Hormone-induced loss of surface membrane during maturation of starfish oocytes: Differential effects on potassium and calcium channels

DEVELOPMENTAL

BIOLOGY

112,396-404

(1985)

Hormone-Induced Loss of Surface Membrane during Maturation of Starfish Oocytes: Differential Effects on Potassium and Calcium Channels WILLIAM Department

J. MOODY AND MARTHA

M. BOSMA

of Zoology, University of Washington, Seattle, Washington 98195 and Department University of California, Los Angeles, California 90024

of Physiology,

Received March 26, 1985: accepted in revised form June ;?I, 1985 Prior to fertilization, starfish oocytes undergo meiotic maturation, triggered by the hormone l-methyladenine (lMA). Maturation involves a variety of complex biochemical, morphological, and electrical changes, many of which are similar to those caused by progesterone in vertebrates. Using voltage-clamp and ultrastructural techniques to study maturation in starfish, we have discovered a novel process by which l-MA alters the electrical properties of the oocyte. The surface area of the oocyte decreases by more than 50% during the first hour of maturation, due to the elimination of microvilli, but the calcium and potassium currents present are affected differently by the loss of membrane. The amplitudes of both the transient K current (“A-current”) and the inwardly rectifying K current decrease, following the time course of the decrease in surface area, while the Ca current amplitude remains virtually unaffected, and may even increase in some oocytes. The kinetics of the currents do not change. This selective removal of K channels results in a larger and more rapidly rising action potential in the mature egg, which may aid in the fast block to polyspermy. The differential accessibility of various ion channels to mechanisms of membrane removal and insertion may play an important role in the development of excitable cells. 8 1985 Academic press, Inc. INTRODUCTION

A variety of complex changes in voltage-dependent ion currents occurs during the development of excitable cells, including the partitioning of specific types of ion channels into different cell lineages early in embryogenesis (e.g., Hirano et al., 1984) and the sequential appearance and disappearance of currents in the later stages of nerve and muscle development (Baccaglini and Spitzer, 197’7; Kano and Yamamoto, 1977). These processes endow each excitable cell in the mature organism with the electrical properties required for its particular function. Relatively little is known, however, about the mechanisms by which such developmental modifications in the electrical properties of cells occur. Starfish oocytes are electrically excitable and have populations of voltage-gated ion channels similar to those commonly associated with adult neurons (see Hagiwara and Jaffe, 1979, for review). Prior to fertilization in starfish, the hormone 1-methyladenine is released from follicle cells in the ovary and interacts with receptors on the oocyte surface to initiate meiotic maturation (Kanatani, 1973). Maturation is characterized by changes in protein phosphorylation (Mazzei and Guerrier, 1982), membrane ultrastructure (Hirai and Shida, 1979; Schroeder and Stricker, 1983), ion transport (Moreau et al., 1978), and membrane electrophysiology (see below), and generally serves to prepare the oocyte for fertilization. The sequence of events in starfish resembles in 0012-1606185 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

396

many respects that initiated by progesterone in the amphibian (Maller and Krebs, 1980). Changes in the electrical properties of the starfish oocyte during maturation have been reported in several species (Miyazaki et al., 1975a,b; Shen and Steinhardt, 1976). These changes include an increase in the resting input resistance of the oocyte, and an increase in the amplitude and rate of rise of the action potential. Each of these changes serves to increase the excitability of the egg, and there is evidence that this is at least in part responsible for the increased ability of the mature egg to prevent polyspermic fertilization (Miyazaki and Hirai, 1979; Miyazaki, 1979). We previously showed that in the starfish Leptasterias, this increased excitability occurs because both types of K currents present in the oocyte are reduced during maturation whereas Ca currents are not (Moody and Lansman, 1983). We now present evidence that the mechanism of this selective effect involves the removal of a large percentage of the surface membrane of the oocyte, triggered by l-MA. K channels appear to be lost concomitantly with the membrane, but Ca channels do not. A preliminary report of this work has been published (Moody and Bosma, 1984). METHODS

Specimens of the starfish collected from the intertidal

Leptasteria hexactis

were zone around San Juan Is-

land, Washington, and were maintained at lo-12°C in natural seawater tanks. Ovaries were removed from the animal and placed in Ca-free artificial seawater (ASW) for 30 min. When the ovaries were returned to normal ASW, immature oocytes were released, free of surrounding follicle cells. No spontaneous maturation of oocytes was seen. Solutions. Standard ASW contained (mm: NaCl, 430; MgC12, 50; KCl, 10; CaClz, 10; Hepes, 10; pH 8.0. Ca-free ASW (Mg substitution) contained 1 mMEGTA. Na-free ASW was made by substituting either Tris or choline for Na; when Tris was used, 150 mM sucrose was added to balance osmolarity. l-MA (Sigma) was stored frozen as a 2 mM stock solution in distilled water which was diluted to a final concentration of 2 X lo--” M in ASW just prior to use. Voltage-clamp. Standard two-microelectrode voltageclamp methods were used. Both electrodes were initially pulled to resistances of 15-20 M12 and then broken back to <4 MQ. A grounded shield was placed between the two electrodes during the experiment. Under voltageclamp, capacitative transients settled in l-3 msec. Cell capacitance was measured by applying a 10 mV, 50 Hz triangle waveform to the command input. Total capacitance was calculated as C;r, = I/2(dV/dt), where I is the amplitude of the square wave current signal which is produced as the slope of the triangle wave command voltage changes from +(dV/dt) to -(dV/dt). Since the measurement is made at a point where there is an instantaneous change in dV/dt, but not in V itself, this method is independent of membrane conductance. Voltage signals were recorded as the difference between intracellular and extracellular microelectrodes. Currents were recorded from an I-r/converter placed in the bath ground circuit. Experiments were done at 12°C with the preparation continuously superfused with ASW. Data were recorded on FM magnetic tape for later analysis. Electrow microscopy. Oocytes taken from the same ovaries were exposed either to ASW or to ASW + l-MA for 1 hr at 12°C before fixation. To obtain adequate preservation of the surface membrane, we found it necessary to use the mixed glutaraldehyde-osmium prefixative method of Eisenman and Alfert (1982). Eggs were “prefixed” in solution of 0.05%~ osmium tetroxide, 4%~glutaraldehyde, 0.2 M Na cacodylate, 11 mM CaC12, 56 mM MgClz, 0.35 M sucrose for 10 min, followed by 2 hr in the same solution without OsOl (“main fix”). Osmium was added to the prefix within 5 min of use. A 1.5 hr postfixation was done in 1% OsO*, 0.3 M NaCl, 0.2 M Na cacodylate. Eggs were dehydrated in an alcohol series and infiltrated overnight and embedded in Epon-Araldite. Sections (silver-gold, 600-750 A) were cut on a Sorvall microtome with a diamond knife, mounted on Formvarcoated slot grids, and stabilized with carbon. Sections

were examined in a Zeiss 109 microscope, and photographed at a magnification of 21,000 for measurements of surface area (see below). RESULTS

Membran~e

Cuwrents

iz Imm,ature

and Matuwe Eggs

Under voltage-clamp conditions, three major ion currents are seen in the immature Leptasterias oocyte (Fig. 1, left panels). The general properties of these currents 1 HR

PRE - HORMONE

POST-HORMONE

in rect

-I---

Ca current i

F’

iti7

capacitance

FIG. 1. Voltage-clamp records of ion currents and membrane capacitance in a single oocyte before (left panels) and 1 hr after (right panels) addition of l-MA. The top three pairs of records represent currents recorded during voltage-clamp steps to -20, -10, 0, and +lO mV (A-current), -100, -120, and -140 mV (inward rectifier), and -70, -60, -50, and -40 mV (Ca current). The holding potential was -80 mV. The A-currents were recorded in Ca,Na-free ASW (Mg, choline). The records under “capacitance” show the square-wave current generated in response to a 50 Hz, 10 mV triangle wave voltage command. The amplitude of the square wave is directly proportional to total membrane capacitance (see Methods). The vertical calibration represents 100 nA for the A-currents, and 50 nA for all other records. The horizontal calibration represents 20 msec for the A-currents, 50 msec for the Ca current and inward rectifier, and 10 msec for the capacitance traces

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DEVELOPMENTAL BIOLOGY

were described in Moody and Lansman (1983) and are summarized briefly below: (1) A fast transient K current, activated at potentials positive to -20 mV. This current is sensitive to external 4-aminopyridine, is inactivated by a prepulse to -35 mV, and appears identical to the “A-current” seen in other oocytes and molluscan neurons (Hagiwara et al., 1961; Connor and Stevens, 1971; Neher, 1971; Hagiwara et al., 1981). (2) An inwardly rectifying K current, activated by negative pulses from the holding potential of -80 mV. This current appears identical to that in other starfish oocytes in voltage and time dependence, and block by low concentrations of external barium ions (Hagiwara et ah, 1978). However, the current density is about 10 times lower in Leptasterias than another starfish, Mediaster aequalis, in which this channel has been extensively studied (see Hagiwara et al, 1976). This current shows some steady-state activation at -70 to -80 mV, and represents most if not all of the resting membrane conductance (see Hagiwara and Jaffe, 1979). (3) An inward current activated at potentials positive to -60 mV, which is carried primarily by calcium ions. The inward current appears to have two distinct components: a rapidly inactivating component (T = 200 msec), which is a “pure” calcium current, and a slowly inactivating component (7 = ca. 1 set), which appears to be carried primarily by Na ions flowing through a Caactivated channel perhaps similar to the Ca-dependent nonspecific cation channel (Colquhoun et ah, 1981;Yellen, 1982). Ca-free (Mg) ASW blocks nearly all of the inward current, whereas Na-free ASW or Ca-free (Ba) ASW blocks only the slow component. We found little change in these two components of the inward current during maturation, and the combined inward current is simply referred to as “Ca current” below. A more complete analysis of these two components of the inward current is presented in the following paper (Moody, 1985), which describes changes in the inward current during oogenesis. These three currents can be readily separated: the inwardly rectifying K current is the only current present at potentials negative to -80 mV; the Ca current reaches its peak at potentials more negative than required for significant A-current activation; the A-current can be measured by blocking the inward current with Ca- and Na-free external solution, and measuring the transient component of the remaining current. Currents were measured in individual immature oocytes which were then exposed to l-MA for 1 hr while the impalement was maintained. Figure 1 shows currents recorded from the same oocyte before and after 1 hr of exposure to l-MA: Both the A-current and the inwardly rectifying K current were decreased-the A-

VOLUME112,1985

current by a somewhat greater amount-while the Ca current remained virtually unchanged. A significant increase in Ca current (>lO%)occurred in about half the cells. We never saw a decrease in Ca current during lMA exposure. The decrease in K currents during maturation has two principal effects on the electrical behavior of the oocyte under current clamp conditions. The reduced inward rectification increases the membrane resistance around the resting potential, making the cell more responsive to applied current. The decrease in A-current broadens the range of potentials at which the membrane generates net inward current. In the immature oocyte, activation of the A-current shunts the Ca current at early times and the membrane generates net outward current at potentials positive to about -10 mV (Fig. 2, top left). Net current is outward at all times when the membrane is stepped to 0 mV or above. After 1 hr of l-MA exposure, the shunting effect of the A-current is greatly reduced, and net inward current is produced at potentials up to about +15 mV (Fig. 2, top right). As a result, there are substantial changes in the action potential, as shown in the bottom panels of Fig. 2. The prominent notch on the rising phase caused by the transient activation of the

PRE - HORMONE

1 HR. POST-HORMONE

FIG. 2. Currents recorded under voltage-clamp, and action potentials in a single oocyte before and after a l-hr exposure to l-MA. Voltage steps from -40 to +20 mV in lo-mV increments elicited the currents shown in the upper panels. The termination of the voltage step is not shown. In the lower records action potentials in response to a brief depolarizing current pulse (top trace) are shown. Calibrations: 100 nA for all current traces; 20 mV for the voltage traces; 20 msec for the upper records; 100 msec for the lower records. Note the reduction in the duration of the capacitative transients in the current records taken after l-MA treatment.

A-current is eliminated, and the spike rises more rapidly to more positive potentials. In cells in which the Ca current increases during maturation, there is also a substantial reduction in spike threshold. Similar changes in the action potential of Asterina oocytes during maturation have been reported, and Miyazaki (1979) suggested that this increased electrical excitability of the mature egg is at least in part responsible for its decreased susceptibility to polyspermic fertilization. We were particularly interested in the mechanism by which K currents could be selectively reduced during maturation. We were able to rule out several possibilities. Neither the A-current nor inward rectifier currentvoltage relations showed shifts along the voltage axis during maturation which could account for the reduction in current amplitude. The voltage dependence of inactivation of the A-current was examined, and showed no change during maturation. The kinetics of inactivation of the A-current were not speeded during maturation, and in fact were slowed slightly in many oocytes. Our measurements indicated a substantial decrease in total membrane capacitance during maturation (see Fig. 1, bottom panels); we speculated that if in fact the decrease in capacitance reflected an actual loss of surface membrane, as apparently occurs in at least some other starfish oocytes during maturation (Hirai and Shida, 1979; Schroeder and Stricker, 1983), then this might result in a loss of K channels. Consequently, the next experiments were designed to quantify the relationship between changes in capacitance and the two K currents during maturation, and to determine using ultrastructural techniques whether the decrease in capacitance reflected an actual loss of surface membrane.

Measurement of K Currents and Capacitance during the First Hour of Maturation In seven oocytes we measured total membrane capacitance (CM) and the limiting chord conductance for both the inward rectifier and A-current before and 1 hr after the beginning of l-MA exposure. Oocytes from the same animals were prepared for electron microscopy so that the capacitance values could be compared to direct measurements of membrane area (see below). Inwardly rectifying K conductance (GIR) was measured directly from the recorded currents, since no other channels are open in this voltage range. A-current conductance (GA) was measured from the transient component of the outward current in Na-free (Tris or choline), Ca-free (Mg) seawater to eliminate interference from inward currents. In the seven cells, the values of the three parameters 1 hr after the onset of l-MA exposure, normalized to before l-MA application, were (mean & SD): C, = 0.48 + 0.06; GA = 0.47 ? 0.1; GIR = 0.72 f 0.06. Thus the A-

current decreased by precisely the same amount as the capacitance, whereas the inwardly rectifying K current decreased by only about half as much. In the same seven oocytes, the peak Ca current increased slightly during the first hour of maturation to 1.17 & 0.08 times its initial value. [Note that in a previous paper, we estimated that the A-current decreased by somewhat more than the above value (Moody and Lansman, 1983). This was because we measured A-currents in normal ASW, and failed to realize that even at positive potentials, contamination of the A-current by inward currents became significant after maturation.] We next compared the time courses of the capacitance, K currents, and Ca currents during the first hour of maturation. In order to avoid possible deleterious effects of repeated exposure of the oocyte to Na-, Ca-free solutions, we adopted the following procedure to measure A-current conductance. From the seven oocytes from which I-V relations were obtained in both normal and Na-, Ca-free seawater, we calculated the I-V relation for the inward current from difference currents between the two solutions, and normalized this to the peak inward current. This derived I-V relation was quite consistent between cells. We could then use the peak inward current in any oocyte at any time after l-MA addition to derive the full I-V relation for the inward current, and thus obtain by subtraction the limiting conductances for the A-current at various times after the beginning of l-MA exposure. Figure 3 compares the time courses of the changes in A-current conductance, Ca current, and capacitance during the first hour of maturation. The A-current followed the capacitance precisely, both in magnitude and

1.2

Ca

r

current

lA

I

o’6-

‘k

_

f;-currenl

--a-------* I memb.

0.4-

I

0

60 TIME

AFTER

120 l-MA

area

100

(min)

FIG. 3. Plots from a single oocyte of limiting A-current chord conductance (conductance at a voltage where all A-current channels are open), total membrane capacitance (“memb. area”), and peak inward Ca current vs time after l-MA addition. All values are normalized to their prematuration levels.

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DEVELOPMENTAL

BIOLOGY

time course, so that the A-current density expressed in nA/nF is held constant at all times. The decrease in inwardly rectifying K conductance, although less, also follows the same time course as the capacitance (see Fig. 4). The Ca current increases slowly and steadily during maturation, so that its current density shows a substantial increase. The ultrastructural results described below indicate that total capacitance is an accurate measure of membrane area. These results imply, therefore, that the selective loss of K currents during maturation occurs because both types of K channels reside in the membrane which is removed during maturation. As discussed below, one possible explanation for Ca currents being relatively unaltered during maturation is that the Ca channels are located in a region of the membrane which is not affected by l-MA.

Effects of Long-Term l-MA Exposure Fertilization in Leptasterias normally takes place about 40 min to 1 hr after exposure of the oocytes to the hormone in vivo, judging from the induction of spawning in intact animals by l-MA injection, and by the acquisition of fertilizability in isolated oocytes exposed to lMA (Stevens, 1971; Kanatani, 1973). In Asterina oocytes exposed to l-MA for 2 hr or more, some of the electrical changes associated with maturation began to reverse, and the incidence of polyspermic fertilization began to increase (Miyazaki, 1979; Miyazaki and Hirai, 1979). We were therefore interested in determining whether in Leptasterias membrane area would increase back toward its original value in oocytes exposed to l-MA for prolonged periods, and if so, whether the close relationship between K currents and area would be maintained. To answer this question, we maintained impalements in individual oocytes for up to 6 hr of l-MA exposure,

1.2 1

co current

A A-A’

\A-A-~-AyA

I -I

0 TIME

I AFTER

2 l-MA

3

4

5

Chrs)

FIG. 4. Plots from a single oocyte of the same parameters as in Fig. 3 plus limiting inward rectifier chord conductance during long-term exposure to l-MA.

VOLUME

112. 1985

and obtained capacitance measurements and I-V relations at regular intervals. Figure 4 shows the results of one such experiment. The capacitance showed considerable, but not complete, recovery beginning at about 2 hr after the beginning of l-MA exposure. This corresponds well with the ultrastructural evidence of Schroeder and Stricker (1983), who showed a partial restoration of microvilli in Pisaster oocytes exposed for long periods to l-MA. Both the A-current and the inwardly rectifying K conductance also began to recover after about 2 hr, and followed a time course similar to the recovery of capacitance. The quantitative relationship between the A-current and capacitance was not as close as during the first 2 hr of maturation, but the direction of the discrepancy was not consistent between oocytes: in some cells, the A-current showed more recovery than the capacitance, in others, less. The percentage recovery of the inward rectifier was always greater than that of either the capacitance or A-current. The Ca current, on the other hand, cont.inued the pattern established during the first hour of maturation: if it remained constant initially, it continued to do so at long times; if it increased during the first hour, it continued to increase throughout long-term l-MA exposure, in some cases reaching four to five times its initial value.

Chan,yesin Membrane during

Ultrastructure

Maturation

In order to confirm that changes in capacitance during maturation reflected changes in membrane area, and to quantify the relationship between the two, we prepared oocytes for electron microscopy, before and 1 hr after the beginning of l-MA exposure. Aside from avoiding the region overlying the germinal vesicle, sections were taken randomly from various regions of the oocytes. Oocytes from the same females were also studied electrophysiologically to confirm that the capacitance decrease and other aspects of maturation had proceeded normally. Electron micrographs of plasma membranes before and after 1 hr of l-MA exposure are shown in Fig. 5. A profound decrease in area clearly occurs during maturation, and results from the almost complete loss of microvilli. This finding agrees with results obtained in Asterina and Pisaster oocytes using scanning electron microscopy (Hirai and Shida, 1979; Schroeder and Stricker, 1983). The smoothing of the surface membrane corresponds well with the values of apparent specific capacitance, which decreased from 3.41 +- 0.41 pF/cm” to 1.56 t 0.13 pF/cm’, near the value of 1 pF/cm’ expected for a smooth sphere. The ratio of surface areas of immature and mature eggs was estimated from the micrographs by counting intersections of the plasma membrane with isotropic curvilinear lines on a transparent overlay (see

FIG. 5. Electron micrographs of sections of oocyte plasma membrane before oocytes are shown, but pairs A, B and C, D were processed in parallel from magnification (calibration bar = 1 pm). The gray area at the top of each picture across the center of the picture. The large dark circles in the cytoplasm are yolk oocyte (ill. C, D: Higher magnification pictures of immature and mature oocytes 401

and after a 1-hr exposure to 1-methyladenine. Four different the same animal. A, before and B, after l-MA taken at low is the vitellinr layer. The plasma membrane cuts horizontally droplets. Note the large number of microvilli in the immature (Cal. bar = 0.5 pm).

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DEVELOPMENTAL BIOLOGY

Tidball, 1984). One hundred photographs of membranes of 10 paired experimental and control oocytes from four animals were measured. The apparent membrane surface area estimated from these photographs decreased during maturation to between 0.38 and 0.41 of its prel-MA value. These values are slightly but significantly smaller than the estimates based on membrane capacitance. This discrepancy is consistent with the idea of a region of the surface membrane which does not undergo morphological changes upon exposure to l-MA. DISCUSSION

Our results show that meiotic maturation in starfish oocytes is accompanied by substantial and closely related changes in membrane morphology and electrophysiology. During the first hour of exposure to l-methyladenine, there is a marked reduction in the two voltagedependent K conductances normally present in the oocyte, and a decrease in membrane surface area of slightly more than 50%. Electron micrographs of the surface membrane indicate that the loss of surface area occurs by the elimination of microvilli which cover the surface of the immature oocyte. In spite of this large decrease in surface area, the inward Ca current, which mediates the rising phase of the action potential, remains unchanged or sometimes increases. There have been several previous studies of electrophysiological changes during maturation of starfish (Miyazaki et al., 1975a,b; Shen and Steinhardt, 1976; Miyazaki and Hirai, 1979; Miyazaki, 1979; Moody and Lansman, 1983) and amphibian (Kado et ah, 1981; Taglietti et al., 1984; Baud and Barish, 1984) oocytes. Miyazaki et al. (1975b) show voltage-clamp records indicating the presence of an A-current in the starfish Asterina, but this seems to change little during the first hour of maturation. Upon prolonged l-MA exposure, the inactivation of this current appeared to diminish. Shen and Steinhardt (1976) report little change in the action potential of Patiria oocytes during maturation, whereas in Asterina, Miyazaki et al. (1975b) reported an increase in both rate of rise and spike duration. In both Patiria and Asterina, inward rectification decreased during maturation. The data obtained in amphibian oocytes show a fairly close analogy to our results in Leptasterias. In Rana, both the inward rectifier (a Cl channel) and the delayed K current (no A-current was reported), but not the inward current (carried by sodium), decrease during maturation (Taglietti et al., 1984). These authors suggest that the decrease in surface area known to occur in amphibian oocytes during maturation (Kado et al., 1981) might cause the decrease in resting input resistance, but they did not quantify the relation between the two or attempt to relate the change in surface area

VOLUME 112, 1985

conductances. In Amcurrent [carried by hydrogen ions (Barish and Baud, 1984; see Byerly et al., 1984)] decreases during maturation, while the inward sodium current increases (Baud and Barish, 1984). These results suggest that there may be substantial common features between the action of 1-methyladenine in asteroids and progesterone in amphibians in terms of their effects on membrane morphology and voltage-dependent ion channels. No mechanisms have previously been suggested to explain the changes in active electrical properties during maturation. We believe our results indicate that in Leptasterias the selective loss of K currents during maturation occurs because K channels, but, not Ca channels, are removed as part of the plasma membrane as the surface area is decreased. The reduction in A-current conductance during the first hour of maturation matches the loss of membrane area precisely. The decrease in inward rectification is smaller, but its time course also follows that of the decline in area. Both currents recover during long-term exposures to l-MA, as microvilli are restored. If our interpretation of the data is correct, and Ca channels are not lost during maturation, then there must be some mechanism operating during maturation which protects Ca channels, but allows K channels to be included in the loss of surface membrane. One possibility is that the Ca channels are spatially segregated in a region of the oocyte membrane which is either devoid of microvilli in the immature state, and thus not subject to further loss of membrane, or which is simply not affected by l-MA. As mentioned above, and analyzed in detail in the following paper (Moody, 1985), the inward current is actually composed of two components: a “pure” Ca current and a Ca-dependent inward current. Since the relative contributions of these two components to the total inward current did not change during maturation, we have referred to the total inward current as “Ca current” for simplicity. It should be emphasized, however, that the retention of the Ca-dependent component during maturation does not necessarily imply that those channels, like the Ca channels, are “protected” from the l-MA-induced decrease in surface area. If Ca channels are indeed clustered in a specific region of the membrane, then by activating the Ca-dependent current with depolarizing voltage steps, we may only be seeing the behavior of those channels which are in close physical proximity to the Ca channels, and thus activatable by Ca influx. If Ca-dependent channels also existed in other regions of the oocyte membrane, we might not detect their loss, since they would never be activated in our voltage-clamp paradigms. It is relevant to this point that in Xenopus oocytes, only a small fraction of the Cadependent Cll current can be activated by depolarizing to any of the voltage-dependent

bystoma, the delayed outward

MOODY

AND BOSMA

Ion Channels

voltage steps, as compared to direct Ca injection (Miledi and Parker, 1984). Tight control over the spatial distribution of ion channels in oocytes would not be surprising. Clustering of ion channels in adult cells is well documented (see, e.g., Stuhmer and Almers, 1982), especially during periods of insertion of channels into the surface membrane (frog slow muscle: Lehouelleur and Schmidt, 1980), and in the embryo of the mollusc Dent&urn there is differential distribution of excitability between the polar lobe and blastomeres (Jaffe and Guerrier 1981). l-MA is known to trigger rapid changes in the cytoskeletal elements of the cortex (Schroeder and Stricker, 1983; Schroeder and Otto, 1984), and the association of ion channels proteins with these elements could provide a means for controlling the behavior of channels during early development. There are other interpretations of our data which, although possible, we consider less likely. The parallel changes of K currents and capacitance could also result if single channel conductance were decreased, rather than channel numbers, but it seems unlikely that a gradual decrease in channel conductance would follow the time course of the decrease in area so closely. Alternatively, the loss of membrane could include Ca as well as K channels, and the apparent protection of Ca channels could result from a second compensatory mechanism which increased Ca current. We know such a mechanism must operate during maturation in some oocytes, since the magnitude of the Ca current increases in many cells even as membrane area decreases. To account for our data, however, such a mechanism would have to increase the Ca currents by an amount always greater than or equal to the area loss, and in many oocytes would have to match the time course and magnitude of the area decrease within a few percent, so as to yield Ca currents which do not vary during maturation. The close temporal association between this mechanism and the area decrease would have to end after the first hour of maturation, when the area loss begins to reverse while Ca currents in many oocytes continue to increase. We therefore prefer the hypothesis that Ca channels are protected from loss during the decrease in surface area rind that some additional mechanism operates in some oocytes to increase the calcium current, even throughout long-term l-MA exposure. In Leptasterius, we have found that Ca currents appear at a much later time in oogenesis than do either of the K currents, and the time when the greatest increase in Ca current occurs corresponds with the migration of the germinal vesicle to the periphery of the cell (Moody, 1985). In mouse oocytes, the portion of the plasma membrane overlying the second meiotic metaphase is virtually devoid of microvilli, which cover the membrane

during

403

Egg Maturation

in other regions (Phillips and Shalgi, 1980). Therefore it is conceivable that during maturation the Ca channels are associated structurally in some way with the area near the germinal vesicle, and participate differently than K channels in structural reorganization of the membrane. We are at present trying to map the distribution of Ca and K channels over the oocyte membrane before and after maturation. We believe that the differential accessibility of ion channels during periods of addition or removal of surface membrane, and the participation of nonrandom spatial distribution of channels in creating such differences, may be important mechanisms regulating the development of the electrical properties characteristic of different cells of the early embryo. We thank Bibbi Wolowski, Scott Smiiey, and Karen Mesce for assistance with sectioning of materials for electron microscopy, Scott Smiley for suggesting improvements in the fixation, and Tom Daniel for pointing out the isotropic curvilinear overlay method of estimating surface areas. This work was supported by NIH Grant HD 174% and an NIH Research Career Development Award to W.J.M. and by NS 09012 to S. Hagiwara. REFERENCES P. I., and SPITZER, N. C. (1977). Developmental changes in the inward current of the action potential of Rohon-Beard neurones. J. Ph,ysiol. 271, 93-117. BARISH, M. E., and BAUD, C. (1984). A voltage-gated hydrogen ion current in the oocyte membrane of the axolotl, Amhystcrma. X Physiol. 352,243-263. BAUD, C., and BARISH, M. E. (1984). Changes in membrane hydrogen and sodium conductances during Progesterone-induced maturation of Ambystoma oocytes. Dev. Biol. 105, 423-434. BYERLY, L., MEECH, R. W., and MOODY, W. J. (1984). Rapidly activating hydrogen ion currents in perfused neurones of the snail, Lymvwu stqmalis. J. Ph ysiol. 351, 199-216. COLQUHOUN, D., NEHER, E., REUTER, H., and STEVENS, C. F. (1981). Inward current channels activated by intracellular Ca in cultured cardiac cells. Natwe (London) 294, 752-754. CONNOR, J. A., and STEVENS, C. F. (1971). Voltage clamp studies of a transient outward membrane current in gastropod neural somata. J. Physiol. 213, 21-30. EISENMAN, E. A., and ALFERT, M. (1981). A new fixation procedure for preserving the ultrastructure of marine invertebrate tissues. .I. Microsc. 125, 117-120. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeny. 8, 385-416. HAGIWARA, S., K~JSANO, K., and SAITO, S. (1961). Membrane changes in Onchidium nerve cell in potassium-rich media. J Physiol 155, 470-489. HAGIWARA, S., MIYAZAKI, S-I., MOODY, W., and PATLAK, J. (1978). Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg. J. Physiol. 279, 167-185. HAGIWARA, S., MIYAZAKI, S-I., and ROSENTHAL, N. P. (1976). Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish. J. G~?z.Ph ysiol. 67,621-638. HAGIWARA, S., YOSHIDA, S., and YOSHII, M. (1981). Transient and deBACCAGLINI,

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