Biotechnol. Prog.

2000, 16, 497505

497

Alginate Coating of Xenopus laevis Embryos

N. Kampf, C. Zohar, and A. Nussinovitch*
The Hebrew University of Jerusalem, Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, P.O. Box 12, Rehovot 76100, Israel

Xenopus laevis eggs were coated, immediately after squeeze-stripping and fertilization, with a thin layer (50 m) of film based on one of three different types of alginates which varied in their mannuronic/guluronic acid ratio. The alginate was cross-linked with either Ca or Ba ions at three different concentrations. The developmental, survival, and hatching of these embryos and the swelling of their natural jelly coats or hydrocolloid coatings were studied over 7 days, while embryos were maintained in flowing aerated water at a ratio of 85 mL per embryo or at a very diminished ratio of 1.6 mL of sterile or nonsterile MMR solution per embryo. All experiments were conducted in triplicate at 20 ( 1 C. Oxygen was monitored continuously. Mineral content was determined in the alginate-jelly coat and within the embryos over time. The coating conferred major advantages when the ratio between the embryos and the surrounding medium was at a minimum under nonsterile conditions, perhaps as a result of the films resistance to diffusion. In the studied systems, the coating seemed to postpone embryo hatching to a more developed stage. In addition, the coating served as a barrier to microbial contamination and thus improved survival prospects.

Introduction
Cells can be entrapped within a gel matrix. Wide ranges of characteristics are attributed to gels as an entrapment medium. On one hand, they include macromolecules held together by relatively weak intermolecular forces, such as hydrogen bonding or ionic crossbonding by divalent or multivalent cations. On the other hand, strong covalent bonding, where the lattice in which the cells are entrapped is considered as one vast macromolecule, is limited only by the particle size in the immobilized cell preparation (Tampion and Tampion, 1987; Nussinovitch et al., 1994). The major categories of entrapment have been reviewed (Cheetham, 1980; Bucke, 1983; Mattiasson, 1983; Nussinovitch et al., 1994; Jen et al., 1996; Nussinovitch, 1997). They include some commonly used, single-step entrapment methods, such as the simple gelation of macromolecules by lowering or raising temperatures using hydrocolloids such as agar (Brodelius and Nilsson, 1980; Wiksstorm et al., 1982; Banerjee et al., 1982; Dobreva et al., 1996), agarose (Khachatourians et al., 1982; Wiksstrome et al., 1982), -carrageenan (Chibata, 1981; Wang and Hettwer, 1982; Krouwel et al., 1982; Kim et al., 1982; Brodelius and Nilsson, 1980; Deriso et al., 1996; Walsh et al., 1996; Green et al., 1996), chitosan (Vorlop and Klein, 1981; Kluge et al., 1982; Stocklein et al., 1983; Deriso et al., 1996), gelatin, and egg whites (Deriso et al., 1996), among others. These preparations regularly suffer from low mechanical strength and possible heat damage. Another simple single-step entrapment method is the ionotropic gelation of macromolecules by di- and multivalent cations, using alginate (Hannoun and Stephanopoulos, 1986; Dainty et al., 1986; Deriso et al., 1996; Krisch and
* Tel: 972-8-948-9016. Fax: 972-8-936-3208. E mail: nussi@ agri.huji.ac.il.
10.1021/bp990153u CCC: $19.00

Szajani, 1996; Dacunzo et al., 1996; Kamboj et al., 1996) and LMP, among others. The limitations of such systems are low mechanical strength and breakdown in the presence of chelating agents. During immobilization, 104-109 microorganisms (bacteria, yeast, or fungal spores having a maximal diameter of 5 m) can be entrapped within 1 mL of gelling agent (Nussinovitch et al., 1994; Nussinovitch, 1994). In such cases, the microorganisms occupy a maximal 6.5% of the volume. In other words, 93.5% of the volume is not occupied by the cells, or if the cells are evenly distributed throughout the gel volume, then each individual cell is coated by a very thick layer of gel in comparison to its own natural dimensions. In contrast, the idea of coating a single cell with a thin layer of film, comprising only a fraction of its diameter, could be of interest. The difference between coating and entrapping is the thickness of the coating layer, being very thin in the former and thick in the latter. Taking this definition into account, it seems that most if not all reports on coating are in fact describing cell entrapment within a gel matrix (Kojima et al., 1990; Redenbaugh et al., 1986; Hatanaka et al., 1994). To obtain a true coating, special microcoating procedures need to be invented, or at least as a start, attempts need to be made with very large cells. For this study, we chose one of the biggest cells in nature: the fertilized frog egg. The objective of this research was to study the influence of different alginate coatings (thin films that are glued to the outer surface of the egg) on the survival of Xenopus laevis embryos, in terms of their biological advantages and disadvantages under different conditions of cross-linking and storage. Moreover, additional goals were to study the impact of the coating on the developmental aspects of the cell and to check the degree of protection the coating provides to the coated embryo and its ability to resist natural hazards.

2000 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/14/2000

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Table 1. Alginate Compositions (provided by the manufacturers) company Sigma Chemical Co. St. Louis, MO Pronova Biopolymer Drommer, Norway Pronova Biopolymer Drommer, Norway
a

Biotechnol. Prog., 2000, Vol. 16, No. 3

product name alginic acid Na salt low viscosity alginic acid Na salt type A alginic acid Na salt type B

origin Macrocystis pyrifera Laminaria hyperborea Laminaria hyperborea

molecular mass (Dalton) 60 000-70 000 123 000 185 000

viscosity (cP) 50 at a concn of 1% 50 at a concn of 1% 126 at a concn of 1%

gel strength not detected 59.9 g (FIRA)a 56.9 g (FIRA)a

% dry solids 88 87.8 86.5

G:M ratio 39:61 71:29 65:35

FIRA jelly tester.

Materials and Methods

Frog Maintenance. Sexually mature Xenopus laevis (South African clawed toads) were maintained in the laboratory under constantly controlled conditions. Room and water temperatures were maintained at 18 ( 1 C using an air conditioner. Animals were exposed to a 12/ 12 h light/dark period, to keep oocytes at a mature stage. Animals were fed with chick liver or heart twice a week, and water was changed after feeding with aged tap water (Wu and Gerhart, 1991). Egg Ovulation. Females were intramuscularly injected with 1000 IU of human chorionic gonadotropin (hCG) (N.V. Organon Oss, Holland). Egg-laying began 18 h after injection. When signs of laying were observed, some of the eggs were squeeze-stripped into a Petri dish and immediately fertilized. Fertilization Procedure. Fertilized eggs were obtained as follows. Fresh testes were dissected from an X. laevis male and kept in full-strength Modified Marcs Ringer (MMR) solution (full-strength MMR ) 100 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, adjusted to pH 7.4). Testes were then mixed with ovulated eggs for 10 s, and 1/3-strength MMR solution was added. Fertilized and nonfertilized eggs were separated by visual inspection, and only eggs showing the first cleavage (1.5 h after fertilization) were chosen for further manipulation. Embryo developmental stages were monitored under a binocular lens and compared to the Normal Table of X. laevis (Daudin) (Nieuwkoop and Farber, 1994). Coating Procedure. After fertilization, the embryos were dropped into a 1% alginate solution, made by dissolving Na-alginate in 1/3-strength calcium-adjusted MMR (CAMMR) solution (same concentration as 1/3 MMR except with reduced calcium content to 0.22 mM to eliminate accidental cross-linking). Alginate compositions, supplied by the manufacturer, are given in Table 1. Embryos were then sucked into a 1.5-mm diameter tube and dropped into the cross-linking agent. The alginates were cross-linked with either Ca or Ba ions (available as CaCl2 or BaCl2 salts; Sigma Chemical Co., St. Louis, MO) at three different concentrations: 0.25%, 0.5%, or 1% (w/w) (equal to 25, 50, and 100 mM CaCl2, respectively, or 12.5, 25, and 50 mM BaCl2, respectively). The salts were dissolved in 1/3-strength CAMMR solution to maintain the eggs physiological osmotic pressure. After dipping in the cross-linking agent for 20 s, coated embryos were washed once and then stored in a sterile 1/3-strength CAMMR solution. Storage Conditions. All embryos were kept for 196 h under one of three different storage conditions: 1. Closed (sterile) Petri dishes containing 30 embryos in 50 mL of 1/3-strength CAMMR solution at a volume ratio of 1.6 mL per embryo. 2. Open Petri dishes containing 30 embryos in 50 mL of 1/3-strength CAMMR solution at a volume ratio of 1.6 mL per embryo.

3. Aerated, circulated (stirred), and dechlorinated tap water at a volume ratio of 85 mL per embryo. All experiments were conducted in triplicate at 20 ( 1 C (maintained by air conditioner), and the embryos developmental stages were monitored as during fertilization. Every 4-8 h, larval hatching from the natural jelly coat or alginate coating was determined. Survival of the larvae was determined by observing movement. Dead or nonhatched embryos were not included in the survival calculations. Percent survival after hatching was calculated as the surviving hatched larvae out of the total number of embryos. Changes in the dimensions of the eggs natural jelly coat or the thickness of the artificial alginate coat were measured up to 48 h under a binocular using a gridmeasuring lens. Scanning electron microscopy of eggs was performed in a JEOL JSM 35C (Kyoto, Japan). Immediately after laying, the egg was glued to a polypropylene stub and tested under low-vacuum conditions. Microbial mass in the embryos 1/3-strength CAMMR medium was assayed as presence of microbial ATP using a dairy products sterility test kit (LUMAC B. V. Landgraaf, The Netherlands). Free ATP was degraded by adding 10 L of ATPase enzyme (SOMASETM) to 50 L of embryo medium and incubating at room temperature for 15 min. Then, the enhancement of microbial cell wall and membrane permeability to ATP was established by adding L-NRB reagent for 30 s. Finally, the presence of microbial ATP was assayed for 10 s by coupled reaction of luciferin-luciferase enzymes:

luciferin + luciferase + ATP 9 8 (luciferin-luciferase-AMP) + PPi f 8 (luciferin-luciferase-AMP) 9 luciferin + luciferase + CO2 + AMP + light
Emitted light was measured by luminescence photometer (BIOCOUNTER, M 2500, Landgraaf, The Netherlands). The correlation between the actual number of microorganisms and the light emitted from the above-mentioned assay was found using a total plate count culture composed of 1% agar (Difco, MI), 0.5% yeast extract (Difco), and 3% tryptic soy broth (Difco). Biological oxygen demand (BOD) was measured every 24 h during the 196-h experiments. An oxygen-temperature electrode was used for BOD detection and was connected to a portable printing and logging dissolvedoxygen meter model HI 9141 (Hanna Instruments, Woonsocket, RI). Oxygen levels in the embryo medium (( 0.01 ppm) were recorded at the specified times. Changes in the pH values of the embryo storage medium were detected using a pH meter model HI 9141 (Hanna Instruments). Mineral Determination. Sample Preparation. Mineral content was determined in the alginate-jelly coat
O2

Mg2+

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Ruddock and Ruffle, 1972

Table 2. Handling and Care Conditions Reported in the Literature for X. laevis Embryos and Hatched Larvae (tadpoles)

winter 1 12/12 15-17 summer 21-23 23 ( 0.1 4-day-old nr larvae 18 3 daysb 8/16

water temp C

Results and Discussion

In a first set of experiments, X. laevis fertilized eggs were coated with three different types of alginate. The properties of these alginates are summarized in Table 1. They differed with respect to their molecular weights, viscosities at a concentration of 1%, gel strengths, and the content ratios of guluronic (G) to mannuronic (M) acid. The specifications, including viscosity, were evaluated and provided by the manufacturers. The molecular weight and the proportion and arrangement of M and G are expected to affect a particular alginates behavior. The percentage of M in the alginates used for coating ranged from 29 to 35 in the alginates extracted from Laminaria hyperborea to 61 in the alginate extracted from Macrocystis pyrifera. Each egg was covered with a thin layer of calcium- or barium-alginate gel. Alginate was chosen for this study because its coatings are easy to produce (see Materials and Methods), and they have been used successfully for many products (Nussinovitch and Kampf, 1993; Nussinovitch and Hershko, 1996; Hershko and Nussinovitch, 1998a,b; Kampf and Nussinovitch, 1998). Moreover, as can be assumed from the vast experience accumulated from cell-entrapment experiments, alginate gels maintain cell viability (Lim and Sun, 1980). The conditions reported in the literature for X. laevis embryo breeding differ (Table 2). We performed our first coating and storage experiments under so-called harsh conditions, thereby making it easy to conclude whether a particular coating is beneficial relative to uncoated embryos; the conditions were modified from those recommended by Wu and Gerhart (1991) and Phillips (1979) (storage conditions 1). However, we increased the proportion of embryos to medium solution such that instead of including 10 embryos per 50 mL of medium, we introduced 30 embryos per 50 mL and allowed only passive natural aeration to take place, thereby increasing the

10-15/L

33/L

embryo/liquid ratio

nr

30/50 mL

10/50 mL

30/50 mL

Ringers soln tap water chemical exchange after 2 weeks dechlorination 90% every day

exchange 8 L/h constant slow running tap water after 2 weeks, tadpoles moved to a new tank nr

nr

1/85 mL
a

nr

nr

nr

nr

no change after 20 ( 1 hatching no change after 20 ( 1 hatching no change after 20 ( 1 hatching Time after fertilization. b Time after hatching. c Not reported. unchanged unchanged 72 h aging circulated (stirred)

25

20-23

(removed manually from the embryos) and within the embryos over time elapsed from fertilization and coating. Each sample was prepared from five embryos and kept in a microfuge tube at -20 C until analysis. Preparations of 1/3 CAMMR, dechlorinated tap water, and crosslinking agents were also analyzed. Each result is the mean of three determinations ( SD. The contents of each microfuge tube were defrosted, dissolved in concentrated nitric acid, and transferred to graduated 50-mL polypropylene vessels. The microfuge tubes were further rinsed with a fresh portion of acid, adding a total volume of 1 mL to each sample. Two blanks were processed in parallel. The vessels were fitted with screw caps and transferred to a temperature-controlled microwave oven. Samples were subjected to three digestion cycles of 20 min each, at 450 W and 95 C. The vessels were allowed to cool for 10 min between cycles, and at the conclusion of the digestion program were brought to room temperature and uncapped. The volume was brought to 10 mL with deionized water. Analytical Method. Analysis was conducted on portions of these solutions, versus multielement standards prepared using the same matrix. All elements were determined in the tested solutions by inductively coupled plasma atomic emission spectrometry (ICP-AES), using a model Spectroflame Modula E ICP-AES from Spectro (Kleve, Germany), with a standard cross-flow nebulizer and a fixed End-On-Plasma torch. The power level was 1.2 kW, with a coolant flow of 15 L/min, an auxiliary flow of 0.5 L/min, and a nebulizer flow of 0.5 L/min.

20-30% Wu and Gerhart, 1991

Douglas et al., 1992

Henriques, 1964

ref

Phillips, 1979

current study

current study 55% weeka 1 48-72 ha

hatching time survival

Davys, 1986

48-72 ha 30%

48-72

light/ dark period

24-48 hb 12/12

start of feeding

weeka

1 weeka

5 daysa

larva/liquid ratio

1/0.5 L

water changes

10/L

250/L

tap water treatment

not treated

not treated

24 h aging

24 h aging

dechlorinated tap water

dechlorinated tap water Stearns soln dechlorinated tap water 1/3 CAMMR none (storage 1) 1/3 CAMMR none (storage 2) tap water dechlorinated (storage 3) tap water FETAX soln

environ changes

culture medium

none

tap water

none

tap water

tap water

nr

nr

20

108

1 weeka

ha

room aeration 12/12 room aeration 12/12 room aeration 12/12 + nr

water aeration

nr

12/12

48-72 ha 58%

48-72 ha nr

nr

48-72 ha nr

ha

nr

nrc

72

nr

ha

nr

current study

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Figure 1. The effect of alginate type on the survival of X. laevis embryos vs elapsed time. The (5% bar indicates the experimental uncertainty.

stress on the coated embryos. Embryo medium was contained within sterile containers under sterile conditions. Coated embryos were also introduced into the same medium, except that the sterile medium was exposed to nonsterile conditions (storage conditions 2). Coated embryos were also maintained under the ideal conditions reported by Wu and Gerhart (1991) and Phillips (1979), to check their performance in a more favorable environment (storage conditions # 3). The survival of embryos vs time under storage conditions 1 is shown in Figure 1. The survival percentage is equivalent to the accumulated number of hatching embryos to a maximal or asymptotic survival value and is the number of embryos left after they begin to die. The accumulated survival percentage of noncoated embryos was 4.6, 54 h after fertilization, increasing to 66 after 60 h (Figure 1). Percent survival then decreased to 41 after 78 h and reached an asymptotic value of 30 between 84 and 196 h. Reduced survival percentages could be due to the secretion of nitrates or other substances into the medium by the developing embryos (Wu and Gerhart, 1991). In parallel to the survival-prospects study, embryo developmental stages were monitored (observed through a binocular lens) and compared to those of noncoated embryos (Nieuwkoop and Farber, 1994). No difference between the two was observed, implying that the coating film does not hamper embryo development. A large difference between the alginates was observed. The alginate with a high proportion of M held better prospects for embryo hatching. The asymptotic survival value for the high-M coating was ca. 53-56%, vs 2232% for the high-G coatings. This is due to the fact that the higher the G content, the stronger the gel (i.e., the film coating the embryo). In other words, a high G content and long G blocks confer high calcium reactivity and the strongest gel-forming potential to the alginates. Coated embryos appeared to develop in a normal fashion, similar to noncoated embryos. However, the strong coating (high G) prevented hatching embryos from bursting the thin coating film, and thus 120 h after fertilization, they

perished. No significant differences were found between the two alginates extracted from the L. hyperborea. Significant differences in survival rate were observed between the high-M and high-G alginates. The hatching process in X. laevis embryos consists of two temporally distinct phases (Carroll and Hedrick, 1974). Phase 1 appears to be a physical process, which ruptures jelly coat layers J3 and J2. This exposes J1 to the outside medium, in which it is partially soluble, thus permitting its gradual dissolution. Phase 2 is a result of both physical and chemical (proteolytic enzyme secretion) processes. Mobility helps the embryo emerge from its jelly coat but is not enough to break through a high-G coating film. An additional difference was observed between the uncoated and coated embryos. The former reached their maximal survival rate a short time after hatching began. For the coated systems, a maximal value was reached 25 h later. This means that some delay in hatching was effected by the coating process. This delay is important for laboratories interested in performing longer-term experiments with embryos. Another advantage is that the embryo hatches at a much more developed stage relative to noncoated embryos. Thus the embryo is less prone to mechanical damage or microbial contamination. Bacteria have been reported to stick to the surface of the J3 outer layer of the jelly coat, and its removal greatly reduces their number. Coating embryos could therefore eliminate the need for including neomycin sulfate in the media (Carroll and Hedrick, 1974). In addition, one of the roles of the natural jelly coat in amphibians is to serve as a heat accumulator, especially at high altitudes where the fertilized eggs are exposed to lower temperatures (Beattie, 1980). Coating the embryo with an artificial gel layer would decrease heat loss by insulating the embryo from its surroundings. Moreover, the artificial gel coating could condense the light rays as they heat the embryo. As stated by Beattie (1980), larger gelatinous capsules around the eggs may increase their chances of survival. On the basis of these preliminary coating experiments and the conclusion that embryos are not capable of

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Figure 2. The effect of cross-linking agents on the survival of X. laevis embryos vs elapsed time in the case of storage condition 1. Boundary areas emphasize coatings with which no significant difference between percent survival was detected.

breaking through films with a high G content, further coating experiments were carried out only with the high-M alginate. Sodium alginate can be cross-linked with several divalent ions. We checked the performance of the high-M alginate coating after cross-linking with different concentrations of Ca or Ba. The embryos were immersed in the same medium (1/3 CAMMR solution), but the conditions were not sterile and the embryos were prone to microbial contamination. Figure 2 demonstrates the relative successes of the different coatings. Coatings produced with alginate cross-linked with 0.25% and 0.5% CaCl2 were most successful, i.e., a higher percentage of hatching and survival was observed relative to the controls (noncoated) or the other variously coated embryos. Lower concentrations of Ba or Ca, i.e., 0.06250.125%, were avoided because they did not produce a nice coating (as per the definition of a coating, see Introduction). Ba is known to produce stronger gels with alginate than Ca at the same alginate concentration (McNeely and Pettitt, 1973). In addition, the higher the concentration of the cross-linking agent with the same predetermined alginate concentration, the stronger the gel. As noted earlier, a stronger coating limits the percent of hatched embryos. Another explanation for our findings is that diffusivity decreases with increasing alginate concentration or gel strength (Dainty et al., 1986). A third, potentially more important explanation is the toxicity of Ba ions to embryos, as reported by Spangenberg and Cherr (1966). Figure 3 presents the thickness of the film and jelly coat for coated embryos. Coating thickness was never more than 16% of the embryos natural Ferret diameter, including the coating (from 0.07 to 0.2 mm) and, in general, not thicker than the embryos natural jelly coat. During the course of natural fertilization, the jelly coat swells when it is immersed in water (Seymour, 1994). In this study, the alginate coating limited the swelling of the jelly coat. After 4 h of observation, we noted that the

Figure 3. Influence of salt type and concentration on the thickness of the alginate coating and the embryos jelly coat.

weaker the coating, the more swollen the natural jelly coat. The amount of cross-linking agent in the system was much higher than the stoichiometric amount necessary to cross-link the alginate (Glicksman, 1969; Nussinovitch, 1997). After the spontaneous cross-linking, the strength of the coating film increased and its thickness decreased. After 24 h, film thickness was reduced by ca. 10-40% for the different cross-linking agents used, while the film strengthened. The final outcome of this effect was a limitation of the natural jelly coats swelling, which was either slowed or prevented by the strengthening of the coating. After 24 h, the film appears to reach its maximal strength (Nussinovitch, 1997), and the jelly coat stops swelling. The coating prevents the jelly coat from reaching its optimal thickness, as compared to noncoated embryos. The medium in our case was prone to microbial contamination because the Petri dishes were stored open, under nonsterile conditions. It was interesting to note the effect of the alginate coating on the microorganisms development as recorded in relative light units (RLU) vs time. RLU can easily be transformed to microbial counts

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Figure 4. SEM micrograph of X. laevis embryo. (a) Cross section. (b) Enlargement of a section of Figure 4a: (1) alginate coating, (2) jelly coat, (3) embryo.

Figure 5. Schematic illustration of the possible interactions between the alginate and the outer jelly coat layer (J3) of the X. laevis embryo. (a) Direct interaction through positively and negatively charged side-chain residues. (b) Interaction between jelly coat and alginate gel through mediation by Ca ions.

with a conversion factor (Waes, 1984). Using such a conversion we found that about 20 h after the coating experiments began, total counts were on the order of 101102, reaching values of 2-5 103 after 48 h and average values of 0.7-1.5 104 after 72 h. One striking observation was that the noncoated embryos were much more contaminated than their coated counterparts. Normally, microorganisms are glued to the jelly coat, causing considerable contamination of the noncoated embryo (Davys, 1986). The thin film coating the embryo prevented microorganisms from being glued directly to the jelly coat, thereby reducing contamination. In addition, it is important to note that the alginate-based coating is not a good medium for microorganism development. Moreover, the fact that the coated embryos hatched at a more mature stage than their noncoated counterparts made them more resistant to microbial contamination. Finally, it must be remembered that bacterial growth, which naturally results in oxygen inhibition, causes death, particularly in newly emerged young frogs (Davys, 1986); in this light, the contribution of the coating becomes much more important. Using a literature search, we tried to construct a hypothetical model for alginates reactivity with the natural jelly coat. Light and electron microscopy observation indicated that the alginate coating is glued directly to the exterior of the embryos, i.e., the J3 layer, with no observable gap between the two (Figure 4a and b). The coated eggs are immersed at a pH of 7.4; pKa values for alginic acid may range from 3.4 to 4.4. The pKa for the sialic acids of the jelly coat is 2.6. Furthermore, the pKa for the glycoprotein amine groups comprising the jelly coat is 7.8-7.95. These values leave us with two possible hypothetical alginate interactions: direct interaction between NH3+ on the jelly coat glycoproteins with alginates COO- (Figure 5a) or with calcium as a bridge between acid residues of the alginate and the jelly coat (Figure 5b). In addition, hydrogen bonds between the jelly

coat and the alginate are a real possibility (Kampf and Nussinovitch, 1999). To study the effect of different conditions on the coated embryos survival, they were introduced into the same medium, which this time was sterile. The results of these experiments are shown in Figure 6. Two main treatment groups appear to emerge, the first reaching asymptotic survival rates of 64-70% from 70 h after fertilization and the second reaching smaller asymptotic survival values of 34-52% at the same time point. This latter group was comprised of coatings cross-linked with 0.5% and 1% BaCl2, again demonstrating bariums toxicity. Since the medium was sterile, the advantages of successful coating were less salient. Although the controls (noncoated) had an initially higher hatching percentage than the coated embryos, the survival prospects of the embryos coated with alginate cross-linked with calcium (0.25%, 0.5%, or 1%) or barium (0.25%) were better. This is maybe due to defense against mechanical damage and hatching at a later stage when the embryo is more developed. The final storage experiments consisted of immersing the coated embryos into dechlorinated, aerated, circulating tap water (see Materials and Methods). This situation more closely resembles that found in nature. A significant difference between the controls and coated systems was observed. The control exhibited an asymptotic survival percentage of nearly 58, whereas the coated embryos reached no more than 31%. However, in this case the coated embryos held a unique advantage. Survival reached an asymptotic value at least 40 h after the control. These results can be explained by ICP studies of element content in the different media (Table 3) in which the coated and noncoated embryos were incubated. Because of the concentrations of potent cross-linking agents, the 1/3 CAMMR solution appeared to be most conducive to achieving a weaker alginate-coating gel layer (Table 3).

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Figure 6. The effect of cross-linking agents on the survival of X. laevis embryos vs elapsed time in the case of storage condition 2. Boundary areas emphasize coatings with which no significant difference between percent survival was detected.
Table 3. Element Content in Mediums Used for Storage of X. laevis Embryosa barium (mg/L) reactivity with alginate (alphabetical order) tap waterb 1/3 MMRb tap water:1/3 MMR ion ratio
a

copper (mg/L) B 0.021 ( 0.000 0.004 ( 1 10-4 5.3

(

strontium (mg/L) C 0.54 ( 0.08 0.008 ( 2 10-4 68 D

calcium (mg/L) E

zinc (mg/L) F

ferro (mg/L)

A 0.10 ( 0.03 0.009 ( 5 10-4 11

96.40 ( 17.00 32 ( 1.60 3

0.50 ( 0.09 0.01 ( 3 10-4 50

0.17 ( 0.03 0.036 ( 7 10-4 5

Each result is the mean of three determinations

SD. b Determined by ICP.

Table 4. Element Content in Jelly Coat (JC), Alginate Coating Including the Jelly Coat, or within the Embryo versus Time after Coatinga content (mM) element Na K Ca Zn Mg P S
a

site of detectionb coatings embryo coatings embryo coatings embryo coatings embryo coatings embryo coatings embryo coatings embryo

JC before coating (stage 2) 82.3 ( 4.1 73.1 ( 3.6 2.5 ( 0.1 72.5 ( 3.6 11.4 ( 0.5 12.7 ( 0.6 0.24 ( 0.01 4.7 ( 0.2 5.3 ( 0.3 34.1 ( 1.7 2.7 ( 0.1 402.9 ( 20.1 12.6 ( 0.6 169.7 ( 8.5

0 h after coating (stage 3) 95.1 ( 4.7 229.7 ( 11.5 0.19 ( 0.0 83.6 ( 4.2 9.14 ( 0.4 17.5 ( 0.8 0.01 ( 0.0 9.3 ( 0.5 0.22 ( 0.01 38.6 ( 1.9 0.83 ( 0.04 475.3 ( 23.7 5.6 ( 0.3 240.8 ( 12.0

2 h after coating (stage 7-9) 8.4 ( 0.4 83.2 ( 4.2 0.2 ( 0.0 81.3 ( 4.0 3.3 ( 0.1 19.7 ( 1.0 0.03 ( 0.0 5.3 ( 0.2 0.4 ( 0.0 38.0 ( 2.0 0.26 ( 0.0 440.4 ( 22.0 0.25 ( 0.0 195.8 ( 9.8

22 h after coating (stage 18-20) 18.2 ( 1.9 31.7 ( 8.0 0.2 ( 0.0 75.1 ( 5.0 6.7 ( 1.2 16.2 ( 4.5 0.1 ( 0.0 4.3 ( 0.6 1.3 ( 0.0 27.1 ( 0.5 2.0 ( 1.2 392.3 ( 8.0 9.5 ( 0.2 150.2 ( 27.5

46 h after coating (stage 25-29) 25.5 ( 1.2 0.7 ( 0.0 9 ( 0.0 0.03 ( 0.0 1.3 ( 0.1 1.0 ( 0.3 5 ( 0.6

Each result is the mean of three determinations ( SD. b Coatings referred to alginate coating including the JC.

In other words, since the CAMMR solution contains less calcium, barium, copper, zinc, or strontium, reaction with non-crosslinked regions within the gel layer are less likely. A spontaneous cross-linking reaction between alginate and excess calcium salt (as happens here) is known to produce a less ordered gel relative to a slow cross-linking reaction, which yields a potentially stronger, more ordered network (Nussinovitch et al., 1990). The embryos coated with the stiffer alginate gel coating developed normally within the coating but exhibited lower hatching rates. This is due to the stronger barrier and hence the more energy the embryo needs to invest

in bursting both the jelly coat and the alginate coating via enzymatic and mechanical activity (see previous discussion). Such coating systems, which postpone embryo hatching, can therefore be useful in long-term laboratory experiments. For such uses it is crucial to optimize the working parameters, such as alginate type and concentration, cross-linking agent type and concentration, time of alginate exposure to the cross-linking agent and the composition of the medium in which the embryos are stored. Other conditions, such as temperature, pH, etc. need to be kept constant and as close as possible to normal biological conditions.

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After coating, the excess minerals (observed by ICP) contained within the alginate gel coating are prone to diffusion. Immediately after cross-linking, excess minerals, particularly calcium and sodium, have a tendency to diffuse into the embryo (Table 4), presumably through the ion channels or the membrane itself (Gillespie, 1983; Gillo et al., 1996). Thus, those minerals are expected to increase within the embryo and decrease in the coating membrane. After a while, this increase slows as a result of the specific activity of the ion channels and the potential ionic diffusion through the gel coat to the surrounding medium (Dascal and Boton, 1990).

Conclusions
Alginate coating of embryos is different from entrapment in that the coating around the embryo is thinner, comprising no more than 16% of the embryos diameter. The thin alginate film is glued to the natural jelly coat directly or via a calcium bridge. It protects the embryo against bacterial contamination and mechanical damage and, under stressful conditions, improves its performance. Under comfortable conditions, its main contribution is that it extends the time for which the embryos can be utilized in the laboratory. Coated embryos hatch at a later stage and are therefore more developed and better able to resist natural hazards.

References and Notes

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Accepted for publication January 7, 2000. BP990153U