- Email: [email protected]
The development of calcium and potassium currents during oogenesis in the starfish, Leptasterias hexactis
DEPELOPMENTAL
BIOLOGY
112, 405-413
The Development
(19%)
of Calcium and Potassium Currents during Oogenesis in the Starfish, Leptasterias hexactis WILLIAM
J. MOODY
electrical properties of oocytes of the starfish Lrptasterias hcmctis during oogenesis The development of memhrane was studied using voltage- and current-clamp techniques. Two voltage-dependent K currents-the fast transient and inwardly rectifying-are present early in oogenesis, before the rapid growth phase, and are maintained throughout oogenesis at the same current density and kinetics. The inward current, which is composed of a Ca current and a slower Ca-dependent inward sodium current, is also present early in oogenesis, but at very low current density. Late in oogenesis, after the oocrte has grown to full size, the inward current increases in amplitude by about fivefold, and undergoes major changes in kinetics. These changes are closely associated with the migration of the germinal vesicle to the cell periphery. The relationship of these events to electrophysiological changes during subsequent maturation and fertilization of the oocytes is discussed. C 1985Academic Press, Inc INTRODUCTION
tivated by briefer depolarizations (Kado et ul., 1979; Baud ef al., 1982). Baud (1983) has shown that this channel appears rather abruptly during oogenesis-at Dumont stage V-after oocytes have reached about 90% of their final diameter. In the present study, the electrical properties of Lepfasterias oocytes were examined under current- and voltage-clamp conditions during the final year of the 2year oogenesis cycle. This period includes oocytes from 250 to 800 pm in diameter, before, during, and after the phase of major vitellogenic activity. The results indicate that both types of K currents (inwardly rectifying and fast transient; see Moody and Bosma, 1985) are present early in oogenesis in essentially their final forms, whereas the inward Ca current undergoes major changes in both current density and kinetics late in oogenesis, after the oocyte has reached its full size.
In the previous paper (Moody and Bosma, 1985) we showed that calcium and potassium currents in oocytes were affected quite of the starfish Lepfasterias hexactis differently during meiotic maturation: both the inwardly rectifying and the fast transient K currents decreased substantially in magnitude, whereas the inward Ca current was unchanged. Moreover, the data indicated that the partial loss of both types of K currents was a result of the decrease in membrane surface area during maturation, and that by some mechanism, Ca channels were protected from loss during this process. In this paper I examine the development of Ca and K channels during oogenesis in Leptasterias. The purpose of these experiments was to compare the behavior of these channels during the addition of plasma membrane associated with cell growth to their behavior during the membrane loss associated with maturation. From this comparison, it might be possible to better understand the mechanisms controlling the relationship of the properties of these ion channels to the cellular events of early development. There have been few studies of the electrical properties of oocytes during oogenesis. Holland and Gould-Somero (1981) reported that Urechis oocytes in various stages of oogenesis all showed a similar two-phase action potential in response to injected current, as well as similar resting potentials and input resistances. In mouse oocytes both the overshoot and rate of rise of the Ca-dependent action potential increase between preantral and metaphase II oocytes (Eusebi et al., 1983). Xewqms oocytes possess an unusual Na channel which is “induced” by prolonged depolarizations, and once induced, is ac-
MATERIALS
AND
METHODS
Methods of animal collection, maintenance, voltage clamping, and data acquisition were all identical to those described in the previous paper (Moody and Bosma, 1985). Recordings were made from 68 oocytes during two separate seasons. For experiments with smaller oocytes (200-350 pm), ovaries were torn open with forceps and cut into small pieces with fine scissors. A piece of ovary containing several oocytes was pinned in the experimental chamber and oocytes were impaled while still loosely attached to the ovarian tissue. This did not create a significant series resistance or diffusion problem. Microelectrodes used to impale and voltage-clamp the smaller oocytes had somewhat higher resistances than 405
0012-1606/85 Copyright All rights
$3.00
C 1985 by Academic Press. Inc. of reproduction in any form reserwd
406
DEVELOPMENTAL BIOLOGY
those used for the larger cells, but in all cases resistances were kept below 10 MQ, to ensure adequate voltageclamp control. Resting potentials of -60 mV or greater could be obtained at all stages, but only with electrodes which were of too high resistance to permit adequate voltage-clamp control. (Because of the voltage dependence of the resting conductance in starfish and many other oocytes, a small leakage at the site of impalement can create a disproportionately large loss of resting potential. See Hagiwara and Jaffe, 1979). For voltage clamp, impalements were judged by the amplitude of leakage currents relative to the time- and voltage-dependent currents. Oocytes which showed significant leakage currents under voltage-clamp were rejected; leakage currents have not been subtracted from any of the records shown. For each cell, the following data were collected: an 1-V relation under voltage-clamp for both hyperpolarizing and depolarizing voltage pulses, the response of the oocyte to depolarizing current pulses under current clamp, total membrane capacitance, and cell diameter. In the later stages of oogenesis, when oocytes developed significant inward current (see below), I-V relations were also recorded with the inward currents eliminated in Na, Ca-free external solution, so that the A-current could be studied in isolation. In the sections below, total capacitance is taken as a measure of the membrane surface area, both to determine current densities and to measure oocyte growth in terms of the amount of surface membrane. All experiments were done at 12°C. RESULTS
Although Leptasterias has an annual spawning cycle, oogenesis requires 2 years (Chia, 1968). There is a phase of slow growth from January of the first year to February of the second year, during which the diameter of the oocyte increases from about 30 to about 200 pm. A period of rapid growth follows, from March through July of the second year, during which oocyte diameter increases from 200 to 800 pm, its final value. From July to November (or later) of the second year, the only obvious morphological change in the oocytes is the migration of the germinal vesicle from a central to a peripheral position, in September. Oocytes become responsive to lmethyladenine-the maturation hormone-at about this time. Spawning occurs in November-April. Thus in a single animal there are always two populations of oocytes, which can readily be distinguished on the basis of diameter: those which will be spawned during the next breeding season, and those which will be spawned a year later (see Chia, 1968, his Fig. 1). This study included oocytes 200-800 pm in diameter, which covers the second year of oogenesis, beginning just before the period of rapid growth.
VOLIJME 112, 1985
There are three principal voltage-dependent currents in fully grown, l-MA responsive Leptasterias oocytes (Moody and Lansman, 1983; Moody and Bosma, 1985): the fast-transient K current, or A-current; the inwardly rectifying K current; and the inward Ca current, which is actually composed of two separate components (see below). No other currents were seen at any stage of oogenesis. In the sections below, the development during oogenesis of each of these currents is described.
The Fast-Transient K Current (A-Current) The A-current is activated at voltages positive to -40 mV. The conductance-voltage relation shows half-maximal activation at about -15 mV and saturates at $10 to +20 mV. At +lO mV the current reaches peak amplitude in ca. 10 msec and inactivates with a time constant of about 25 msec (Fig. 1). In molluscan neurons the A-current controls the interspike interval during repetitive firing (Connor and Stevens, 1971). In Leptasterias oocytes, the A-current shunts the inward current and controls the overshoot of the action potential (Moody and Lansman, 1983). Figure 1 summarizes the development of the A-current during oogenesis. In the left portion of the figure, the current (at +lO mV, where conductance is saturated) per unit membrane capacitance is plotted vs total membrane capacitance, a measure of membrane area. (A plot of current density vs oocyte diameter is virtually identical.) Sample voltage-clamp records from various stages are also shown; the current gains in these records have been set inversely proportional to cell capacitance, so that the size of the displayed current is proportional to current density. At the earliest stages examined, in which membrane area was only about 8% of its final value, the A-current was already present at its final (prematuration) density. During the entire final year of oogenesis, including the period of rapid growth, the magnitude of the A-current closely followed oocyte growth, so that current density was maintained constant. In oocytes of about 550 pm in diameter (C,,, = 40 nF), which corresponds to midpoint of the rapid growth phase, the current density of the A-current tended to be slightly higher than at either earlier or later times. Comparison of A-currents from recordings made before and after September 1 indicated that germinal vesicle migration in the full grown oocytes was not accompanied by any change in A-current amplitude (Fig. 1). This is in contrast to the inward current, which shows dramatic changes in both magnitude and kinetics during this time (see below). Plots of conductance-voltage relations and inactivation curves at various stages indicated no significant changes in the voltage dependence of activation or in-
WILLIAM
J. MOODY
Electrophysiology
407
of Oogenesis
May
June
14nF
38nF
L I
Aug 65nF
Ott
65nF
LJ
20ms
FIG. 1. Development of the A-current during oogenesis. The left panel shows a plot of A-current density (nA/nF) vs total capacitance (nF). The + symbols distinguish measurements made from oocytes after Sept. 1. The right four panels show sample voltage-clamp records of A-currents at various stages of oogenesis. Each record represents the current during a voltage step from -80 to +lO mV, and is labeled with the month it was recorded and the total capacitance of the oocyte. The gains have been adjusted to be inversely proportional to capacitance, so that the amplitudes of the currents as displayed are proportional to current density. For May, June, Aug., and Oct., the vertical calibration bar represents 22, 58, 100, and 100 nA, respectively. The time scale is the same for all records.
activation of the A-current during oogenesis. The twomicroelectrode voltage-clamp did not permit sufficient resolution of the rising phase of the current to determine activation kinetics, but time to peak did not change during oogenesis. There was a slight slowing of inactivation in some, but not all, fully grown oocytes. These results indicate that A-current channels are present at normal density in the membrane of the oocyte before it enters the period of rapid growth during the second year of oogenesis, and that during the remainder of oogenesis the properties of A-current channels do not change. During the period of rapid growth, A-current channels are apparently added along with new membrane so that A-current density remains roughly constant. The tendency of current density to go through a maximum near the midpoint of the rapid growth phase, although small, was also seen for the inward current (see Fig. 3), and may indicate a slight lag between channel and lipid addition to the surface membrane during the rapid growth phase. The Inwardly
Rectifying
K Current
The inwardly rectifying K current is activated at potentials negative to -70 mV. The small steady-state activation of this channel around -70 mV represents most, if not all, of the resting conductance of Leptasterias (and many other) oocytes (see Hagiwara and Jaffe, 1979). Because of its voltage dependence, the inward rectifier creates a resting conductance which turns off as the mem-
brane is depolarized, creating a high input resistance in the voltage range -10 to -60 mV and thus allowing long duration action potentials and fertilization potentials to be generated with relatively small inward currents and ionic fluxes. The inwardly rectifying K current behaved much the same as the A-current during oogenesis. It was present in oocytes before the period of rapid growth and its current density was maintained throughout oogenesis. No changes in either voltage dependence or kinetics were detected. The Inward
Current in Fully
Grown Oocytes
In order to understand the complex developmental changes in the inward current during oogenesis, it was first necessary to analyze the inward current in oocytes which had completed oogenesis. The inward current is composed of at least two components, which can be most easily separated on the basis of their different rates of inactivation. Figure 2 shows an example of the separation of these two components at V, = -40 mV. Inactivation of the total current follows a two-exponential time course (Fig. 2A), with time constants of 120-200 and 900-1200 msec (-40 mV; 12°C). The slow component is particularly obvious in the lower panel of Fig. 2B, which shows a 5-set voltage pulse to -40 mV. In ASW in which all Ca was replaced with Ba, or in Na-free ASW, the current decreased in amplitude by about 50% and showed only the fast component of inactivation (Figs. 2A, B). A
408
DEVELOPMENTAL BIOLOGY
VOLUME 112, 1985
1 OOms
FIG. 2. Analysis of two components of the inward current in an oocyte which has completed oogenesis. (A) Semi-log plot of the inactivation of the inward current in ASW (0). A straight line was fit to the slow component by eye, and then this relation subtracted from the total plot to obtain the fast component (steeper solid line). These two lines indicate time constants of 1060 and 120 msec, as indicated. A plot of inactivation in Ca-free (Ba) ASW (+) shows only the fast component. The Ba data has been shifted to the right by about 0.15 set so the slope can be compared more easily with the steeper solid line. (B) Voltage-clamp records (-40 mV) of the inward current from this cell. The top panel shows records in ASW and Ca-free (Ba) ASW, as well as the difference current between the two obtained electronically. The lower panel shows the current during a 5-set voltage pulse to -40 mV, to illustrate the slow component of inactivation.
similar analysis of the inward current has been done by Lansman (1985). These results suggest the following model to describe the inward current: The fast component represents a rapidly inactivating “pure” Ca current, and the slow component a Ca-activated nonspecific inward current (Colquhoun et al., 1981; Yellen, 1982), which at -40 mV is carried primarily by Na ions. The two contribute about equally to the total peak inward current. Ca-free (Ba) ASW eliminates the slow component presumably because barium ions entering through the Ca channel fail to activate the Ca-dependent inward current channel. The fact that Ba supports inactivation at exactly the rate of the fast component in normal ASW suggests that inactivation of the Ca channel is voltage-dependent (see Eckert and Chad, 1984, for a review of Ca channel inactivation), which seems to be a common feature of Ca channels in oocytes (see Fox, 1981; Hirano and Takahashi, 1984). The fact that Ca-free (Ba) and Na-free ASW reduce the total inward current by approximately the same amount indicates that Ca and Ba are nearly equally permeant through the Ca channel, which is also consistent with Ca channel permeability in other oocytes (Hirano and Takahashi, 1984). The full time course of the Ca-dependent inward current can be obtained by subtracting the current in Ca-free (Ba) from that in
normal ASW (Fig. 2B). The kinetics of the resulting current are consistent with a Ca-dependent process. During activation the slow component approximates the integral of the “pure” Ca current, and its very long time constant of inactivation is on the order of that of the Ca-dependent K current in molluscan neurons (Barish and Thompson, 1983), which in that preparation is taken to represent the removal of Ca from the submembrane space. An alternative hypothesis-that the slow component of the inward current represents Na permeation throught the Ca channel-has been considered, but tentatively rejected. Divalent ions, especially Ca, are powerful inhibitors of Na permeation through the Ca channel (Hess and Tsien, 1984; Almers et al., 1984), and thus in seawater one would not expect to see a large Na component through the Ca channel. In addition, Ba should be less powerful than Ca in blocking Na permeation (Hess and Tsien, 1984), whereas in Leptasteria,s Ba appears to eliminate the slow component of the inward current. As a further test of this hypothesis, Na currents through the Ca channel were measured in divalent-free ASW. Although very large Na currents could be recorded under these conditions, they showed kinetics which approximated the fast-not the slow-component of the inward current in normal ASW.
WILLIAM
J. MOODY
Electrophysiology
409
of Oogenesis
been adjusted inversely to the cell capacitance, so that the displayed traces show current density. The bottom two records are from two cells of the same diameter and membrane area (and therefore are displayed at the same gain), the one on the left before September 1, and the one on the right after. A significant morphological event which occurs at about this time in oogenesis, after the oocytes reach full size, is the migration of the germinal vesicle (GV) (Chia, Changes in the Inward Current during Oogenesis 1968). In order to determine more exactly the temporal relationship between the increase in inward current Figure 3 shows current density for the total inward amplitude and germinal vesicle migration, 14 oocytes current plotted vs total membrane capacitance (surface from August 24 to September 27 were fixed and sectioned area) as it was for the A-current in Fig. 1. Throughout and the position of the germinal vesicle was measured. the period of rapid growth, the inward current behaved much the same as the A-current. Approximately the In 8 of the 9 oocytes from after September 1, GV migration was complete and the GV was closely apposed same current density was maintained throughout this period (February-August), with a tendency for oocytes (<50 grn) to the plasma membrane. GV migration had with capacitances near 40 nF-about half-way through not been completed in any of the (5) oocytes from before the rapid growth phase-to have somewhat larger curSeptember 1, and the minimum distance from the GV rent densities. There was, however, a major difference to the plasma membrane ranged from 75 to 300 pm. This between the inward current and A-current: the inward suggests a close relationship between the migration of current density that was maintained through the period the germinal vesicle and in the increase in Ca current amplitude. of growth was only about 20% of the final value achieved at the end of oogenesis. The large increase in the inward The sudden increase in amplitude of the inward curcurrent which made up this difference occurred abruptly rent at the time of germinal vesicle migration was acat the beginning of September, after the oocytes had companied by major changes in its kinetics. As is apcompleted the period of rapid growth and reached full parent in the records in Figs. 2 and 3, inward currents size. Figure 3 emphasizes the abruptness of this increase recorded before the period of germinal vesicle migration, in inward current amplitude. Among 26 full-grown oo- even in full-grown oocytes, inactivated much more rapcytes studied after August 1 (700-900 ym diameter; C,,, idly than the large inward currents recorded in later = 65-90 nF), inward currents recorded from oocytes after oocytes. The appearance of slow inactivation was very September 1 were significantly larger than those re- strongly correlated with the increase in current amplicorded earlier, showing an almost nonoverlapping dis- tude during oogenesis. Oocytes with large inward curtribution. This pattern of development is illustrated in rents with rapid inactivation kinetics were only very the records in Fig. 3. As in Fig. 1, the current gains have rarely encountered, and a plot of the slow time constant
As described below, there are dramatic changes in the kinetics of the inward current during oogenesis, mainly involving the slow component. Although these changes are more easily interpreted considering the slow component to be a Ca-mediated inward current, it should be kept in mind that the evidence on this point is not conclusive.
May
+
2OnF
15nA
f Aug
7OnF
7OnA
I
Ckt
70nF
v -.w
325nA
___
1OOms
FIG. 3. Development of the inward current during oogenesis. This figure is analogous to Fig. 1 for the A-current. In the right panels, the gains of the current traces have been adjusted to be inversely proportional to capacitance, so current density is displayed. The voltage-clamp pulse is from -80 to -40 mV. Each record is labeled also with the peak current amplitude. In the bottom two records, a dashed line near the end of the voltage pulse indicates zero current, in order to emphasize the appearance of the slow inactivation.
410
DEVELOPMENTAL
BIOLOGY
vs capacitance (not shown here) showed a distinct break around September 1 identical to that seen in Fig. 3A for current amplitude. At first, these data seemed to suggest that before germinal vesicle migration, oocytes simply did not have the slowly inactivating Ca-dependent component of the inward current. However, as the analysis below indicates, younger oocytes do have the slow component, and it comprises about the same proportion of the total inward current as in the older oocytes, but its kinetics of inactivation are much more rapid and consequently difficult to separate from those of the pure calcium current. Figure 4 shows analysis of inactivation kinetics and the effects of Ca-free (Ba) ASW for a typical oocyte before the period of germinal vesicle migration. (For comparison see Fig. 2, which shows a similar analysis for the inward current of a later oocyte.) The time course of inactivation follows two exponentials, but the slow component is about five times faster than in later oocytes. In 19 oocytes in which the slow time constant of inactivation was determined, it averaged 233 ? 42 msec (n = 10) before September 1 as compared to 948 + 205 msec (n = 9) after. Ca-free (Ba) ASW had the same effect on the inward current in these oocytes as it did in the later oocytes: the slow component of inactivation was eliminated, and the total inward current was reduced by almost 50% (Fig. 4B). The Ca-dependent component obtained by subtraction in the early oocytes showed much more rapid inactivation relative to its activation kinetics than in oocytes after germinal vesicle migration (Fig. 4B).
VOLUME 112, 1985
These results indicate that the Ca-dependent inward current which is probably responsible for the slow phase of inactivation is present well before the period of germinal vesicle migration, and perhaps throughout oogenesis (in oocytes less than about 400 pm in diameter, inward currents were too small for accurate analysis of inactivation kinetics). The major changes which occurred near the time of germinal vesicle migration were a marked slowing of the inactivation kinetics of this current and an increase in its absolute magnitude, although not in its relative contribution to the total inward current. As discussed below, this could result directly from the increase in amplitude and slight slowing of the Ca current itself, or could reflect a change in intracellular Ca buffering or the response of the Ca-dependent channel to internal Ca. Changes in the Action Potential
during
Oogenesis
The development of the action potential during oogenesis generally reflected the development of the inward current. Active responses to depolarizing current pulses were small or nonexistent in early oocytes and became large, and in many cases overshooting, later in oogenesis, at the time when the inward currents became large (Fig. 5). The marked slowing of the kinetics of inactivation of the inward current resulted in a substantial increase in the duration of the action potential in later oocytes (Fig. 5, compare bottom two panels). Since the A-current does not become significant until the membrane potential reaches ~20 to 0 mV, it plays
A
FIG. 4. Analysis of the two components of the inward the left panel, the steeper solid line is the mathematically the raw data in Ca-free (Ba) ASW.
current before germinal vesicle migration. See legend for Fig. 2. Note again that in obtained fast time constant inactivation in normal ASW, and the + symbols indicate
WILLIAM
411
J. MOODY
B
A May
13nF
R
R
July
C Aug
D Nov
70nF
R
40nF
65nF
Rr--
FIG. 5. Development of the action potential during oogenesis. Current clamp records. In each case, the resting potential has been adjusted to -80 mV with holding current so spike waveforms can be accurately compared. In each oocyte, the maximum obtainable response is shown. The top traces indicate the current pulses, and at 0 current mark the bath potential. Note the increased duration and the appearance of the notch on the rising phase in panel D
little if any role in the electrically elicited response of the oocyte until near the period of germinal vesicle migration, when the action potential becomes large enough to reach this voltage range. It is not until this time that the action potential develops the fast and slow rising phases separated by a notch in the waveform, which is characteristic of the fully grown Leptasterias oocyte after germinal vesicle migration. DISCUSSION
The results described above indicate that Ca and K currents show quite different patterns of development during oogenesis in Leptasterias. During the final 12 months of the 24-month oogenesis cycle the oocytes undergo a rapid growth phase, increasing in diameter from 200 to 800 pm, and then enter a “resting period” (Chia, 1968), during which the germinal vesicle migrates to a peripheral position. After the migration of the germinal vesicle, the oocytes become responsive to the maturation hormone (unpublished observation), and are subsequently capable of undergoing development in response to insemination. Both K currents-the A-current and the inward rectifier-were present at normal current density in the smallest oocytes studied (200 pm), before the rapid growth phase. Throughout the remainder of oogenesis, the increase in both K currents matched the
addition of new membrane, so that the current density remained constant. This implies a strict regulation of the density of K channels in the membrane, even during the rapid addition of membrane during growth, although there was some indication of a small transient increase in the density of all currents during the time of most rapid membrane addition. The inward current behaved quite differently. Although a constant current density was maintained throughout rapid growth, this density was much less than the final value at the end of oogenesis. The inward current increased markedly in amplitude after the end of the growth phase, at a time which closely correlated with the approach of the germinal vesicle to a position near the plasma membrane. The inward current is composed of two components, a Ca current and a slowly inactivating Ca-dependent inward current carried primarily by Na. These two components contributed about equally to the peak inward current throughout oogenesis. However, at the time of germinal vesicle migration, when the total inward current increased abruptly, the slowly inactivating component became much slower, by about a factor of five. The development of Ca and K currents during oogenesis is analogous to their behavior during hormone-induced maturation (Moody and Bosma, 1985). During the first hour of maturation, about 50% of the surface membrane is lost, and the A-current is decreased in the same
412
DEVELOPMENTAL BIOLOGY
VOLUME 112. 1985
proportion. During oogenesis, A-current channels are It is also possible that a change in the sequence of present early, at normal density, and channel density events subsequent to Ca entry led to the slowing of the keeps pace with the addition of new membrane. On the Ca-dependent component of the inward current. A pool other hand, Ca currents are unaffected by membrane of calcium in the cortex whose release is triggered by removal during maturation. During oogenesis, the major Ca entry might have appeared or greatly increased in increase in Ca current amplitude occurs after completion size late in oogenesis, or the response of the Ca-depenof oocyte growth, when no new surface membrane is dent channel itself to Ca ions might have been altered. being added. So during both oogenesis and maturation, The activation of Ca-dependent channels is thought to underly the fertilization potential in a variety of oocytes Ca currents appear to be modulated somewhat independently of membrane addition and removal, whereas A- (see Miledi and Parker, 1984; Kline et al, 1985; Steinhardt and Epel, 1974; Miyazaki, 1982). Changes during oogencurrent channels are added or deleted with membrane during both oogenesis and maturation. Our data are not esis in the response of the Ca-dependent channel to cytoplasmic Ca ions, or in the buffering of Ca in the subcomplete regarding the development of the Ca-depenmembrane space, might represent an important stage dent channel (slow component of the inward current). in the preparation of the oocyte for fertilization. In adSince this current was activated only by Ca entering during depolarizing voltage steps, it is possible that we dition, the slowing of the action potential which results from the change in inward current kinetics might allow are examining only a small fraction of the activatable poconductance (possibly represented by those channels in the transition from action potential to fertilization tential to occur without substantial repolarization of the close physical proximity to the Ca channels). In Xenopus membrane, and thus permit a more successful polyoocytes (Miledi and Parker, 1984), only a small portion of the Ca-dependent Cl- current can be activated by de- spermy block (see Miyazaki and Hirai, 1979; Miyazaki, 1979). polarizing voltage steps, in contrast to direct injection Both the increase in amplitude of the Ca current and of Ca buffers. Thus, without using Ca injection to actithe slowing of the kinetics of the Ca-dependent current vate this channel, we cannot determine whether the total late in oogenesis were correlated closely in time with activatable conductance changes during oogenesis. the approach of the germinal vesicle to a position near As with maturation, there is an interesting analogy the plasma membrane. Cytoarchitectural elements between these results and those obtained in amphibian which anchor the germinal vesicle might also participate oocytes. Baud (1983) reported that the voltage-dependent Ca channels in this region, oocytes was present, but at low in inserting or activating Na current in Xenops density, early in oogenesis, and was maintained at ap- altering the properties of Ca-dependent channels, or proximately constant current density throughout most changing cytoplasmic Ca buffering in this portion of the of the growth of the oocyte (see Baud, 1983, Fig. 2B, cytoplasm. The position of the germinal vesicle might also be important in the electrical changes associated noting that current-not current density-is plotted). Later, after the oocyte had reached almost its full size, with maturation. It is possible that the membrane overthere was an abrupt increase in the Na current, by about lying the germinal vesicle is not subject to loss during maturation because of its particular structural relaa factor of five. There are several possible interpretations of the tionship to the nucleus. The clustering of Ca channels in this membrane might provide a physical basis for the change in inward current kinetics which accompanied of Ca channels from loss during the increase in current amplitude. The slowing of the observed “protection” (Moody and Bosma, 1985). This would be Ca-dependent component might have been caused by the maturation increase in Ca entry resulting from the increase in Ca especially interesting since the position of the nucleus current which occurred at the same time during oogen- marks the presumptive animal pole of the egg, and clustering of Ca channels there might play a role either in esis. However, models of Ca diffusion in the cytoplasm affecting polarity or in determining the later partitionand experimental data on the Ca-dependent K channel in molluscan neurons indicates that an increase in the ing of different electrical properties into early blastoamount of Ca entry increases the amplitude, but does meres. not markedly alter the time course of submembrane Ca These experiments were supported by NIH Grant HD 17486 and by activity (see, e.g., Barish and Thompson, 1983). Furthermore, although small Ca currents early in oogenesis a NIH Research Career Development Award to the author. were correlated with rapid inactivation kinetics, in ooREFERENCES cytes with large Ca currents after germinal vesicle migration there was a large range of Ca current amplitudes ALMERS, W., MCCLESKEY, E. W., and PALADE, P. T. (1984). A non(150-750 nA) within which there was no relationship selective cation conductance in frog muscle membrane blocked by micromolar external calcium ions. J. Phgslsiol. 353, 565-583. between Ca current size and inactivation kinetics.
WILLIAM
BACCAGLINI, P. I., and SPITZER, N. C. (1977). Developmental changes in the inward current of the action potential of Rohon-Beard neurones.1 Physiol. 271, 93-117. BARISH, M. E., and THOMPSON, S. H. (1983). Calcium buffering and slow recovery kinetics of calcium-dependent outward current in molluscan neurones. J. Phgsiol. 337, 201-219. BAUD, C. (1983). Developmental change of a depolarization-induced sodium permeability in the oocyte of Xenopus laevis. Dev. Biol. 99, 524-528.
Baut), C., KADO, R. T., and MARCHER, K. S. (1982). Sodium channels induced by depolarization of the Xenopus lae71is oocyte. Proc. N&l. Acu
Land.
154, 453-461.
COI,QI:HOUN, D., NEHER, E., REUTER, H., and STEVENS, C. F. (1981). Inward current channels activated by intracellular Ca in cultured cardiac cells. Kutura (Lmdon) 294, 752-754. CONPZOR,J. A., and STEVENS, C. F. (1971). Voltage-clamp studies of a transient outward membrane current in gastropod neural somata. .I. Ph,ysiol. 213, 21-30. ECKER’I’, R., and CHAD, J. E. (1984). Inactivation of Ca channels. Proq Bioph {)S. Mol. Bid.
413
J. MOODY
44, 215-267.
EUSEBI, F., COLONNA, R., and MANGIA, F. (1983). Development of membrane excitability in mammalian oocytes and early embryos. Ganzete RPS. 7, 39-47. Fox. A. P. (1981). Voltage-dependent inactivation of a calcium channel. Proc. Nutl. Acud. Sci. USA 78, 953-956. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng 8, 385-416. HESS, P., and TSIEN, R. W. (1984). Mechanism of ion permeation through calcium channels. Xoture (London) 309, 453-456. HIRANO, T., and TAKAHASHI, K. (1984). Comparison of properties of calcium channels between the differentiated l-cell embryo and the egg cell of ascidians. J. Physiol. 347, 32’7-344. HOLLAX;D, L. Z., and GOULD-SOMERO, M. (1981). Electrophysiological
responses to insemination
in oocytes of Urechis cnupo. Dew. Biol. 83,
90-100.
KADO, R. T., MARCHER, K., and OZON, R. (1981). Electrical membrane properties of thexenopus laevis oocyte during progesterone-induced meiotic maturation. Dev. Biol. 84, 471-476. KLINE, D., JAFFE, L. A., and KADO, R. T. (1985). The fertilization potential of the nemertian worm Cerebrutulus Zacteus depends on a calcium-activated sodium conductance. Cell Differ., in press LANSMAN, J. 8. (1985). Two components of the inward current in oocytes of the starfish, Leptasterias hem&s. J. Physiol., in press. MILEDI, R., and PARKER, I. (1984). Chloride current induced by injection of calcium into Xenonus oocytes. J. Phgsiol. 357, 173-183. MIYAZAKI, S-I (1979). Fast polyspermy block and activation potential. Electrophysiological bases for their changes during oocyte maturation of a starfish. Deu. Biol. 70, 341-354. MIYAZAKI, S-I. (1982). Ca-mediated activation of a K current at fertilization of golden hamster eggs. Proc. N&l. Acad. Sci. USA 79,931935. MIYAZAKI, S-I., and HIRAI, S. (1979). Fast polyspermy block and activation potential. Correlated changes during oocyte maturation of a starfish. Dew. Biol. 70, 327-340. MOODY, W. J., and BOSMA, M. M. (1985). Hormone-induced loss of surface membrane during maturation of starfish oocytes: Differential effects on potassium and calcium channels. Dev. Biol. 112, 396-404. MOODY, W. J., and LANSMAN, J. B. (1983). Developmental regulation of Ca and K currents during hormone-induced maturation of starfish oocytes. Proc. N&l. Acad. Sci. USA 80,3096-3100. STEINHARDT, R. A., and EPEL, D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. USA 71, 1915-1919. TAKAHASHI, K., and YOSHII, M. (1981). Development of sodium, calcium and potassium channels in the cleavage-arrested embryo of an ascidian. J. Physiol. 315, 515-529. WARNER, A. E. (1973). The electrical properties of the ectoderm in the amphibian embryo during induction and early development. J. Physiol. 235, 267-286.
YELLEN, G. (1982). Single Ca-activated nonselective in neuroblastoma. Nuture (London) 296.357-359.
cation channels
BIOLOGY
112, 405-413
The Development
(19%)
of Calcium and Potassium Currents during Oogenesis in the Starfish, Leptasterias hexactis WILLIAM
J. MOODY
electrical properties of oocytes of the starfish Lrptasterias hcmctis during oogenesis The development of memhrane was studied using voltage- and current-clamp techniques. Two voltage-dependent K currents-the fast transient and inwardly rectifying-are present early in oogenesis, before the rapid growth phase, and are maintained throughout oogenesis at the same current density and kinetics. The inward current, which is composed of a Ca current and a slower Ca-dependent inward sodium current, is also present early in oogenesis, but at very low current density. Late in oogenesis, after the oocrte has grown to full size, the inward current increases in amplitude by about fivefold, and undergoes major changes in kinetics. These changes are closely associated with the migration of the germinal vesicle to the cell periphery. The relationship of these events to electrophysiological changes during subsequent maturation and fertilization of the oocytes is discussed. C 1985Academic Press, Inc INTRODUCTION
tivated by briefer depolarizations (Kado et ul., 1979; Baud ef al., 1982). Baud (1983) has shown that this channel appears rather abruptly during oogenesis-at Dumont stage V-after oocytes have reached about 90% of their final diameter. In the present study, the electrical properties of Lepfasterias oocytes were examined under current- and voltage-clamp conditions during the final year of the 2year oogenesis cycle. This period includes oocytes from 250 to 800 pm in diameter, before, during, and after the phase of major vitellogenic activity. The results indicate that both types of K currents (inwardly rectifying and fast transient; see Moody and Bosma, 1985) are present early in oogenesis in essentially their final forms, whereas the inward Ca current undergoes major changes in both current density and kinetics late in oogenesis, after the oocyte has reached its full size.
In the previous paper (Moody and Bosma, 1985) we showed that calcium and potassium currents in oocytes were affected quite of the starfish Lepfasterias hexactis differently during meiotic maturation: both the inwardly rectifying and the fast transient K currents decreased substantially in magnitude, whereas the inward Ca current was unchanged. Moreover, the data indicated that the partial loss of both types of K currents was a result of the decrease in membrane surface area during maturation, and that by some mechanism, Ca channels were protected from loss during this process. In this paper I examine the development of Ca and K channels during oogenesis in Leptasterias. The purpose of these experiments was to compare the behavior of these channels during the addition of plasma membrane associated with cell growth to their behavior during the membrane loss associated with maturation. From this comparison, it might be possible to better understand the mechanisms controlling the relationship of the properties of these ion channels to the cellular events of early development. There have been few studies of the electrical properties of oocytes during oogenesis. Holland and Gould-Somero (1981) reported that Urechis oocytes in various stages of oogenesis all showed a similar two-phase action potential in response to injected current, as well as similar resting potentials and input resistances. In mouse oocytes both the overshoot and rate of rise of the Ca-dependent action potential increase between preantral and metaphase II oocytes (Eusebi et al., 1983). Xewqms oocytes possess an unusual Na channel which is “induced” by prolonged depolarizations, and once induced, is ac-
MATERIALS
AND
METHODS
Methods of animal collection, maintenance, voltage clamping, and data acquisition were all identical to those described in the previous paper (Moody and Bosma, 1985). Recordings were made from 68 oocytes during two separate seasons. For experiments with smaller oocytes (200-350 pm), ovaries were torn open with forceps and cut into small pieces with fine scissors. A piece of ovary containing several oocytes was pinned in the experimental chamber and oocytes were impaled while still loosely attached to the ovarian tissue. This did not create a significant series resistance or diffusion problem. Microelectrodes used to impale and voltage-clamp the smaller oocytes had somewhat higher resistances than 405
0012-1606/85 Copyright All rights
$3.00
C 1985 by Academic Press. Inc. of reproduction in any form reserwd
406
DEVELOPMENTAL BIOLOGY
those used for the larger cells, but in all cases resistances were kept below 10 MQ, to ensure adequate voltageclamp control. Resting potentials of -60 mV or greater could be obtained at all stages, but only with electrodes which were of too high resistance to permit adequate voltage-clamp control. (Because of the voltage dependence of the resting conductance in starfish and many other oocytes, a small leakage at the site of impalement can create a disproportionately large loss of resting potential. See Hagiwara and Jaffe, 1979). For voltage clamp, impalements were judged by the amplitude of leakage currents relative to the time- and voltage-dependent currents. Oocytes which showed significant leakage currents under voltage-clamp were rejected; leakage currents have not been subtracted from any of the records shown. For each cell, the following data were collected: an 1-V relation under voltage-clamp for both hyperpolarizing and depolarizing voltage pulses, the response of the oocyte to depolarizing current pulses under current clamp, total membrane capacitance, and cell diameter. In the later stages of oogenesis, when oocytes developed significant inward current (see below), I-V relations were also recorded with the inward currents eliminated in Na, Ca-free external solution, so that the A-current could be studied in isolation. In the sections below, total capacitance is taken as a measure of the membrane surface area, both to determine current densities and to measure oocyte growth in terms of the amount of surface membrane. All experiments were done at 12°C. RESULTS
Although Leptasterias has an annual spawning cycle, oogenesis requires 2 years (Chia, 1968). There is a phase of slow growth from January of the first year to February of the second year, during which the diameter of the oocyte increases from about 30 to about 200 pm. A period of rapid growth follows, from March through July of the second year, during which oocyte diameter increases from 200 to 800 pm, its final value. From July to November (or later) of the second year, the only obvious morphological change in the oocytes is the migration of the germinal vesicle from a central to a peripheral position, in September. Oocytes become responsive to lmethyladenine-the maturation hormone-at about this time. Spawning occurs in November-April. Thus in a single animal there are always two populations of oocytes, which can readily be distinguished on the basis of diameter: those which will be spawned during the next breeding season, and those which will be spawned a year later (see Chia, 1968, his Fig. 1). This study included oocytes 200-800 pm in diameter, which covers the second year of oogenesis, beginning just before the period of rapid growth.
VOLIJME 112, 1985
There are three principal voltage-dependent currents in fully grown, l-MA responsive Leptasterias oocytes (Moody and Lansman, 1983; Moody and Bosma, 1985): the fast-transient K current, or A-current; the inwardly rectifying K current; and the inward Ca current, which is actually composed of two separate components (see below). No other currents were seen at any stage of oogenesis. In the sections below, the development during oogenesis of each of these currents is described.
The Fast-Transient K Current (A-Current) The A-current is activated at voltages positive to -40 mV. The conductance-voltage relation shows half-maximal activation at about -15 mV and saturates at $10 to +20 mV. At +lO mV the current reaches peak amplitude in ca. 10 msec and inactivates with a time constant of about 25 msec (Fig. 1). In molluscan neurons the A-current controls the interspike interval during repetitive firing (Connor and Stevens, 1971). In Leptasterias oocytes, the A-current shunts the inward current and controls the overshoot of the action potential (Moody and Lansman, 1983). Figure 1 summarizes the development of the A-current during oogenesis. In the left portion of the figure, the current (at +lO mV, where conductance is saturated) per unit membrane capacitance is plotted vs total membrane capacitance, a measure of membrane area. (A plot of current density vs oocyte diameter is virtually identical.) Sample voltage-clamp records from various stages are also shown; the current gains in these records have been set inversely proportional to cell capacitance, so that the size of the displayed current is proportional to current density. At the earliest stages examined, in which membrane area was only about 8% of its final value, the A-current was already present at its final (prematuration) density. During the entire final year of oogenesis, including the period of rapid growth, the magnitude of the A-current closely followed oocyte growth, so that current density was maintained constant. In oocytes of about 550 pm in diameter (C,,, = 40 nF), which corresponds to midpoint of the rapid growth phase, the current density of the A-current tended to be slightly higher than at either earlier or later times. Comparison of A-currents from recordings made before and after September 1 indicated that germinal vesicle migration in the full grown oocytes was not accompanied by any change in A-current amplitude (Fig. 1). This is in contrast to the inward current, which shows dramatic changes in both magnitude and kinetics during this time (see below). Plots of conductance-voltage relations and inactivation curves at various stages indicated no significant changes in the voltage dependence of activation or in-
WILLIAM
J. MOODY
Electrophysiology
407
of Oogenesis
May
June
14nF
38nF
L I
Aug 65nF
Ott
65nF
LJ
20ms
FIG. 1. Development of the A-current during oogenesis. The left panel shows a plot of A-current density (nA/nF) vs total capacitance (nF). The + symbols distinguish measurements made from oocytes after Sept. 1. The right four panels show sample voltage-clamp records of A-currents at various stages of oogenesis. Each record represents the current during a voltage step from -80 to +lO mV, and is labeled with the month it was recorded and the total capacitance of the oocyte. The gains have been adjusted to be inversely proportional to capacitance, so that the amplitudes of the currents as displayed are proportional to current density. For May, June, Aug., and Oct., the vertical calibration bar represents 22, 58, 100, and 100 nA, respectively. The time scale is the same for all records.
activation of the A-current during oogenesis. The twomicroelectrode voltage-clamp did not permit sufficient resolution of the rising phase of the current to determine activation kinetics, but time to peak did not change during oogenesis. There was a slight slowing of inactivation in some, but not all, fully grown oocytes. These results indicate that A-current channels are present at normal density in the membrane of the oocyte before it enters the period of rapid growth during the second year of oogenesis, and that during the remainder of oogenesis the properties of A-current channels do not change. During the period of rapid growth, A-current channels are apparently added along with new membrane so that A-current density remains roughly constant. The tendency of current density to go through a maximum near the midpoint of the rapid growth phase, although small, was also seen for the inward current (see Fig. 3), and may indicate a slight lag between channel and lipid addition to the surface membrane during the rapid growth phase. The Inwardly
Rectifying
K Current
The inwardly rectifying K current is activated at potentials negative to -70 mV. The small steady-state activation of this channel around -70 mV represents most, if not all, of the resting conductance of Leptasterias (and many other) oocytes (see Hagiwara and Jaffe, 1979). Because of its voltage dependence, the inward rectifier creates a resting conductance which turns off as the mem-
brane is depolarized, creating a high input resistance in the voltage range -10 to -60 mV and thus allowing long duration action potentials and fertilization potentials to be generated with relatively small inward currents and ionic fluxes. The inwardly rectifying K current behaved much the same as the A-current during oogenesis. It was present in oocytes before the period of rapid growth and its current density was maintained throughout oogenesis. No changes in either voltage dependence or kinetics were detected. The Inward
Current in Fully
Grown Oocytes
In order to understand the complex developmental changes in the inward current during oogenesis, it was first necessary to analyze the inward current in oocytes which had completed oogenesis. The inward current is composed of at least two components, which can be most easily separated on the basis of their different rates of inactivation. Figure 2 shows an example of the separation of these two components at V, = -40 mV. Inactivation of the total current follows a two-exponential time course (Fig. 2A), with time constants of 120-200 and 900-1200 msec (-40 mV; 12°C). The slow component is particularly obvious in the lower panel of Fig. 2B, which shows a 5-set voltage pulse to -40 mV. In ASW in which all Ca was replaced with Ba, or in Na-free ASW, the current decreased in amplitude by about 50% and showed only the fast component of inactivation (Figs. 2A, B). A
408
DEVELOPMENTAL BIOLOGY
VOLUME 112, 1985
1 OOms
FIG. 2. Analysis of two components of the inward current in an oocyte which has completed oogenesis. (A) Semi-log plot of the inactivation of the inward current in ASW (0). A straight line was fit to the slow component by eye, and then this relation subtracted from the total plot to obtain the fast component (steeper solid line). These two lines indicate time constants of 1060 and 120 msec, as indicated. A plot of inactivation in Ca-free (Ba) ASW (+) shows only the fast component. The Ba data has been shifted to the right by about 0.15 set so the slope can be compared more easily with the steeper solid line. (B) Voltage-clamp records (-40 mV) of the inward current from this cell. The top panel shows records in ASW and Ca-free (Ba) ASW, as well as the difference current between the two obtained electronically. The lower panel shows the current during a 5-set voltage pulse to -40 mV, to illustrate the slow component of inactivation.
similar analysis of the inward current has been done by Lansman (1985). These results suggest the following model to describe the inward current: The fast component represents a rapidly inactivating “pure” Ca current, and the slow component a Ca-activated nonspecific inward current (Colquhoun et al., 1981; Yellen, 1982), which at -40 mV is carried primarily by Na ions. The two contribute about equally to the total peak inward current. Ca-free (Ba) ASW eliminates the slow component presumably because barium ions entering through the Ca channel fail to activate the Ca-dependent inward current channel. The fact that Ba supports inactivation at exactly the rate of the fast component in normal ASW suggests that inactivation of the Ca channel is voltage-dependent (see Eckert and Chad, 1984, for a review of Ca channel inactivation), which seems to be a common feature of Ca channels in oocytes (see Fox, 1981; Hirano and Takahashi, 1984). The fact that Ca-free (Ba) and Na-free ASW reduce the total inward current by approximately the same amount indicates that Ca and Ba are nearly equally permeant through the Ca channel, which is also consistent with Ca channel permeability in other oocytes (Hirano and Takahashi, 1984). The full time course of the Ca-dependent inward current can be obtained by subtracting the current in Ca-free (Ba) from that in
normal ASW (Fig. 2B). The kinetics of the resulting current are consistent with a Ca-dependent process. During activation the slow component approximates the integral of the “pure” Ca current, and its very long time constant of inactivation is on the order of that of the Ca-dependent K current in molluscan neurons (Barish and Thompson, 1983), which in that preparation is taken to represent the removal of Ca from the submembrane space. An alternative hypothesis-that the slow component of the inward current represents Na permeation throught the Ca channel-has been considered, but tentatively rejected. Divalent ions, especially Ca, are powerful inhibitors of Na permeation through the Ca channel (Hess and Tsien, 1984; Almers et al., 1984), and thus in seawater one would not expect to see a large Na component through the Ca channel. In addition, Ba should be less powerful than Ca in blocking Na permeation (Hess and Tsien, 1984), whereas in Leptasteria,s Ba appears to eliminate the slow component of the inward current. As a further test of this hypothesis, Na currents through the Ca channel were measured in divalent-free ASW. Although very large Na currents could be recorded under these conditions, they showed kinetics which approximated the fast-not the slow-component of the inward current in normal ASW.
WILLIAM
J. MOODY
Electrophysiology
409
of Oogenesis
been adjusted inversely to the cell capacitance, so that the displayed traces show current density. The bottom two records are from two cells of the same diameter and membrane area (and therefore are displayed at the same gain), the one on the left before September 1, and the one on the right after. A significant morphological event which occurs at about this time in oogenesis, after the oocytes reach full size, is the migration of the germinal vesicle (GV) (Chia, Changes in the Inward Current during Oogenesis 1968). In order to determine more exactly the temporal relationship between the increase in inward current Figure 3 shows current density for the total inward amplitude and germinal vesicle migration, 14 oocytes current plotted vs total membrane capacitance (surface from August 24 to September 27 were fixed and sectioned area) as it was for the A-current in Fig. 1. Throughout and the position of the germinal vesicle was measured. the period of rapid growth, the inward current behaved much the same as the A-current. Approximately the In 8 of the 9 oocytes from after September 1, GV migration was complete and the GV was closely apposed same current density was maintained throughout this period (February-August), with a tendency for oocytes (<50 grn) to the plasma membrane. GV migration had with capacitances near 40 nF-about half-way through not been completed in any of the (5) oocytes from before the rapid growth phase-to have somewhat larger curSeptember 1, and the minimum distance from the GV rent densities. There was, however, a major difference to the plasma membrane ranged from 75 to 300 pm. This between the inward current and A-current: the inward suggests a close relationship between the migration of current density that was maintained through the period the germinal vesicle and in the increase in Ca current amplitude. of growth was only about 20% of the final value achieved at the end of oogenesis. The large increase in the inward The sudden increase in amplitude of the inward curcurrent which made up this difference occurred abruptly rent at the time of germinal vesicle migration was acat the beginning of September, after the oocytes had companied by major changes in its kinetics. As is apcompleted the period of rapid growth and reached full parent in the records in Figs. 2 and 3, inward currents size. Figure 3 emphasizes the abruptness of this increase recorded before the period of germinal vesicle migration, in inward current amplitude. Among 26 full-grown oo- even in full-grown oocytes, inactivated much more rapcytes studied after August 1 (700-900 ym diameter; C,,, idly than the large inward currents recorded in later = 65-90 nF), inward currents recorded from oocytes after oocytes. The appearance of slow inactivation was very September 1 were significantly larger than those re- strongly correlated with the increase in current amplicorded earlier, showing an almost nonoverlapping dis- tude during oogenesis. Oocytes with large inward curtribution. This pattern of development is illustrated in rents with rapid inactivation kinetics were only very the records in Fig. 3. As in Fig. 1, the current gains have rarely encountered, and a plot of the slow time constant
As described below, there are dramatic changes in the kinetics of the inward current during oogenesis, mainly involving the slow component. Although these changes are more easily interpreted considering the slow component to be a Ca-mediated inward current, it should be kept in mind that the evidence on this point is not conclusive.
May
+
2OnF
15nA
f Aug
7OnF
7OnA
I
Ckt
70nF
v -.w
325nA
___
1OOms
FIG. 3. Development of the inward current during oogenesis. This figure is analogous to Fig. 1 for the A-current. In the right panels, the gains of the current traces have been adjusted to be inversely proportional to capacitance, so current density is displayed. The voltage-clamp pulse is from -80 to -40 mV. Each record is labeled also with the peak current amplitude. In the bottom two records, a dashed line near the end of the voltage pulse indicates zero current, in order to emphasize the appearance of the slow inactivation.
410
DEVELOPMENTAL
BIOLOGY
vs capacitance (not shown here) showed a distinct break around September 1 identical to that seen in Fig. 3A for current amplitude. At first, these data seemed to suggest that before germinal vesicle migration, oocytes simply did not have the slowly inactivating Ca-dependent component of the inward current. However, as the analysis below indicates, younger oocytes do have the slow component, and it comprises about the same proportion of the total inward current as in the older oocytes, but its kinetics of inactivation are much more rapid and consequently difficult to separate from those of the pure calcium current. Figure 4 shows analysis of inactivation kinetics and the effects of Ca-free (Ba) ASW for a typical oocyte before the period of germinal vesicle migration. (For comparison see Fig. 2, which shows a similar analysis for the inward current of a later oocyte.) The time course of inactivation follows two exponentials, but the slow component is about five times faster than in later oocytes. In 19 oocytes in which the slow time constant of inactivation was determined, it averaged 233 ? 42 msec (n = 10) before September 1 as compared to 948 + 205 msec (n = 9) after. Ca-free (Ba) ASW had the same effect on the inward current in these oocytes as it did in the later oocytes: the slow component of inactivation was eliminated, and the total inward current was reduced by almost 50% (Fig. 4B). The Ca-dependent component obtained by subtraction in the early oocytes showed much more rapid inactivation relative to its activation kinetics than in oocytes after germinal vesicle migration (Fig. 4B).
VOLUME 112, 1985
These results indicate that the Ca-dependent inward current which is probably responsible for the slow phase of inactivation is present well before the period of germinal vesicle migration, and perhaps throughout oogenesis (in oocytes less than about 400 pm in diameter, inward currents were too small for accurate analysis of inactivation kinetics). The major changes which occurred near the time of germinal vesicle migration were a marked slowing of the inactivation kinetics of this current and an increase in its absolute magnitude, although not in its relative contribution to the total inward current. As discussed below, this could result directly from the increase in amplitude and slight slowing of the Ca current itself, or could reflect a change in intracellular Ca buffering or the response of the Ca-dependent channel to internal Ca. Changes in the Action Potential
during
Oogenesis
The development of the action potential during oogenesis generally reflected the development of the inward current. Active responses to depolarizing current pulses were small or nonexistent in early oocytes and became large, and in many cases overshooting, later in oogenesis, at the time when the inward currents became large (Fig. 5). The marked slowing of the kinetics of inactivation of the inward current resulted in a substantial increase in the duration of the action potential in later oocytes (Fig. 5, compare bottom two panels). Since the A-current does not become significant until the membrane potential reaches ~20 to 0 mV, it plays
A
FIG. 4. Analysis of the two components of the inward the left panel, the steeper solid line is the mathematically the raw data in Ca-free (Ba) ASW.
current before germinal vesicle migration. See legend for Fig. 2. Note again that in obtained fast time constant inactivation in normal ASW, and the + symbols indicate
WILLIAM
411
J. MOODY
B
A May
13nF
R
R
July
C Aug
D Nov
70nF
R
40nF
65nF
Rr--
FIG. 5. Development of the action potential during oogenesis. Current clamp records. In each case, the resting potential has been adjusted to -80 mV with holding current so spike waveforms can be accurately compared. In each oocyte, the maximum obtainable response is shown. The top traces indicate the current pulses, and at 0 current mark the bath potential. Note the increased duration and the appearance of the notch on the rising phase in panel D
little if any role in the electrically elicited response of the oocyte until near the period of germinal vesicle migration, when the action potential becomes large enough to reach this voltage range. It is not until this time that the action potential develops the fast and slow rising phases separated by a notch in the waveform, which is characteristic of the fully grown Leptasterias oocyte after germinal vesicle migration. DISCUSSION
The results described above indicate that Ca and K currents show quite different patterns of development during oogenesis in Leptasterias. During the final 12 months of the 24-month oogenesis cycle the oocytes undergo a rapid growth phase, increasing in diameter from 200 to 800 pm, and then enter a “resting period” (Chia, 1968), during which the germinal vesicle migrates to a peripheral position. After the migration of the germinal vesicle, the oocytes become responsive to the maturation hormone (unpublished observation), and are subsequently capable of undergoing development in response to insemination. Both K currents-the A-current and the inward rectifier-were present at normal current density in the smallest oocytes studied (200 pm), before the rapid growth phase. Throughout the remainder of oogenesis, the increase in both K currents matched the
addition of new membrane, so that the current density remained constant. This implies a strict regulation of the density of K channels in the membrane, even during the rapid addition of membrane during growth, although there was some indication of a small transient increase in the density of all currents during the time of most rapid membrane addition. The inward current behaved quite differently. Although a constant current density was maintained throughout rapid growth, this density was much less than the final value at the end of oogenesis. The inward current increased markedly in amplitude after the end of the growth phase, at a time which closely correlated with the approach of the germinal vesicle to a position near the plasma membrane. The inward current is composed of two components, a Ca current and a slowly inactivating Ca-dependent inward current carried primarily by Na. These two components contributed about equally to the peak inward current throughout oogenesis. However, at the time of germinal vesicle migration, when the total inward current increased abruptly, the slowly inactivating component became much slower, by about a factor of five. The development of Ca and K currents during oogenesis is analogous to their behavior during hormone-induced maturation (Moody and Bosma, 1985). During the first hour of maturation, about 50% of the surface membrane is lost, and the A-current is decreased in the same
412
DEVELOPMENTAL BIOLOGY
VOLUME 112. 1985
proportion. During oogenesis, A-current channels are It is also possible that a change in the sequence of present early, at normal density, and channel density events subsequent to Ca entry led to the slowing of the keeps pace with the addition of new membrane. On the Ca-dependent component of the inward current. A pool other hand, Ca currents are unaffected by membrane of calcium in the cortex whose release is triggered by removal during maturation. During oogenesis, the major Ca entry might have appeared or greatly increased in increase in Ca current amplitude occurs after completion size late in oogenesis, or the response of the Ca-depenof oocyte growth, when no new surface membrane is dent channel itself to Ca ions might have been altered. being added. So during both oogenesis and maturation, The activation of Ca-dependent channels is thought to underly the fertilization potential in a variety of oocytes Ca currents appear to be modulated somewhat independently of membrane addition and removal, whereas A- (see Miledi and Parker, 1984; Kline et al, 1985; Steinhardt and Epel, 1974; Miyazaki, 1982). Changes during oogencurrent channels are added or deleted with membrane during both oogenesis and maturation. Our data are not esis in the response of the Ca-dependent channel to cytoplasmic Ca ions, or in the buffering of Ca in the subcomplete regarding the development of the Ca-depenmembrane space, might represent an important stage dent channel (slow component of the inward current). in the preparation of the oocyte for fertilization. In adSince this current was activated only by Ca entering during depolarizing voltage steps, it is possible that we dition, the slowing of the action potential which results from the change in inward current kinetics might allow are examining only a small fraction of the activatable poconductance (possibly represented by those channels in the transition from action potential to fertilization tential to occur without substantial repolarization of the close physical proximity to the Ca channels). In Xenopus membrane, and thus permit a more successful polyoocytes (Miledi and Parker, 1984), only a small portion of the Ca-dependent Cl- current can be activated by de- spermy block (see Miyazaki and Hirai, 1979; Miyazaki, 1979). polarizing voltage steps, in contrast to direct injection Both the increase in amplitude of the Ca current and of Ca buffers. Thus, without using Ca injection to actithe slowing of the kinetics of the Ca-dependent current vate this channel, we cannot determine whether the total late in oogenesis were correlated closely in time with activatable conductance changes during oogenesis. the approach of the germinal vesicle to a position near As with maturation, there is an interesting analogy the plasma membrane. Cytoarchitectural elements between these results and those obtained in amphibian which anchor the germinal vesicle might also participate oocytes. Baud (1983) reported that the voltage-dependent Ca channels in this region, oocytes was present, but at low in inserting or activating Na current in Xenops density, early in oogenesis, and was maintained at ap- altering the properties of Ca-dependent channels, or proximately constant current density throughout most changing cytoplasmic Ca buffering in this portion of the of the growth of the oocyte (see Baud, 1983, Fig. 2B, cytoplasm. The position of the germinal vesicle might also be important in the electrical changes associated noting that current-not current density-is plotted). Later, after the oocyte had reached almost its full size, with maturation. It is possible that the membrane overthere was an abrupt increase in the Na current, by about lying the germinal vesicle is not subject to loss during maturation because of its particular structural relaa factor of five. There are several possible interpretations of the tionship to the nucleus. The clustering of Ca channels in this membrane might provide a physical basis for the change in inward current kinetics which accompanied of Ca channels from loss during the increase in current amplitude. The slowing of the observed “protection” (Moody and Bosma, 1985). This would be Ca-dependent component might have been caused by the maturation increase in Ca entry resulting from the increase in Ca especially interesting since the position of the nucleus current which occurred at the same time during oogen- marks the presumptive animal pole of the egg, and clustering of Ca channels there might play a role either in esis. However, models of Ca diffusion in the cytoplasm affecting polarity or in determining the later partitionand experimental data on the Ca-dependent K channel in molluscan neurons indicates that an increase in the ing of different electrical properties into early blastoamount of Ca entry increases the amplitude, but does meres. not markedly alter the time course of submembrane Ca These experiments were supported by NIH Grant HD 17486 and by activity (see, e.g., Barish and Thompson, 1983). Furthermore, although small Ca currents early in oogenesis a NIH Research Career Development Award to the author. were correlated with rapid inactivation kinetics, in ooREFERENCES cytes with large Ca currents after germinal vesicle migration there was a large range of Ca current amplitudes ALMERS, W., MCCLESKEY, E. W., and PALADE, P. T. (1984). A non(150-750 nA) within which there was no relationship selective cation conductance in frog muscle membrane blocked by micromolar external calcium ions. J. Phgslsiol. 353, 565-583. between Ca current size and inactivation kinetics.
WILLIAM
BACCAGLINI, P. I., and SPITZER, N. C. (1977). Developmental changes in the inward current of the action potential of Rohon-Beard neurones.1 Physiol. 271, 93-117. BARISH, M. E., and THOMPSON, S. H. (1983). Calcium buffering and slow recovery kinetics of calcium-dependent outward current in molluscan neurones. J. Phgsiol. 337, 201-219. BAUD, C. (1983). Developmental change of a depolarization-induced sodium permeability in the oocyte of Xenopus laevis. Dev. Biol. 99, 524-528.
Baut), C., KADO, R. T., and MARCHER, K. S. (1982). Sodium channels induced by depolarization of the Xenopus lae71is oocyte. Proc. N&l. Acu
Land.
154, 453-461.
COI,QI:HOUN, D., NEHER, E., REUTER, H., and STEVENS, C. F. (1981). Inward current channels activated by intracellular Ca in cultured cardiac cells. Kutura (Lmdon) 294, 752-754. CONPZOR,J. A., and STEVENS, C. F. (1971). Voltage-clamp studies of a transient outward membrane current in gastropod neural somata. .I. Ph,ysiol. 213, 21-30. ECKER’I’, R., and CHAD, J. E. (1984). Inactivation of Ca channels. Proq Bioph {)S. Mol. Bid.
413
J. MOODY
44, 215-267.
EUSEBI, F., COLONNA, R., and MANGIA, F. (1983). Development of membrane excitability in mammalian oocytes and early embryos. Ganzete RPS. 7, 39-47. Fox. A. P. (1981). Voltage-dependent inactivation of a calcium channel. Proc. Nutl. Acud. Sci. USA 78, 953-956. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng 8, 385-416. HESS, P., and TSIEN, R. W. (1984). Mechanism of ion permeation through calcium channels. Xoture (London) 309, 453-456. HIRANO, T., and TAKAHASHI, K. (1984). Comparison of properties of calcium channels between the differentiated l-cell embryo and the egg cell of ascidians. J. Physiol. 347, 32’7-344. HOLLAX;D, L. Z., and GOULD-SOMERO, M. (1981). Electrophysiological
responses to insemination
in oocytes of Urechis cnupo. Dew. Biol. 83,
90-100.
KADO, R. T., MARCHER, K., and OZON, R. (1981). Electrical membrane properties of thexenopus laevis oocyte during progesterone-induced meiotic maturation. Dev. Biol. 84, 471-476. KLINE, D., JAFFE, L. A., and KADO, R. T. (1985). The fertilization potential of the nemertian worm Cerebrutulus Zacteus depends on a calcium-activated sodium conductance. Cell Differ., in press LANSMAN, J. 8. (1985). Two components of the inward current in oocytes of the starfish, Leptasterias hem&s. J. Physiol., in press. MILEDI, R., and PARKER, I. (1984). Chloride current induced by injection of calcium into Xenonus oocytes. J. Phgsiol. 357, 173-183. MIYAZAKI, S-I (1979). Fast polyspermy block and activation potential. Electrophysiological bases for their changes during oocyte maturation of a starfish. Deu. Biol. 70, 341-354. MIYAZAKI, S-I. (1982). Ca-mediated activation of a K current at fertilization of golden hamster eggs. Proc. N&l. Acad. Sci. USA 79,931935. MIYAZAKI, S-I., and HIRAI, S. (1979). Fast polyspermy block and activation potential. Correlated changes during oocyte maturation of a starfish. Dew. Biol. 70, 327-340. MOODY, W. J., and BOSMA, M. M. (1985). Hormone-induced loss of surface membrane during maturation of starfish oocytes: Differential effects on potassium and calcium channels. Dev. Biol. 112, 396-404. MOODY, W. J., and LANSMAN, J. B. (1983). Developmental regulation of Ca and K currents during hormone-induced maturation of starfish oocytes. Proc. N&l. Acad. Sci. USA 80,3096-3100. STEINHARDT, R. A., and EPEL, D. (1974). Activation of sea urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. USA 71, 1915-1919. TAKAHASHI, K., and YOSHII, M. (1981). Development of sodium, calcium and potassium channels in the cleavage-arrested embryo of an ascidian. J. Physiol. 315, 515-529. WARNER, A. E. (1973). The electrical properties of the ectoderm in the amphibian embryo during induction and early development. J. Physiol. 235, 267-286.
YELLEN, G. (1982). Single Ca-activated nonselective in neuroblastoma. Nuture (London) 296.357-359.
cation channels