Changes in voltage-dependent currents and membrane area during maturation of starfish oocytes: Species differences and similarities

DEVELOPMENTAL

BIOLOGY

138, 194-201 (1990)

Changes in Voltage-Dependent Currents and Membrane Area during Maturation of Starfish Oocytes: Species Differences and Similarities LUCIANA SIMONCINI Department

of Zoology, University

J. MOODY

AND WILLIAM of Wadingtmz,

Accepted November

Seattle, Washingtrm 98195

87, 1989

Full grown starfish oocytes are arrested at meiotic prophase I in the ovary. The natural hormone 1-methyladenine triggers oocyte maturation which involves meiosis reinitiation along with a variety of morphological, biochemical, and electrical changes. In studying oocytes of two species, Henricia leviuscula and Asterina miniata, using the voltage-clamp technique, we found interesting differences and similarities in the electrophysiological changes which occurred during maturation. Oocytes of both species have three major voltage-dependent currents: an inward Caa+ current, an inwardly rectifying K+ current, and a transient outward K+ current (A-current). The Caa+ current and the A-current were similar in the two species but the inward rectifier in Henricia had activation kinetics that were more than lo-fold slower than in Aster&a. Nonetheless, all three currents were affected similarly during maturation: the inward Ca2+ currents remained constant in both species, while the two K+ currents decreased in amplitude. In Henricia the membrane surface area decreased substantially during maturation, while in Asterina it remained constant. This may be explained by the more highly infolded state of the membrane in the immature Hew-i& oocyte. The selective loss of K+ current followed the time course of the area decrease in Hwricia, but the same percentage decrease in current occurred in Astetina without a net membrane loss. o 1990 Academic press. 1ne.

membrane surface area, however, decreases only in Henricia, the species with the larger diameter oocytes. These electrical events have a similar time course in both species despite the fact that nuclear membrane breakdown occurs at very different times after hormone addition.

INTRODUCTION

Starfish oocytes remain arrested in the first prophase stage of meiosis in the ovary. During the spawning period the follicle cells surrounding the oocytes release a hormone, 1-methyladenine (l-MA), which induces meiosis reinitiation or maturation (Kanatani, 1973). Maturation can be induced in isolated oocytes by exposing them in vitro to l-MA. Maturation involves changes in protein phosphorylation (Mazzei and Guerrier, 1982), membrane ultrastructure (Hirai and Shida, 1979; Schroeder and Stricker, 1983), and membrane electrophysiology (Miyazaki et al., 1975a,b; Shen and Steinhardt, 19’76; Moody and Bosma, 1985). Voltageclamp studies on Leptasterias (Moody and Lansman, 1983; Moody and Bosma, 1985) have shown that the increase in excitability during maturation occurs because both types of K+ currents present in the oocyte membrane are reduced during maturation, whereas the Ca2’ current remains unaffected. The selective loss of K+ current is closely correlated with removal of a large percentage of the surface membrane of the oocyte (Moody and Bosma, 1985). In the present study we use oocytes of two starfish species to try to define common and divergent features of maturation. We have found that the electrical changes in both species are due to selective loss of K+ currents, while Ca2+ currents remain unaffected. The 0012-1606/90 $3.00 Copyright All rights

B 1990 by Academic Press. Inc. of reproduction in any form reserved.

METHODS

Specimens of the starfish Henricia leviuscula and (old nomenclature, Patiria miniata) Asterina miniata were obtained commercially and maintained at 12-14°C in natural seawater tanks. Ovaries were removed and placed in Ca-free artificial seawater (Ca-free ASW) for 30 min. When the ovaries were returned to normal artificial seawater (ASW), immature oocytes were released, free of surrounding follicle cells. Solutions. ASW contained (in mM): NaCl, 460; KCl, 10; MgC12, 50; CaCl,, 10; Hepes, 10; pH 8.50-Sr ASW (or 50-Ba ASW or 50-Ca ASW) contained 50 SrCl, (or BaCl, or CaC12) and 10 MgCl, replacing 50 MgC12 and 10 CaCl,. In Ca-free ASW CaCl, was replaced by 10 MgC& and 1 mM EGTA. 0-Na, 50-Sr ASW contained 460 choline-Cl replacing 460 NaCl and was adjusted to pH 8 using tris(hydroxymethyl)amminomethane (Tris). 1-Methyladenine (l-MA) was stored frozen as a 2 mM stock solution in distilled water which was diluted to a final concentration of 2 x 10e5 M in ASW. 194

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RESULTS

Voltage-clamp. Standard two-electrode voltage-clamp

methods were used. Electrodes were pulled to resistances of 4-10 M!J for impalement of Asterina oocytes, while they were broken back to <2 MR for impalement of the larger Henricia oocytes to optimize voltageclamp control. Cell capacitance was measured by applying a lo-mV, 50-Hz triangle waveform to the command input. The current response to a triangle wave voltage command is a square wave whose amplitude is proportional to capacitance. Total capacitance was calculated as CM = 1/(2dV/dt), where I is the amplitude of the square step in 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 were there is an instantaneous change in dV/dt, but not Vitself, this method is independent of membrane conductance. Experiments were done at 12-14°C (Henricia) or 14-17°C (Asterina). Data were stored both on FM magnetic tape and in digital form on a computer.

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Currents in Asterina

Oocytes

The starfish A. miniata produces small oocytes with a diameter of 170-200 pm. Under voltage-clamp conditions, three major ion currents were seen in Asterina oocytes: (1) A transient inward CaZ+current activated at potentials positive to -60 mV (Fig. 1). This current was blocked by addition of 1 mM Cd” to the external solution. The channel has a permeability sequence Sr2+ G Ba2+ ti Ca”+ as judged by current amplitudes (Fig. 1A); we therefore performed our experiments using Sr2+ as the charge-carrying ion to obtain larger currents and more accurate measurements. The mean current amplitude, using 50-Sr ASW, was 21.2 + 8.3 nA which corresponds to a current density of 12.9 + 4.3 nA/nF (71 = 36). This inward current appears to be a “pure” calcium current without a Na component because it remained unchanged when 50 Sr ASW was substituted

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FIG. 1. Characterization of currents through the Ca*’ channel. (A) Left panel shows currents recorded in 50 mM Ca”+, Ba”, or SP during voltage-clamp steps to -70 and -40 mV for Ca2+ and -70, -60, -50, -40, and -30 for Ba” and Sr2+. The holding potential was -80 mV. The 2 nA calibration refers to Ca, 10 nA to Ba and Sr currents. (B) Current-voltage relation measured with 50 Sr ASW (0) or 0-Na 50 Sr ASW (0) from a holding potential of -80 mV. (C) Inactivation curve (0). Maximum available current (ordinate) at -40 mV was measured from different holding potentials (abscissa). A current-voltage curve (A) was also plotted to show the overlap of the two curves (see text).

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with 0-Na, 50 Sr ASW (Fig. 1B). This differs from the inward current in oocytes of the starfish Leptasterias which has a substantial Na component activated by Ca2+ entry through voltage-gated Ca2+ channels (Moody, 1985; Lansman, 1987). The inactivation versus voltage curve (Fig. 1C) indicates that at the resting potential (-80 mV) the maximum number of Ca2+ channels is available for opening by depolarization. The available current decreased sharply for holding potentials more positive than -70 mV. In Fig. 1C the inactivation curve (filled circles) is plotted together with the I-V relation (filled triangles) to show the overlap of these two curves. This overlap implies that at potentials between -60 and -45 mV there is a steady-state Cazf current that allows tonic Ca2+ influx into the cell. (2) A transient K’ current (Fig. 2A), activated at potentials positive to +lO mV. This current is sensitive to 4-aminopyridine. Maximum currents are achieved only when conditioning prepulses to potentials more negative than -90 mV are used (Figs. 2C and 2D). This current appears identical to the “A-current” seen in other oocytes and molluscan neurons. (3) An inwardly rectifying K+ current (Fig. 3A, top panel), activated at voltages negative to -80 mV. This current is blocked by barium ions and is similar to the

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VOLUME 138, 1990

inward rectifier of Mediaster and Leptasterias oocytes in voltage-dependence and kinetics (Hagiwara et al., 1978; Moody and Lansman, 1983; see also Standen and Stanfield, 1978). Current and Capacitance Measurements Maturation in Asterina Oocytes

during

Voltage-clamp measurements of current and capacitance were made in individual oocytes before and during exposure to l-MA. Figure 3A shows inwardly rectifying K+ currents (top panel) and Ca2+ currents (lower panel) recorded from the same oocyte before and after 1 hr of exposure to l-MA. The inwardly rectifying K’ current decreased during maturation together with the A current (Figs. 2A and 2B), while the Ca2+ current remained unchanged. Figure 3B shows current-voltage relations for this oocyte before (solid line) and after (dashed line) l-MA application. No substantial shifts in voltage dependence of any of the currents were seen during maturation. Figure 4 shows the time course of changes in the inwardly rectifying K+ current during maturation. The amplitude of this current was stable in the absence of the hormone but began to decline within about 5 min after the beginning of l-MA exposure,

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FIG. 2. Characterization of the transient outward K+ current. Upper panels show currents recorded under voltage-clamp in a single oocyte before (A) and after (B) 1 hr of exposure to I-MA. Voltage steps from -90 mV to +30, +40, and +50 mV. The termination of the voltage step is not shown. (C) Oocyte currents recorded at f20 mV from holding potentials between -95 and -40 mV. These current values were used to plot the inactivation curve in D.

Ion Channels

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membrane capacitance remained constant (open symbols). In the first 5 min after l-MA application, capacitance measurements were made at 1-min intervals to ensure that rapid changes were not missed. After reaching its minimum value, the current began to increase again. This is consistent with results of prolonged l-MA exposure in other starfish (Moody and Bosma, 1985; Miyazaki and Hirai, 1979; Miyazaki, 1979). In seven experiments the inward rectifier K+ current decreased to 60 ? 4% of the initial value, while both the

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FIG. 3. Inwardly rectifying K+ and Ca2’ currents before and after maturation in Astetina. (A) Upper panel shows K+ currents recorded in a single oocyte before and 1 hr after l-MA application. Currents were recorded during voltage steps to ~60, -87, -100, and -113 mV in ASW. The vertical calibration represents 10 nA. The lower pair of records show currents through Caa’ channels in 50-Sr ASW. Voltage steps to ~60, -50, -40, and -30 mV from a holding potential of -90 mV. The vertical calibration represents 4 nA. (B) Current&voltage relation for the same oocyte before (solid line) and 1 hr after (dashed line) exposure to l-MA. The circles represent points taken in ASW and the diamonds points taken in 50-Sr ASW.

reaching a minimum at 20-30 min. In another starfish, Leptasteriao (Moody and Bosma, 1985), application of l-MA to the oocyte induces a similar loss of K+ currents leaving the Ca”’ current unaffected. The time course of the changes in the K+ currents in Leptasterias followed precisely a loss of membrane surface area, measured as capacitance (see Moody and Bosma, 1985, their Fig. 4). In Asterina, however, no decrease in membrane surface area was seen even though the percentage decrease in inward rectifier Ki current was similar to that of Leptasterias. Figure 4B shows the results of such an experiment in three Asterina oocytes. The inwardly rectifying K+ current (filled symbols) decreased, while the

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FIG. 4. Time course of the effect of l-MA on the inwardly rectifying K+ current and membrane area in Astetinu. (A) Current-voltage relation from 15 min before to 15 min after l-MA application. (B) Normalized slope conductance (filled symbols) and capacitance (open symbols) plotted versus time before and after l-MA exposure for three oocytes. All experiments were done at 17°C.

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FIG. 5. (A) Inwardly rectifying K’ (top records) and Ca2+ currents (bottom records) before and after maturation in Henricia. Voltage steps for the inward rectifier were to -60, -70, -80, and -90 mV from a holding potential of -50 mV. Voltage steps for the Ca” current were to -50, -40, -30, and -20 mV from a holding potential of -70 mV. Both currents were recorded in ASW. (B) Current-voltage relation for the same oocyte before (solid line) and 1 hr after (dashed line) exposure to l-MA.

capacitance and the Ca2+ current within 10% of their initial values. Membrane Currents in Henricia Mature Eggs

remained

Immature

constant

Oocytes and

The action of l-MA in Asterina, shows an interesting contrast to its action in Leptasterias (Moody and Bosma, 1985): even though the changes in voltage-dependent currents induced by the hormone are very similar in the two species, they are correlated with a decrease in surface area only in Leptasterias, not in Aster&a. Previous work in another starfish species with small oocytes

VOLUME 138, 1990

(Asterina pectinifera) also found no decrease in surface area (Miyazaki et al., 1975b). To determine if hormoneinduced membrane loss was indeed correlated with large oocyte size and to define other common and divergent features of l-MA action, we repeated our experiments on oocytes of H. leviuscula which, like Leptasterias, produces large yolky eggs (1-1.2 mm in diameter). Under voltage clamp conditions three major ion currents are seen in Henricia oocytes, which are qualitatively similar but quantitatively different from those seen in Aster&a: (1) A transient inward current activated at potentials positive to -50 mV (Fig. 5A, bottom records). This current can be blocked by addition of 1 mM Cd’+ to the external solution. By criteria similar to those used for the inward current in Asterina, this was determined to be a voltage-dependent Ca2+ current. (2) A transient outward K+ current activated at potentials positive to -10 mV. This current appears similar to the A-current seen in Asterina. We did not investigate the properties of this current further because its activation kinetics were fast compared to the charging time of the voltage-clamp, which is slower in Henricia oocytes because of their large size. (3) A slowly activating inwardly rectifying K+ current, activated at voltages negative to -70 mV (Fig. 5A, top records). The inwardly rectifying K+ current of Henricia oocyte is much larger than the same current seen in Aster&a but when normalized to membrane area their densities are similar. The maximum slope conductance for Henricia is 50-200 nS/nF (four oocytes, mean surface area = 5.3 mm2), while for Aster&a it is 400-2000 nS/nF (four oocytes, mean surface area = 0.105 mm2). The kinetics of this inwardly rectifying K+ current are about 20 times slower than those of inwardly rectifying Kt currents in Aster&a and other starfish previously studied (compare Figs. 5A & 3A) (Hagiwara et al., 1976). Figure 6A shows activation time courses for the inward rectifier in Henricia (dashed line) and Asterina (solid line) at -90 mV. Both activate with a single exponential time course, but the activation time constant in Asterina is 260 msec (17”C), while in Henricia it is 4.1 see (14°C). This difference is far too large to be explained by the temperature dependence of activation kinetics, which is very small at -90 mV (Hagiwara and Yoshii, 1980). Despite its slow kinetics of activation, the inwardly rectifying K+ current in Henricia shares several properties in common with more rapidly activating inward rectifying K+ currents in other cells. The current-voltage relation shifts with changes in external K+ concentration, so that the current shows an apparent dependence on (V-V,) rather than on Valone (Fig. 6B) (Hagiwara et al., 1976; Stan-

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FIG. 6. Properties of the slow inward rectifier in Henriciu. (A) Comparison of activation kinetics Henricia (dashed line) and Asterinu (solid line) for a voltage step to -90 mV. The normalized difference between the actual current and its steady-state value (I - I,,,,,) was plotted against the time from the onset of a voltage pulse. (B) Current-voltage relations of the Henriciu inward rectifier at four different K’ concentrations. Holding potential was -50 mV.

den and Stanfield, 1978). In addition the inwardly rectifying K+ current in Henricia is blocked by low concentrations of external Ba2+ ions (not shown), as are inwardly rectifying K’ currents in other cells (Hagiwara et al., 1978; Standen and Stanfield, 1978). As shown below, it also behaves similarly to other inward rectifying K+ currents during maturation. Current and Capacitance Measurements Maturution in Henricia Oocytes

during

Voltage-clamp measurements of current and capacitance were made in individual Henricia oocytes before

and during exposure to l-MA. Figure 5A shows inwardly rectifying K+ currents (top panel) and Ca2+ currents (lower panel) recorded from the same oocyte before and after 1 hr of exposure to l-MA. The inwardly rectifying K+ current decreased to 25% of its initial amplitude during maturation, while the Ca”+ current increased slightly. Figure 5B shows current-voltage relations for the same oocyte before (solid line) and after (dashed line) l-MA. No substantial shifts in the voltage dependence of any currents were seen during maturation. Figure ‘7 shows the time course of changes in the inwardly rectifying K+ current and capacitance during

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DEVELOPMENTAL BIOLOGY

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oJ,,,1,,,,,,,,,,,,,,, -30 0

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FIG. 7. Time course of the effect of l-MA on the inwardly rectifying K’ current and membrane area in Henriciu. (A) Current-voltage relations from 23 min before to 145 min after hormone application. (B) Normalized slope conductance (0) and capacitance (+) plotted against time during maturation.

maturation. The amplitude of the current began to decline a few minutes after l-MA application, stabilizing after approximately 90 min (12°C) at 25% of its initial value. The decline of inwardly rectifying K+ current in Henricia after l-MA application followed a time course similar to that of Asterina (Fig. 7B; see Fig. 4B). However, unlike Asterina, the decline in inwardly rectifying K+ current (filled circles) was accompanied in Henricia by a decrease in membrane surface area measured as a decrease in membrane capacitance (crosses; Fig. ‘7B). In four other experiments the inward rectifier K+ current (Ii,) and capacitance (C,,,) decreased to 55 + 5.7% and 67 +- 5% (n = 4), respectively. DISCUSSION

We have studied changes in the electrical properties of oocytes of two starfish species, A. miniata and H.

VOLUME 138, 1990

leviuscula during meiotic maturation and compared the results to previous work in other starfish and amphibian oocytes to try to understand the common and divergent features of this process. One common feature in all oocytes studied is a substantial decrease in inwardly rectifying conductance activated at potentials negative to rest. In starfish this is an inwardly rectifying Kt conductance that decreases by about half in all the species studied (A. miniata and H. leviuscula, present study and Shen and Steinhardt, 1976; A. pectinifera, Miyazaki et al., 1975b; Leptasterias hezactis, Moody and Lansman, 1983). Note that Henricia shares this feature even though the current is kinetically very different than in other species. In amphibians, in which maturation is triggered by the steroid hormone progesterone, the inward rectifier of at least one species is carried by Cl-, and it too decreases during maturation (Taglietti et al., 1984). A second common feature of maturation is the pattern of changes in depolarization-activated currents that subserve the action potential. Outward currents decrease in amplitude, while inward currents remain constant or increase. This is true for A-currents and calcium currents in three starfish species (present results; Leptasterias, Moody and Bosma, 1985). In amphibians, the outward current is carried in various species by Ki (Taglietti et al., 1984), Cll (Schlichter, 1983), or Hi (Barish and Baud, 1984; Baud and Barish, 1984). All decrease during maturation. Depolarization-activated inward currents in amphibians, commonly carried by Na+ ions, are either retained or increase with maturation (see above references). All of the above electrophysiological changes tend to increase the excitability of the mature egg. Inwardly rectifying currents serve to make the resting conductance of the oocyte voltage-dependent. This provides sufficient conductance to create a stable resting potential, while allowing the resting channels to close whenever a significant depolarizing stimulus is received. In this way the oocyte can generate large depolarizing responses with minimal ion fluxes and metabolic load. The decrease of this conductance during maturation increases the membrane resistance of the oocyte, making it more likely that depolarizing inputs will reach threshold for the action potential. Once generated, the action potential in the mature egg will be larger and/or longer because of the decrease of outward relative to inward currents. The relationship between these changes and the ability to produce an effective fast polyspermy block is supported by experiments done on overmature eggs (Miyazaki, 1979). With prolonged exposure to l-MA the electrical changes seen in maturation start to reverse (see Fig. 4C). These overmature eggs became highly polyspermic. The application of

Blocking effect of barium and hydrogen ions on the potassium curBa”’ ions, which reduce the resting K’ conductance of rent during anomalous rectification in star&h egg. J. Physiol. 279, the membrane back to its level in the mature state, lti7-185. reestablishes monospermic fertilization. HAGIWARA, S., MIYAZAKI, S-I., and ROSF,NTIIAL, N. P. (19’76). PotasEven though the loss of the A-current and the insium current and the effect of cesium on this current during anomwardly rectifying K’ current is common to all starfish alous rectification of the egg cell membrane of a starfish. J. Gwl. Ph ysiol. 67, 621-638. studied, only in Hewiciu and Lepfnsterias is this loss correlated with a net loss of surface membrane area, as H~(;IwAKI\, S., and YOSHII, M. (1980). Effect of temperature on the anomalous rectihcation of the mrmhrane of the egg of the starfish, seen by a decrease in capacitance. In A. minicrfu and A. MfYlitr.stcr trtYJutr/Ls. ,I. P//+siOl. 307, 517527. pecfin~brcx (Miyazaki et crl., 1975b) no decrease in memHIKAI, S. AND SHII~A, H. (1979). Shortening of microvilli during the brane surface area occurs. We calculated the degree of maturation of starfish oocvtr from which vitellinr coat was removed. 8,111. Mtrr. Biol. St. ..lsccn~rtshi. Tohuku LT?ric,.16, 161-167. infolding as the ratio between the surface area meaKAIW, R. T., MAWHW, Ii., and OZON, R. (1981). Electrical memhrane sured from capacitance, assuming a specific capaciproperties of the Xi~/op~.s (CIC,I~~.S oocyte during progesteronr-intance of 1 wF/cm”, and the surface area estimated from duced meiotic maturation. &v. Kiol. 84, 471-476. the oocyte diameter. For Lepfusterius oocytes the ratio KAN.&T~\NI, II. (1973). Maturation-inducing substance in starfishes. is 3.37 & 0.7 before and 1.51 ? 0.26 after maturation (rz zut. XPI’. (‘!/to/. 35, X-298. LANSM,\N, J. B. (1987). Calcium current and calcium-activated inward = lo), (from Bosma and Moody, unpublished results). current in the oocvtr of the starfish Lvptrrslericls hr,.roctis. J. Ph+sFor Hc)~ricicr we accurately measured the geometrical id. 390, 39741:1. diameter only for three maturing oocytes and the ratio MAZEI, G., and GTIWRIER, I’. (19x2). Changes in the pattern of prowas 5.7 + 0.35 before and 2.5 i 0.2 after. In contrast for tein phosphorvlation during meiosis reinitiation in starlish ooA. ntiniufu the ratio was 1.6 +- 0.35 before and after rvtes. 11c~r7.Bio/. 91, 24G256. maturation (sn = lo), and for A. pecfin~fera the ratio was MI~AUKI, S-I. (1979). Fast polq’spermq’ block and activation potential. Ele~trol)hysioloRical basis for their changes during maturation reported as approximately 1 before and after maturaof a starfish. I)c~I~,Hiol. 70, 341-354. tion (Miyazaki et u.L., 19’75b). These indicate that the MIY.Z\KI, S-I., and HIKIII, S. (l!W). Fast pol~spermg block and actilarge yolky oocytes have a much more infolded memvation potential. Correlated changes during oocgte maturation of brane and that the infolding decreases during the matustarfish. &‘I,. i?iol. 70, 3277340. MIY,UAL;I, S-I., OIIMORI, H., and SASAKI, S. (1975a). Action potential ration process reaching a value similar to that of small and non-linear current-voltage relation in starfish oocgtes. J. oocytes. Three hypotheses could account for these difPh,//siol. 246, 37-54. ferences in net membrane loss: (a) the basic mechaMI~.UAKI, S-I., OHMORI, II., and SASAKI, S. (1975h). Potassium rectinisms of l-MA action are different in different species; fication of the starfish oocgte memhrane and their changes during (b) the electrical actions of l-MA are not secondary to ooc’gtr maturation. J. l’h!jsio/. 246, 55-78. membrane loss in any starfish, being explained, for ex- Moot)y. W. J. (19%). The development of calcium and potassium currents during oogencsis in the starfish, Lcytostc~ritrs h(~.rcrc~is. Lk11. ample, by the release into the cytoplasm of a K’ chanBid. 112, 405-413. nel-blocking molecule; or (c) they are explained by MooI)~. W. J., and BOWA, M. M. (1985). Hormone-induced loss of membrane loss in all species, but this appears as net surface membrane during maturation of starfish oocytes: Differloss only in Henricicx and Lepfusterias, because oocytes ential effects on potassium and calcium channels. Dc,t!. Biol. 112, 396-404. with a less infolded membrane before maturation may not have sufhcient surface area to support a net loss MooI)~, R. J., and LANSMAN, J. B. (19X3). Developmental regulation of C:<” and K ’ currents during hormone-induced maturation of starequal to the required loss of K’ current and so recycle fish oocvtes. I’rw iVo11. ~lcurl. Sci. 1CU 80, 3096-3100. membrane, effectively replacing membrane that con- SC~HFWEWR,T. E., and STRWKEK, S. A. (19%). Morphological changes tains K’ channels with that which does not. during maturation of starfish oocytes: Surface ultrastructure and Supported by NIH Grant HI)17186 ment Award to \V.J.M.

anti

a

Research Career Develop

REFERENCES BAI’I), C’., and BAKISH, M. (l!IX4). Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of A rnbysfottrtr oocytes. L)r8%.Kid 105, 423-434. BAKISH, M., and BAITI), C. (1984). A voltage-gated hydrogen ion current in the oocvte membrane of the axolotl, ilrvtb~~stow~u. ,J. Physiol. 3.52, 243-263. IIac:rw~~., S.. M1y.4z.4~1, S., MooI)~, U’., and PATLAR, J. (197X).

cortical act in. Df,c,. Bio(. 98, 373-384. SCIII~IITICR, L. C. (1983). Spontaneous action potentials produced bv Na and (‘1 channels in maturing Row ~,l~,iwt.s oocytes. 1)~. Riol. 98, 47-59. SII~~N, S., and S’rF:tNl+.ktxrr, R. A. (1976). An elcctrophysiological studs of the membrane properties of the immature and mature oocyte of the batstar, Ptr(iricc rrtiniutu. Lkr. Bid. 48, 148-162. STANL)F,P~,N. R., and STANE’IELI~, P. R. (197X). Inward rectification in skeletal muscle: A blocking particle model. P.fllrq~rs ;1rch. 378, 173-176. TAGI.WTI, \:., TA~ZI, F. R~MWO, R., and SIMUNCINI, L. (19X4). Maturation involves suppression of voltage-Eated currents in the frog oocyte. J. (b/l. m&sir,/. 121, 576x%X.