Biol Cell (1991) 72, 269-273 © Elsevier, Paris
269
Original article
Effect o f maitotoxin on sea urchin egg fertilization and on Ca 2÷ permeabilities o f eggs and intracellular stores* Danielle Pesando I Jean-Pierre Girard 2-, Monique Durand-Cl6ment ~, Patrick Payan 2 Simone Puiseux-Dao ~ ] Unit~ 303 INSERM, BP 3, 06230 Viilefranche-sur-Mer; 2Laboratoire de Physiologic Cellulaire et Compar~e, UA 651 CNRS, Facultd des Sciences, 06034 Nice, France (Received I I February 1991; accepted 8 July 1991) Summary - Maitotoxin (MTX), a potent marine toxin involved in ciguatera poisoning, inhibited sea urchin egg fertilization in a dose-dependent manner with an ICs0 of 7.5 x 10- 3 MU (mouse-unit)/ml. It did not affect male gametes fertilizing capabilities but provoked exocytosis in female gametes. It induced a K + loss simultaneously with a Na + entry into unfertilized eggs and increased the Ca 2+ influx at higher concentrations. On isolated cortex preparations, high concentrations of MTX reduced the rate of ATPdependent Ca2+ accumulation into reticulum compartments and caused a leakage of Ca '+ from a preparation pre-loaded with 45Ca2+. Verapamil (10 -4 M) similarly blocked the increase of egg permeability to Ca2+ and the effect on Ca2+ sequestering into intracellu. lar compartment, induced by MTX. Ion transport perturbations which evolved relatively slowly are probably not the direct cause of fertilization inhibition which could be related to a modification of the plasma membrane of the female gametes by this hydrophilic toxin. maitotoxin I sea urchin egg fertilization I Ca2÷
Introduction
Maitotoxin (MTX), a non-peptidic marine toxin, is one of the compounds involved in ciguatera seafood poisoning, a human disease caused by the ingestion of fish inhabiting or feeding on coral reefs [1, 36]. The toxin which is highly lethal to mice [34], was found to be synthetized by the epiphytic dinoflagellate Gambierdiscus toxicus [2]. MTX is extensively used as a pharmaceutical tool to study biological processes triggered by Ca 2+. Some chemical features of the toxin were elucidated by Yokoyama et al [38]: the molecular weight of MTX (disodium salt) was determined to be 3 424.5 + 0.5 Da and the presence of two sulfate ester groups was detected. Maitotoxin appears to stimulate a large spectrum of calcium-dependent physiological processes; it is a potent activator of calcium infux in a large variety of cells. It increases Ca 2+ influx into aortic smooth muscle cells [4, 13], in cardiac cells [18, 27], parathyroid cells [8], liver cells [19] and synaptosomes [31]. In all cells studied MTXinduced Ca 2+ influx is partially or completely abolished by both inorganic calcium channel blockers (manganese, cobalt) and by verapamil [9, 21, 32]. Stimulation of Ca 2+ uptake by MTX has been mainly attributed to an activation of Ca 2+ voltage-dependent channels [21]. However, MTX action has been linked to other mechanisms including influx of Na + [26], and pore forming or channel opening activity [29, 39]. MTX also stimulates phospholipid breakdown in a large variety of cells [4, 13], including Xenopus oocytes [3]. In a preliminary study, we showed that MTX could modify the growth of unicellular marine algae (Diatoms, * In memoriam tO Daniel Sandoz ** Correspondence and reprints
Volvocales and DinoflageUates) and could inhibit the fertilization and influence cell division of sea urchin eggs (Paracentrotus Iividus) [6]. Sea urchin eggs have been recommended as material for routine bioassay by several workers [5, 10, 15, 16, 22, 35]. The aim of the present work was to investigate the action of maitotoxin on sea urchin egg fertilization in relation to egg and non-mitochondrial intracellular compartment permeability to Ca 2+. In our experiments, fertilization was inhibited in a dosedependent manner by MTX. The toxin did not affect the male gamete fertilization capabilities but was able to induce exocytosis in the unfertilized egg. The toxin increased the permeability to Ca 2+ of both plasma and intraceUular membranes and modified K + and Na + distribution in the female gametes. The toxin induced ion permeabilities were observed at concentrations much higher than those inhibiting fertilization and did not evolve rapidly. Thus the fertilization block may not be related to ion permeability perturbations caused by MTX. A modification of the unfertilized egg plasma membrane by this hydrophilic toxin is more likely. Material and m e t h o d s
Biological material Eggs and sperm were collected from the gonads by dissecting Paracenlrotus lividus. Sperm was kept dry at 4°C. The ovaria were suspended in artificial sea water (ASW, Tropen Meersalz). Eggs were rinsed and filtered through a mesh five time to remove the jelly coat. Eggs were diluted at a concentration of 4% by volume in ASW (approx 2 × 104 eggs per ml) and maintained in suspension at 20°C by gentle stirring with a three blade propeller. Fertilization was obtained by adding 20 ~1 of diluted sperm (X50, in ASW) to 1 ml of egg suspension.
270
D Pesando et at
Preparation and quantification o f maitotoxin Maitotoxin was obtained from cultures of the benthic dinoflagellate Gambierdiscus toxicus [7]. Extraction was performed as described by Berta et al [4]. Briefly, cells were harvested by centrifugation, extracted with methanol and the residue was resuspended in water and extracted with diisopropylether. The aqueous phase was extracted with butanol and the butanol phase was dried. Purification was performed using high performance liquid chromatography on a reverse phase column (Cyano, Waters 0.4 × 30) eluted with a gradient of methanol-water. The toxic fraction was dried, resuspended in methanol as a stock solution and frozen. Toxicity was evaluated using the mouse bioassay. Serially diluted fractions were dried under a nitrogen stream and emulsified in 0.5% Tween 60 solution. Each fraction was injected intraperitoneally into two mice (19-21 g, Swiss, female). Toxicity was expressed in MLD (minimal lethal dose capable of killing the two mice within 24 h, expressed in mg/kg) [2]. In these experiments, the concentration of maitotoxin (MTX) is expressed as MU/ml (one mouse-unit (MU) equals MLD: 50).
solution resembling intracellular medium: KGM [25]. This preparation was used to measure ATP-dependent uptake of 45Ca2+ into the non-mitochondrial store evaluated by the difference between 4SCa2+ accumulated with or without 2 mM Mg-ATP, in the presence or absence of various concentrations of MTX. Free Ca 2+ concentration in the labelling medium was 0.5 tiM. To study Ca 2+ release, cortices previously loaded with 45Ca2+ for 30 min were quickly rinsed and exposed to KGM, from which, at appropriate times, samples were taken and counted [25].
Results Effect o f M T X on gametes and sea urchin egg fertilization Prior to our experiments we assessed whether MTX could affect fertilization by inhibiting sperm activity. Sperm activated by dilution in sea water a n d then treated for 5 min with 5 M U / m l MTX, was still able to fertilize eggs.
Meosurement of egg fertilization The extent of fertilization was determined by counting the number of eggs showing a complete elevation of the fertilization envelope when examined under the light microscope. The relationship between the MTX treatment duration just before insemination and the rate of fertilization was determined by adding MTX (15 × 10-3 MU/ml final concentration) at different times before sperm addition. Experiments where membrane elevation was below 90% for controls were rejected. Eggs from the same female were used simultaneously to compare the effects of various MTX concentrations with a control batch in which only ethanol was added, The maximal ethanol concentration of 1% used in the experiments did not affect cell fertilization. Male and female gametes were treated by high concentrations of MTX (5-10 MU/ml) at varying times. The ability of the sperm to fertilize the eggs was observed by the examination of the fertilization envelope of the eggs in contact with the treated sperm. The unfertilized eggs were observed with light microscopy after different MTX treatment durations to examine the cortical exocytosis induced by the toxin.
;E • • :.E
60
!i.0__ g
i
o
2'o
1'o MTX
(MUIml
10-3)
100
Ca~÷ uptake by eggs, Na + and K + contents o f eggs Uptake of Ca =+ by the eggs was studied according to the pulselabelling experiments adapted from Biyiti et al [5]. Experiments were carried out by incubating unfertilized eggs, previously attached to polylysine-coatedFalcon dishes, during 20 min in I ml of ASW containing 1 t~Ci of 4SCa2+ (Commissariat/~ l'Energie Atomique CEN, Saclay). After rinsing, the eggs were removed from the dishes [5] for determination of protein content and radioactivity; Ca2+ uptake was expressed as nmol per mg of protein. Labelling was performed in the absence or presence of various concentrations of MTX. Treatment with verapamil (10 -4 M) started 10 min before radioactive labelling in the presence of MTX. Eggs were filtered following the technique of Payan et al [24], which allows a rapid elimination of the external medium with less than !% error. The Na ÷ and K + contents were determined after homogenization, by flame photometry (Eppendor0 and were expressed as/~ eq per mg of protein. Caz÷ uptake and release by isolated cortices The isolated cortices, consisting of a preparation of plasma membrane with associated cortical organelles, mainly endoplasmic reticulum, were obtained by attaching eggs to polylysine-coated dishes and shearing away the bulk of the egg with a stream of
~g
:! ~t
B=
Ji
1i
m
i
time (rain)
Fig 1. A. Effect of increasing concentration of MTX on fertilization of sea urchin eggs. Toxin was added 2 min (o) and 10 min (o) prior to insemination. Fertilization rate was determined by counting the eggs with an elevated fertilization membrane. Values are the means of three experiments. B. Relationship between the length of MTX treatment (15 min × 10 -3 MU/ml) prior to insemination and the rate of sea urchin egg fertilization.
271
Effect o f maitotoxin on sea urchin egg
Addition of MTX to a sea urchin egg suspension before insemination inhibited fertilization in a dosedependent manner. When eggs were incubated in the presence of the toxin for 2 or 10 min before sperm addition, separate dose-response curves were found with those exposure times, but the ICs0 remained in the same order of magnitude (fig IA). As shown in figure IB, the degree of inhibition was dependent on the time of egg incubation in the presence of toxin. Thus, the MTX concentration of 15 × 10 -3 MU/mi, which totally inhibited fertilization in the first experiment (fig IA), must be present for at least 1 min before sperm addition to give a 50% inhibition (fig IB). The treatment of unfertilized eggs with high concentrations of MTX (5 to 10 MU/ml) provoked an elevation of the fertilization envelope (exocytosis), within 30 min.
Those effects of MTX on intracellular calcium stores (fig 3) occurred in the same range of concentrations as those observed for Ca 2+ influx of eggs (_> 1 MU/ml). On isolated cortices pre-loaded with 4SCa2+, MTX (5 MU/ml) enhanced without delay the rate of Ca 2+ discharge releasing 100% of the sequestered Ca 2+ within 15 min after drug addition (fig 4).
Maitotoxin increased Ca2+ permeability o f egg membranes
|
Unfertilized eggs maintain their cytosolic free calcium at a low level [30] when compared to the mM Ca z+ concentration in sea water or in intracellular stores where Ca z + is sequestered by energy
269
Original article
Effect o f maitotoxin on sea urchin egg fertilization and on Ca 2÷ permeabilities o f eggs and intracellular stores* Danielle Pesando I Jean-Pierre Girard 2-, Monique Durand-Cl6ment ~, Patrick Payan 2 Simone Puiseux-Dao ~ ] Unit~ 303 INSERM, BP 3, 06230 Viilefranche-sur-Mer; 2Laboratoire de Physiologic Cellulaire et Compar~e, UA 651 CNRS, Facultd des Sciences, 06034 Nice, France (Received I I February 1991; accepted 8 July 1991) Summary - Maitotoxin (MTX), a potent marine toxin involved in ciguatera poisoning, inhibited sea urchin egg fertilization in a dose-dependent manner with an ICs0 of 7.5 x 10- 3 MU (mouse-unit)/ml. It did not affect male gametes fertilizing capabilities but provoked exocytosis in female gametes. It induced a K + loss simultaneously with a Na + entry into unfertilized eggs and increased the Ca 2+ influx at higher concentrations. On isolated cortex preparations, high concentrations of MTX reduced the rate of ATPdependent Ca2+ accumulation into reticulum compartments and caused a leakage of Ca '+ from a preparation pre-loaded with 45Ca2+. Verapamil (10 -4 M) similarly blocked the increase of egg permeability to Ca2+ and the effect on Ca2+ sequestering into intracellu. lar compartment, induced by MTX. Ion transport perturbations which evolved relatively slowly are probably not the direct cause of fertilization inhibition which could be related to a modification of the plasma membrane of the female gametes by this hydrophilic toxin. maitotoxin I sea urchin egg fertilization I Ca2÷
Introduction
Maitotoxin (MTX), a non-peptidic marine toxin, is one of the compounds involved in ciguatera seafood poisoning, a human disease caused by the ingestion of fish inhabiting or feeding on coral reefs [1, 36]. The toxin which is highly lethal to mice [34], was found to be synthetized by the epiphytic dinoflagellate Gambierdiscus toxicus [2]. MTX is extensively used as a pharmaceutical tool to study biological processes triggered by Ca 2+. Some chemical features of the toxin were elucidated by Yokoyama et al [38]: the molecular weight of MTX (disodium salt) was determined to be 3 424.5 + 0.5 Da and the presence of two sulfate ester groups was detected. Maitotoxin appears to stimulate a large spectrum of calcium-dependent physiological processes; it is a potent activator of calcium infux in a large variety of cells. It increases Ca 2+ influx into aortic smooth muscle cells [4, 13], in cardiac cells [18, 27], parathyroid cells [8], liver cells [19] and synaptosomes [31]. In all cells studied MTXinduced Ca 2+ influx is partially or completely abolished by both inorganic calcium channel blockers (manganese, cobalt) and by verapamil [9, 21, 32]. Stimulation of Ca 2+ uptake by MTX has been mainly attributed to an activation of Ca 2+ voltage-dependent channels [21]. However, MTX action has been linked to other mechanisms including influx of Na + [26], and pore forming or channel opening activity [29, 39]. MTX also stimulates phospholipid breakdown in a large variety of cells [4, 13], including Xenopus oocytes [3]. In a preliminary study, we showed that MTX could modify the growth of unicellular marine algae (Diatoms, * In memoriam tO Daniel Sandoz ** Correspondence and reprints
Volvocales and DinoflageUates) and could inhibit the fertilization and influence cell division of sea urchin eggs (Paracentrotus Iividus) [6]. Sea urchin eggs have been recommended as material for routine bioassay by several workers [5, 10, 15, 16, 22, 35]. The aim of the present work was to investigate the action of maitotoxin on sea urchin egg fertilization in relation to egg and non-mitochondrial intracellular compartment permeability to Ca 2+. In our experiments, fertilization was inhibited in a dosedependent manner by MTX. The toxin did not affect the male gamete fertilization capabilities but was able to induce exocytosis in the unfertilized egg. The toxin increased the permeability to Ca 2+ of both plasma and intraceUular membranes and modified K + and Na + distribution in the female gametes. The toxin induced ion permeabilities were observed at concentrations much higher than those inhibiting fertilization and did not evolve rapidly. Thus the fertilization block may not be related to ion permeability perturbations caused by MTX. A modification of the unfertilized egg plasma membrane by this hydrophilic toxin is more likely. Material and m e t h o d s
Biological material Eggs and sperm were collected from the gonads by dissecting Paracenlrotus lividus. Sperm was kept dry at 4°C. The ovaria were suspended in artificial sea water (ASW, Tropen Meersalz). Eggs were rinsed and filtered through a mesh five time to remove the jelly coat. Eggs were diluted at a concentration of 4% by volume in ASW (approx 2 × 104 eggs per ml) and maintained in suspension at 20°C by gentle stirring with a three blade propeller. Fertilization was obtained by adding 20 ~1 of diluted sperm (X50, in ASW) to 1 ml of egg suspension.
270
D Pesando et at
Preparation and quantification o f maitotoxin Maitotoxin was obtained from cultures of the benthic dinoflagellate Gambierdiscus toxicus [7]. Extraction was performed as described by Berta et al [4]. Briefly, cells were harvested by centrifugation, extracted with methanol and the residue was resuspended in water and extracted with diisopropylether. The aqueous phase was extracted with butanol and the butanol phase was dried. Purification was performed using high performance liquid chromatography on a reverse phase column (Cyano, Waters 0.4 × 30) eluted with a gradient of methanol-water. The toxic fraction was dried, resuspended in methanol as a stock solution and frozen. Toxicity was evaluated using the mouse bioassay. Serially diluted fractions were dried under a nitrogen stream and emulsified in 0.5% Tween 60 solution. Each fraction was injected intraperitoneally into two mice (19-21 g, Swiss, female). Toxicity was expressed in MLD (minimal lethal dose capable of killing the two mice within 24 h, expressed in mg/kg) [2]. In these experiments, the concentration of maitotoxin (MTX) is expressed as MU/ml (one mouse-unit (MU) equals MLD: 50).
solution resembling intracellular medium: KGM [25]. This preparation was used to measure ATP-dependent uptake of 45Ca2+ into the non-mitochondrial store evaluated by the difference between 4SCa2+ accumulated with or without 2 mM Mg-ATP, in the presence or absence of various concentrations of MTX. Free Ca 2+ concentration in the labelling medium was 0.5 tiM. To study Ca 2+ release, cortices previously loaded with 45Ca2+ for 30 min were quickly rinsed and exposed to KGM, from which, at appropriate times, samples were taken and counted [25].
Results Effect o f M T X on gametes and sea urchin egg fertilization Prior to our experiments we assessed whether MTX could affect fertilization by inhibiting sperm activity. Sperm activated by dilution in sea water a n d then treated for 5 min with 5 M U / m l MTX, was still able to fertilize eggs.
Meosurement of egg fertilization The extent of fertilization was determined by counting the number of eggs showing a complete elevation of the fertilization envelope when examined under the light microscope. The relationship between the MTX treatment duration just before insemination and the rate of fertilization was determined by adding MTX (15 × 10-3 MU/ml final concentration) at different times before sperm addition. Experiments where membrane elevation was below 90% for controls were rejected. Eggs from the same female were used simultaneously to compare the effects of various MTX concentrations with a control batch in which only ethanol was added, The maximal ethanol concentration of 1% used in the experiments did not affect cell fertilization. Male and female gametes were treated by high concentrations of MTX (5-10 MU/ml) at varying times. The ability of the sperm to fertilize the eggs was observed by the examination of the fertilization envelope of the eggs in contact with the treated sperm. The unfertilized eggs were observed with light microscopy after different MTX treatment durations to examine the cortical exocytosis induced by the toxin.
;E • • :.E
60
!i.0__ g
i
o
2'o
1'o MTX
(MUIml
10-3)
100
Ca~÷ uptake by eggs, Na + and K + contents o f eggs Uptake of Ca =+ by the eggs was studied according to the pulselabelling experiments adapted from Biyiti et al [5]. Experiments were carried out by incubating unfertilized eggs, previously attached to polylysine-coatedFalcon dishes, during 20 min in I ml of ASW containing 1 t~Ci of 4SCa2+ (Commissariat/~ l'Energie Atomique CEN, Saclay). After rinsing, the eggs were removed from the dishes [5] for determination of protein content and radioactivity; Ca2+ uptake was expressed as nmol per mg of protein. Labelling was performed in the absence or presence of various concentrations of MTX. Treatment with verapamil (10 -4 M) started 10 min before radioactive labelling in the presence of MTX. Eggs were filtered following the technique of Payan et al [24], which allows a rapid elimination of the external medium with less than !% error. The Na ÷ and K + contents were determined after homogenization, by flame photometry (Eppendor0 and were expressed as/~ eq per mg of protein. Caz÷ uptake and release by isolated cortices The isolated cortices, consisting of a preparation of plasma membrane with associated cortical organelles, mainly endoplasmic reticulum, were obtained by attaching eggs to polylysine-coated dishes and shearing away the bulk of the egg with a stream of
~g
:! ~t
B=
Ji
1i
m
i
time (rain)
Fig 1. A. Effect of increasing concentration of MTX on fertilization of sea urchin eggs. Toxin was added 2 min (o) and 10 min (o) prior to insemination. Fertilization rate was determined by counting the eggs with an elevated fertilization membrane. Values are the means of three experiments. B. Relationship between the length of MTX treatment (15 min × 10 -3 MU/ml) prior to insemination and the rate of sea urchin egg fertilization.
271
Effect o f maitotoxin on sea urchin egg
Addition of MTX to a sea urchin egg suspension before insemination inhibited fertilization in a dosedependent manner. When eggs were incubated in the presence of the toxin for 2 or 10 min before sperm addition, separate dose-response curves were found with those exposure times, but the ICs0 remained in the same order of magnitude (fig IA). As shown in figure IB, the degree of inhibition was dependent on the time of egg incubation in the presence of toxin. Thus, the MTX concentration of 15 × 10 -3 MU/mi, which totally inhibited fertilization in the first experiment (fig IA), must be present for at least 1 min before sperm addition to give a 50% inhibition (fig IB). The treatment of unfertilized eggs with high concentrations of MTX (5 to 10 MU/ml) provoked an elevation of the fertilization envelope (exocytosis), within 30 min.
Those effects of MTX on intracellular calcium stores (fig 3) occurred in the same range of concentrations as those observed for Ca 2+ influx of eggs (_> 1 MU/ml). On isolated cortices pre-loaded with 4SCa2+, MTX (5 MU/ml) enhanced without delay the rate of Ca 2+ discharge releasing 100% of the sequestered Ca 2+ within 15 min after drug addition (fig 4).
Maitotoxin increased Ca2+ permeability o f egg membranes
|
Unfertilized eggs maintain their cytosolic free calcium at a low level [30] when compared to the mM Ca z+ concentration in sea water or in intracellular stores where Ca z + is sequestered by energy
