DEVELOPMENTAL

BIOLOGY

137,258-265

(1990)

The Major Structural Proteins of Cod (Gadus morhua) Eggshells and Protein Crosslinking during Teleost Egg Hardening’ DAG 0. OPPEN-BERNTSEN~ JON V. HELVIK, AND BERNT T. WALTHER Department

of Biochemistry,

University

of Bergen, ~rstadveien

19, N-5009 Bergen, Nwway

Accepted October 16, 1989

The highly hydrophobic protein aggregate which constitutes the fish eggshell has for the first time been quantitatively solubilized. This study shows that the nonactivated eggshell from cod is composed primarily of only three protein monomers, designated ~~(74kDa) /3(54 kDa) and y (47 kDa). Protein extraction studies of the eggshells before and after egg activation demonstrate that egg hardening is accompanied by a lo-fold decline in total protein solubility, which is due to nonextraction of the (Y,p, and y chains. When present during the egg activation process monodansylcadaverine (MDC-a fluorescent lysine analog) inhibits eggshell hardening and at the same time becomes covalently incorporated into the eggshell. This MDC incorporation is calcium-dependent and suggests the induction of a perivitelline transglutaminase activity after egg activation. (Transglutaminases catalyze the formation of an amide bond (isopeptide bond) between the y-earbonyl group of glutamine and the t-amino group of lysine with release of ammonia. Crosslinks between proteins are generated when the two amino acid residues are located on different proteins.) Protein solubilization studies and NaDodS04 gel analysis of the eggshell proteins from eggs subjected to 5 mM MDC during egg activation, reveal that when eggshell hardening is blocked by MDC, the three main eggshell proteins remain extractable even after egg activation. Simultaneously we observed a covalent incorporation of MDC into the y protein. o isao Academic

Press, Inc.

after fertilization, suggesting that the increase in mechanical strength may be due to the formation of isoLight and electron microscopic studies have during peptide bonds during the hardening process. In contrast the past decades revealed that the eggshell is a complex the hardening process in Echinoderms (Sea urchin) and of several concentric lamellae (zones) with different Lepidoptera (Butterfly) eggs is reported to be due to the electron density (Arndt, 1960; Cherr and Clark, 1982; generation of di- and tri-tyrosyls through the action of Kudo, 1982, Hosokawa, 1985). The nomenclature used to a peroxidase (Shapiro et al, 1981; Griffith and Lai-Fook, describe the extracellular egg membranes is rather 1986). confusing not only between different phyla, but also Different solvents have been reported to be effective between teleosts. Often homologous structures are desfor solubilizing fish eggshell proteins. Treatment with ignated differently, and different structures are someSweitzer’s reagent followed by titration with 1 N NaOH times given the same name (Ginzburg, 1968). In this was reported by Kaighn (1964) to bring about a slow paper the term xona radiata is used for the innermost, dissociation of eggshells isolated from fertilized Funoften perforated, zone of the eggshell. The second and dulus heteroclitus eggs. Certain carbohydrate-containmore homogenous layer together with the third extering buffers have been reported to be adequate solvents nal jelly layer will be referred to as xona pellucida. for the proteins of the xcmapellucida (Hjerten and Wu, After fertilization or after parthenogenic activation 1985; Kudo and Inoue, 1986). &ma pellucida is a minor the eggshell undergoes a calcium-dependent hardening structure of the fish eggshell, and its glycoproteins may process, which induce a lo-fold increase in the mechanical strength of the eggshell (Davenport et al., 1981, not be representative of the proteins of xona radiata. Lenning et al. 1984). The mechanism of this process is The low solubility of the fish eggshell proteins under unknown, but Hagenmaier et al. (1976) found isopeptide nondestructive conditions has from the start complibonds3 in the eggshell proteins of rainbow trout only cated biochemical analysis of these proteins (Young and Inman, 1938). Characterization has therefore been limited to amino acid composition (performed on acid hyi Supported by a grant from the Norwegian Fisheries Research drolysates of the whole eggshell) (Young and Smith, Council (NFFR). INTRODUCTION

’ To whom correspondence should be addressed. 3Transglutaminases catalyze the formation of an amide bond (isopeptide bond) between the y-carbonyl group of glutamine and the t-amino group of lysine with release of ammonia. Crosslinks between 0012-1606190$3.00 Copyright All rights

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

proteins are generated when the two amino acid residues are located on different proteins. 258

OPPEN-BERNTSEN,HELVIK, AND WALTHER

Cod Eggshell Proteins

259

1955; Kaighn, 1964; Hagenmaier et al, 1976), histochem- Preparation of Dried Eggshell Proteins ical studies (Hagenmaier, 1973; Kudo, 1982), and identiTo avoid hardening resulting from parthenogenic acfication of the major soluble degradation products of tivation, unfertilized eggs were immediately immersed the eggshell. Studies of proteins derived from the in a large volume of ice cold solution of 100 mM EDTA hatching process have shown that these proteins are and 500 mM NaCl, (pH 8.5) (Buffer A). Homogenization glycosylated (Iuchi and Yamagami, 1976; Hagenmaier, of fertilized and unfertilized eggs was performed in the 1985). The amino acid compositions of fish eggshell same solution using a Dounce homogenizer with a proteins differ from that of other structural proteins loosely fitted glass piston. The eggshells were rinsed in such as collagen, elastin, keratin (wool), and silk fibroin five changes of 500 mM NaCl for 24 hr, followed by five (Kaighn, 1964; Hagenmaier 1985) and has substantiated changes of distilled water to remove salts, all at 5°C. their classification by Block (1937) as pseudokeratins The purity of the eggshells was checked by monitoring (or ichtulokeratins). the washwater absorbance at 280 nm. The eggshells The aim of this paper is to define the nma radiata in were subsequently placed on a watchglass and dried in terms of a macromolecular model of its constituent an incubator at 35°C or lyophilized. Both procedures monomeric proteins. We have therefore developed ex- gave eggshells with stable proteins. Proteins were traction procedures for integral eggshell proteins which stored at room temperature or at -80°C. Eggshells rely on prior blocking of the egg hardening process. from fertilized eggs were prepared the same way, but Methods applying a fluorescent lysine analog (Mono- the treatment with Buffer A was omitted. dansyl cadaverine) has made it possible for the first time to demonstrate the mechanism and molecular kiSolubilixation of Eggshell Proteins netic of hardening and identify one of the eggshell protein monomere involved in crosslinking. The eggshells were solubilized (1 mg/ml) in 100 mM Tris/HCl (pH 8.8) containing 8 M urea, 1% NaDodSO,, 300 mM mercaptoethanol and 10 mM EGTA (Buffer B: MATERIALS AND METHODS extraction buffer) for lo-20 min using a waterbath at Materials 70°C. The sample was sonicated at ambient temperaAll reagents used were of analyttical grade. MDC4 tures for 30-60 see using a Branson B-15 cell disruptor with a 50% duty cycle and an output of 60 W. Then the was obtained from Sigma. Sexually mature cods (Gadus morhua) were kept in a sample was replaced in the waterbath for about 5 min spawning pen at Austevoll Marine Aquaculture Station before the sonication procedure were repeated. Solubinear Bergen. Fertilized eggs were collected as described lized protein was measured in the extract according to by Huse and Jensen (1983), while unfertilized eggs were Lowry et al (1951) after extensive dialysis against 50% removed from 5-kg female cods by stripping and treated glycerol and 50 mM Tris/HCl (pH 8.8) at ambient temas described below. Unfertilized and fertilized eggs peratures and centrifugation to remove possible unsolfrom halibut (Hippoglossus hippoglossus) were collected ubilized eggshell protein. weekly at Austevoll during stripping of the halibut production stock. Fertilized and unfertilized eggs from sal- In Viva Incorporation of Monodansylcadaverine (MDC) mon (Salmo salar) were obtained from Matre AquaculMDC (0.67 g) was added to 1 ml ethanol (absolute) ture Station near Bergen, and transported in a styro- and 0.1 M HCl was added dropwise until MDC was comfoam container under melting ice. Eggs from pletely dissolved. This MDC stock was added to 200 ml lumpsucker (Cyclopterus lumpus) were obtained from of seawater with constant stirring. The pH was adProfessor S. Lenning, University of Tromse, and were justed to 8.5 by dropwise adding 0.1 N NaOH. The careairfreighted on ice. Unfertilized eggs were transported ful pH titration was necessary in order to avoid precipiin ovarian fluid, while the fertilized eggs were trans- tation of MDC. ported in seawater. Eggs were stripped from several female cods to get a 4Abbreviation used: MDC: monodansylcadaverine, fluorescent lysine analog; Buffer A: isolation buffer for unactivated eggs, see Materials and Methods section; Buffer B: extraction buffer for eggshell proteins, see Materials and Methods section; TRIS: tris-(hydroxymethyl)-methylamine; NaDodSOl: sodium dodecyl sulphate; EDTA: ethylenediaminetetraacetic acid; EGTA: ethylene glycol bis (p-aminoethylether)-N,N,N’,N’-tetraacetic acid TEM and SEM: transmission and scanning electron microscopy, respectively.

sufficient amount of unfertilized (and unactivated) eggs. The eggs were gently blended to obtain a homogenous egg batch, which was divided into eleven aliquots of 50 ml each. Activation was started by addition of an equal volume of the MDC solution. The hardening process was carried out at 5 C” under gentle aeration, allowing for incorporation of MDC under in vivo conditions. The incubations were stopped at different times

260

DEVELOPMENTALBIOLOGYV0~~~~137,1990

by washing the eggs in large amounts of cold Buffer A. Electron Microsco(py The eggs were homogenized as described above, and The eggshells used for scanning electron microscopy eggshells were separated by filtration from the yolk and were fixed in 100 mM cacodylate buffer, pH 7.2, concytoplasma before storage at -80C. Aliquots of the isotaining 8 mM CaCl,, 4% sucrose and 2.5% glutaraldelated eggshells from the different egg groups (2-3 ml) hyde for 24 hr. The fixative was removed by washing in were thawed in 20 ml of Buffer A and rinsed as deseveral changes of 10 mM phosphate-buffered saline, scribed under “Preparation of dried eggshell proteins.” pH 7.5, for 24 hr. Osmication was performed in a 1% The eggshells were extracted for 24 hr and five changes OsOI solution for 60-90 min, followed by dehydration in of ethanokether (1:l) to remove unbound MDC (Lorand increasing concentrations of acetone; 1X 70%) 1X 80%) et al. 1969). The washed precipitates were isolated by 1X 90%, 3X loo%, with 20-30 min for each step. Criticentrifugation and dried under nitrogen. The solubility cal-point drying was performed under COZ, of proteins in these eggshells was tested in Buffer B. RESULTS

Control Experiments

Eggshell Solubilixation Studies

As control experiments to the in vivo incorporation of MDC, two experiments were carried out: Incubation of fertilized eggs in the presence of MDC and Ca-ions, and fertilization of eggs in the presence of MDC in a medium devoid of Ca-ions containing 1 mM EDTA.

The eggshells from unfertilized (and unactivated) cod eggs are solubilized with relative ease in Buffer B when heated to 70°C and sonicated. These conditions gave translucent protein solutions without unsolubilized material. Protein determination performed after dialysis of the extracts indicated that more than 80% of the total dry weight protein had been solubilized (Fig. lA, a columns). Some of the remaining (20%) eggshell proteins were shown to be peptides of low molecular weight by direct NaDodSOl gel electrophoresis of eggshell extracts (data not shown), and such proteins appear to be lost during dialysis (cut-off 10 kDa). Similar results were also obtained with eggshells isolated from unfertilized (an unactivated) eggs from halibut, lumpsucker, and Atlantic salmon (Fig. lA, a columns). In contrast, when identical solubilization studies were carried out with eggshells isolated from fertilized (or activated) and hardened cod eggs, the solubility of the eggshell proteins had decreased about lo-fold (Fig. lA, b columns). Again, similar results were obtained with fertilized (or activated) and hardened eggs from halibut, lumpsucker, and Atlantic salmon. The electrophoretic migration pattern of the proteins present in the cod eggshell extracts is shown in Fig. 1B. Three major bands were observed and are designated (Y, 6 and y (Fig. lB, lane a), with apparent molecular weights of 74,54, and 47 kDa. These three bands are not seen in the extracts from fertilized and hardened cod eggshells, which contained predominantly polypeptides (6 chains) of lower molecular weight (Fig. lB, lane b). The same amount of a-chains are also present before fertilization, and may be visualized in heavily overloaded gels (data not shown). The lo-fold decrease in total extractable protein from fertilized compared to unfertilized eggs (Fig. 1A) is therefore the result of a selective loss of protein extractability. The major proteins of the eggshells are no longer extractable from hardened eggshells, while the minor proteins remain extractable from such eggshells. These smaller (6) pro-

Gel Electrophoresis NaDodS04-polyacrylamide gel electrophoresis was performed according to Laemmli (1970). Prior to electrophoresis the dried or lyophilized proteins were solubilized as described above. Silver staining of the gels was carried out using the procedure of Wray et al. (1981), while staining with Coomassie was performed in a 0.2% (w/v) Coomassie brilliant blue in acetic acid:methanol:water (1:lO:lO) solution. Stained gels were photographed on a conventional light box. Gels containing fluorescent proteins were photographed on a UVP Transilluminator, TM 20,302 nm, using a Wratten no. 8 (yellow) filter with Polaroid high speed 4 X 5 instant sheet film. Molecular weights of silver-stained and fluorescent proteins were estimated relative to Pharmacia low molecular weight protein calibration kit. Measurement of MDC Incorporation The incorporation of MDC was measured in the different protein solutions using a Perkin-Elmer (LS-5) Luminescence spectrometer with excitation at 335 nm and emission at 525 nm. Relative fluorescence per mg protein was plotted against time with the Buffer B as blank. Amino Acid Analysis Amino acid analysis was performed according to Vasstrand et al. (1980). Proline was detected in the same regime at 440 nm.

OPPEN-BERNTSEN,HELVIK, AND WALTHER

261

Cod Eggshd Proteins

A a

salmon

cod

100

lumpsucker

halibut

a

a

CY

b

78

F z 80 B pjI % x 20

a

D

a

t3

TV

t3

FIG. 1. (A) The figure demonstrates the general nature of the phenomenon that results in reduced solubility of eggshell proteins upon fertilization; (a) before, and (b) after, fertilization and complete hardening. The four different fish species span the teleostean phylogenetic tree, i.e., Salmon represents a primitive teleost, cod belongs to the intermediate teleosts, while lumpsucker and halibut represent the more advanced families in this infraclass. The histograms show the amounts of extracted protein recovered (measured according to Lowry et al, 1951) as the percentage of total dry weight protein subjected to extraction. Number of extractions was 4 (n = 4). The largest empirical standard deviation was 3.5% (s = 3.5%). Bar indicates two standard deviations. (B) NaDodSO,-polyacrylamide gel electrophoresis (10%) of extracts from (a) unfertilized and (b) fertilized and completely hardened cod eggshells. The same amount of protein (5 pg) was applied to each well. Molecular weights in kilodaltons are indicated. The gel was silverstained according to Wray et al, 1981. Molecular weights were calculated as described under Material and Methods.

teins were also found to be readily solubilized by extraction buffers containing carbohydrates according to the procedures of Kudo and Inoue (1986). Such buffers also solubilized some /3chains but no (Yor y chains from fertilized cod eggshells.

tered solubility of eggshell proteins even after egg actiin the presence of calcium ions (Fig. 3A). At the same time the eggshells became strongly fluorescent. Total protein extracts from such eggshells demonstrated a progressive increase in relative fluorescence per mg protein with time (Fig. 3B) from incorporated MDC. The increase of fluorescence reveals identical kiEggshell Morphology netics to the reported increase in mechanical strength The external surface of cod eggshells seen on the miof the cod eggshell reported by Lsnning et al. (1984). The crograph in Fig. 2A, is quite smooth but exhibits pores covalent incorporation of MDC into eggshell protein is arranged in a regular pattern as described for several substantiated by the presence of a major high molecuspecies (Groot and Alderdice, 1985). In contrast, the lar weight fluorescent band after NaDodSOl gel electrointerior surface of cod eggshells exhibits a nonsmooth phoresis (Fig. 4A, lanes 12 and 24) even after extensive structure. Large variations between different species extraction with ethanol and ether. However, if the are apparent. While the inside of cod eggshell shows no MDC-inhibition of eggshell hardening was attempted in particular pattern (Fig. 2B), the inside of a salmon egg- absence of calcium ions, no covalent incorporation of shell (Fig. 2C) clearly shows a fibrillar structure. In the MDC was detected (and no hardening of the eggshell case of cod and halibut the X(WM.radiata shows no oroccurred). Also the control experiment performed on dered fibers when examined by scanning electron mifertilized eggs in the presence of calcium ions revealed croscopy even at higher magnification. no incorporation of MDC. The highly hydrophobic cod eggshell proteins apThe Hardening Process peared to be even more hydrophobic after MDC incorpoThe hardening process in cod eggs was completely ration, possibly because of the hydrophobicity of MDC. inhibited in the presence of MDC and resulted in unal- This sometimes caused precipitation of the proteins in vation

262

DEVELOPMENTAL BIOLOGY

VOLUME 137,199O

FIG. 2. Scanning electron micrographs of (a) the exterior (Zone pellucida), and (h) interior (Mona radiata) surfaces of an eggshell from cod. (c) Scanning electron micrograph of the interior (Mona radiate) surface of salmon eggshell. All micrographs are equally magnified and both bars represent 1 pm.

the well during NaDodSOI gel electrophoresis. Overloaded gels tended to yield progressively less relative fluorescence in the gel and could occasionally cause severe problem during the performance of quantitative PAGE analysis such as Fig. 4A due to precipitation in some wells and not in other. MDC-labeled proteins extracted from cod eggshells, activated in the presence of MDC, were separated by one-dimensional electrophoresis in NaDodSO,-polyacrylamide gels (Fig. 4A). The molecular weight of the highly fluorescent band corre-

spond to the y-chain of the eggshell proteins. In contrast, Coomassie-stained NaDodSOl gels of such extracts document that the (Y(faint), 6, and y chains are all present in such extracts (Fig. 4B). We are not sure on the explanation as to why the (Y chain becomes faint relative to the 0 and y chain, but the relatively high hydrophobicity of this chain can from time to time cause precipitation when this chain enters the gel. The phenomenon is observed frequently also in preparations that are not incubated with MDC. The same pherB

0

5

10 Time,

15 hours

20

25

o”“‘l’.““.“~““~.‘.., 0

5

10 Time,

15 hours

20

25

FIG. 3. Eggs were collected directly from female cods as described under Materials and Methods. Eleven egg groups (egg volume 200 ml each) were incubated after addition of 200 ml of seawater containing 10 mA4 MDC at 5°C. Calcium ions in sea water initiate egg activation. The incubation was stopped at indicated times after activation. Eggshell proteins from the different groups were solubilized in Buffer B, and protein and fluorescence measurements were performed on the same extracts. (A) Amounts of solubilized cod eggshell proteins (measured according to Lowry et &, 1951) from eggs activated in the presence of 5 mMMDC, as a function of time after egg activation. (B) Incorporation of MDC into cod eggshell proteins as a function of time after egg activation. Incorporation of MDC is plotted as relative fluorescence/mg protein.

OPPEN-BERNTSEN,HELVIK, AND WALTHER

Cod Eggshell Proteins

263

completely inhibited in media without calcium ions. The fluorescent lysine analog MDC inhibits the mechanical hardening of cod eggs (Lonning, Oppen-Berntsen and Walther, unpublished observations) as well as the decrease in solubility of eggshell proteins (Fig. 1A). MDC apparently becomes covalently linked to the eggshell proteins when present together with Ca-ions during the hardening process, since it is not removed from the eggshell proteins even after extensive extraction with ethanol and ether, by gel filtration or by NaDodSO1polyacrylamide gel analysis. Such procedures have previously been used to demonstrate MDC incorporation into fibrin (Lorand et al, 1969; Lorand and Campbell, 1971). The inhibition of the hardening process with concurrent covalent incorporation of MDC into eggshell proteins (Fig. 4) lends support to the hypothesis of HaFIG. 4. NaDodSO,-polyacrylamide gel electrophoresis (10%) of genmaier et al. (1976) that the hardening enzyme is a enzyme. MDC fails to be incorprotein extracts from cod eggshells which had been activated in sea- transglutaminase-type water containing a final concentration of 5 mMMDC. Numbers above porated into the eggshell in absence of Ca-ions or into each lane indicates the time in hours after egg activation until the the eggshell of already fertilized eggs. This strongly incubation was stopped. (A) Unstained gel photographed when transsuggests that the incorporation of MDC is limited in illuminated by a uv source. The main fluorescent band exhibits a time to the following 24 hr after fertilization in a calmolecular weight corresponding to the y chain. (B) Extract identical cium-dependent manner, thereby further supporting to lane 24 (from Fig. 5A) after staining with Coomassie blue.

A nomenon is also observed in the fibrin system despite the fact that the molar ratio between the monomers of fibrin are shown to be unity. Likewise, the appearance of some new bands has to be delineated. Eggshell Amino Acid Composition There are relatively small differences between the amino acid compositions of the eggshells from the four different species; salmon, cod, lumpsucker, and halibut. Also it is difficult to discern any systematic changes in the amino acid composition before and after fertilization within the same species. The mean content of hydrophobic amino acids for all four species is about 43% (including Ala, Gly, Pro, Val, Leu, Ile, and Phe). We detected no post-translationally modified amino acids, such as dityrosine, hydroxyproline, or hydroxylysine. In addition, no unidentified peaks were observed in the chromatograms. The two sulphur-containing amino acids are present in relatively small amounts (about 3%), and similarly glycine accounts for only about 6% of the amino acids in eggshell structural proteins. DISCUSSION

The decrease in solubility of eggshell proteins after egg activation (Fig. 1A) coincides with the reported increase in mechanical strength of the eggshell after egg activation (Lonning et al., 1984). Both processes are

B

FIG. 5. Postulated mechanism of hardening. (A) The drawing shows two polypeptide chains, one with a lysine residue and the other with a glutamine. The scheme demonstrates the generation of a crosslink between these protein chains upon the catalytic action of a transglutaminase. The reaction mechanism is an elimination of ammonia in which a crosslink is generated between the e-amino group of lysine to the y-amido group of glutamine. (B) The drawing demonstrates the competitive action of monodansyl cadaverine (MDC), where the cadaverine residue mimicks the side chain of lysine thereby replacing the lysine donor protein. MDC is by this mechanism suggested to be incorporated into the eggshell protein causing the glutamine donor protein to be fluorescently labeled. The eggshell will stay solubilizable since the crosslink is competitively inhibited. Lys, Lysine. Gln, Glutamine. R, Monodansyl; this fluorescent residue is coupled to the depicted diamine: cadaverine, which mimicks the side chain of lysine. Transgln., Transglutaminase.

264

DEVELOPMENTALBIOLOGY

the hypothesis that the hardening enzyme is a calciumdependent transglutaminase (Fig. 5). Three polypeptides designated (Y, @, and y are observed following one-dimensional electrophoresis in NaDodS04-polyacrylamide gels of extracts of unfertilized (and unactivated) cod eggshells. These proteins are the major components of the eggshell and have apparent molecular weights of 78, 54, and 47 kDa (Fig. 2). Hence, the huge lamellar structure (zcma radiata), depicted in earlier works on the ultrastructure of cod eggshells (Davenport et ah, 1981; Kjesbu, 1988), rather surprisingly consists primarily of only three proteins. The fact that the eggshell proteins become insoluble after activation (fertilization) and hardening, and that specifically the (11,0, and y chains (unlike the 6 chains) no longer can be extracted from such eggshells, suggests that these three proteins are selectively crosslinked in the eggshell after egg activation. This hypothesis is further supported by the covalent incorporation of MDC principally into the y chain (Fig. 4). Early workers suggested that the generation of disulphide bonds might be responsible for the hardening process (Ohtsuka, 1960). The present data do not support this hypothesis since hardened eggshells are not solubilized by reducing agents such as mercaptoethanol or dithiothreithol. Disulphide bridges may be important in the protein interchain stabilization in eggshells but are apparently not the cause of the reduced protein solubility after eggshell hardening in activated eggs which is also suggested by the relatively low content of cysteine (l.O-1.5%). The fact that it is difficult to detect any differences in the amino acid compositions between noncrosslinked and crosslinked eggshells can easily be explained by the nature of the postulated crosslink: The isopeptide bond is acid labile, hence acid hydrolysates of crosslinked protein will generate the initial amino acids. Several reports show that eggshells from certain species belonging to the families of Salmonidae, Mug& dae, Cyprinodontidae, and Acipenseridue, exhibit large fibers in the xcma radiata ordered in concentric lamellae (Sterba and Muller, 1962; Brusle, 1985; Groot and Alderdice, 1985; Cherr and Clark, 1982; Hagenmaier, 1985). See also Fig. 2C. However, other reports describe only concentric lamellae in the xona radiata, without references to fibrous structures, in species belonging to the families of Gadidae, Labridae, and Pleuronectidae including cod and halibut (Lsnning, 1972). See also Fig. 2B. However, rescently a closer TEM examination of the cod eggshell (Kjesbu, 1988) reveals that this species has a fibrous xonu radiata, although the fibers are much smaller. It thus appears that fish in general have a fibrous .zcmaradiata, although these fibers are of different sizes. It remains to be delineated how these

VOLUME137,199O

fibers are composed from only three monomers, but a hydrophobic aggregation of rod-like monomers is an attractive hypothesis. The low solubility of eggshell proteins in water is likely to be explained by their conformation, i.e., being rod-like with exposed hydrophobic residues rather than globular with internal hydrophobic domains, since the content of hydrophobic amino acids is not exceptionally high. The amino acid composition of fish eggshell proteins is different from collagen, elastin, (Y-and /I-kertins but not unlike (human) fibrin. The three major proteins of the cod eggshell have molecular weights 78,54, and 47 kDa, respectively (Fig. lB, lane a), which resembles the molecular weights of the three monomeric chains of human fibrin (Doolittle, 1981; Henschen et aZ., 1983). Furthermore, the suggested eggshell protein polymerization into an insoluble matrix during egg activation by the action of a (calcium-dependent) transglutaminase is reminiscent of the role of Factor-XIII in mammalian blood coagulation. The crosslinking of human fibrin chains is initiated by the y chain and followed by the a! chain (McDonagh Jr. et aZ., 1971; Lorand et al, 1972), while the /3 chain is not considered to participate in the crosslinking process. This polymerization pattern may pertain to crosslinking of eggshell proteins since we found a somewhat greater extractability of p chains (compared to the (Yand y chains). Our results may indicate either an ancient common ancestry, i.e., homology, between fibrin and the fish eggshell proteins, or merely mechanistic analogies. Answers to such questions are presently sought through further characterization of the eggshell proteins, particularly their amino acid sequences and mode of synthesis during oogenesis. We thank Drs. E. Vasstrand and H. B. Jensen for the amino acid analysis, and Dr. Per Flood for help with the electron microscopy. We also thank professor Sunniva Lenning and cand. mag. Sverre Ulvik for many helpful discussions and Drs. Holm Holmsen, Anders Gokseyr, and Mariann Rand-Weaver for helpful suggestions during the preparation of this manuscript. Finally, we thank Ingvar Huse and his staff at the Austevoll Marine Aquaculture Station for their professional help and patient cooperation during the last 5 years when sampling the material used in this work. REFERENCES ARNDT, E. A. (1960). Untersuchungen uher die Eihulle von Cypriniden. 2. Zellforsch. 52,315-32’7. BLOCK,R. J. (1937). Chemical studies on the neuroproteins IV. On the nature of the proteins of the ectoderm: Eukeratins and pseudokeratins. J. Biol. Chem 121, ‘761-770. BRUSLE, S. (1985). Fine structure of oocytes and their envelopes in Chelm labrosus and Liza aura&s (Teleostei, Mugilidae). Zooll Sci. 2, 681-693. CHERR, G. N., and CLARK, W. H. JR. (1982). Fine structure of the envelope and micropyle in the eggs of the white sturgeon, Acipenser transmontanus Richardson. Dev. Growth D@w. 24(4), 341-352.

OPPEN-BERNTSEN,HELVIK, AND WALTHER DAVENPORT,J., INNING, S., and KJ~RSVIK, E. (1981). Osmotic and structural changes during early development of eggs and larvae of the cod, Gadus morhuu L J. Fish BioL 19,31’7-331. DOOLI~E, R. F. (1981). Fibrinogen and Fibrin. Sci. Amer. 245 (Dec.), 92-101. GINZBLJRG,A. S. (1968). “Fertilization in fishes and the problem of polyspermy.” In (Israel Program for Scientific Translations, Jerusalem, 1972. T. A. Detlaf, Ed.), pp. l-366. GRIFFITH, C. M., and LAI-FOOK, J. (1986). Structure and formation of the chorion in the butterfly Calpodes. Tissue Cell 18(4), 589-601. GROOT,E. P., and ALDERDICE, D. F. (1985). Fine structure of the external egg membrane of five species of pacific salmon and steelhead trout. Canaa! J. Zool. 63,552-566. HAGENMAIER,H. E. (1973). The hatching process in fish embryos. III. The structure, polysaccharide and protein cytochemistry of the chorion of the trout egg, Salmo gairdneri (Rich.). Acta Histochem, 47,61-69. HAGENMAIER, H. E. (1985). The hatching process in fish embryos. VIII. The chemical composition of the trout chorion (Zone radiatu) and its modification by the action of the hatching enzyme. Zool Jb Physiol. 89,509-520. HAGENMAIER, H. E., SMITZ, I., and F~HLES, J. (1976). Zum Vorkommen von Isopeptidbindungen in der Eihulle der Regenbogenforelle (Salmo gairdneri Rich.). Hoppe-Seyler’s Z. Physiol. Chem. 357, 1436-1438. HENSCHEN,A., LOTTSPEICH,F., KEHL, M., and SOUTHAN,C. (1983). Covalent structure of fibrinogen. Ann N. I: Acad Sci. 408.28-43. HJERTEN,S., and WV, B. L. (1985). Studies of the fish zonupeUwida by high performance ionexchange chromatography on agarose columns and free zone electrophoresis. J. Chromatogr. 341,295-304. HOSOKAWA,K. (1985). Electron microscopic observation of chorion formation in the teleost, Nat&on modestus. Zool. Sci. 2,513-522. HUSE, I., and JENSEN,P. A. (1983). A simple and inexpensive spawning and egg collection system for fish with pelagic eggs. Aquacult. Eng. 2,165-171. IUCHI, I., and YAMAGAMI, K. (1976). Major glycoproteins solubilized from the teleostean egg membrane by the action of the hatching enzyme. B&him Biophys. Acta 453,240-249. KAIGHN, M. E. (1964). A Biochemical study of the hatching process in Fund&us heteroclitus. Dev. BioL 9,56-80. KJESBU, 0. S. (1988). “Aspects of the Reproduction in Cod (Gadus morhua I*): Oogenesis, Fecundity, Spawning in Captivity and Stage of Spawning.” Dr. Scient. Thesis, Univ. of Bergen, Norway. KUDO, S. (1982). Ultrastructure and ultracytochemistry of fertilization envelope formation in the carp egg. Dev. Growth D&-r. 24(4), 327-339.

Cod Eggshell Proteins

265

KUDO, S., and INOUE, M. (1986). A Bactericidal effect of fertilization envelope extract from fish eggs. ZooL Sti 3,323-329. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. LORAND,L., and CAMPBELL,L. K. (1971). Transamidating enzymes: I. Rapid chromatographic assays. Anal Biochem. 44,207-220. LORAND, L., CHENOWETH,D., and GRAY, A. (1972). Titration of the acceptor crosslinking sites in fibrin. Ann. N. Y. Acad Sci. 202, 155-171. LORAND,L., URAYAMA, T., DE KIEWIET, J. W. C., and NOSSEL,H. L. (1969). Diagnostic and genetic studies on fibrin stabilizing factor with a new assay based on amine incorporation. J. Clin Invest. 48, 1054-1064. LOWRY,0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurements by the folin reagent. J. Biol. Chews. 193,371-374. INNING, S. (1972). Comparative electron microscopic studies of teleostean eggs with special reference to the chorion. Sarsia 49,41-48. I&NNING, S., KJ~RSVIK, E., and DAVENPORT,J. (1984). The Hardening process of the egg chorion of the cod, Gadus morhua L. and lumpsucker, Cyclopterus lumpus L, J. Fish Biol. 24,505-522. MCDONAGH,R. P., JR., MCDONAGH,J., BLOMB~CK,M., and BLOMBACK, B. (1971). Crosslinking of human fibrin: Evidence for intermolecular crosslinking involving alpha-Chains. FEBS I&t. 14(l), 33-36. OHTSUKA,E. (1960). On the hardening of the chorion of the fish egg after fertilization. III. The mechanism of chorion hardening in Orytzias lutipes. Biol Bull. WoodsHole Mass. 118, 120-128. SHAPIRO,B. M., SCHACKMANN,R. W., and GABLE, C. A. (1981). Molecular approches to the study of fertilization. Ann Rev. B&hem 50, 815-843. STERBA,G., and MULLER, H. (1962). Elektronmikroskopische Untersuchungen uber Bildung und Struktur der Eihullen bei Knochenfishen. I. Die Eihullen junger Oozyten von Cynolebias belotti Steindachner (Cyprinodontidae). Zoul. Jb. Anat. 80,65-80. VASSTRAND, E., JENSEN, H. B., and MIRON, T. (1980). Microbore single-column analysis of amino acids and amino sugars specific to bacterial cell wall peptidoglycans. Anal Biochem 105,X%-158. WRAY, W., BOULIKAS,T., WRAY, V. P., and HANCOCK,R. (1981). Silver staining of proteins in polyacrylamide gels. Anal. B&hem 118, 197-203. YOUNG,E. G., and INMAN, W. R. (1938). The protein of the casings of salmon eggs. J. Biol. Chem. 124,189-193. YOUNG,E. G., and SMITH, D. G. (1955). The Amino acids in the icthulokeratin of salmon eggs. J. BioL Chem 219,161-U%.