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Effect of diesel fuel hydrocarbons on embryogenesis and 45Ca2+ uptake by unfertilized eggs of sea urchin, Strongylocentrotus intermedius
Camp. Biochem. Physiot.
Vol. 105C,No. 3,
pp. 543-548,
1993 0
Printed in Great Britain
EFFECT OF DIESEL EMBRYOGENESIS UNFERTILIZED
FUEL AND EGGS
0306~4492/93 $6.00 + 0.00 1993 Pergamon Press Ltd
HYDROCARBONS ON 45Ca2+ UPTAKE BY OF SEA URCHIN,
STRONGYLOCENTROTUS
ZNTERMEDZUS
P. M. ZHADAN* and M. A. VASCHENKO~ *Laboratory of Physical-Chemical Ecology, Pacific Oceanological Institute; and fLaboratory of Cellular Physiology and Pharmacology of the Institute of Marine Biology, Far East Branch, Russian Academy of Sciences, Vladivostok, 690032 Russia (Fax 423-222-4552) (Received
4 December
1992; accepted for publication
20 January
1993)
Abstract-l. The quality of unfertilized eggs of the sea urchin Strongylocentrotus intermedius, kept for a long time (50 days) in a sea water containing water soluble hydrocarbons of diesel fuel in sublethal of embryogenesis and the intensity of concentrations (0.34.04 mg/l), was assessed through observation 45Ca2+ uptake. 2. It has been shown that such treatment led to delay and asynchronism of embryonal and larval development and to appearance of a greater number of abnormalities compared to the control. 3. Unfertilized eggs of sea urchins exposed to the hydrocarbons in sublethal concentrations accumulated 3&60% more 45Ca2C than those of control animals. Short-term incubation (2 hr) of eggs at the same hydrocarbon concentrations did not change 45Ca2+ uptake by unfertilized eggs of control animals. 4. The increase of hydrocarbon concentration up to 1 mg/l (i.e. to a concentration causing disturbance of embryogenesis in acute experiments) in short-term experiments caused a small elevation in the “‘Ca2+ uptake by unfertilized eggs of control animals (30% more than in untreated eggs). 5. Ionomycin-induced (concentration 10-8P10-9M) increase of 45Ca2+ uptake by unfertilized eggs (SCrlOO% more than the untreated eggs) caused the same disturbance of embryogenesis as under hydrocarbon exposure. 6. It is suggested that one of the mechanisms inducing the deleterious effect of hydrocarbons in sea urchin gametes is related to the increase of membrane permeability to calcium ions.
INTRODUCTION
Previously, it has been established that a long-term (during gametogenesis) exposure of sea urchins to water soluble hydrocarbons of diesel fuel in sublethal
concentrations yields low-quality eggs (Vaschenko, 1980, 1985). Subsequent development of such eggs in an unpolluted sea water is characterized by a decrease in the rate of fertilization and cleavage, by asynchronous and delayed development, and also by the appearance of numerous abnormalities (Vaschenko et al., 1988; Vaschenko and Naidenko, 1989). Exactly how hydrocarbons affect eggs is still unknown. Previously, it was suggested, that deleterious effects of hydrocarbons might be related to changes in cell membrane permeability (Van Overbeek and Blondeau, 1954; Rowlands et al., 1977; Reddin and Prendeville, 1981; Balantine et al., 1983) although at that time the question was not clearly understood. Taking into consideration the essential role of 4JCa2+ in the control of gametogenesis and early embryogenesis (Whitaker and Steinhardt, 1982; Swann and Whitaker, 1990) we may assume that a change in the membrane permeability to calcium ions is one of the factors causing disturbances in
intracellular regulatory processes. Increase of the intracellular concentration of free calcium due to fertilization triggers a number of important processes, such as cortical reaction (Epel, 1978; Eisen et al., 1984; Iwamatsu et al., 1985), rise in mitochondrial respiration (Steinhardt and Epel, 1974), cytoplasmic pH (Shen and Steinhardt, 1978), and activation of some enzymes, NAD-kinase, proteinkinase, ATP-ase (Petzelt, 1972; Epel et al., 1981; Ribon et al., 1984). The role of calcium ions in mitotic regulation is surely significant (Petzelt, 1972; Petzelt and Wulroth, 1984; Poenie et al., 1985; Steinhardt and Alderton, 1988). Since many important processes in early embryogenesis are calcium-dependent, disturbances in the transport of these ions through cell membranes might be one of the factors responsible for developmental abnormalities. In our work we introduce some data evidencing to the fact that the long-term effect of low concentrations of hydrocarbons (0.040.3 mg/l) on parent specimens of sea urchin S. intermedius and the short-term influence of hydrocarbon higher concentrations (about 1 mg/l) on unfertilized eggs of untreated animals provoke the rise in 45Ca2+ uptake and the embryogenesis violations.
P. M. ZHADANand M. A.
544
MATERIALS AND Experiment
METHODS
condition
Sea urchins were collected on 1 August 1986, in Peter the Great Bay (Sea of Japan) and then were incubated for 50 days in aerated 100 1 aquaria with non-flowing water. The control group was kept in clean sea water, and the experimental one, in sea water containing a water soluble fraction of diesel fuel (WSFDF). Water was changed daily; the animals were fed on Laminariu. The water temperature in the aquaria fell gradually from 21 “C in August to 165°C by the end of September. Eggs were obtained using electrostimulation immediately before the experiments. Then eggs were washed twice with filtered (pore diameter 0.23 pm) sea water and put into a refrigerator for l-2 hr at 4°C. Preparation
of WSFDF
Light diesel fuel (type L) was used for the experiment. For the chronic experiment it was mixed with sea water (1:9) in the 20 1 vessel and left to infuse for 24 hr. WSFDF was separated from under a pellicled layer of water and diluted 20-fold in an experimental aquarium. Thereafter, to the vessel with diesel fuel sea water was added up to the initial level. Oil product in the vessel was changed weekly. The hydrocarbon concentration in the original WSFDF was determined using IK-spectrophotometry (Nesterova and Nemirovskaya, 1978). During a week the concentration of hydrocarbons in the original WSFDF and in the experimental aquarium fell from 5.2 to 0.8 mg/l and from 0.3 to 0.4 mg/l, respectively. For a short-term incubation of eggs with WSFDF a mixture of diesel fuel with filtered sea water (1 :9) was gently stirred with a magnetic stirrer during 2 hr. The hydrocarbon concentration in the resulting solution was about 1 mg/l. Embryological
analysis
The development of embryos in the chronic experiments proceeded in clean natural sea water using a standard technique (Buznikov and Podmarev, 1975). Development of the offsprings from five parental pairs (in two parallels for each pair) both of control and experimental groups was run down from zygote to 4-armed pluteus. Unfertilized sea urchin eggs (obtained from untreated animals) in acute experiments were prelimmary incubated in the media with WSFDF at concentration 1 mg/l for 120 min or with ionomycin (10-6-10-‘o M) for 30 min at temperature 19-20°C. The density of egg suspension in all embryological experiments was about 200 cell/ml. Then eggs were fertilized by sea urchin sperm (final dilution of “dry” sperm was 40,000 times). The number of normal embryos and larvae was calculated at the stages of fertilization (within 5 min of insemination), first cleavage (within 90min of the same process), blastula (within 8 hr), gastrula (within 22 hr) and
VASCHENKO
4-armed pluteus method described Determination
(within 72 hr) according by Kobayashi (1971).
to
of 45Ca2+ uptake in unfertilized
the
eggs
A mixture of equal batches of eggs from 4-5 females both of control and experimental groups was used for the experiments. Three equal portions of gametes were picked out from each mixture and subjected synchronously to all required operations. The eggs (cell density about 4000egg/ml) were incubated in clean sea water in the presence of 20 p Ci/ml 45Ca2f. Three 100 p 1 suspension aliquotes were taken up within 5, 15, 30 and 90min of incubation Eggs were rinsed out three times with ice cold sea water, resedimented by means of hand centrifuge and then homogenized in 200 ~1 of 50 mM Tris-HCI buffer (pH 7.5). The homogenate was dissolved in a mixture of ZhS-1 scintillation liquid (Reachim, C.I.S.) and Triton X-100 (3: 1) and the 45Ca2+ radioactivity in eggs was determined by a scintillation counter. The 45Ca2f content of the eggs was counted per 1 mg of egg protein. The 100 ~1 suspension of eggs was analyzed for protein concentration by the modified method of Lowry et al. (Markwell et al., 1978) using bovine serum albumin as a standard. In the short-term experiments, the eggs from control animals were transfered to sea water containing 45Ca2+(20 pCi/ml) and hydrocarbons (about 1 mg/ml) or ionomycin (10-6-10-9 M). The next procedure was as described above.
RESULTS
AND DISCUSSION
Chronic exposure of adult sea urchins S. intermedius to water soluble hydrocarbons of diesel fuel caused disturbances in embryogenesis and early larval development (Table 1). The number of fertilized eggs obtained from experimental sea urchins was reduced by 15% as compared to the control group. By the time of the first cleavage (90 min after fertilization) the number of two-cell embryos in the experimental group was three times less than in the control one while the number of noncleavaged zygotes was 60%. This indicates some delay in fertilization and cleavage. The delay and asynchronism in the development of experimental sea urchin progeny showed up vividly at the gastrula stage since the formation of larvae archenteron was inhibited. At all the developmental stages the number of abnormalities was found to be higher in the experimental sea urchin progeny. Previously, similar disturbances were observed in the embryonal and larval development of Strongylocentrotus nudus subjected to long-term effects of diesel fuel hydrocarbons (Vaschenko, 1980, 1985; Vaschenko et al., 1988). Experimental cross fertilization, ascertained by the early embriogenesis (before hatching of blastulae), seems to be much dependent on the quality of female sex cells.
s5Ca2+ uptake into hydrocarbon-treated
sea urchin eggs
545
Little attention was paid to calcium transport across the plasma membrane in sea urchin unfertilized eggs under normal conditions. In accordance with existing data, Ca*+ uptake by unfertilized eggs of the sea urchin Anthocidaris crassipina is close to zero and this process increases sharply after fertilization (Fujino et al., 1985). A similar result was obtained also from our experiments on peculiarities of 4sCa2+ uptake by unfertilized eggs of the sea urchin S. intermedius. The radioactivity of unfertilized eggs increased slowly through the whole incubation period (Fig. 1). The addition of sperm led to a dramatic increase in calcium uptake. It is well known that calcium channel antagonists, verapamil and its derivatives, 14-dihydropyridines and benzodiazepines, inhibit calcium uptake by fertilized eggs and embryos of sea urchin (Komukai et al., 1985; Chin, 1986). In our experiments a calcium channel antagonist D-600 at the concentration of 80 PM also inhibited 45Ca2C uptake in unfertilized eggs (Fig. 1). The decrease in calcium uptake 60 min after incubation was 30%. This shows that calcium uptake by eggs is accomplished at least partially through the calcium channel. The calcium uptake rate was strongly dependent on the temperature (Fig. 2). The optimal thermal range for development of S. intermedius is 17-21°C (Buznikov and Podmarev, 1975). 45Ca2+ uptake by unfertilized eggs at 21°C was 4-6 times than at 17°C implying participation of metabolic processes in the regulation of calcium uptake by unfertilized eggs. The temperature of 19-20°C was found to be optimal for our experiments. In that case the rate of calcium
e+r+---
90
Time, min
Fig. 1. The effects of D-600 and insemination on 45Ca2+ uptake by unfertilized eggs of the sea urchin S. intermedius. 1: normal 45Ca2+uptake; 2: upon incubation of eggs in the presence of 80 pm D-600; 3: 1min after sperru addition. The arrow shows the time of sperm addition. The temperature is 19°C. Each point is the mean of the values obtained from three experiments. Vertical range bars, SE. Abscissa: time of incubation, min; ordinate: radioactivity of 45Ca2+ in eggs, cpm/mg egg protein.
P. M. ZHADANand M. A. V&XENKO
546
T
P
6000 r
21
Time, min Fig. 2. The effect of temperature on %a2+ uptake by unfertilized eggs of the sea urchin S. intermedius. To the right of the curves is the temperature of incubation. The rest of the designations are the same as in Fig. 1.
uptake by eggs was sufficiently high and their quality did not worsen during incubation. Long-term exposure of parent sea urchins to hydrocarbons increased the rate of 45Ca2+ uptake by unfertilized eggs. The radioactivity of eggs obtained from experimental sea urchins 90min after incubation in the presence of 45Ca2+ was 25-60% higher than in the control ones (Fig. 3). Fall in the incubation temperature diminished, to some extent, to a relative increase in 4sCa2+ uptake by the eggs of experimental sea urchins as compared to control animals. At the same time, the total 45Ca2t uptake has been reduced several times (cf. Fig. 3a and 3b) both in experimental and control groups. In our previous works we discussed the fact that similar disturbances in embryonic and larval development resulted from both long-term treatment of the parent sea urchins with hydrocarbons at low concentrations and a short-term exposure of gametes to high concentration of hydrocarbons, more than 1 mg/l (Vaschenko, 1985; Vaschenko et al., 1988). As reported by a number of authors, the asynchronism and delay in embryonic and larval development of sea urchins were exhibited when eggs were treated with hydrocarbons at concentrations of l-10 mg/l and higher, depending on the sea urchin species and on oil product grade (Lonning and Hagstrom, 1975; Nicol et al., 1977; Falk-Peterson, 1978; Kobayashi, 1981). We performed several experiments on the short-term (2 hr) effect of hydrocarbons on 45Ca2+ uptake by unfertilized eggs from control animals. Experimental results showed that hydrocarbons in concentration, used for parent sea urchins exposure, did not affect 45Ca2+ uptake by eggs. It was not until concentration was increased to 1 mg/ml, producing a small rise in calcium uptake (by 30% for 90 min of incubation) by sea urchin eggs (Fig. 4).
5
15
5
15
60 30 Time, min
90
4ooc
2000
II
I
I
I
60 30 Time, min
I
90
Fig. 3. The effect of long-term exposure of parents sea urchins S. intermedius, to diesel fuel hydrocarbons (0.04-0.3 mg/l) on Ya2+ uptake by unfertilized eggs. 1: control; 2: experiment. A: incubation temperature of 17°C; B: incubation temperature of 19°C. The rest of the designations are the same as in Fig. 1.
Experimentally, the relation between the disturbances in embryonic development and 45Ca2f uptake by unfertilized egg cells was studied using ionomycin,
**r 2000-
50 Time, min Fig. 4. The effect of short-term exposure of sea urchin S. intermedius unfertilized eggs to diesel fuel hydrocarbons (the concentration 1 mg/l) on 45Ca2+ uptake. 1: control; 2: experiment. The incubation temperature = 17°C. The rest of the designations are the same as in Fig. I.
45Ca2+ uptake
into hydrocarbon-treated
% 3.0 0
“;t !: “0 ” 1 & f
2.5 -
2.0 -
1.5 -
*$ 2
LO10”s
103
10-s
lo-’
104
Ionomycin concentration, M Fig. 5. Relative increase of 45Ca2+ uptake into unfertilized sea urchin S. intermedius eggs during 30 min incubation with ionomycin. Abscissa: ionomycin concentration in incubation medium. mohl. Ordinate: relative increase of “Ca2+ uptake into urchin eggs.
calcium ionophore. The increase of calcium uptake by the eggs and the level of disturbances in embryonic development are strongly dependent on ionomycin concentration (Figs 5 and 6). Ionomycin at 10-6-10-7 M (3-4 fold elevation of calcium ingress) was heavily toxic and arrested the division of embryos. Ionomycin concentrations from lo-* to 10m9M increased 45Ca2+uptake into unfertilized eggs by 70-120% (Fig. 5). At such concentrations, the embryogenesis was accompanied by the disturbances similar to those caused by the effects of WSFDF in the chronical experiments, i.e. the delay in cleavage and in the formation of archenterone. Thus, the results of our investigation show that the diesel fuel hydrocarbons may induce an increase in the rate of 4sCa2+ uptake by unfertilized eggs in
sea urchin
eggs
547
chronic exposure (of parent specimens) as well as in acute exposure (of eggs) experiments. Prolonged exposure of the sea urchins to diesel fuel hydrocarbons at low concentrations exerted a stronger effect on 4sCa2+ deposition in eggs than a short-term incubation of eggs at high hydrocarbon concentrations. In both cases this fact might indicate the existence of various mechanisms responsible for the increase of 45Ca2+. In the case of a long-term exposure, the rise in membrane permeability of sea urchin eggs due to alteration in their biochemical composition might be the cause of the enhanced 45Ca2+uptake. Experimental data, although scarce, testifies in favour of supposition that the action of hydrocarbons bring about the damage to lipid metabolism, resulting in an increase in the amount of unsaturated fatty acids of gill phospholipids in crustaceans (Morris et al., 1982) and elevation of the level of cholesterol and other sterols in lobster embryos and larvae (Capuzzo et al., 1984). However, it is known that a rise in cholesterol concentration leads to an enhancement of membrane permeability to calcium ions (Lecher ef al., 1984). Elevation of the calcium uptake level caused by the breaks in calcium metabolism in the tissues of M. edulis, dwelling in sea water polluted with hydrocarbons and heavy metals was reported by Viarengo and co-authors (1988). The increased 4sCa2+ uptake by eggs during their short-term exposure to hydrocarbons at high concentrations might be stipulated by another mechanismthe rise in membrane permeability due to dissolution of hydrocarbons in membrane lipid phase. The scope of such an oil product effect was mentioned in studies of kerosene impact on calcium permeability of the sarcoplasmic reticulum membranes (Garcia and Gonzales, 1985). As to the relation between two phenomena caused by the hydrocarbon effects-the delay and asynchronism at the early embryonic and larval developmental stages and increased 45Ca2f uptake by the eggs-it should be emphasized that both effects showed up synchronously after prolonged exposure of sea urchin parental individuals to hydrocarbons at low concentrations and after a short-term treatment of unfertilized eggs with ionomycin and hydrocarbons at high concentrations. This allows us to conclude that one of the mechanisms responsible for deleterious effects of hydrocarbons on sea urchin female sex cells is related to changes in membrane permeability to calcium ions. REFERENCES
Ionomycin concentration, M Fig. 6. Inhibition of first clevage sea urchin eggs with ionomycin - Abscissa: concentration of . ionomycin, mol/l. . . __ Wrdmate: number of Z-cell embryos, %.
Ballantine J. A., Lavis A. and Morris R. J. (1983) A seasonal survey of the sterol composition of the cocle Garastoderma edule: a composition of polluted and non-polluted environments. Mar. Enuir. Res. 9, I I l-122. Buznikov G. A. and Podmarev V. K. (1975) Sea urchins (Srrogylocentrorus drobachiensis, S. nudus, S. inrermedius). In Objects of Developmental Biology (Edited by Astaurov B. L. and Detlaf T. A.), 188-216. Nauka, Moscow (In Russtan).
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ZUADAN
and M. A.
Capuzzo J. M., Lancaster B. A. and Sasaki G. C. (1984) The effects of petroleum hydrocarbons on lipid metabolism and energetics of larval development and metamorphosis in the American lobster (Homarus americanus Mime Edwards). Mar. Envir. Res. 14, 201-228. Chin J. H. (1986) Differential sensitivity of the calcium channel to dihydropyridines. The modulated receptor hypothesis. Biochem. Pharmac. 35, 4115-4120. Eisen D. P., Wieland S. J. and Reynolds G. T. (1984) Temporal sequence and spatial-distribution of early events of fertilization in single sea urchin eggs. J. Cell -Biol. 39, 1647-1654.
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Epel D. (1978) Mechanism of activation of sperm and eggs during fertilization of sea urchin gametes. Curr. T. Dev. 12, 185-286. Epel D., Patton C., Wallance R. W. and Cheung W. Y. (1981) Calcium activated NAD-kinase of sea urchin eggs. An early event of fertilization. Cell 23, 543-549. Falk-Peterson I. B. (1978) Toxic effects of aqueous extracts of Ekofisk crude oil, crude oil fractions and commercial products on the development of sea urchin eggs. Sursiu. 64, 161-170. Fujino Y., Mitsunaga K. and Fujiwara A. (1985) Inhibition of Ca2+ uptake in the eggs and embryos of the sea urchin. Anthocidaris crussispina, by several calcium antagonists, anion transport inhibitor, and chloride transport inhibitors. J. Exp. Zool. 235, 281-288. Garcia M. and Conzales R. (1985) Uncouoline of the sarcoplasmic reticulum by kerosene. Toxicol. Lft. 28, 59964.
Iwamatsu T., Ohta T., Oshima E. and Sugiura T. (1985) Requirement of extracellular calcium ions for the early fertilization events in the medaka egg. Devl. Growth Differ. 27, 751-762. Izant J. G. (1983) The role of calcium ions during mitosiscalcium participates in the anaphase trigger. Chromosoma 88, l-10. Kobayashi N. (1971) Fertilized sea urchin eggs as an indicatory material for marine pollution bioassay, preliminary experiments. Publ. Mar. Biol. Lab. 18, 3799406. Kobayashi N. (1981) Comparative toxicity of various chemicals, oil extracts and oil dispersant extracts to Canadian and Japanese sea urchin eggs. Publ. Seto Mar. Biol. Lab. 26, 123-133. Komukai M., Fujiwara A., Fijino Y. and Yasumase I. (1985) \ , The effects of several ions channel blockers and calmodulin antagonists on fertilization-induced acid release and Ca uptake in sea urchin eggs. Exp. Cell Res. 159, 463-472. Lecher R., Neyses L., Stimpel M., Kuffer B. and Vetter W. (1984) The cholesterol content of the human erythrocyte influences calcium influx through the channel. Biochem. biophys. Res. Commun. 124, 822-828.
Lonning S. and Hagstrom B. E. (1975) The effects of crude oils and the dispersant Corexit 8664 on sea urchin gametes and embryos. Norv. J. Zool. 23, 121-129. Markwell M. A. K., Haas S. M., Bieber L. L. and Tolbert N. E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analyt. Biochem. 87, 206-206.
Morris R. J., Lockwood A. (1982) Changes in the fatty phospholipids in Camarus contamination. Mar. Pollut.
P. M. and Dswson M. E. acid composition of the gill duebeni with degree of gill Bull. 13, 345-348.
VASCHENKO
Nesterova M. P. and Nemirovskaya I. A. (1978) Identification of oil-product in sea-water. In Methods of Hydrochemical Studies of the Ocean (Edited by Bordovsky 0. K., Ivanenkov V. I.), pp. 165-178. Nauka, Moscow (In Russian). Nicol J. A. C., Donahue W. H., Wang R. T. and Winters K. (1977) Chemical composition and effects of water extract of petroleum on eggs of the sand dollar Mel&a quinquiensperforata.
Mar. Biol. 40, 309-316.
Petzelt Ch. (1972) Ca-activated ATP-ase during the cell cycle of the sea urchin Slronnvlocentrotus .purpuratus. . &p.
Ceil. Res. 70, 3333339.
_.
Petzelt Ch. and Wulroth P. (1984) Cell cycle specific variations in transport capacity of an isolated Ca-transport system. Cell Biol. Intern. Rep. 8, 823-840. Poenie M., Alderton J., Tsien R. Y. and Steinhardt R. A. (1985) Changes of free calcium levels with stages of the cell division cycle. Nature 315, 147-149. Reddin A. and Prendeville G. V. (1981) Effects of oil cell membrane permeability in Fucus cerratus and Lnminaria digital. Mar. Pollut. Bull. 12, 339-342.
Ribon H. D., Eiseman E. A. and Kinsey W. H. (1984) Fertilization result in increased tyrosine phosphorylation of eggs proteins. J. biol. Chem. 259, 5333-5338. Rowlands J. R., Allen C. J. and Cause E. M. (1977) Effects of environmental agents on membrane dynamics. In Biochemical Effects of Environmental Pollutants (Edited by Lee S. D.), Ann. Arbor., Michigan, 203-246. Shen S. S. and Steinhardt R. A. (1978) Direct measurement of intracellular pH during metabolic depression of the sea urchin egg. Nature 272, 253-254. Steinhardt R. A. and Alderton J. (1988) Intracellular free calcium rise triggers nuclear envelope breakdown in the sea urchin embryo. Nature 332, 364366. Steinhardt R. A. and Epel D. (1974) Activation of sea urchin eggs by a calcium ionophore. Proc. Narl. Acud. Sci. U.S.A. 77, 1915-1919.
Swann K. and Whitaker M. T. (1990) Second messengers at fertilization in sea-urchin eggs. J. Reprod. Fert. 42, 141-154.
Van Overbeek J. and Blondeau R. (1954) Mode of action phytotoxic oils. Weeds 3, 33-65. Vaschenko M. A. (1980) The effects of water soluble hydrocarbons of diesel fuel on development of sex cells and quality of progeny of the sea urchin, Strongylocentrotus nudus. Marine Biology (C.I.S.) 4, 68-73 (In Russian). Vaschenko M. A. (1985) The effect of water soluble hydrocarbons of diesel fuel on gametogenesis of the sea urchin, Srrongylocenrrotus nudus, Cand. thesis. Vladivostok. (In Russian). Vaschenko M. A., Durkina V. B. and Gnezdilova S. M. (1988) A comparative characteristics of the effect of diesel fuel hydrocarbons and cadmium on the quality of progeny in sea urchins. Orthogenesis (C.I.S.) 19, 82288. (In Russian). Vaschenko M. A. and Naidenko T. Kh. (1989) The effect of long-term expozure of sea urchins to hydrocarbons on the development of their larvae. Marine Biology (C.I.S.) 1, 59-65. (In Russian). Viarengo A., Mancinel G., Martin0 G., Pertica M., Canesi L. and Mazzucot A. (1988) Integrated cellular stress indexes in trace-metal contamination - critical evaluation in a field study. Mar. Ecol. Prog. Ser. 46, 65570. Whitaker M. J. and Steinhardt R. A., (1982) Ionic regulation of egg activation. Q. Rev. Eiophys. 15, 593-666.
Vol. 105C,No. 3,
pp. 543-548,
1993 0
Printed in Great Britain
EFFECT OF DIESEL EMBRYOGENESIS UNFERTILIZED
FUEL AND EGGS
0306~4492/93 $6.00 + 0.00 1993 Pergamon Press Ltd
HYDROCARBONS ON 45Ca2+ UPTAKE BY OF SEA URCHIN,
STRONGYLOCENTROTUS
ZNTERMEDZUS
P. M. ZHADAN* and M. A. VASCHENKO~ *Laboratory of Physical-Chemical Ecology, Pacific Oceanological Institute; and fLaboratory of Cellular Physiology and Pharmacology of the Institute of Marine Biology, Far East Branch, Russian Academy of Sciences, Vladivostok, 690032 Russia (Fax 423-222-4552) (Received
4 December
1992; accepted for publication
20 January
1993)
Abstract-l. The quality of unfertilized eggs of the sea urchin Strongylocentrotus intermedius, kept for a long time (50 days) in a sea water containing water soluble hydrocarbons of diesel fuel in sublethal of embryogenesis and the intensity of concentrations (0.34.04 mg/l), was assessed through observation 45Ca2+ uptake. 2. It has been shown that such treatment led to delay and asynchronism of embryonal and larval development and to appearance of a greater number of abnormalities compared to the control. 3. Unfertilized eggs of sea urchins exposed to the hydrocarbons in sublethal concentrations accumulated 3&60% more 45Ca2C than those of control animals. Short-term incubation (2 hr) of eggs at the same hydrocarbon concentrations did not change 45Ca2+ uptake by unfertilized eggs of control animals. 4. The increase of hydrocarbon concentration up to 1 mg/l (i.e. to a concentration causing disturbance of embryogenesis in acute experiments) in short-term experiments caused a small elevation in the “‘Ca2+ uptake by unfertilized eggs of control animals (30% more than in untreated eggs). 5. Ionomycin-induced (concentration 10-8P10-9M) increase of 45Ca2+ uptake by unfertilized eggs (SCrlOO% more than the untreated eggs) caused the same disturbance of embryogenesis as under hydrocarbon exposure. 6. It is suggested that one of the mechanisms inducing the deleterious effect of hydrocarbons in sea urchin gametes is related to the increase of membrane permeability to calcium ions.
INTRODUCTION
Previously, it has been established that a long-term (during gametogenesis) exposure of sea urchins to water soluble hydrocarbons of diesel fuel in sublethal
concentrations yields low-quality eggs (Vaschenko, 1980, 1985). Subsequent development of such eggs in an unpolluted sea water is characterized by a decrease in the rate of fertilization and cleavage, by asynchronous and delayed development, and also by the appearance of numerous abnormalities (Vaschenko et al., 1988; Vaschenko and Naidenko, 1989). Exactly how hydrocarbons affect eggs is still unknown. Previously, it was suggested, that deleterious effects of hydrocarbons might be related to changes in cell membrane permeability (Van Overbeek and Blondeau, 1954; Rowlands et al., 1977; Reddin and Prendeville, 1981; Balantine et al., 1983) although at that time the question was not clearly understood. Taking into consideration the essential role of 4JCa2+ in the control of gametogenesis and early embryogenesis (Whitaker and Steinhardt, 1982; Swann and Whitaker, 1990) we may assume that a change in the membrane permeability to calcium ions is one of the factors causing disturbances in
intracellular regulatory processes. Increase of the intracellular concentration of free calcium due to fertilization triggers a number of important processes, such as cortical reaction (Epel, 1978; Eisen et al., 1984; Iwamatsu et al., 1985), rise in mitochondrial respiration (Steinhardt and Epel, 1974), cytoplasmic pH (Shen and Steinhardt, 1978), and activation of some enzymes, NAD-kinase, proteinkinase, ATP-ase (Petzelt, 1972; Epel et al., 1981; Ribon et al., 1984). The role of calcium ions in mitotic regulation is surely significant (Petzelt, 1972; Petzelt and Wulroth, 1984; Poenie et al., 1985; Steinhardt and Alderton, 1988). Since many important processes in early embryogenesis are calcium-dependent, disturbances in the transport of these ions through cell membranes might be one of the factors responsible for developmental abnormalities. In our work we introduce some data evidencing to the fact that the long-term effect of low concentrations of hydrocarbons (0.040.3 mg/l) on parent specimens of sea urchin S. intermedius and the short-term influence of hydrocarbon higher concentrations (about 1 mg/l) on unfertilized eggs of untreated animals provoke the rise in 45Ca2+ uptake and the embryogenesis violations.
P. M. ZHADANand M. A.
544
MATERIALS AND Experiment
METHODS
condition
Sea urchins were collected on 1 August 1986, in Peter the Great Bay (Sea of Japan) and then were incubated for 50 days in aerated 100 1 aquaria with non-flowing water. The control group was kept in clean sea water, and the experimental one, in sea water containing a water soluble fraction of diesel fuel (WSFDF). Water was changed daily; the animals were fed on Laminariu. The water temperature in the aquaria fell gradually from 21 “C in August to 165°C by the end of September. Eggs were obtained using electrostimulation immediately before the experiments. Then eggs were washed twice with filtered (pore diameter 0.23 pm) sea water and put into a refrigerator for l-2 hr at 4°C. Preparation
of WSFDF
Light diesel fuel (type L) was used for the experiment. For the chronic experiment it was mixed with sea water (1:9) in the 20 1 vessel and left to infuse for 24 hr. WSFDF was separated from under a pellicled layer of water and diluted 20-fold in an experimental aquarium. Thereafter, to the vessel with diesel fuel sea water was added up to the initial level. Oil product in the vessel was changed weekly. The hydrocarbon concentration in the original WSFDF was determined using IK-spectrophotometry (Nesterova and Nemirovskaya, 1978). During a week the concentration of hydrocarbons in the original WSFDF and in the experimental aquarium fell from 5.2 to 0.8 mg/l and from 0.3 to 0.4 mg/l, respectively. For a short-term incubation of eggs with WSFDF a mixture of diesel fuel with filtered sea water (1 :9) was gently stirred with a magnetic stirrer during 2 hr. The hydrocarbon concentration in the resulting solution was about 1 mg/l. Embryological
analysis
The development of embryos in the chronic experiments proceeded in clean natural sea water using a standard technique (Buznikov and Podmarev, 1975). Development of the offsprings from five parental pairs (in two parallels for each pair) both of control and experimental groups was run down from zygote to 4-armed pluteus. Unfertilized sea urchin eggs (obtained from untreated animals) in acute experiments were prelimmary incubated in the media with WSFDF at concentration 1 mg/l for 120 min or with ionomycin (10-6-10-‘o M) for 30 min at temperature 19-20°C. The density of egg suspension in all embryological experiments was about 200 cell/ml. Then eggs were fertilized by sea urchin sperm (final dilution of “dry” sperm was 40,000 times). The number of normal embryos and larvae was calculated at the stages of fertilization (within 5 min of insemination), first cleavage (within 90min of the same process), blastula (within 8 hr), gastrula (within 22 hr) and
VASCHENKO
4-armed pluteus method described Determination
(within 72 hr) according by Kobayashi (1971).
to
of 45Ca2+ uptake in unfertilized
the
eggs
A mixture of equal batches of eggs from 4-5 females both of control and experimental groups was used for the experiments. Three equal portions of gametes were picked out from each mixture and subjected synchronously to all required operations. The eggs (cell density about 4000egg/ml) were incubated in clean sea water in the presence of 20 p Ci/ml 45Ca2f. Three 100 p 1 suspension aliquotes were taken up within 5, 15, 30 and 90min of incubation Eggs were rinsed out three times with ice cold sea water, resedimented by means of hand centrifuge and then homogenized in 200 ~1 of 50 mM Tris-HCI buffer (pH 7.5). The homogenate was dissolved in a mixture of ZhS-1 scintillation liquid (Reachim, C.I.S.) and Triton X-100 (3: 1) and the 45Ca2+ radioactivity in eggs was determined by a scintillation counter. The 45Ca2f content of the eggs was counted per 1 mg of egg protein. The 100 ~1 suspension of eggs was analyzed for protein concentration by the modified method of Lowry et al. (Markwell et al., 1978) using bovine serum albumin as a standard. In the short-term experiments, the eggs from control animals were transfered to sea water containing 45Ca2+(20 pCi/ml) and hydrocarbons (about 1 mg/ml) or ionomycin (10-6-10-9 M). The next procedure was as described above.
RESULTS
AND DISCUSSION
Chronic exposure of adult sea urchins S. intermedius to water soluble hydrocarbons of diesel fuel caused disturbances in embryogenesis and early larval development (Table 1). The number of fertilized eggs obtained from experimental sea urchins was reduced by 15% as compared to the control group. By the time of the first cleavage (90 min after fertilization) the number of two-cell embryos in the experimental group was three times less than in the control one while the number of noncleavaged zygotes was 60%. This indicates some delay in fertilization and cleavage. The delay and asynchronism in the development of experimental sea urchin progeny showed up vividly at the gastrula stage since the formation of larvae archenteron was inhibited. At all the developmental stages the number of abnormalities was found to be higher in the experimental sea urchin progeny. Previously, similar disturbances were observed in the embryonal and larval development of Strongylocentrotus nudus subjected to long-term effects of diesel fuel hydrocarbons (Vaschenko, 1980, 1985; Vaschenko et al., 1988). Experimental cross fertilization, ascertained by the early embriogenesis (before hatching of blastulae), seems to be much dependent on the quality of female sex cells.
s5Ca2+ uptake into hydrocarbon-treated
sea urchin eggs
545
Little attention was paid to calcium transport across the plasma membrane in sea urchin unfertilized eggs under normal conditions. In accordance with existing data, Ca*+ uptake by unfertilized eggs of the sea urchin Anthocidaris crassipina is close to zero and this process increases sharply after fertilization (Fujino et al., 1985). A similar result was obtained also from our experiments on peculiarities of 4sCa2+ uptake by unfertilized eggs of the sea urchin S. intermedius. The radioactivity of unfertilized eggs increased slowly through the whole incubation period (Fig. 1). The addition of sperm led to a dramatic increase in calcium uptake. It is well known that calcium channel antagonists, verapamil and its derivatives, 14-dihydropyridines and benzodiazepines, inhibit calcium uptake by fertilized eggs and embryos of sea urchin (Komukai et al., 1985; Chin, 1986). In our experiments a calcium channel antagonist D-600 at the concentration of 80 PM also inhibited 45Ca2C uptake in unfertilized eggs (Fig. 1). The decrease in calcium uptake 60 min after incubation was 30%. This shows that calcium uptake by eggs is accomplished at least partially through the calcium channel. The calcium uptake rate was strongly dependent on the temperature (Fig. 2). The optimal thermal range for development of S. intermedius is 17-21°C (Buznikov and Podmarev, 1975). 45Ca2+ uptake by unfertilized eggs at 21°C was 4-6 times than at 17°C implying participation of metabolic processes in the regulation of calcium uptake by unfertilized eggs. The temperature of 19-20°C was found to be optimal for our experiments. In that case the rate of calcium
e+r+---
90
Time, min
Fig. 1. The effects of D-600 and insemination on 45Ca2+ uptake by unfertilized eggs of the sea urchin S. intermedius. 1: normal 45Ca2+uptake; 2: upon incubation of eggs in the presence of 80 pm D-600; 3: 1min after sperru addition. The arrow shows the time of sperm addition. The temperature is 19°C. Each point is the mean of the values obtained from three experiments. Vertical range bars, SE. Abscissa: time of incubation, min; ordinate: radioactivity of 45Ca2+ in eggs, cpm/mg egg protein.
P. M. ZHADANand M. A. V&XENKO
546
T
P
6000 r
21
Time, min Fig. 2. The effect of temperature on %a2+ uptake by unfertilized eggs of the sea urchin S. intermedius. To the right of the curves is the temperature of incubation. The rest of the designations are the same as in Fig. 1.
uptake by eggs was sufficiently high and their quality did not worsen during incubation. Long-term exposure of parent sea urchins to hydrocarbons increased the rate of 45Ca2+ uptake by unfertilized eggs. The radioactivity of eggs obtained from experimental sea urchins 90min after incubation in the presence of 45Ca2+ was 25-60% higher than in the control ones (Fig. 3). Fall in the incubation temperature diminished, to some extent, to a relative increase in 4sCa2+ uptake by the eggs of experimental sea urchins as compared to control animals. At the same time, the total 45Ca2t uptake has been reduced several times (cf. Fig. 3a and 3b) both in experimental and control groups. In our previous works we discussed the fact that similar disturbances in embryonic and larval development resulted from both long-term treatment of the parent sea urchins with hydrocarbons at low concentrations and a short-term exposure of gametes to high concentration of hydrocarbons, more than 1 mg/l (Vaschenko, 1985; Vaschenko et al., 1988). As reported by a number of authors, the asynchronism and delay in embryonic and larval development of sea urchins were exhibited when eggs were treated with hydrocarbons at concentrations of l-10 mg/l and higher, depending on the sea urchin species and on oil product grade (Lonning and Hagstrom, 1975; Nicol et al., 1977; Falk-Peterson, 1978; Kobayashi, 1981). We performed several experiments on the short-term (2 hr) effect of hydrocarbons on 45Ca2+ uptake by unfertilized eggs from control animals. Experimental results showed that hydrocarbons in concentration, used for parent sea urchins exposure, did not affect 45Ca2+ uptake by eggs. It was not until concentration was increased to 1 mg/ml, producing a small rise in calcium uptake (by 30% for 90 min of incubation) by sea urchin eggs (Fig. 4).
5
15
5
15
60 30 Time, min
90
4ooc
2000
II
I
I
I
60 30 Time, min
I
90
Fig. 3. The effect of long-term exposure of parents sea urchins S. intermedius, to diesel fuel hydrocarbons (0.04-0.3 mg/l) on Ya2+ uptake by unfertilized eggs. 1: control; 2: experiment. A: incubation temperature of 17°C; B: incubation temperature of 19°C. The rest of the designations are the same as in Fig. 1.
Experimentally, the relation between the disturbances in embryonic development and 45Ca2f uptake by unfertilized egg cells was studied using ionomycin,
**r 2000-
50 Time, min Fig. 4. The effect of short-term exposure of sea urchin S. intermedius unfertilized eggs to diesel fuel hydrocarbons (the concentration 1 mg/l) on 45Ca2+ uptake. 1: control; 2: experiment. The incubation temperature = 17°C. The rest of the designations are the same as in Fig. I.
45Ca2+ uptake
into hydrocarbon-treated
% 3.0 0
“;t !: “0 ” 1 & f
2.5 -
2.0 -
1.5 -
*$ 2
LO10”s
103
10-s
lo-’
104
Ionomycin concentration, M Fig. 5. Relative increase of 45Ca2+ uptake into unfertilized sea urchin S. intermedius eggs during 30 min incubation with ionomycin. Abscissa: ionomycin concentration in incubation medium. mohl. Ordinate: relative increase of “Ca2+ uptake into urchin eggs.
calcium ionophore. The increase of calcium uptake by the eggs and the level of disturbances in embryonic development are strongly dependent on ionomycin concentration (Figs 5 and 6). Ionomycin at 10-6-10-7 M (3-4 fold elevation of calcium ingress) was heavily toxic and arrested the division of embryos. Ionomycin concentrations from lo-* to 10m9M increased 45Ca2+uptake into unfertilized eggs by 70-120% (Fig. 5). At such concentrations, the embryogenesis was accompanied by the disturbances similar to those caused by the effects of WSFDF in the chronical experiments, i.e. the delay in cleavage and in the formation of archenterone. Thus, the results of our investigation show that the diesel fuel hydrocarbons may induce an increase in the rate of 4sCa2+ uptake by unfertilized eggs in
sea urchin
eggs
547
chronic exposure (of parent specimens) as well as in acute exposure (of eggs) experiments. Prolonged exposure of the sea urchins to diesel fuel hydrocarbons at low concentrations exerted a stronger effect on 4sCa2+ deposition in eggs than a short-term incubation of eggs at high hydrocarbon concentrations. In both cases this fact might indicate the existence of various mechanisms responsible for the increase of 45Ca2+. In the case of a long-term exposure, the rise in membrane permeability of sea urchin eggs due to alteration in their biochemical composition might be the cause of the enhanced 45Ca2+uptake. Experimental data, although scarce, testifies in favour of supposition that the action of hydrocarbons bring about the damage to lipid metabolism, resulting in an increase in the amount of unsaturated fatty acids of gill phospholipids in crustaceans (Morris et al., 1982) and elevation of the level of cholesterol and other sterols in lobster embryos and larvae (Capuzzo et al., 1984). However, it is known that a rise in cholesterol concentration leads to an enhancement of membrane permeability to calcium ions (Lecher ef al., 1984). Elevation of the calcium uptake level caused by the breaks in calcium metabolism in the tissues of M. edulis, dwelling in sea water polluted with hydrocarbons and heavy metals was reported by Viarengo and co-authors (1988). The increased 4sCa2+ uptake by eggs during their short-term exposure to hydrocarbons at high concentrations might be stipulated by another mechanismthe rise in membrane permeability due to dissolution of hydrocarbons in membrane lipid phase. The scope of such an oil product effect was mentioned in studies of kerosene impact on calcium permeability of the sarcoplasmic reticulum membranes (Garcia and Gonzales, 1985). As to the relation between two phenomena caused by the hydrocarbon effects-the delay and asynchronism at the early embryonic and larval developmental stages and increased 45Ca2f uptake by the eggs-it should be emphasized that both effects showed up synchronously after prolonged exposure of sea urchin parental individuals to hydrocarbons at low concentrations and after a short-term treatment of unfertilized eggs with ionomycin and hydrocarbons at high concentrations. This allows us to conclude that one of the mechanisms responsible for deleterious effects of hydrocarbons on sea urchin female sex cells is related to changes in membrane permeability to calcium ions. REFERENCES
Ionomycin concentration, M Fig. 6. Inhibition of first clevage sea urchin eggs with ionomycin - Abscissa: concentration of . ionomycin, mol/l. . . __ Wrdmate: number of Z-cell embryos, %.
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