Evidence of collagen in the egg capsule of the dogfish, scyliorhinus canicula

Comp. Biochem. Physiol., 1976, Vol. 53B, pp, 539 to 543. Peroamon Press. Printed in Great Britain

EVIDENCE OF COLLAGEN IN THE EGG CAPSULE OF THE DOGFISH, SCYLIORHINUS C A N I C U L A M. RUSAOU~N1, J.-P. PUJOL 2, J. BOCQUET2, A. VEILLARD3 AND J.-P. BOREL3 i Laboratoire de Cytologie, Universit6 Paris VI, 7 Quai St. Bernard, 75005 Paris Laboratoire de Biologie Marine, 14530 Luc-sur-Mer a Laboratoire de Biochimie M+dicale, U.E.R. de MOdecine, 51095 Reims, France

(Received 16 January 1975) Abstract--1. The amino acid composition of the dogfish egg capsule and X-ray patterns of samples are conclusive regarding the evidence of collagen in this structure. 2. Labelling of the egg capsule secreting gland (nidamental gland) with radioactive proline demonstrates that this collagen is secreted by very narrow cells of tubules located in the areas D and E of the glands. 3. These cells alternate with large prismatic cells secreting at least another protein, which contains a high amount of tyrosine and SH groups.

INTRODUCTION

Oligochetes cuticle, according to the sole evidence THE FIRST significant studies on the nature of the egg provided by Faur6-Fremiet et al. (1938). As a first step in investigations undertaken to elucicapsule of Selacians were made by Faur6-Fremiet and his collaborators in the period 1937-1939 when work- date the process of Selacian egg capsule formation, ing on scleroproteins. The presence of sulphur in the we made an attempt to determine the nature of prosecretory granules of the nidamental gland as well teins involved. In recent years our knowledge of as in the egg shell led these authors to range that fibrous proteins has significantly increased and we pliant, elastic, resistant structure among "secreted ker- have applied current methodology to ascertain atins" and they called it "ovokeratin" (Faur6-Fremiet whether collagen was present in the egg capsule or not. & Baudouy, 1938). However, further investigations of the physical properties of the capsule (water content, X-ray diffracMATERIALS AND METHODS tion pattern: Baudouy, 1938; Champetier & Faur6Materials used in .the following study are Scyliorhinus Fremiet, 1938; Garrault & Filhol, 1937) change that view and these authors came to believe that this struc- canicula egg capsules and the capsule-secreting nidamental glands. The shell containing the dogfish egg appears as ture was more closely related to collagen-type proa small convex rectangular-shaped capsule. The two anterteins than to cutaneous keratins. ior corners elongated into a pair of horns and the two In a comprehensive study of quinone tanning pro- posterior ones are each provided with a long, coiled threadcess, Brown (1950) draws a parallel between byssus like filament (Fig. 1). The newly-laid eggs are fixed by the formation in the mussel whose collagen component female onto an available support, mostly sea-weeds. has been studied by Pujol (1967, 1970) and that of The twin glands which secrete these capsules surround the dogfish egg-case. The presence of phenolic com- the oviducts at the first third upper level. Each gland is pounds and phenoloxydase was demonstrated by ana- composed of two symmetrical halves which roughly look like kidneys (Fig. 2). The volume of the gland undergoes lysing shell fragments. Threadgold's (1957) histochemical study of the important changes throughout the sexual cycle: it reaches 3.5 cm in length and 2 cm in diameter at egg-laying time, nidamental gland has strengthened this theory which but only appears as a slight bulge of the oviduct during is not inconsistent with the presence of sulphur-con- the period of sexual inactivity. taining proteins (Vovelle, 1965). A longitudinal section of the gland (Fig. 3) shows a The presence of collagen-like compound in the structure composed of similar-looking secretory tubules structure was also suggested by Krishnan (1959) who (Henneguy, 1893; Nalini, 1940). However, they are set in analysed the egg capsule of Chiloscyllium with a wide variety of patterns and secrete various substances, roughly methods. He failed to detect hydroxylysine, so that one can distinguish five different areas within each leucine and valine in paper chromatograms of acid half of the gland (Fig. 4) (Threadgold, 1957). The tubules hydrolysates but revealed the presence of trypto- from zone D open into the internal folds of the glandular mucosis. These lamellae-shaped folds are parallel and horiphane. Taking into account the variety of collagen zontal (Fig. 3), and make a clearly visible band on the in the animal life, he concluded that the egg capsule internal upper part of the gland, below area A (Fig. 2b). protein might be allied to the collagen group. Unaware of Krishnan's studies, Gross & Piez Methods (1960) range the protein secreted by the nidamental The egg capsules were slit open, carefully washed and gland among collagens along with Mytilus byssus a n d kept in distilled water at 4°C. They were allowed to dry 539

M. RUSAOU~Net al.

540

The spots of the separated sugars were identified by comparison with standards run in the same condition. They were cut out and sugars were recovered by clution for further titration with the potassium ferricyanide technique of Montreuil et al. (1965).

Wide-angle X-ray diffraction The intensity of X-rays diffracted by samples of the egg shell was measured in a plane by means of a proportional counting device set on a motor-rotated goniometer (C.G.R.). The primary monochromatic beam is produced in a narrow-focus tube (C.G.R.G.C. 342.0) fitted with a long focal distance (510mm) monochromator (C.GR. Presse R-1400). Samples are placed at the centre of thc goniometer perpendicular to the X-ray incident beam.

Autoclaving procedure Fragments of the egg shell were minced and ground m distilled water using an Ultra-Turrax homogenizer. The mixture was then heated up to 123°C for 3 hr in an autoclave. After cooling, it was centrifuged at 40,000 ,q for l hr and the supernatant dialyzed several times against distilled water.

0.5¢

Semi-thin sections 0[' the shell

3

=~

~v

4

Fig. 1. Egg-capsule of the dogfish Scyliorhmus canicula. Fig. 2. (a) Half nidamental gland, external view. tb) The same half gland, internal view. La. Lamellae, Fig. 3. Longitudinal section of a half gland. La, Lamellae; A-D-E. areas A, D and E. Areas B and C are not visible here. ov. oviduct. Fig. 4. Skctch of a half gland showing the different secretor 3 areas, ov, oviduct: A-B-C-D-E, the areas (after Threadgold. 1957. modified).

at room temperature before use. They can equally be ground for assays. Hydroxyproline content. Al~er hydrolysis of the samples in 6 N HC1 at 1IffC for 24 hr, the Prockop & Udenfriend's (1960) method of titration was used. Amino acid analyfls. Analyses were performed on a Technicon amino acid auto-analyser, on samples obtained after hydrolysis at I I0'C for 24hr in 6N HC1 (Moore & Stein, 1963). Values were not corrected and were not extrapolated to the zero time of hydrolysis, Neutral sugars analysis. Samples were hydrolysed for 10 hr at 100'C in 2 N HCh The hydrolysates were evaporated to dryness, the residue diluted in distilled water and passed down through a column of Dowex 50 (Dowex 50, × 12, 200~400 mesh, form H+). The eluate was concentrated and determination carried out by the sulphuric orcinol method (Rimington, 1940).

Determination and quantitative chromatography o['neutral sugars. Hydrolysates processed as before were concentrated and applied onto a thin cellulose plate (Dc. Plastikfolier cellulose, Merck). Chromatography was carried out in the mixture propanol:ethyl:acetate water (5:1: 1). Neutral sugars were detected on the plates by spreading aniline oxalate or ammoniacal silver nitrate solutions. Titration of neutral sugars was performed after chromatographic separation on Whatman No. 1 paper. Solvents used butanol:acetic acid:water (4:1:5) (Partridge, 1948) and pyridine: ethyl acetate : water ( I : 2: 2) (Jermyn & lsherwood. 1949).

For morphological studies, small strips of egg shells were fixed at 4 C in 2% glutaraldehyde in cacodylate buffer (pH 7.4) for 1 hr, rinsed several times in cold buffer, then postfixed, at 4 C , in 1% osmium tetroxyde (same buffer) for a further hour. After dehydration, specimens were imbedded in Epon. Sections (0.5 1 #m) were stained with methylene blue and Azure A.

Histoautoradiography of the nidamemal gland 500/~Ci of tritium labelled proline (L-[4-3H]-proline. S.A. 15 20Ci/mM--C.E.A.. France) diluted with l ml of physiological liquid for Selacians (Bocquet & Pujol, 1973) were injected into the heart of animals. The nidamental glands were removed l hr after injection and each hall" gland kept in Carnoy fixative for 2 hr, or neutral salted formol for 24 hr. The organs were in full secretory activity since a capsule-wrapped egg was extracted from each oviduct when removing the gland. After paraplast embedding, longitudinal sections of each half gland (5-7-5 #m) were mounted on glass slides. After drying, the slices were dipped into llford K emulsion diluted 50",~; with distilled water and exposed at 4'C for 3 wk. The radioautographs were revealed using Kodak D 19 developer. After washing, the slices were stained with nuclear red and picric indigo carmin (Montreuil & Langlois, 1962),

RESULTS

Egg shell structure The dogfish egg shell appears as a stratified structure (Widakowitch, 1906) tanned by a polyphenol oxydase secretion (Brown. 1950). According to Widakowitch (1906) and Borcea (1906), the shell would contain as many layers as interlamellar spaces present in nidamental gland (Figs. 3 and 5). In the optical microscope, a transversal semi-thin section of the shell stained with methylene blue and Azure A shows that the capsule wall is divided into four different looking areas (Fig. 6). The outer layer L~, 25 tim thick, displays a finely fibrillous surface and numerous granules in its inner portion. The next layer L,_ is the thickest (225 tim approximately) and is built of regularly repeated laminae; the most cyanophilic p,,rtion of those laminae rapidly decreases in thick-

Evidence of collagen in the egg capsule of the dogfish

541

teins present in the shell partly conceal the composition of pure collagen; they might account for the unusually high rate of tyrosine, the presence of sulphur containing amino acids, or the low level of glycine. The detection of methionine and cysteine is not surprising since larger cells than those which secrete collagen can be found in the secretory tubules of the glandular areas D and E, and they secrete a sulphurcontaining protein (Fig. 9). This protein was called "prokeratin" by Faur6-Fremiet et al. (1938). A few unidentified ninhydrin positive components also appear on the analyzer trace; some of them were also mentioned in connection with other tanned structures: Buccinum operculum (Hunt, 1970) or Fig. 5. Longitudinal section of area D at the lamellae level Mytilus byssus (Pikkarainen et al., 1968). The signifishowing the secreted filaments. F, filaments; La, lamellae cant peak eluted before glycin might be interpreted (after Borcea, 1906). as desmosin (Hunt, 1970). This peak is absent in autoFig. 6. Transversal section of the egg-shell. L1, outer layer; clave extracts analysis (Table 1). Extraction by autoclaving procedure at 123°C L2, second layer; L3, third layer; L4, inner layer. proved to be quite efficient in the case of mussel byssus, a collagen-containing tanned structure (Pikness from the second third of the layer (Fig. 6). The karainen et al., 1968). The yield reached 11% of solunumerous granules of the outer layer (L1) are also ble products which represents 47% of the total colpresent in the upper part of the second layer (L2), lagen. Unfortunately, the amount of soluble products and their number gradually decreases to reach zero obtained from the egg shell through the same proat the level of the 7th lamina of this second layer. cedure does not exceed 4~o which roughly represents The third layer is slightly thicker than the second 3'4% of the total shell collagen content. In fact, we one (approximately 30 #m), it looks more homo- do not find a marked difference in amino acid compogeneous than the previous ones. However, it is very sition between these extracts and the complete egg finely stiated, the striae being orthogonal to the shell (Table 1); in addition, the extracts do not display second layer laminae. At last, we find the extremely an increase in the amino acids characteristic of colthin inner layer (4~5 #m) which appears homogeneous lagen; sulphur containing amino acids are equally in light microscopy. These observations were made present. after dehydration; dehydration should reduce the thickness of layers by 15% (Faur6-Fremiet, 1938). Besides, we must mention that the egg shells studied T a b l e 1 - A m i n o a c i d c o m p o s i t i o n of S. c a n i c u l a here were tanned. Tanned shells seem to be thinner e g g shell ( R e s i d u e s / 1 0 0 0 t o t a l r e s i d u e s ) * than those removed from the oviducts, and the appearance of layers also differs significantly Amino acids Egg s h e l l Autoclave extract (Rusaou~n, in preparation). Evidence Jbr collagen Colorimetric titrations show that the shell contains 1.9mg of hydroxyproline/100mg. Thus, we may assume that collagen represents from 13 to 14% of its global composition. We tried to separate the fours layers of the shell under binocular lens, but only tx~. layers were actually isolated, L1 and L4. The two m ternal layers remained partly intermingled. Then we carried out three different titrations: LI, L2 +L3, L4.

Hydroxyproline content regularly increases from the outer to the inner layer: L~ = l167/~g/100mg; L2 + L3 = 1622/~g/100mg; L4 = 2131 #g/100mg. We are therefore led to believe that collagen is more abundant in the inner layers of the capsule. The network of laminae and orthogonal fibrils of L2 and L 3 may account for the cohesion of these two layers. Their respective amounts of hydroxyproline remain to be found. Amino acid composition The results of the amino acid assay (Table 1) evidenced that collagen is present in the egg shell since hydroxyproline, high content of proline and traces of hydroxylysine could be detected. But the other pro-

O H Pro Asp Thr Ser Glu Pro

39 125 41 33 50 81 ? 159 70 49

40 139 35.5 63.6 70.9 112.2

1/2 Cys Cys S O 3 H

10

19.6

Met Met SO 2

13

GIy Ala Val

Ileu Leu Tyr Phe O H Lys Lys His

Arg

30 33 106 31 ?? Trace 46 43

41

171 48.9 17.3

Trace

13.6 14.8 125.5 22.5 ? 0 29.6 47. Z ? 28.7

* The i n t e r r o g a t i v e points show out the unknown p e a k s .

542

M. RUSAOU~!Net al.

It seems likely that the autoclave method of extraction only releases an incompletely tanned fl'action of the shell.

IL

Neutral sugars titration The egg capsule contains approximately 0"21~i; of neutral sugars. We have detected the presence of glucose, galactose, mannose, fucose and xylose on chromatograms. The molar ratio is: xylosc I, fucose 2, mannose 2, galactose 2.4, glucose 3.4. These figures cannot be accounted for by the sole presence of collagen: mannose, fucose and xylose suggest the presence of glycoproteins.

X-ray d!flJ'action X-ray diffraction shows that the three-chain coiled helix of collagen is present in the egg shell. A first peak is visible at 7 5 and corresponds to the distance between the parallel rows of collagen molecules. The second shoulder at 31 reflects the distance between two successive amino acid rcsidues. The high peak at 20' mainly indicates the diffraction of amorphous regions of the macromolecule (Fig. 9t.

H istoautoradiography The Scyliorhimcs canicula nidamental gland does not diffcr from those of other Selacians. It is composed of secretory tubules formed by large prismatic cells interspaced with very narrow ciliated cells (Henneguy, 1893: Nalini. 1940). These two types of cells are found everywhere in the gland and are not specific to any particular area. The labelled proline injection has led to the localization of the collagenic protein secretory areas, i.e. areas D and E (Fig. 4). Some proline labelled tubules are also visible in area C but the incorporation is very weak (Fig. 7). Areas A and B are not labelled at all, which is perfectly consistent with all histochemical tests previously carried out on the nidamental gland. A more detailed study of each tubule in the collagen-secreting areas shows that the synthesis takes place inside the very narrow cells of the tubules (Fig. 8). The large prismatic cells which were up to now

Fig. 7. Longitudinal section of the ghmd showing the incorporation of tritium labelled proline. Notice the unlabelled tubules in area A. I.s.t, labelled secretory tubules A-C-D. areas A, C and D. Fig. 8. Transversal section of a secretory tubule of area D showing the labelled narrow cells. I.n.c, labelled narrow cell: p.c, prismatique cell: In., lumen.

t

L II

~,

t i

~•

g +.

v::& ",52

]5

', ,

,&

5

I0

15

20

25

30

5

35

Angles

Fig. 9. X-ray diffraction pattern ol S. canicula egg shell for two orthogonal planes. No special direction of the fibres had been detected by this procedure.

regarded as the shell protein secretory cells show no trace of labelling. But they are, on the other hand, the privileged localization of sulphur containing protein and tyrosine secretion. Thus we are inclined to believe that the dogfish egg capsule is not composed of a single tanned protein, but of at least two distinct ones: a collagenic protein secreted by the narrow cells, and a second one, originated in the large prismatic cells, which is characterized by a very high content of tyrosine and the presence of SH and SS groups. DISCt SSION The presence of collagen in the dogfish Scyliorhim~s canicula egg shell has been conclusively demonstrated in this preliminary study. The X-ray diffraction pattern clearly shows the characteristic helicoidal structure of this protein. The collagen is secreted by cpithelial cells located in the gland tubules of areas D and E. We find here a type of exocrine process which differentiates these cellular elements from true fibroblasts, and suggests similarity to collagen formation in Mytilus byssus filaments (Pujol, 1967; Pujol et al., 1972; Tamarin & Keller, 1972). In both cases the collagen is associated with quinone tanned proteins. Regarding collagenic protein adaptation to external environment, here sea water, we can note the convergence of natural processes resulting in structures able to withstand this environment: Mytilus, fixation on the substrate, and Scyliorhinus, protection of embryonic development. Variations in secreted collagens could thus be attributed to their environment (Bairati, 1972). The common striated structure of fibrous collagen is not found in the egg shell layers. This may be due to associated proteins on quinone-tanning. In this regard the internal structure of byssus filament pro-

Evidence of collagen in the egg capsule of the dogfish vides significant information (Bairati & Vitellaro, 1973). Discrete filamentous formations have been recently detected in the proximal portion of the filament. They display an alternance of light and dark bands ("filamentous banded elements"). They escaped observations in a Randall et al. study (1952). We are not far from thinking that such a localization is connected with the limited area of the phenol gland at that level, whereas it is much more important at the distal end of the foot. At this localization, the relative collagen content compared to its associated protein is much higher, while quinone tanning is reduced. This would explain why collagen appears here as elements displaying alternate bands. Furthermore, the figures supplied by Bairati & Vitellaro (1973) are to be compared with those of collagen intracellular granule seen in the electron microscope (Pujol et al., 1972; Tamarin & Keller, 1972). Amino acid composition of the egg capsule of Scyliorhinus canicula reveals the presence of one or two non-collagenic proteins. Can this fraction be identifield with Faur6-Fremiet et al. "'ovokeratin'. so-called because of the presence of sulphur in the granules of the gland zone D? Acknowledgement~We thank Dr. Herbage and Dr. Huc (Laboratoire de Chimie des Macromol6cules, Universit6 de Lyon) who kindly carried out X-ray analyses.

REFERENCES

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