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Free calcium in full grown Xenopus laevis oocyte following treatment with ionophore A 23187 or progesterone
Molecular and Cellular Endocrinology, 0 Elsevier/North-Holland
8 (1977)
65-72
Scientific Publishers, Ltd.
FREE CALCIUM IN FULL GROWN XENOPUS TREATMENT
LAEVZS OOCYTE FOLLOWING
WITH IONOPHORE A 23 187 OR PROGESTERONE
R. BELLE *, R. OZON * and J. STINNAKRE ** * Laboratoire de Physiologic de la Reproduction, Universitk Pierre et Marie Curie, 9 Quai Saint-Bernard, 75005 Paris, and ** Laboratoire de Neurobiologie Gif-sur- Yvette, France
Cellulaire, 91190
Received 12 January 1977; accepted 5 April 1977
Free intracellular Ca*+ was monitored in isolated Xenopus laevis oocyte during induced maturation using the Ca*“- sensitive luminescent protein, aequorin. Internal free Ca*+ was not precisely measured but data suggest it was quite low (in the micromolar range). No change in internal free Ca*+ was detected during maturation induced either by progesterone or byp-chloromercuribenzoate. By contrast, the ionophore A 23187 gave an increase in the free Ca*+ level when there was a raised external Ca*+ (10 mM), cond’t’ t ions which also induce oocyte maturation. About 3 h after progesterone or p-chloromercuribenzoate stimulation, the oocyte membrane potential decreased by about 50 mV while the membrane resistance increased transitorily . Keywords:
Xenopus
laevis; oocyte;
free calcium; aequorin; progesterone;
ionophore
A
23187.
In the Amphibian full grown oocyte the resumption of meiosis is induced in vivo and in vitro by progesterone (Smith, 1976) and depends upon the presence of divalent cations in the external medium (Ecker and Smith, 1971; Merriam, 1971a); progesterone does not induce maturation of Xenopus oocyte in the presence of 10 mM EDTA (Marot et al., 1976). Further recent reports suggest that divalent cations are indeed involved in oocyte maturation: - exposing the Xenopus oocyte to divalent cations at high concentration in the presence of the ionophore A 23187 initiates maturation (Wasserman and Masui, 1975); - p-chloromercuribenzoate increases Ca 2+ influx into the Xenopus oocyte (Marot et al., 1976) and also induces maturation (Brachet et al., 1975); - propanolol-like drugs which can interfere, at high concentration, with Ca*+ fixation to the membrane, can induce Xenopus oocyte maturation (SchorderetSlatkine and Schorderet, 1976). However, no direct evidence of Ca*+ movements triggered by progesterone has been reported. On the contrary, no change of influx or efflux of 4sCa2+ has been 65
66
R. Beflc?et al.
detected during progesterone treatment until there is germinal vesicle breakdown associated with maturation; moreover, external Ca2+ is not necessary, since the presence of EGTA in excess relative to Ca*+ ions does not suppress the progesterone-induced maturation (Marot et al., 1976) whereas the presence of magnesium ions is required (Marot et al., 1976). Also, it has been indicated that exchange of divalent cations between the oocyte and the extracellular medium is not necessary for the completion of maturation (Merriam, 1971 b). It appears then that sequestered divalent cations could be released from intracellular store(s) during the maturation process. In order to test the hypothesis of internal Ca2+ movements initiated by progesterone in the full grown oocyte, we have studied the free internal Ca*+ concentration using the Ca*+-sensitive luminescent protein aequorin (Shimomura et al., 1962) as it has already been used for early embryos of Xenopus faevis (Baker and Warner, 1972). We also analysed the effect of ionophore A 23 187 on free Ca*+ concentration.
MATERIAL
AND METHODS
Animal and oocyte preparations ~enop~s Zaevis (de Rover, The Ne~erl~ds) were bred and m~ntained under laboratory conditions. Full grown oocytes (stage 6, diameter >1.2 mm) were isolated from the ovary and treated by collagenase (Worthington), 1 mg/ml, at 30°C for 4-6 h, which removed the follicular envelope that could interfere with the measurement of membrane potential. This procedure also facilitated the penetration of intracellular micropipettes. Chemicals and buffers The oocytes were left to equilibrate with the following incubation medium (medium A) overnight (in mM): NaCl, 88; KCl, 1; Ca(NOs)*, 0.33; CaC12, 0.41; MgS04, 0.82; Tris, 2; pH = 7.4. Medium A was diluted to 70% to give a hypotonic medium which was used to facilitate electrode penetration. All electrical and light emission measurements were performed in a hypertonic medium which was made from medium A in which NaCl was raised to 132 mM. Progesterone (pregn-4-ene-3,20 dione) purchased from Sigma was chromatographically pure; p-chloromercuribenzoate was purchased from Nutritional Biochemicals Corporation. The ionophore A 23187 was a gift of the Lilly Research Laboratories. It was dissolved in d~ethylsulfoxide (5 mgfml) and diluted to 2.5 &g/ml in the test medium before use. Electrical measurements ‘Classical’ electrophysiological equipment tial and apparent membrane resistance.
was used to record membrane
poten-
Free Ca*+ in Xenopus oocyte
67
The oocytes were immobilized in an indentation in a plasticine-coated Petri dish; the pigmented area was orientated upward or tilted by 90” from this position. One oocyte was impaled by 2 or 3 micropipettes under microscopic examination after the chamber had been perfused for several minutes with the diluted medium. In these conditions, the oocyte membrane was less depressed under the tip of the electrodes and the penetration was found to be easier and less damaging to the cell. One double-barrelled pipette (3 X 6 pm) contained the aequorin solution to be injected in one channel, and the other one contained 3 M KC1 to record the membrane potential (Stinnakre and Taut, 1973, 1974). One KC1 pipette ($, 2 /_fm)was used to pass current pulses for membrane resistance measurement. One 0.39 M CaCl, pipette either replaced the KC1 pipette or was used in addition to ensure proper aequorin injection and to monitor changes in aequorin concentration by recording light emission from Ca*+ electrophoretic pulses (Kusano et al., 1975a,b). A small retaining current (2-5 nA) prevented release of Ca*+ between injections. All currents flowing through the Ca*+ pipette crossed the membrane. Membrane resistance measurements were taken by recording the voltage drop due to a small hyperpolarizing current (2-4 nA) passed across the membrane through either the KC1 or the CaC12 pipette. Injections After the electrodes penetrated into the oocyte, the hypotonic medium was replaced by the hypertonic solution which permitted larger injection. Injections were performed in the dark using air pressure pulses while monitoring light emission and membrane potential. No attempts were made to estimate the injected volume. Injections were stopped when Ca*+ pulses gave stable light responses (see fig. 1B). Light measurements The light emitted from the injected oocyte was collected photocathode of a photomultiplier (HTV R-374, cathode located a few millimeters above the level of the physiological amplification and filtering, the light signal together with the was displayed on a paper recorder (Brush 280). All experiments were performed at 20-22°C.
in the dark on the potential -1100 V) saline. After proper membrane potential
RESULTS Electrical characteristics of defolliculated Xenopus oocytes The membrane potential of isolated oocytes left overnight in the incubation medium was -63 mV (SD 16 mV, 17 determinations) as measured from the maximum deflexion obtained when the first electrode (usually KCl) penetrated the cell (i.e. in the hypotonic solution). No significant variation was seen when the medium was changed to the normal or the hypertonic solution. The mean value for the
68
R. Bell& et al.
apparent membrane resistance was 2.6 MOhms (SD 1.4 MOhms, 8 determinations). As has been described for Rana pipiens oocytes (Morrill et al., 1966) Xenopus oocytes membrane potential is sensitive to external Ca2’ ions. Perfusion of the chamber with a medium containing 10 mM EGTA decreased the membrane potential to a low value in a few minutes (for example from -76 to -1.5 mV). Perfusion with the control medium slowly restored the membrane potential. In other experiments addition of CaCl, to 10 mM resulted in a similar reversible depolarization whereas MgCl, (10 mM) had no effect. During Ca2+ perfusion the membrane resistance dropped about lo-fold. Injection ofaequorin Before injecting aequorin, a background light signal was recorded coming from the aequorin pipette itself plus the dark current of the photomultiplier. In 3 exper-
A 10.4 nA
-.....~
10 J-1omv
7-----
J
!A
8
1nA
i
0 1-2ordJ
Fig. 1. Effect of perfusion of the ionophore A 23187 (A) and electrophoretic Ca2+ pulses (B) on light emission (top traces) and membrane potential (lower traces) of an aequorin injected Xenopus oocyte. The ionophore in the presence of high Ca2+ was perfused between the arrows; there is a time-lag of about 2 min due to the dead space of the perfusion system, as can be seen when normal Ca*+ solution without ionophore was perfused (t). The broken line corresponds to a 3 min interruption of the record during which the electrophoretic Ca2+ pulse shown in B2 was applied. Ca2+ inJections Bl and B3 were made respectively before and after the record shown in A (injection parameters: 500 ms, 25 nA). The origin of the fast transients in the A light record is unknown (time constant of the light recording system: 110 ms).
Free Ca*+ in Xenopus oocyte
69
iments injections were accompanied by a slight decrease of the PM current while no change was detected in the others. Then the Ca*+ concentration inside the oocyte was equal to or less than in the pipette. Electrophoretically injected Ca*+ pulses produced changes in light emission (fig. 1B). After the end of the pulse, return to the base line occurred with a half decay time of about 0.6-2 s. Responses to Ca*+ pulses were seen for several hours without further injection of aequorin. These observations suggest that the internal Ca*+ concentration is well buffered and, in all likelihood, similar to that found in Molluscan neurons (i.e. less than lop7 M). The amplitude of the light transients was measured for different Ca*+ currents and duration (from 10 nC to 200 nC) and was found to vary linearly with the electrophoretic current as it has been observed in the squid axon (Kusano et al., 1975b). In control injected oocytes the light decreased slowly to a very low level. It is not clear whether this is due to the PM and/or the aequorin pipette or to the cell itself. No changes were observed in light emission during EGTA (10 mM), CaC12 (10 mM) or MgC12 (10 mM) perfusion of the chamber. Effect
of the
ionophore A 2318 7
The ionophore A 23 187 (2.5 pg/ml) had no effect when perfused in normal Ca*+ buffer. Raising CaCl, to 10 mM in the presence of the ionophore resulted in a progressive increase in light emission (fig. 1A). Washing with normal Ca*+ medium restored base line in a few minutes. No change was observed when MgCl, was raised to 10 mM in the presence of the ionophore and normal Ca*+ concentration. Effect of progesterone No changes in light emission were observed from the initial progesterone (lop6 M) stimulation until germinal vesicle breakdown. During the same period electrophoretic Ca2+ pulses gave light responses indicating that exhaustion of aequorin was not responsible for the lack of detectable [Ca*+]t change. The same result was obtained when aequorin was injected near the margin of the pigment. The membrane potential and membrane resistance did not show any significant variation during the 2-4 h following progesterone application (fig. 2). After this quiet period, the membrane potential dropped and stabilized at about -16 mV (SD 2.5 mV, 5 determinations), this change taking place during approximately 1 h. At about the time the membrane depolarized, the apparent membrane resistance showed a transitory increase (2-S-fold) and later decreased to a value which was beneath the initial one (see fig. 2). After these changes were observed, the incidence of germinal vesicle breakdown was’determined as judged from the presence of a white spot on the center of the pigmented hemisphere. Three experiments were performed using p-chloromercuribenzoate (10e4 M) instead of progesterone. Again no significant changes occurred in the light emission
70
R. Belli et al.
Fig. 2. Time course of membrane potential (continuous line) and resistance changes (broken line) of a Xenoppus oocyte following stimulation with progesterone (lo@ M).
although potential one did occur.
and resistance
changes similar to those observed with progester-
DISCUSSION Xeno~~s irrevis oocyte maturation is induced by several compounds: by progesterone which is the natural stimulus (Smith, 1976) by p-chloromercuribenzoate (Bachet et al., 1975) and by the ionophore A 23187 in the presence of raised Ca2+ (Wasserman and Masui, 1975). The two latter substances are known to modify Ca2+ fluxes across the oocyte membrane (Wasserman and Masui, 1975; Marot et al., 1976). it was then postulated that progesterone acts through an intraceIluIar change of free Ca2+ concentration, for Ca2+ influx and efflux are not modified by the hormone (Marot et al., 1976). Using aequorin we have observed that the ionophore A 23187 is indeed able to increase free internal Ca2+ provided the external concentration of this ion is raised. But no change of free Ca *+ has been detected during either progesterone or p-chloromercuribenzoate treatment. Unfortunately it is impossible it is probably less to give a precise figure for the free Ca2+ CoIlcentration (~thou~ than IOe6 M) and for the minimal change which can be detected. A calibration is rather difficult to establish and will not be curve (lightfCa’+ concentration) useful due to the strong absorption given by the yolk and the pigment. From the length of the tip of the Ca2+ electrode entering the cell and the radial diffusion of Ca2’, it can be assumed that the light emitted in response to Ca2+ pulses originates from the first tens of a micrometer of cytoplasm under the membrane. Therefore it is likely that the change in fCa2’] i observed in the presence of A 23 I87 and high Ca2+ occurs in this layer. The absence of light response during progesterone or p-chloromercuribenzoate action might be interpreted as an indication that an increase of free Ca 2*, if any, is not taking p lace in the vicinity of the oocyte membrane during the maturation induced by these substances. However, it remains possible that such a Ca2+ change is too small to be detected by our method or it occurs
Free Ca2+ in Xenopus oocyte
71
in another compartment than the aequorin diffusion compartment. in that part of the The regulatory mechanisms of the free Ca2+ concentration cell which is accessible to aequorin examination appears to be very efficient, for the resting light emission was rapidly restored following Ca2+ injections or washing out the ionophore and the excess external Ca 2+. On the other hand, it has been observed that CaZf influx is increased either by A 23187 in normal Ca2+ (i.e. 0.74 mM) or by raised Ca2+ (10 mM) (R. Belle, J. Marot and R. Ozon, unpublished data); since no change of emission is seen in these conditions, the cells should be able to get rid of this influx and maintain the resting Ca2+ concentration. When the influx is increased by applying simultaneously the ionophore and raised Ca2+, [Ca2+]i starts to increase and in some way can trigger the maturation. Progesterone as well as p-chloromercuribenzoate modifies plasma membrane electrical characteristics as has already been observed (Moreau et al., 1976). But in contrast with these previous results, we observed a delay before these modifications occur; also the increase in membrane resistance is only transitory, the value reached after the germinal vesicle breakdown being lower than the initial value. The origin of these discrepancies is unknown. In fact, oocyte maturation can be obtained in different chemical environments where membrane potential and membrane resistance are quite different - for example, in the presence of EGTA (Marot et al., 1976). Therefore, modifications of these parameters induced by progesterone or p-chloromercuribenzoate treatment reflect internal modifications (which cannot be related to detectable Ca2+ movements) but do not seem to play a causative role in the initiation of this phenomenon as has been previously suggested (WaIlace and Steinhardt, 1975). In conclusion, our results have shown that in the experimental conditions where oocyte maturation is obtained by the ionophore A 23187 in the presence of high Ca2+, there is a detectable increase of internal free Ca2+ concentration. This increase may be sufficient for the induction of maturation, but may not be necessary since progesterone which is the natural stimulus does not provoke the same effect, unless it occurs in a place where it cannot be detected by our method.
ACKNOWLEDGEMENTS We are indebted of aequorin.
to Professor 0. Shimomura
(Princeton
University)
for the gift
REFERENCES Baker, P.F. and Warner, A.E. (1972) J. Cell Biol. 53,579. Brachet, J., Baltus, E., De Schutter-Pays, A., Hanocq-Quertier, (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1574.
J., Hubert,
E. and Steiner&
G.
72
R. Belle! et al.
Ecker, R.E. and Smith, L.D. (1971) J. Cell. Physiol. 77,61. Kusano, K., Miledi, R. and Stinnakre, J. (1975a) Proc. R. Sot. London Ser. B 189, 39. Kusano, K., Miledi, R. and Stinnakre, J. (1975b) Proc. R. Sot. London Ser. B 189,49. Marot, J., Belle, R. and Ozon, R. (1976) C.R. Acad. Sci. Paris 282, 1301. Merriam, R.W. (1971a) Exp. Cell Res. 68,75. Merriam, R.W. (1971b) Exp. Cell Res. 68, 81. Moreau, M., Guerrier, P. and Dorce, M. (1976) C.R. Acad. Sci. Paris 282, 1309. Morrill, G.A., Rosenthal, J. and Watson, D.E. (1966). J. Cell. Physiol. 67, 375. Schorderet-Slatkine, S. and Schorderet, M. (1976) C.R. Acad. Sci. Paris 282, 1733. Shimomura, O., Johnson, F.H. and Saiga, Y. (1962) J. Cell. Comp. Physiol. 59, 223. Smith, L.D. (1976) In: The Biochemistry of Animal Development. Ed.: R. Weber (Academic Press, New York) Vol. 3, p. 1. Stinnakre, J. and Taut, L. (1973) Nature (London), New Biol. 242, 113. Stinnakre, J. and Taut, L. (1974) C.R. Acad. Sci. Paris 278, 1409. Wallace, R.A. and Steinhardt, R.A. (1975) J. Cell Biol. 67,443a. Wasserman, W.J. and Masui, Y. (1975) J. Exp. Zool. 193, 369. Wasserman, W.J. and Masui, Y. (1976) Science 191,1266.
8 (1977)
65-72
Scientific Publishers, Ltd.
FREE CALCIUM IN FULL GROWN XENOPUS TREATMENT
LAEVZS OOCYTE FOLLOWING
WITH IONOPHORE A 23 187 OR PROGESTERONE
R. BELLE *, R. OZON * and J. STINNAKRE ** * Laboratoire de Physiologic de la Reproduction, Universitk Pierre et Marie Curie, 9 Quai Saint-Bernard, 75005 Paris, and ** Laboratoire de Neurobiologie Gif-sur- Yvette, France
Cellulaire, 91190
Received 12 January 1977; accepted 5 April 1977
Free intracellular Ca*+ was monitored in isolated Xenopus laevis oocyte during induced maturation using the Ca*“- sensitive luminescent protein, aequorin. Internal free Ca*+ was not precisely measured but data suggest it was quite low (in the micromolar range). No change in internal free Ca*+ was detected during maturation induced either by progesterone or byp-chloromercuribenzoate. By contrast, the ionophore A 23187 gave an increase in the free Ca*+ level when there was a raised external Ca*+ (10 mM), cond’t’ t ions which also induce oocyte maturation. About 3 h after progesterone or p-chloromercuribenzoate stimulation, the oocyte membrane potential decreased by about 50 mV while the membrane resistance increased transitorily . Keywords:
Xenopus
laevis; oocyte;
free calcium; aequorin; progesterone;
ionophore
A
23187.
In the Amphibian full grown oocyte the resumption of meiosis is induced in vivo and in vitro by progesterone (Smith, 1976) and depends upon the presence of divalent cations in the external medium (Ecker and Smith, 1971; Merriam, 1971a); progesterone does not induce maturation of Xenopus oocyte in the presence of 10 mM EDTA (Marot et al., 1976). Further recent reports suggest that divalent cations are indeed involved in oocyte maturation: - exposing the Xenopus oocyte to divalent cations at high concentration in the presence of the ionophore A 23187 initiates maturation (Wasserman and Masui, 1975); - p-chloromercuribenzoate increases Ca 2+ influx into the Xenopus oocyte (Marot et al., 1976) and also induces maturation (Brachet et al., 1975); - propanolol-like drugs which can interfere, at high concentration, with Ca*+ fixation to the membrane, can induce Xenopus oocyte maturation (SchorderetSlatkine and Schorderet, 1976). However, no direct evidence of Ca*+ movements triggered by progesterone has been reported. On the contrary, no change of influx or efflux of 4sCa2+ has been 65
66
R. Beflc?et al.
detected during progesterone treatment until there is germinal vesicle breakdown associated with maturation; moreover, external Ca2+ is not necessary, since the presence of EGTA in excess relative to Ca*+ ions does not suppress the progesterone-induced maturation (Marot et al., 1976) whereas the presence of magnesium ions is required (Marot et al., 1976). Also, it has been indicated that exchange of divalent cations between the oocyte and the extracellular medium is not necessary for the completion of maturation (Merriam, 1971 b). It appears then that sequestered divalent cations could be released from intracellular store(s) during the maturation process. In order to test the hypothesis of internal Ca2+ movements initiated by progesterone in the full grown oocyte, we have studied the free internal Ca*+ concentration using the Ca*+-sensitive luminescent protein aequorin (Shimomura et al., 1962) as it has already been used for early embryos of Xenopus faevis (Baker and Warner, 1972). We also analysed the effect of ionophore A 23 187 on free Ca*+ concentration.
MATERIAL
AND METHODS
Animal and oocyte preparations ~enop~s Zaevis (de Rover, The Ne~erl~ds) were bred and m~ntained under laboratory conditions. Full grown oocytes (stage 6, diameter >1.2 mm) were isolated from the ovary and treated by collagenase (Worthington), 1 mg/ml, at 30°C for 4-6 h, which removed the follicular envelope that could interfere with the measurement of membrane potential. This procedure also facilitated the penetration of intracellular micropipettes. Chemicals and buffers The oocytes were left to equilibrate with the following incubation medium (medium A) overnight (in mM): NaCl, 88; KCl, 1; Ca(NOs)*, 0.33; CaC12, 0.41; MgS04, 0.82; Tris, 2; pH = 7.4. Medium A was diluted to 70% to give a hypotonic medium which was used to facilitate electrode penetration. All electrical and light emission measurements were performed in a hypertonic medium which was made from medium A in which NaCl was raised to 132 mM. Progesterone (pregn-4-ene-3,20 dione) purchased from Sigma was chromatographically pure; p-chloromercuribenzoate was purchased from Nutritional Biochemicals Corporation. The ionophore A 23187 was a gift of the Lilly Research Laboratories. It was dissolved in d~ethylsulfoxide (5 mgfml) and diluted to 2.5 &g/ml in the test medium before use. Electrical measurements ‘Classical’ electrophysiological equipment tial and apparent membrane resistance.
was used to record membrane
poten-
Free Ca*+ in Xenopus oocyte
67
The oocytes were immobilized in an indentation in a plasticine-coated Petri dish; the pigmented area was orientated upward or tilted by 90” from this position. One oocyte was impaled by 2 or 3 micropipettes under microscopic examination after the chamber had been perfused for several minutes with the diluted medium. In these conditions, the oocyte membrane was less depressed under the tip of the electrodes and the penetration was found to be easier and less damaging to the cell. One double-barrelled pipette (3 X 6 pm) contained the aequorin solution to be injected in one channel, and the other one contained 3 M KC1 to record the membrane potential (Stinnakre and Taut, 1973, 1974). One KC1 pipette ($, 2 /_fm)was used to pass current pulses for membrane resistance measurement. One 0.39 M CaCl, pipette either replaced the KC1 pipette or was used in addition to ensure proper aequorin injection and to monitor changes in aequorin concentration by recording light emission from Ca*+ electrophoretic pulses (Kusano et al., 1975a,b). A small retaining current (2-5 nA) prevented release of Ca*+ between injections. All currents flowing through the Ca*+ pipette crossed the membrane. Membrane resistance measurements were taken by recording the voltage drop due to a small hyperpolarizing current (2-4 nA) passed across the membrane through either the KC1 or the CaC12 pipette. Injections After the electrodes penetrated into the oocyte, the hypotonic medium was replaced by the hypertonic solution which permitted larger injection. Injections were performed in the dark using air pressure pulses while monitoring light emission and membrane potential. No attempts were made to estimate the injected volume. Injections were stopped when Ca*+ pulses gave stable light responses (see fig. 1B). Light measurements The light emitted from the injected oocyte was collected photocathode of a photomultiplier (HTV R-374, cathode located a few millimeters above the level of the physiological amplification and filtering, the light signal together with the was displayed on a paper recorder (Brush 280). All experiments were performed at 20-22°C.
in the dark on the potential -1100 V) saline. After proper membrane potential
RESULTS Electrical characteristics of defolliculated Xenopus oocytes The membrane potential of isolated oocytes left overnight in the incubation medium was -63 mV (SD 16 mV, 17 determinations) as measured from the maximum deflexion obtained when the first electrode (usually KCl) penetrated the cell (i.e. in the hypotonic solution). No significant variation was seen when the medium was changed to the normal or the hypertonic solution. The mean value for the
68
R. Bell& et al.
apparent membrane resistance was 2.6 MOhms (SD 1.4 MOhms, 8 determinations). As has been described for Rana pipiens oocytes (Morrill et al., 1966) Xenopus oocytes membrane potential is sensitive to external Ca2’ ions. Perfusion of the chamber with a medium containing 10 mM EGTA decreased the membrane potential to a low value in a few minutes (for example from -76 to -1.5 mV). Perfusion with the control medium slowly restored the membrane potential. In other experiments addition of CaCl, to 10 mM resulted in a similar reversible depolarization whereas MgCl, (10 mM) had no effect. During Ca2+ perfusion the membrane resistance dropped about lo-fold. Injection ofaequorin Before injecting aequorin, a background light signal was recorded coming from the aequorin pipette itself plus the dark current of the photomultiplier. In 3 exper-
A 10.4 nA
-.....~
10 J-1omv
7-----
J
!A
8
1nA
i
0 1-2ordJ
Fig. 1. Effect of perfusion of the ionophore A 23187 (A) and electrophoretic Ca2+ pulses (B) on light emission (top traces) and membrane potential (lower traces) of an aequorin injected Xenopus oocyte. The ionophore in the presence of high Ca2+ was perfused between the arrows; there is a time-lag of about 2 min due to the dead space of the perfusion system, as can be seen when normal Ca*+ solution without ionophore was perfused (t). The broken line corresponds to a 3 min interruption of the record during which the electrophoretic Ca2+ pulse shown in B2 was applied. Ca2+ inJections Bl and B3 were made respectively before and after the record shown in A (injection parameters: 500 ms, 25 nA). The origin of the fast transients in the A light record is unknown (time constant of the light recording system: 110 ms).
Free Ca*+ in Xenopus oocyte
69
iments injections were accompanied by a slight decrease of the PM current while no change was detected in the others. Then the Ca*+ concentration inside the oocyte was equal to or less than in the pipette. Electrophoretically injected Ca*+ pulses produced changes in light emission (fig. 1B). After the end of the pulse, return to the base line occurred with a half decay time of about 0.6-2 s. Responses to Ca*+ pulses were seen for several hours without further injection of aequorin. These observations suggest that the internal Ca*+ concentration is well buffered and, in all likelihood, similar to that found in Molluscan neurons (i.e. less than lop7 M). The amplitude of the light transients was measured for different Ca*+ currents and duration (from 10 nC to 200 nC) and was found to vary linearly with the electrophoretic current as it has been observed in the squid axon (Kusano et al., 1975b). In control injected oocytes the light decreased slowly to a very low level. It is not clear whether this is due to the PM and/or the aequorin pipette or to the cell itself. No changes were observed in light emission during EGTA (10 mM), CaC12 (10 mM) or MgC12 (10 mM) perfusion of the chamber. Effect
of the
ionophore A 2318 7
The ionophore A 23 187 (2.5 pg/ml) had no effect when perfused in normal Ca*+ buffer. Raising CaCl, to 10 mM in the presence of the ionophore resulted in a progressive increase in light emission (fig. 1A). Washing with normal Ca*+ medium restored base line in a few minutes. No change was observed when MgCl, was raised to 10 mM in the presence of the ionophore and normal Ca*+ concentration. Effect of progesterone No changes in light emission were observed from the initial progesterone (lop6 M) stimulation until germinal vesicle breakdown. During the same period electrophoretic Ca2+ pulses gave light responses indicating that exhaustion of aequorin was not responsible for the lack of detectable [Ca*+]t change. The same result was obtained when aequorin was injected near the margin of the pigment. The membrane potential and membrane resistance did not show any significant variation during the 2-4 h following progesterone application (fig. 2). After this quiet period, the membrane potential dropped and stabilized at about -16 mV (SD 2.5 mV, 5 determinations), this change taking place during approximately 1 h. At about the time the membrane depolarized, the apparent membrane resistance showed a transitory increase (2-S-fold) and later decreased to a value which was beneath the initial one (see fig. 2). After these changes were observed, the incidence of germinal vesicle breakdown was’determined as judged from the presence of a white spot on the center of the pigmented hemisphere. Three experiments were performed using p-chloromercuribenzoate (10e4 M) instead of progesterone. Again no significant changes occurred in the light emission
70
R. Belli et al.
Fig. 2. Time course of membrane potential (continuous line) and resistance changes (broken line) of a Xenoppus oocyte following stimulation with progesterone (lo@ M).
although potential one did occur.
and resistance
changes similar to those observed with progester-
DISCUSSION Xeno~~s irrevis oocyte maturation is induced by several compounds: by progesterone which is the natural stimulus (Smith, 1976) by p-chloromercuribenzoate (Bachet et al., 1975) and by the ionophore A 23187 in the presence of raised Ca2+ (Wasserman and Masui, 1975). The two latter substances are known to modify Ca2+ fluxes across the oocyte membrane (Wasserman and Masui, 1975; Marot et al., 1976). it was then postulated that progesterone acts through an intraceIluIar change of free Ca2+ concentration, for Ca2+ influx and efflux are not modified by the hormone (Marot et al., 1976). Using aequorin we have observed that the ionophore A 23187 is indeed able to increase free internal Ca2+ provided the external concentration of this ion is raised. But no change of free Ca *+ has been detected during either progesterone or p-chloromercuribenzoate treatment. Unfortunately it is impossible it is probably less to give a precise figure for the free Ca2+ CoIlcentration (~thou~ than IOe6 M) and for the minimal change which can be detected. A calibration is rather difficult to establish and will not be curve (lightfCa’+ concentration) useful due to the strong absorption given by the yolk and the pigment. From the length of the tip of the Ca2+ electrode entering the cell and the radial diffusion of Ca2’, it can be assumed that the light emitted in response to Ca2+ pulses originates from the first tens of a micrometer of cytoplasm under the membrane. Therefore it is likely that the change in fCa2’] i observed in the presence of A 23 I87 and high Ca2+ occurs in this layer. The absence of light response during progesterone or p-chloromercuribenzoate action might be interpreted as an indication that an increase of free Ca 2*, if any, is not taking p lace in the vicinity of the oocyte membrane during the maturation induced by these substances. However, it remains possible that such a Ca2+ change is too small to be detected by our method or it occurs
Free Ca2+ in Xenopus oocyte
71
in another compartment than the aequorin diffusion compartment. in that part of the The regulatory mechanisms of the free Ca2+ concentration cell which is accessible to aequorin examination appears to be very efficient, for the resting light emission was rapidly restored following Ca2+ injections or washing out the ionophore and the excess external Ca 2+. On the other hand, it has been observed that CaZf influx is increased either by A 23187 in normal Ca2+ (i.e. 0.74 mM) or by raised Ca2+ (10 mM) (R. Belle, J. Marot and R. Ozon, unpublished data); since no change of emission is seen in these conditions, the cells should be able to get rid of this influx and maintain the resting Ca2+ concentration. When the influx is increased by applying simultaneously the ionophore and raised Ca2+, [Ca2+]i starts to increase and in some way can trigger the maturation. Progesterone as well as p-chloromercuribenzoate modifies plasma membrane electrical characteristics as has already been observed (Moreau et al., 1976). But in contrast with these previous results, we observed a delay before these modifications occur; also the increase in membrane resistance is only transitory, the value reached after the germinal vesicle breakdown being lower than the initial value. The origin of these discrepancies is unknown. In fact, oocyte maturation can be obtained in different chemical environments where membrane potential and membrane resistance are quite different - for example, in the presence of EGTA (Marot et al., 1976). Therefore, modifications of these parameters induced by progesterone or p-chloromercuribenzoate treatment reflect internal modifications (which cannot be related to detectable Ca2+ movements) but do not seem to play a causative role in the initiation of this phenomenon as has been previously suggested (WaIlace and Steinhardt, 1975). In conclusion, our results have shown that in the experimental conditions where oocyte maturation is obtained by the ionophore A 23187 in the presence of high Ca2+, there is a detectable increase of internal free Ca2+ concentration. This increase may be sufficient for the induction of maturation, but may not be necessary since progesterone which is the natural stimulus does not provoke the same effect, unless it occurs in a place where it cannot be detected by our method.
ACKNOWLEDGEMENTS We are indebted of aequorin.
to Professor 0. Shimomura
(Princeton
University)
for the gift
REFERENCES Baker, P.F. and Warner, A.E. (1972) J. Cell Biol. 53,579. Brachet, J., Baltus, E., De Schutter-Pays, A., Hanocq-Quertier, (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1574.
J., Hubert,
E. and Steiner&
G.
72
R. Belle! et al.
Ecker, R.E. and Smith, L.D. (1971) J. Cell. Physiol. 77,61. Kusano, K., Miledi, R. and Stinnakre, J. (1975a) Proc. R. Sot. London Ser. B 189, 39. Kusano, K., Miledi, R. and Stinnakre, J. (1975b) Proc. R. Sot. London Ser. B 189,49. Marot, J., Belle, R. and Ozon, R. (1976) C.R. Acad. Sci. Paris 282, 1301. Merriam, R.W. (1971a) Exp. Cell Res. 68,75. Merriam, R.W. (1971b) Exp. Cell Res. 68, 81. Moreau, M., Guerrier, P. and Dorce, M. (1976) C.R. Acad. Sci. Paris 282, 1309. Morrill, G.A., Rosenthal, J. and Watson, D.E. (1966). J. Cell. Physiol. 67, 375. Schorderet-Slatkine, S. and Schorderet, M. (1976) C.R. Acad. Sci. Paris 282, 1733. Shimomura, O., Johnson, F.H. and Saiga, Y. (1962) J. Cell. Comp. Physiol. 59, 223. Smith, L.D. (1976) In: The Biochemistry of Animal Development. Ed.: R. Weber (Academic Press, New York) Vol. 3, p. 1. Stinnakre, J. and Taut, L. (1973) Nature (London), New Biol. 242, 113. Stinnakre, J. and Taut, L. (1974) C.R. Acad. Sci. Paris 278, 1409. Wallace, R.A. and Steinhardt, R.A. (1975) J. Cell Biol. 67,443a. Wasserman, W.J. and Masui, Y. (1975) J. Exp. Zool. 193, 369. Wasserman, W.J. and Masui, Y. (1976) Science 191,1266.