during this period or preexisting structures might become sensitive to induction by membrane depolarization, or channels could be incorporated in the membrane via vesicle secretion. The sodium content of oocytes of BzGfo and Rum increases during cell growth (Cannon et al., 1974). To account for it, an increase of passive sodium influx has been postulated. It is thus tempting to speculate that the sodium channels found in the membrane allow this
having the capacitance 60, 1’70, and 220 nF from left at +50 mV versus cell capacitance. The corresponding
influx of sodium ions at a certain period of the oocyte development. For this to be true, the channels would have to be made functional under normal conditions. Since it is very unlikely that the cell will experience long-lasting depolarizations to a positive potential, there must exist another physiological way to open these sodium-selective channels. Recently, sodium-selective, electrically gated slow sodium channels have been reported in Rana oocytes (Schlichter, 1983a). These channels do not need to be induced but they open slowly (in the order of several
528
DEVELOPMENTAL BIOLOGY
seconds) and they do not inactivate, this might indicate a similarity with the sodium channels found in Xenopuzx However, they open spontaneously during maturation and serve to load the oocyte with sodium (Schlichter, 1983b); in Xenopus, no spontaneous regenerative depolarization is observed during maturation (Kado et al, 1981), thus it is doubtful that both channels serve the same function. I am grateful to Dr. R. T. Kado, in whose laboratory this work has been performed for advice and encouragement. I thank Drs. M. Barish, D. W. Hilgemann, and R. Gunning for valuable criticisms on the manuscript. The author was supported by a fellowship from the Delegation i la Recherche Scientifique et Technique. REFERENCES BAUD, C., and KADO, R. T. (1983). Induction and disappearance of a sodium selective membrane potential sensitive channel: A voltage clamp study. J. Physid Submitted for publication. BAUD, C., KADO, R. T., and MARCHER, K. S. (1982). Sodium channels induced by depolarization of the Xenopus la&s oocyte. Proc. Nat. Acad Sci USA 79.3188-3192. CANNON,J. D., DICK, D. A. T., and HO-YEN, D. 0. (1974). Intracellular sodium and potassium concentrations in toad and frog oocytes during development. J. Physid 241,497-508. CROSS,N. L., and ELINSON, R. P. (1989). A fast block to polyspermy in frog mediated by changes in the membrane potential. Dev. Biol 75, 187-198. DUMONT, J. N. (1972). Oogenesis in Xelzqpus Zueuis. I. Stages of oocytes development in laboratory maintained animals. J. Morphol 136, 155-179.
VOLUME 99, I983
DUMONT, J. N., and BRUMMETT, A. R. (1978). Oogenesis in Xenopus Levis (Daudin). V. Relationships between developing oocytes and their follicular tissues. J. Morph& 136, 73-97. GREY, R. D., BASTIANI, M. J., WEBB, D. J., and SCHERTEL, E. R. (1982). An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xaspus laewia Dev. Biol. 89.475-484. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8, 385-416. JAFFE, L. A., GOULD-SOMERO, M., and HOLLAND, L. (1979). Ionic mechanism of the fertilization potential of the marine worm, Urechis coup0 (Echiura). J. Gen Physid 73.469-492. KADO, R. T., MARCHER, K. S., and OZON, R. (1979). Mise en evidence dune depolarisation de longue du&e dans l’ovocyte de Xaspus laaria C.R. Ad Sti Park 288D. 1187-1189. KADO, R. T., MARCHER, K., and OZON, R. (1981). Electrical membrane properties of the Xenqpus laevti oocyte during progesterone induced meiotic maturation. Dev. Biol 64, 471-476. MIYAZAKI, S., TAKAHASHI, K., and TSUUA, K. (1972). Calcium and sodium contributions to regenerative responses in the embryonic excitable cell membrane. Science 176, 1441-1443. SCHLICHTER,L. C. (1983a). Spontaneous action potentials produced by Na and Cl channels in maturing Ranu pipiens oocytes. Den Bid 98.47-59. SCHLICHTER, L. C. (1983b). A role for action potential in maturing Rana pipiem oocytes. Lkv. Bid 96, 60-69. Vrrro, A., and WALLACE, R. A. (1976). Maturation of Xenspus oocytes. I. Facilitation by ouabain. Exp. Cell Res. 97, 56-62. WALLACE, R. A., JARED, D. W., DUMONT, J. N., and SEGA, M. W. (1973). Protein incorporation by isolated amphibian oocytes. III. Optimum incubation conditions. J. Exp. Zool. 184, 321-334. WALLACE, R. A., and STEINHARDT, R. A. (1977). Maturation in Xenopus oocytes. II. Observation on membrane potential. Dev. Bid 57,305316.
BIOLOGY
(1983)
99,524-528
Developmental Change of a Depolarization-Induced Sodium Permeability in the Oocyte of Xenopus laevis CHRISTIANE Laboratoire
de Neurobiolcgie
Received March
Cellulaire
18, 1983;
BAUD’ CNRS G+sur- Yvette 91190, France
accepted in revised form May
18, 1983
In the full grown oocyte of Xenqpus luevti, a sodium permeability can be induced by depolarization potential with current injection. Voltage-clamp analysis has shown that depolarization causes a long-lasting of the membrane during which voltage-gated sodium channels become functional. In the present study, for the existence of these channels during cell growth. The channels appear during a very restricted growth, corresponding to Dumont stage V. A possible biological function is discussed. INTRODUCTION
has not been studied, but voltage alone is insufficient to cause them to open (Cross and Elinson, 1980; Grey et cd, 1982). The aim of the present study was to look for the existence of the sodium channels during development of the Xenopus oocyte, with the expectation that this could give a hint at their function. In the ovary, oocytes grow to a final diameter of 1.4 mm. Growth has been morphologically characterized and separated in six successive stages (Dumont, 1972). Channels have been induced in oocytes at different stages of development. Conductances (i.e., the ratio of current to driving force) have been compared. It is shown that sodium channels appear in the membrane during a very restricted period of cell growth, corresponding approximately to Dumont stage V.
Following the first finding by Miyazaki et al. (1972) of excitability in a tunicate egg, membrane potentialgated channels have been found in eggs and oocytes of many animal species (reviewed by Hagiwara and Jaffe, 1979); calcium-selective channels are the most widespread, sodium selective channels have been found only in tunicate and recently in Amphibians eggs (Schlichter, 1983a). In the oocyte of Xenopus luevis, a sodium-selective channel has been described, which displays some unusual features (Kado et aL, 1979; Baud et al, 1982; Baud and Kado, 1983). The channels have to be “induced” by a depolarization of several seconds to a positive potential; after induction they show activation as do other voltagegated channels; in addition, they do not inactivate; if the membrane potential is brought back to a negative value they close, displaying a “tail current”; if the membrane potential is maintained at a negative value, they slowly disappear, over several minutes. In spite of their particularities, these channels are probably similar to other membrane-gated sodium channels since they have a functional selectivity filter and a voltage-gating mechanism. The biological significance of membrane potentialgated channels in most egg cells is not known. In the egg of Urechis, Jaffe et aL (1979) have shown that an action potential due to calcium-selective voltage-gated channels amplifies the fertilization potential, the fertilization potential itself being due to sperm-gated sodium channels. However, in amphibians the fertilization potential is due to the activation of a chloride conductance only; the voltage dependence of these channels i Present address: Jerry Lewis Neuromuscular partment of Physiology UCLA, Los Angeles,
Research Center Calif. 90024.
MATERIAL
Copyright All rights
$3.00
0 1933 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
The animals were kept in laboratory holding tanks filled with tap water, and were lighted on a 12-hr daynight cycle. They had received no hormonal treatment of any kind. They were anesthetized in MS222 and a piece of ovary was surgically removed. Oocytes free of follicular cells were obtained by treatment in a 40 mg/ 100 ml Dispase (Neutral Protease Grade III, Boehringer) in saline solution for 2 hr. Oocytes were dissected free from their follicular cells with fine forceps. The vitelline layer, a net of fibers surrounding the oocyte (Dumont and Brummett, 1978), was not removed in the procedure. The oocytes were then kept in OR2 medium (Wallace et aL, 1973) having the ionic composition Na, 84.5 mM, K, 2.5 mM, Ca, 1 mM; Mg, 1 mM, PO1, 1 mM, Hepes, 5 mM, Cl, 86.5 mM, pH 7.4. The oocytes were generally used up to 3 or 4 days postdissection and kept at 5°C during the night. Electrophysiological measurements
De-
524 0012-1606/83
to positive modification I have looked period of cell
BRIEF
were done at 16-17°C. The bath was cooled by a Peltier cell placed under the chamber. Membrane potential control was achieved by a twomicroelectrode voltage-clamp amplifier. The electrodes were filled with 3 MKCl; their resistance ranged between 1 and 10 MOhms. Membrane potential and current were continuously recorded on a pressure ink pen recorder (Brush, Gould Inc.). The current was measured and the current-voltage curves were plotted by hand. The cell membrane surface area was quantified by the cell electrical capacitance. In each cell, a 1-nA current was injected in constant-current mode and the membrane potential shift was recorded and analyzed for resistance and capacitance measurements. The resistance was calculated as the ratio of the voltage shift to the applied current. The capacitance was calculated from the time constant of the exponential membrane potential change since this time constant is the product of the cell resistance and the cell capacitance. The cell capacitance was chosen as an index of the cell surface rather than the cell diameter, because the cell surface becomes covered with microvilli during cell growth (Dumont, 1972). Thus, cells of the same diameter can have very different membrane surface areas (Marcher and Kado, in preparation). RESULTS
Each cell was impaled with both electrodes and the membrane potential was allowed to recover for 10 to 20 min, until it reached a steady value. Typical membrane resting potentials were between -60 and -80 mV. A lnA pulse was applied and the membrane potential change was analyzed for resistance and capacitance measurements as described under Materials and Methods. Characteristics of the cell population studied are presented in Table 1.
CHARACTERISTICS
Capacitance W) O-50 50-100 loo-150 150-200 200-250 250-300
Dumont stage III IV IV V IV, v VI
TABLE 1 OFTHECELLPOPULATION Diameter (rm) (*SD) 460 + 650 f 880 + 1150 f 1200 f 1300
Resistance (Mohms) (*SD) 150 75 100 150 70
8.5 6.5 2.9 2.6 2.1
f 4.0 + 5.0 -t 0.5 + 0.7 f 0.6 3.05
STUDIED
ENa WV) (*SD) 79 80 84 88 88
+ + f + f 96
N 8.0 9.2 7.5 7.8 3.9
5 4 3 19 6 1
Note. Ceils were classified according to electrical capacitance, which is directly proportional to the membrane surface. Corresponding Dumont stages have been determined from diameter measurements, according to Dumont (1972). ENa is the potential at which the transient current through the induced channels reverses its direction. Cells have been obtained from two different animals.
NOTES
525
As described in the Introduction, the number of voltage-gated channels in a given cell depends on the duration (and possibly on the amplitude) of the inducing depolarization; furthermore, it also depends on the time during which the membrane is kept at a negative potential after induction, since the channels disappear. It was thus important to perform exactly the same sequence of depolarization on each cell. Figure 1A shows a chart recording of a standard experiment. During the first inducing depolarization, the current showed the typical sigmoidal time course corresponding to the induction. A series of 3-set pulses to different membrane potentials was then applied. Induced channels opened at the onset of the pulse and closed at the return to resting potential. The overall procedure lasted less than 3 min after the end of induction. In all traces leakage current was not subtracted. The current at the end of each pulse was measured and plotted against the membrane potential at which it was obtained (Fig. 1B). Extrapolation of the line obtained at negative potentials to the positive potential range gave an estimate of the current due to the passive properties of the cell membrane. The intersection of this line with the I-Vcurve gave the reversal potential. The reversal potential was also estimated by direct observation of the transient current during a test depolarization, as the potential at which this transient reverses its direction (see legend of Fig. 1). Reversal potentials estimated in both ways were in good agreement.
Variability of the Reversal Potential within D#erent Cells As shown in the second to last column of Table 1, the reversal potential showed a large variability within cells at similar stages. It is known that after defolliculation the Na-K ATPase is activated leading to a decrease of internal sodium activity (Vitto and Wallace, 1976; Wallace and Steinhardt, 1977), this extrusion taking place in 6 to 7 hr. Although the cells were allowed to sit for more than 6 hr at room temperature before impalement, a difference in sodium extrusion might account largely for the variability between cells at the same stage. Furthermore, there is a slight increase in the reversal potential during cell growth (a Student t test between the first and the second to last groups gave P < 0.05). This could also be due to a difference in the rate of extrusion, although other possibilities cannot be ruled out before a Na-sensitive electrode is used to clarify this point.
Variation of the Total Sodium Conductance When the Cell Size Increases Comparison of I- Vcurves from cells of different stages showed that the inward current was always smaller in
526
DEVELOPMENTAL BIOLOGY
7
nA
B
FIG. 1. (A) Standard test procedure for inducing and measuring I-Vcharacteristics of the channels in a given cell (upper trace, membrane potential; lower trace, current). A depolarization is applied to +80 mV for 30 sec. The current becomes slowly inward, following a sigmoidal time course (induction and opening). The membrane potential is then stepped to the resting level (-60 mV), the current goes to zero, following a large capacitive transient and an inward tail current. During the following test pulses, a rapid current component is observed, it corresponds to the opening of the channels which have been induced during the first long-lasting depolarization. The potential at which the direction of this current reverses is an estimate of the reversal potential for the sodium current. In this case it is between the potentials applied during the first two short pulses, at about +95 mV. Cell diameter: 1.04 mm, capacitance: 170 nF. (B) I-V curve obtained from the traces in A. Extrapolation of the curve obtained at negative potential to the positive potential (dotted line) allows an estimation of the sodium reversal potential, which is close to the potential estimated in A. However, in some cases such as the one presented here, a small discrepancy was observed, the potential estimated by the transient current being always larger than the crossing point potential. Since the former is more likely to reflect the true reversal potential, a slightly curved extrapolated line was drawn rather than a straight line. This curvature can he explained by a small amount of instantaneous rectification in the resting permeability.
smaller cells. In Fig. 2A, I-V curves obtained in three cells at different developmental stages are shown, corresponding to cell capacitance of 60, 170, and 220 nF. In these cells, the reversal potential was similar. In such a case, there was no indication of a shift of the curves along the voltage axis. Therefore it can be assumed that the channels have the same sensitivity to membrane
VOLUME 99,1983
potential at different stages. Thus, if the single channel conductance is unchanged, the total conductance measured at a given potential is proportional to the number of channels which can be induced in the membrane. The conductance at +50 mV was plotted against cell electrical capacitance. Data from 38 cells, with capacitances ranging from 30 to 280 nF, are shown in Fig. 2B. The numbers under the abscissa give the corresponding Dumont stage of development (Dumont, 19’72), based on diameter measurements. The plot shows that the sodium conductance is not a linear function of the membrane surface. In the early part of the plot (cell capacitance between 20 and 80 nF) the conductance slightly increases. It is followed by a period during which no more channels are added to the membrane although the cell surface undergoes a clear increase between 90 and 150 nF. This plateau period corresponds to the time during which the cell acquires its morphological differentiation between animal and vegetal poles, as observed by the concentration of the pigment at the animal pole (Dumont stage IV). There is a sharp increase of the total conductance when the cell capacitance changes from 150 to 180 nF corresponding to a diameter change between 900 and 1100 pm (end of stage IV and stage V). This is a period of very active yolk accumulation, after the morphological differentiation between animal and vegetal poles has taken place. These sodium channels were not observed in ovulated eggs from females stimulated with human chorionic gonadotropin, nor were they found in embryonic cells at early stages (data not shown). Thus they seem to be inducible only in the immatured oocyte. DISCUSSION
In the full grown oocyte of Xenopus laevis, electrically excitable sodium channels are present in the membrane, although they are not readily functional. They have to be induced by a long-lasting depolarization to a positive potential. This induction probably does not depend on de novo protein synthesis since cycloheximide has no effect on the induction rate (Baud and Kado, 1983). This suggests that the molecular components of the sodium channels do exist in the oocyte membrane or close to the membrane. In this report I have looked for their existence during the growth of the oocyte, by studing cells of different diameters, between 200 and 1300 pm. Assuming that channel sensitivity to membrane potential is the same during development of the cell, the number of channels that can be induced in the membrane is directly proportional to the conductance at a given potential. It is shown that the number of channels that can be induced increases drastically during a very short growth period, corresponding approximately to Dumont
BRIEF
NOTES
527
I-111 , I
FIG. 2. (A) to right. Dumont
I-V curves from three different cells, at different developmental stages, Notice that the current scale is different in each case. (B) Plot of the conductance stages are indicated below the cell capacitance.
stage V. However, from the data reported here, the underlying mechanism cannot be determined. An increased synthesis rate could occur during this period or preexisting structures might become sensitive to induction by membrane depolarization, or channels could be incorporated in the membrane via vesicle secretion. The sodium content of oocytes of BzGfo and Rum increases during cell growth (Cannon et al., 1974). To account for it, an increase of passive sodium influx has been postulated. It is thus tempting to speculate that the sodium channels found in the membrane allow this
having the capacitance 60, 1’70, and 220 nF from left at +50 mV versus cell capacitance. The corresponding
influx of sodium ions at a certain period of the oocyte development. For this to be true, the channels would have to be made functional under normal conditions. Since it is very unlikely that the cell will experience long-lasting depolarizations to a positive potential, there must exist another physiological way to open these sodium-selective channels. Recently, sodium-selective, electrically gated slow sodium channels have been reported in Rana oocytes (Schlichter, 1983a). These channels do not need to be induced but they open slowly (in the order of several
528
DEVELOPMENTAL BIOLOGY
seconds) and they do not inactivate, this might indicate a similarity with the sodium channels found in Xenopuzx However, they open spontaneously during maturation and serve to load the oocyte with sodium (Schlichter, 1983b); in Xenopus, no spontaneous regenerative depolarization is observed during maturation (Kado et al, 1981), thus it is doubtful that both channels serve the same function. I am grateful to Dr. R. T. Kado, in whose laboratory this work has been performed for advice and encouragement. I thank Drs. M. Barish, D. W. Hilgemann, and R. Gunning for valuable criticisms on the manuscript. The author was supported by a fellowship from the Delegation i la Recherche Scientifique et Technique. REFERENCES BAUD, C., and KADO, R. T. (1983). Induction and disappearance of a sodium selective membrane potential sensitive channel: A voltage clamp study. J. Physid Submitted for publication. BAUD, C., KADO, R. T., and MARCHER, K. S. (1982). Sodium channels induced by depolarization of the Xenopus la&s oocyte. Proc. Nat. Acad Sci USA 79.3188-3192. CANNON,J. D., DICK, D. A. T., and HO-YEN, D. 0. (1974). Intracellular sodium and potassium concentrations in toad and frog oocytes during development. J. Physid 241,497-508. CROSS,N. L., and ELINSON, R. P. (1989). A fast block to polyspermy in frog mediated by changes in the membrane potential. Dev. Biol 75, 187-198. DUMONT, J. N. (1972). Oogenesis in Xelzqpus Zueuis. I. Stages of oocytes development in laboratory maintained animals. J. Morphol 136, 155-179.
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DUMONT, J. N., and BRUMMETT, A. R. (1978). Oogenesis in Xenopus Levis (Daudin). V. Relationships between developing oocytes and their follicular tissues. J. Morph& 136, 73-97. GREY, R. D., BASTIANI, M. J., WEBB, D. J., and SCHERTEL, E. R. (1982). An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xaspus laewia Dev. Biol. 89.475-484. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8, 385-416. JAFFE, L. A., GOULD-SOMERO, M., and HOLLAND, L. (1979). Ionic mechanism of the fertilization potential of the marine worm, Urechis coup0 (Echiura). J. Gen Physid 73.469-492. KADO, R. T., MARCHER, K. S., and OZON, R. (1979). Mise en evidence dune depolarisation de longue du&e dans l’ovocyte de Xaspus laaria C.R. Ad Sti Park 288D. 1187-1189. KADO, R. T., MARCHER, K., and OZON, R. (1981). Electrical membrane properties of the Xenqpus laevti oocyte during progesterone induced meiotic maturation. Dev. Biol 64, 471-476. MIYAZAKI, S., TAKAHASHI, K., and TSUUA, K. (1972). Calcium and sodium contributions to regenerative responses in the embryonic excitable cell membrane. Science 176, 1441-1443. SCHLICHTER,L. C. (1983a). Spontaneous action potentials produced by Na and Cl channels in maturing Ranu pipiens oocytes. Den Bid 98.47-59. SCHLICHTER, L. C. (1983b). A role for action potential in maturing Rana pipiem oocytes. Lkv. Bid 96, 60-69. Vrrro, A., and WALLACE, R. A. (1976). Maturation of Xenspus oocytes. I. Facilitation by ouabain. Exp. Cell Res. 97, 56-62. WALLACE, R. A., JARED, D. W., DUMONT, J. N., and SEGA, M. W. (1973). Protein incorporation by isolated amphibian oocytes. III. Optimum incubation conditions. J. Exp. Zool. 184, 321-334. WALLACE, R. A., and STEINHARDT, R. A. (1977). Maturation in Xenopus oocytes. II. Observation on membrane potential. Dev. Bid 57,305316.