The effects of hypoxia, alkalinity and neurochemicals on hatching of Atlantic salmon (Salmo salar) eggs

AC~UCU~~UF~T, 86 (1990) 417-430 Elsevier Science Publishers B.V., Amsterdam -

417 Printed in The Netherlands

The Effects of Hypoxia, Alkalinity and Neurochemicals on Hatching of Atlantic Salmon (Salmo salar) Eggs DAG 0. OPPEN-BERNTSEN,

ARE BOGSNES and BERNT TH. WALTHER

Department of Biochemistry, University of Bergen, 5009 Bergen (Norway) (Accepted 23 August 1989)

ABSTRACT Oppen-Berntsen, D.O., Bogsnes, A. and Walther, B.Th., 1990. The effects of hypoxia, alkalinity and neurochemicals on hatching of Atlantic salmon (Salmo salur) eggs. Aquaculture, 86: 417430. The hatching process of eggs from Atlantic salmon was investigated. Salmon eggs were subjected to hypoxia under different alkaline conditions in the laboratory using different buffers in the test systems. Hypoxia was a highly effective inducer of hatching in mature salmon eggs (2 days before natural hatching at 5-7”C), causing 100% hatching within 2 h of application at physiological pH. Salmon eggs were also induced to hatch by hypoxia 2-3 weeks ahead of normal hatching, but hatching occurred more slowly and asynchronously than in mature eggs and was not complete (86% ) . However, in this situation the induction of salmon egg hatching by hypoxia was greatly facilitated at alkaline pH. Hypoxia induced hatching only when applied less than 1 month prior to normal hatching, i.e. after ca. 300 day degrees (dC) of development. Under normoxic and physiological pH conditions hatching of mature salmon eggs was induced by dopaminergic antagonists and delayed by dopaminergic agonists. Noradrenergic and serotonergic agents also induced hatching, but they apparently act through a terminal dopaminergic mechanism. The data suggest that hypoxia is involved in the natural hatching process, and that physiological signals for hatching in mature salmon eggs may involve specific neural monitors of oxygen tension.

INTRODUCTION

Under common hatchery conditions salmon yolksac larvae normally exit from eggs approximately 450 dC (day-degrees) after fertilization. In salmon aquaculture, hatching success is usually quite high, approximating 95%. However, the process of hatching is highly asynchronous and may take from l-2 days to 1 week for completion in a single egg batch. Consequently the hatching Supported by a grant from the Norwegian Fisheries Research Council (NFFR V: 108.003 ) .

0044-8486/90/$03.50

0 1990 Elsevier Science Publishers B.V.

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period requires considerable labour for removal of hatched eggshells in order to avoid clogging and infections in the hatching trays. Routine neutralization of river water used in hatcheries has removed acid rain-water as a cause of lengthy arrest of salmon egg hatching. Nevertheless, significant benefits would accrue from new procedures allowing a more synchronous hatching of salmon. The sturdy fish eggshell ensures good protection of the embryo during embryogenesis. Because of the mechanical strength of the eggshell, exit of the salmon larvae from the egg requires prior degradation of the eggshell. The eggshell of salmonid eggs is mainly a large protein aggregate composed of approximately 95% protein and 5% carbohydrates (Hagenmaier, 1985), organized in a thin outer layer (zona pellucida) and a thick fibrous inner layer (zona radiata). The fish eggshell is rather impervious to proteolytic degradation from the external side, but is degraded extensively from within by specific proteases during hatching (Yamamoto and Yamagami, 1975; Yamagami 1988). Such hatching enzymes are normally not present in the perivitelline space, but are synthesized during embryogenesis in specific ectodermal cells termed hatching glands. Hatching stimuli provoke the release of the hatching enzyme into the perivitelline space, which causes rapid digestion of the zona radiata. The exact time of hatching depends on specific interactions between the embryo and environmental factors such as oxygen (Kaighn, 1964; Hagenmaier, 1972; DiMichele and Taylor, 1980). During this process the embryo exhibits vigorous muscular activity. However, drastic degradation of the eggshell is required before these movements suffice to tear open the eggshell. Earlier workers have found that low environmental oxygen tension stimulates hatching in fish (Ishida, 1944; Ishida, 1985; DiMichele and Taylor, 1981; DiMichele and Powers, 1984; Rombough, 1988; Yamagami 1988). However, the literature contains divergent reports as regards the effects of hypoxia on the hatching of salmonid eggs. In the case of rainbow trout eggs, l-2 days before natural hatching, Hagenmaier (1972, 1974) obtained nearly complete hatching within 1 h after depleting the incubation medium of oxygen by nitrogen gas bubbling. In the case of mature Atlantic salmon eggs, Haya and Waiwood (1981) reported that a similar procedure did not increase hatchability, although the eggshells became markedly softened during the hypoxic treatment and extensive “turning activity” of the embryos was observed. We have studied the effects of alkaline pH and low oxygen tension in different buffer systems, and how these two parameters are related to the induction of synchronous hatching. Furthermore, we have screened some hormones and neurotransmittors for their ability to induce or delay hatching in order to learn the neural mechanisms involved in normal salmon egg hatching. Some of these agents have been found to participate in the hatching process of other fish species (Schoots et al., 1983a,b, Yamagami, 1988).

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MATERIALS AND METHODS

Materials Salmon eggs from the normal production line of Atlantic salmon stock in 1986 and 1987 were obtained from the Matre Aquaculture Station on the Norwegian west coast. Fertilized eggs were maintained on trays percolated with water from the Matre River, heated to 8”C, aerated and buffered to pH 6.5 with lye. Three weeks before expected natural hatching, the eggs were transported to the laboratory, which entailed egg maintenance for up to 6 h in Styrofoam containers under melting ice. Eggs at different stages of development were used in the experiments as specified. In the experiments involving application of neurochemicals, distilled water was used as solvent if not otherwise specified. Pharmaceuticals were used as follows: (1) control I - 20 mA4 Tris/HCl pH = 7.4; (2) phenylephrine - alfa agonist (Sigma); (3) phentolamine - alfa antagonist (Ciba); (4) isoprenaline - beta agonist (NAF); (5) propranolol - beta antagonist (ICI); (6) epinephrine - alfa/beta agonist (Sigma); (7) norepinephrine - alfa/beta agonist (Sigma); (8) bromocriptine - dopamine agonist (Sandoz); (9) haloperidoldopamine antagonist (Mekos); (10) dopamine (Sigma); (11) 5-methoxy-iV,Ndimethyltryptamine* (Sigma); (12) metergolin - serotonin antagonist** (Pharm Italia); (13) control for metergolin = control I + 0.04 ml ethanol; (14) 4-hydroxytryptamine (serotonin) (Sigma); (15) norepinephrine + bromocriptine. Methods Laboratory egg incubation All eggs were initially placed in a cold room at 4’ C and maintained on plastic trays (50 x 40 cm) under stagnant water conditions throughout the laboratory period. The eggs were covered with a minimum amount of 10 mM NaCl containing 0.005% (w/u) oxytetracycline. All hatching experiments were carried out in the cold room at 4 ’ C. Starting 24 h after arrival in the cold room, eggs to be used in experiments were transferred individually from the holding trays and incubated either in excess of stagnant liquid (over 250 ml in 500 ml glass beakers) or in minimal *Dimethyltryptamine is rather potent in producing inhibition at the presumed presynaptic sites on the dorsal raphe neurons. iV,N-dialkyltryptamines together with indoleacetamidines are principally effective against a variety of se‘rotonin actions on ganglion cells and peripheral nerve endings including those in the carotid body. This means that it is difkult to predict whether 5methoxy-NJ-dimethyltryptamine is an agonist or an antagonist before it is tested in a certain system (Gilman et al., 1980). **Metergolin was solubilized in 0.04 ml ethanol.

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volumes of stagnant liquid (about 10 ml in lo-cm-diameter plastic Petri dishes). All incubation media contained 0.005% (w/u) oxytetracycline. Preliminary test of buffer systems: measurements of pH and oxygen tensions To monitor the response of the different buffer systems on nitrogen gas bubbling with respect to pH and oxygen tension, 500 ml of 50 and 100 mikf ammonium bicarbonate and 100 mA4 Tris/HCl buffer were subjected to nitrogen gas bubbling (about 1 l/min). The gas was introduced into the liquid through a diffusor. Oxygen tension was measured as mm Hg by a Lewis cell coupled to a recorder for continuous monitoring of ~0,. The scale was calibrated with solutions equilibrated at 6°C under a normal atmosphere. The pH was measured continuously with a combination electrode (Radiometer) connected to a Corning 130 pH meter. Buffer pH changes were observed as a function of duration of treatment with nitrogen gas. Hypoxia Experimental groups. The effects on hatching of hypoxia under different pH’s were examined by preparing groups of 60 eggs each in 250 ml of 100 mA4 Tris/ HCl buffers with pH values from 7.0 to 10.0 and with 0.5 pH interval between each group (Fig. 1, El-E7). The eggs were exposed to hypoxia by subjecting the buffers to nitrogen gas bubbling. Tris buffers (100 mM) were chosen for the entire pH spectrum to ensure sufficient buffer capacity and avoid effects of miscellaneous ions. Special attention was paid to the use of Tris/HCl buffers at pH 9.0 to 10.0, and pH was monitored frequently in the experimental groups with Lyphan tri-color indicator paper throughout the experiment. Control groups. As normoxic controls to the above-described pH regimes, complementary groups exposed to identical buffers were set up (Fig. 1, Cl-C7). To ensure that these groups had an adequate oxygen supply, the eggs were covered with a minimum of buffer (about 25 ml on a lo-cm-diameter Petri dish) thereby giving them direct access to atmospheric oxygen by passive diffusion through the moist eggshell. To control the effects of the Tris/HCl buffer on the hatching frequency, one group of 60 eggs was exposed to hypoxia in an approximately isosmotic NaCl solution (250 ml, 150 m.M, Fig. 1,C8). To determine the effect of mechanical stress arising from the gas diffusor on the hatching frequency, a group of 60 eggs was set up with 250 ml of 150 m&f NaCl solution and bubbled with air (Fig. 1, C9 ). The effect of the ionic strength of the Tris/HCl buffer was examined by including two groups of 60 eggs each under normal oxygen conditions covered with 250 ml of 150 mA4 NaCl (Fig. 1, ClO) and distilled water (Fig. 1, Cll) respectively.

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100

mhl Tris/HCI

150 mM NaCl

Fig. 1. The experimental design used for the hypoxia experiment. Experimental groups exposed to hypoxia at different pH’s are designated El-E7. The corresponding normoxic control groups in Petri dishes are designated Cl-C7. The isosmotic control groups with nitrogen bubbling, air bubbling and without bubbling are designated C8-ClO. The hyposmotic group to control the effect of ionic strength on hatching was kept in distilled water and designated Cll.

Neurotransmittor/hormone treatment Groups of 60 eggs were incubated in Petri dishes and covered with 25 ml of buffer (20 mA4Tris/HCl pH = 7.4) containing the different neurochemicals at 1.6 micromolar concentration, using eggs immediately prior (ca. 2 days) to natural hatching. In the case of agents dissolved in non-aqueous solvents in stock solutions, the appropriate control of diluted solvents with no drug was run. The different groups were screened for pharmacological effects on the hatching incidence in the absence of other known hatching inducers, i.e. light and hypoxia. Neurochemicals were tested only for their ability to induce/delay hatching, and not for effects on the development of hatching capability. Hatching scores and statistics Hatching was registered from the onset of experimental treatments. All experimental groups were examined every 30 min. Hatching was scored at the time when a part of the embryo had penetrated the eggshell. Hatched larvae were counted and transferredto separate Petri dishes for independent verifications of hatching scores. Empty eggshells were removed when produced. In the neurotransmittor/hormone experiment the hatching frequencies in the different groups were recorded at the time when 50% had hatched in the control group. The Petri dishes were considered to contain 60 independent

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ETAL.

experiments where the hatching process is a binomial event which is normally distributed around the timepoint of hatching, i.e. when the hatching frequency is 50%. The normal distribution approach was used in testing for statistical significance. The different hatching frequencies were compared with the observed hatching frequencies in the respective control groups, and tested for significance calculating the probability value. The observed hatching frequencies in the experimental groups were considered to be significantly different from the observed hatching frequencies in the control groups when the calculatedprobability value was less than 0.001, i.e. P-c 0.001 (Wannacott and Wannacott, 1977). RESULTS

Preliminmy testing of buffer systems Upon bubbling buffers with nitrogen gas, the p0, of the buffer in our experimental design declined from about 150 mm Hg to less than 5 mm Hg oxygen within 5-10 min regardless of whether the buffer was 50 mM or 100 mA4 ammonium bicarbonate or 100 mA4 Tris/HCl (Fig. 2a,b,c, continuous curves). As shown in Fig. 2a,b the rise in pH (curve with dots) is more dramatic with ammonium bicarbonate buffer than with Tris/HCl buffer (Fig. 2~). The increase in pH is dependent on the initial concentration of the buffer components as demonstrated in Fig. 2a,b (50-100 mM). The results obtained from this preliminary test demonstrate that the pH of a Tris/HCl buffer is minimally influenced by nitrogen bubbling. Tris buffers were therefore used in all subsequent experiments. 151

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Survival Total salmon egg mortality for the duration of the experimental period was 1.9%. No increased mortality of eggs was observed under the various pH conditions, even after a week-long exposure to biologically rather extreme alkaline conditions. However, hatched larvae were seriously affected even by brief exposure to such pH conditions. In addition, no deleterious effects were observed (measured in terms of viability through hatching) of prolonged exposure (up to 24 h) of eggs to severe hypoxia at early times of development, or immediately prior to hatching. Longer exposure to severe hypoxia during embryogenesis has been demonstrated to be lethal (Hamdorf, 1961). No problems of microbial or fungal infections were encountered in any egg batches. Development of hatching capacity Hatching was only induced by hypoxia after the eggs had passed a developmental stage equal to 300 dC or two-thirds of the intraovo period, i.e. 3 weeks before natural hatching at 57°C. When such eggs were exposed to hypoxia, 7-8 h elapsed before the first eggs hatched. The hatching process was highly asynchronous, i.e. it took place over several hours (12 h or more) and was not complete (86% ). In contrast, when eggs were taken 2 days before natural hatching, the process was more synchronous (2 h) and started within 1 h of hypoxia treatment. In this case the hatching process was also complete (100% ) . Hypoxia-induced hatching under different pH conditions The effect of hypoxia (pOz lower than 5 mm Hg) on salmon eggs taken 3 weeks before natural hatching (300 dC ) under different pH regimes (El-E7 ) is plotted three-dimensionally in Fig. 3a. The hatching frequency was greatly enhanced at pH 9.0 (E5) and 9.5 (E6). Maximum hatching frequency (H% =86) was observed at pH 9.5 (E6) which is significantly different from the hatching frequency (H% = 0) observed in the corresponding control group (Fig. 3b) with appropriate oxygen supply (C6) (P< 0.0000001). Minimum hatching frequency (H% = 6) under hypoxic conditions was observed at pH 7.5 (E2) and 8.0 (E3) which was calculated to be significantly different from the frequency (H% = 0) observed in the corresponding control groups C2 and C3 (PC 0.025). Under extreme alkaline conditions, pH = 10.0 (C7), an increased hatching frequency (H% = 10) was also detected under appropriate oxygen supply. However, the hatching frequency (H% = 24) observed in the corresponding experimental group (E7) was found to be significantly different (P
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Fig. 3. Hatching frequency as a function of pH and time. (a) Hatching frequency in experimental groups under different pH’s and hypoxic conditions (referred to as El-E7 in Fig. 1) . (b ) Hatching frequency in control groups under different pH’s and normoxic conditions (referred to as Cl-C7 in Fig. 1).

groups in Tris/HCl (Cl-C6). However, as pointed out earlier, a small increase in hatching incidence was detected where the pH values deviated far from physiological conditions (pH lO.O), C7. The mechanical stress from the diffusor did not cause any eggs to hatch, as no hatching (H% = 0) was observed with air bubbling (C9). Exposing eggs to hypoxia in 150 mA4 NaCl (CS) gave almost the same hatching incidence (H% ~8) as in hypoxia at pH 7.5 (E2) where the observed hatching frequency was H% = 6. Neurotrunsmittor/horm.one experiment The results from the neurochemical experiment (Fig. 4) show that serotonin and norepinephrine both tend to accelerate hatching, while dopamine delays hatching. Epinephrine appears to be without any effect. The results of the tested neurochemicals (agonists and antagonists) indicate no effects of alfaacting drugs, while the beta-acting drug, propranolol, acts in a manner consistent with the norepinephrine action and suggests the presence of beta-norepinephrine receptors. The dopamine agonist, bromocriptine, also acts in a manner consistent with the dopamine inhibition of hatching. The serotonin antagonist tested; metergolin, seems to be without effect. 5-Methoxy-N,N-dimethyltryptamine delays hatching, suggesting a serotonin antagonistic effect of this drug when applied at such concentrations in this system, which is consistent with the ability of serotonin to induce hatching. The ability of 5-methoxy-N,N-dimethyltryp-

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Fig. 4. Hatching frequencies in the different groups exposed to neurochemicals, at the time when 50% had hatched in the control group. (For statistics see Materials and Methods.) The hatching frequencies which were found to be significantly different (PC 0.001) from the expected 50% are represented by a filled histogram. Horizontal lines at k 12.9 represent the 95% confidence interval with respect to the control group. Bars on top of each histogram represent the 95% confidence interval for the respective experimental groups. Co, control group; Pe, phenylephrine; Pa, phentolamine; Ip, isoprenaline; Pp, propranolol; Ep, epinephrine; Ne, norepinephrine; Bc, bromocriptine; Hp, haloperidol; Da, dopamine; Mt, 5-methoxy-NJ-dimethyltryptamine; Mg, metergolin; St, 5-hydroxytryptamine (serotonin); Ne/Bc, norepinephrine + bromocriptine.

tamine to delay hatching might, however, also be explained by other nonspecific effects on the CNS. When bromocriptine and norepinephrine, which have opposite effects on hatching, are applied together, the observed effect is the same as for bromocriptine alone.

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DISCUSSION

Fish egg hatching is a complicated process which involves eggshell components, hatching enzyme, hatching gland and general embryonic development. Environmental signals interact with the embryo to elicit the active exodus of the embryo from the egg. Clearly more molecular information is needed about the components in this process if we are to understand the mechanisms of hatching of fish eggs in their natural environment. The present study documents the pertinence of classical chemistry of pH in volatile and non-volatile buffers subjected to gas bubbling. The pH in the volatile buffer (ammonium bicarbonate) increased during the gas bubbling because of the different solubility of ammonia and carbon dioxide. On the other hand, both buffers were depleted of oxygen when the gas used was nitrogen. The extent of hypoxia achieved creates conditions which are irreconcilable with maintenance of normoxic mitochondrial metabolism in fish embryos (Hamdorf, 1961). However, the extent to which oxygen reserves in primitive embryos, together with low metabolic demand for oxygen, delayed functional hypoxia in the fish eggs was not measured. The general lack of effect on viability by hypoxia under our conditions would suggest that the oxygen effect is a signal effect rather than a metabolic effect. The maintenance of viability of eggs subjected to extreme alkaline pH’s indicates an extra-embryonic target of the effect of pH on hatching. Similarly, J.V. Helvik (pers. commun., 1986) has determined that the perivitelline pH of salmon eggs reflects the pH of the external medium. Extreme alkaline pH can induce hypoxia by increasing the metabolic rates. Similar explanations may be advanced for other stressors such as high ionic strength (diluted seawater, M. Iwata, pers. commun., 1988) and high temperature (Luczynsky, 1984). Thus, of the two parameters studied in this work, only hypoxia, and not alkaline pH, appears to be a candidate for a physiological stimulus for hatching. An increase in the intra-embryonic pH due to metabolic deamination of amino acids during embryogenesis could be a plausible hatching stimulus. However, if alkaline pH were the actual physiological stimulus for hatching, one would expect the same hatching frequency when the eggs were exposed to alkaline pH under normoxic as under hypoxic conditions. This is demonstrated not to be the case as seen from Fig. 3a,b; only at the extreme pH (C7) was hatching slightly stimulated. Hypoxia was proposed to be the physiological hatching signal by Ishida (1944). As the consumption of oxygen by the embryo increase with development (Hamdorf, 1961; Serigstad, 1987a,b; Rombough, 1988), parts of the embryo may experience hypoxia. Hypoxia is most effective in inducing hatching of salmon eggs. The results shown in Fig. 2 demonstrate that the oxygen tension drops below 5 mm Hg after 5 min of bubbling with nitrogen gas. Hence, eggs exposed to nitrogen gas bubbling are subjected to severe external hypoxia

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within minutes, and presumably to severe internal hypoxia shortly thereafter, regardless of the buffer used. We interpret the longer time required for hatching after hypoxia exposure in early, as compared to older, embryos to signify that early and less developed embryos possess smaller amounts of hatching enzyme. A longer time is therefore required for sufficient digestion of the zona radiata before the yolk sac larvae may exit from the eggs. An additional, and possibly more important, explanation for this phenomenon is that early embryos have lower oxygen demands and are less capable of responding to low oxygen tensions; hence, more time elapses before a hypoxic condition is created and hatching is induced in such embryos. Once triggered, it is reasonable to speculate that such embryos have a less developed mechanism to cope with the induction of hatching, e.g. lower levels of hormones and neurotransmittors, and fewer receptors to respond to such signals. Site of action of hypoxia The site of action of hypoxia is unknown (Iuchi et al., 1985; Yamagami, 1988). The embryo may possess chemosensor cells, or hypoxia may be registered in cells with high mitochondrial (aerobic) metabolism. The hypoxia attained in our experimental induction of hatching may render the mitochondria of the hatching gland cells non-functional whereupon a pre-necrotic release of the enzyme might take place. However, the action of neurochemicals on the hatching process tends to suggest that hypoxia is acting through some chemosensor element (Yamagami, 1988). Schoots et al. ( 1982,1983a,b) have suggested that zebrafish hatch upon cancellation of a dopaminergic inhibition of prolactin secretion which was inferred to bring about hatching enzyme release in this species. Our data on hatching of Atlantic salmon eggs are consistent with the neural control mechanism proposed by Shoots et al. (1983a). We suggest that hypoxia leads to a signal (norepinephrine ) that blocks the dopaminergic inhibition which finally results in the secretion of an effector signal to the hatching gland cells. This suggestion is founded on the fact that the observed hatching frequencies when norepinephrine is applied together with bromocriptine are the same as for bromocriptine alone, indicating that a beta-adrenergic signal precedes the dopaminergic signal in the regulatory sequence. The induced hatching resulting from exposing the eggs to &hydroxy-tryptamine (serotonin) indicates that this component could be involved in the regulation of the hatching process. It is often observed in Norwegian salmon hatcheries that even brief exposure to light just prior to hatching can induce hatching within l-2 h. The pineal gland is known to be a light-sensitive brain area, in which under dark conditions serotonin is converted to melatonin. Serotonin is postulated to participate in a light-mediated response which stimulates the embryo to hatch. The data do not exclude more complex explanations.

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The structure of the eggshell and its importance in fish egg hatching Hatching of salmon embryos involves mechanical force in addition to enzymatic action, although no study so far has delineated the relative importance of these two elements in the overall process. The strong body movements of the salmon yolksac larvae at hatching may in the course of normal hatching provide the force which finally tears apart the eggshell, and particularly the rather inert zona pellucida, and allows the embryo to exit. However, the muscular apparatus of the salmon embryo is probably unable to muster a force sufficient to tear apart the zona radiata in the absence of prior enzymatic digestion. Extensive digestion of the rigid zona radiata demands that the hatching enzyme is contained within the perivitelline space until the degradation of the zona radiata has proceeded far enough for the embryo to tear apart the remainder of the eggshell by muscular force. A corollary of this demand is that Zona pellucida;

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Fig. 5. Model of fish egg hatching, demonstrating the different functions of the two components of the eggshell. The zona pellucida is relatively inert to the action of the hatching enzyme during the hatching process, and acts as a diffusion barrier to the hatching enzyme. This ensures sufficient digestion of the rigid zona radiata, and leaves the rather weak zona pellucida to be broken only by muscle force. When the zona pellucida is damaged as a result of low-grade fungal infections (fresh water) or bacteria infections (marine water), severe loss of hatching enzyme may occur before sufficient degradation of the zona radiata has been achieved. This may be a common cause of nonsuccessful hatching.

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the hatching enzyme must be barred from diffusing out of the eggshell until degradation is adequate for mechanical rupture. The zona pellucida is not totally resistant to degradation by the hatching enzyme but the rate at which it is digested is much slower than for the zona radiata. We find almost total disappearance of the zona pellucida after 24-48 h of incubation at 5°C in dilute hatching medium (data not shown). Normally the eggshell provides a barrier to diffusion for all compounds with molecular weights above 500 Dalton (Davenport et al., 1981). It is not known where the barrier to diffusion is located anatomically. The most likely site is the zona pellucida, as diffusion of hatching enzyme into the zona radiata is available through the channels occupied by the oocyte’s microvilli during the accumulation of vitellogenin during oogenesis (Hosokawa, 1985). Hence, the diffusion barrier in the eggshell is directly exposed to the hatching enzyme. Any puncturing of the zona pellucida will permit the hatching enzyme to escape before the zona radiata is sufficiently weakened to be torn open by larval muscular movements. The loss of hatching enzyme through a damaged zona pellucida is caused by high hydrostatic perivitelline pressure due to the high content of perivitelline proteins and colloidal material (Eddy, 1974) in addition to passive diffusion. In our experience, salmon eggs often will not hatch if infected by fungus. Inexplicable non-hatching of salmon eggs was occasionally observed upon long incubations in the laboratory. Such eggs appear to be fully viable despite the absence of hatching. The embryos initially exhibit many facets of hatching behaviour, and there is softening and incipient deformation of the eggshell, a phenomenon observed during four seasons both in the laboratory and at the hatchery. Concerning hatching of marine fish eggs, low hatching rates in some batches of cod (Gadus morhua) and halibut (Hippoglossus hippoglossus ) have been correlated with loss of hatching enzyme and minor microbial damage to the zona pellucida. Inexplicable non-hatching of salmon eggs warrants a closer examination for possible low-grade fungal infections. We suggest that fungusrelated minimal damage of the zona pellucida may permit loss of hatching enzyme before digestion of the zona radiata is completed and hatching possible. A better understanding of fish egg hatching would therefore be aided by a better understanding of the chemical structure and properties of the zona pellucida.

REFERENCES Davenport, J., Lsnning, S. and Kjrarsvik, E., 1981. Osmotic and structural changes during early development of eggs and larvae of the cod, Gadus morhua L. J. Fish. Biol., 19: 317-331. DiMichele, L. and Powers D.A., 1984. The relationship between oxygen consumption rate and hatching in Fund&s heteroclitus. Physiol. Zool., 57 (1): 46-51.

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DiMichele, L. and Taylor, M.H., 1980. The environmental control of hatching in Fund&s heteroclitus. J. Exp. Zool., 214: 181-187. DiMichele, L. and Taylor, M.H., 1981. The mechanism of hatching in Fundulus heteroclitus: development and physiology. J. Exp. Zool., 217: 73-79. Eddy, F.B., 1974. Osmotic properties of the perivitelline fluid and some properties of the chorion of Atlantic salmon (Salmo salar). J. Zool., 174: 237-243. Gilman, A.G., Goodman, L.S. and Gilman, A. (Editors), 1980. The Pharmacological Basis of Therapeutics. Macmillan, New York, NY, 1843 pp. Hagenmaier, H.E. 1972. Zum Schlupfprozess bei Fishen: II. Experentia, 28: 1214-1215. Hagenmaier, H.E., 1974. The hatching process in fish embryos: IV. Comp. Biochem. Physiol., 49B: 313-324. Hagenmaier, H.E., 1985. Zum Schlupfprozess bei Fischen: VIII. Zool. Jahrb. Physiol., 89: 509520. Hamdorf, K., 1961. Die Beinflussung der Embryonal- und Larvalentwicklung der Regenbogenforelle (Salmo iridius Gibb.) durch die Umweltfaktoren oxygen Partiaklruck und Temperatur. Z. Vergl. Physiol., 44: 523-549. Haya, K. and Waiwood, B.A., 1981. Acid pH and chorionase activity of Atlantic salmon (Salmo salar) eggs. Bull. Environ. Contam. Toxicol., 27: 7-12. Hosokawa, K., 1985. Electron microscopic observation of chorion formation in the teleost, Navodon modestus. Zool. Sci., 2: 513-522. Ishida, J., 1944. Hatching enzyme in the fresh-water fish, Oryzias latipes. Annot. Zool. Jpn., 22: 137-154. Ishida, J., 1985. Hatching enzyme: past, present and future. Zool. Sci., 2: l-10. Iuchi, I., Hamazaki, T. and Yamagami, K., 1985. Mode of action of some stimulants of the hatching enzyme secretion in fish embryos. Dev. Growth Differ., 27: 573-581. Kaighn, M.E., 1964. A biochemical study of the hatching process in Fund&s heteroclitus. Dev. Biol., 9: 56-80. Luczynsky, M., 1984. Temperature and electric shock control the secretion of chorionase in Coregoninae embryos. Comp. Biochem. Physiol., 78A (2): 371-374. Rombough, P.J., 1988. Respiratory gas exchange, aerobic metabolism, and effects of hypoxia during early life. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. XI, part A. Academic Press, New York, NY, pp. 59-161. Schoots, A.F.M., De Bount, R.G., Van Eys, G.J.M. and Denuce, J.M., 1982. Evidence for a stimulating effect of prolactin on teleostean hatching enzyme secretion. J. Exp. Zool., 219: 129132. Schoots, A.F.M., Meijer, R.C. and Denuce, J.M., 1983a. Dopaminergic regulation of hatching in fish embryos. Dev. Biol., 100: 59-63. Schoots, A.F.M., Ruijter, J.M., Van Kemenade, J.A.M. and Denuce, J.M., 1983b. Immunoreactive prolactin in the pituitary gland of cyprinodont fish at the time of hatching. Cell. Tiss. Res., 233: 611-618. Serigstad, B., 1987a. Respiratory studies on cod (Gadus morhua L.) with special reference to effects of oil exposure on eggs and larvae. Dr. Scient. Thesis, University of Bergen, N-5000 Bergen, Norway, 85 pp. Serigstad, B., 1987b. Oxygen uptake of developing fish eggs and larvae. Sarsia, 72: 369-371. Wannacott, T.H. and Wannacott, R.J., 1977. Introductory Statistics, 3rd edn. John Wiley & Sons, New York, NY, pp. 250-251. Yamagami, K., 1988. Mechanisms of hatching in fish. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. XI, part A. Academic Press, New York, NY, pp. 447-499. Yamamoto, M. and Yamagami, K., 1975. Electron microscopic studieson choriolysis by the hatching enzyme of the teleost, Oryzias latipes. Dev. Biol., 43: 313-321.