Egg-capsule Proteins of Selachians and Trout
By C. H. BROWN
(From Girton College, Cambridge)
SUMMARY
1. The material of the egg-capsules of selachians and the chorion of trout eggs has
been examined by physical, chemical, and histochemical methods.
2. The material of the egg-capsule of selachians has been found to be a quinonetanned protein.
3. The chorion of trout eggs is not quinone-tanned but its formation and chemical
behaviour allies it with the invertebrate cuticular proteins rather than with the vertebrate keratins.
INTRODUCTION
W
HILE the physics and chemistry of vertebrate epidermal proteins, the
a- and feather-type keratins (Rudall, 1946), have been extensively investigated, the organic membranes surrounding the eggs of oviparous vertebrates have received very little attention. These latter structures, in contrast
to the epidermal keratins, are extracellular secretions, analogous to the cuticles
and egg-membranes of invertebrates. Two such membranes, very different
in origin, the selachian egg-case and the chorion of trout eggs, have been
examined and the results are reported here.
THE SELACHIAN EGG-CAPSULE
Between 1937 and 1938 Faure-Fremiet and his co-workers investigated the
structure and chemistry of the egg-capsules, and the anatomy and histology
of the glands secreting them, in various selachians; and in the course of
publication of their results they have given a full review of earlier work on this
subject.
The egg-capsules are more or less rectangular in outline, with the corners
prolonged into hollow tubes or horns. In detail the structure of the capsule
wall differs in different species. The capsules from different species also
differ in size. That of Raja clavata, the species occurring most commonly at
Plymouth, is on average 7-5 cm. long (excluding the horns) and 5 7 cm. wide
(Clark, 1922). Faure-Fremiet (1938) described the structure of the capsule in
Scyliorhinus, Raja batis, and R. undulata. In these, as indeed in many species,
the wall of the capsule consists of three or four distinct layers. Each layer
consists of a number of birefringent laminae, and each lamina is composed of
parallel fibrils lying in the plane of the lamina, while in successive layers the
fibrils are oriented approximately at right angles to each other, either parallel
to or at right angles to the length of the capsule. In some species one of the
inner layers may be alveolar. In Raja spp. the outer layer is much more
[Quarterly Journal of Microscopical Science, Vol. 96, part 4, pp. 483-488, December 1955.]
484
Brown—Egg-capsule Proteins of Selachians and Trout
coarsely fibrous than the inner layers and in this layer the fibrils run longitudinally.
The anatomy and histology of the shell-gland which secretes the egg-capsule
have been described by Filhol and Garrault (1938) in Raja batis, R. miraletus,
Mustelus vulgaris, Acanthias vulgaris, and Scyliorhinus canicula. While there
are differences between the species, they conform to the same general plan
(figs. 1 and 2). The glands are swellings at the top of the oviducts. Within the
gland it is possible to distinguish two regions; an upper, producing the
albumen that surrounds the egg inside the capsule, and a lower, producing
duct from ovary to qland
shellqland
oviducr
FIG. 1. Shell-gland from Raja spp.
granules that form the capsule. In Raja batis the portion of the gland concerned with the production of the capsule is further divided into two parts: (1)
a part composed of long branching tubes lined by granule-producing cells
along their whole length; these tubes open into troughs between a number of
long parallel ridges on the internal wall of the shell-gland; and (2), the rest of
the capsule-producing portion of the shell-gland; this is composed of groups
of granule-cells, discharging by a duct into tubes lined by mucous cells. These
tubes themselves discharge through pores in the lower portion of the gland.
Faure-Fremiet and his co-workers assumed with Krukenberg (1885) that,
as the material of the capsule is a protein containing sulphur and resistant to
most chemical reagents, it is a keratin. Filhol and Garrault (1938) therefore
called the granules concerned in the formation of the capsule 'prokeratin
granules'. Faure-Fremiet and Garrault (1938) have described a method for
obtaining the granules in a condition suitable for chemical analysis and Faure-
Brown—Egg-capsule Proteins of Selachians and Trout
485
Fremiet and Baudouy (1938) analysed, for nitrogen and sulphur, the 'prokeratin granules' and the different layers of the capsule. The average sulphur
content of the 'prokeratin granules' was found to be 1-4% and of the internal,
middle, and external layers, 1-19%, i'o8%, and 0-85% respectively compared
with the sulphur content of human hair of 5% (Block, 1938).
Filhol and Garrault (1938) comment on the similarity of the histology of
the shell-gland of selachians and the glands in the foot of Mytilus concerned
in the production of the byssus. Brown (1952) showed that the byssus of
Mytilus is quinone-tanned, and in view of this similarity in the histology of
the glands and in the appearance of their products, it seemed desirable to
examine the possibility of quinone-tanning in the egg-capsule of selachians.
Material
Most of the material used was collected through the co-operation of the
skipper of a Newlyn fishing boat, who preserved in 80% alcohol the eggs and
FIG. 2. Diagram of shell-gland from Raja spp. A, lateral longitudinal section. B, dorsoventral
longitudinal section, a, lips through which albumen coat of egg is secreted; b, transverse lips
concerned with formation of capsule; c, tubules carrying products of cells producing 'prokeratin'; d, pores concerned with formation of capsule; e, area in which tubules are lined by
mucous cells.
shell-glands from ripe females found when gutting the fish. In such material
it was not possible to identify the species with any certainty. The material
thus collected was examined by methods which permit the determination of
the types of linkage between protein molecules in any particular structure
(Brown, 1950). On these depend its mechanical and chemical properties.
Results
Material from fully-formed capsules and material from half-formed capsules
removed from the oviducts was tested, and the capsule wall in both cases was
separated into a dark outer portion and a white inner portion before testing.
The properties of the two portions were examined separately. Before the
material was examined it was washed repeatedly in distilled water to remove
the preserving alcohol.
When boiled in distilled water all specimens contracted in length and width
486
Brown—Egg-capsule Proteins of Selachians and Trout
and increased in thickness. This result is to be expected from material composed of fibrillar laminae with a periodic change in the fibril direction through
90° if boiling water hydrolyses some of the electrovalent bonds between the
protein molecules forming the fibrils, allowing them to contract. In dilute
hydrochloric acid in the cold, both layers of mature and partly formed capsules
decrease in length and width and increase in thickness, curl up and become
rubbery. Dilute sodium hydroxide solution in the cold has no obvious effect
on the capsule material, but on boiling the material completely dissolves. All
these results confirm the presence of weak electrovalent bonds holding the
material together.
Alkaline sodium sulphide solution, which dissolves vertebrate keratins by
breaking the disulphide bonds, has little or no effect on either layer of the
fully formed capsule, but it causes the outer layers of the partly formed capsule
to swell and soften, and completely dissolves the inner layers. These results
suggest that disulphide bonds are important in holding the protein molecules
together in the early stages of formation of the capsule, but in the fully formed
capsule some further linkage of the material takes place supplementing the
disulphide bonds and preventing it from dissolving in alkaline sodium sulphide solution. Conceivably, as in Mytilus, this further linkage is a quinonetanning process.
Ferric chloride tests for the presence of orthodiphenols were negative on
fully formed capsules taken from the sea, but on partly formed capsules they
were strongly positive both for inner and outer layers.
Sections of the shell-gland were cut and stained for the presence of quinones
by the argentafHn (Lison, 1936) and diazo techniques (Danielli, 1947). Both
these gave positive staining of the 'prokeratin' granules. While the granules
in the cells of the gland did not stain very darkly, the granules secreted into
the tubes of the gland gave a very intense reaction. The positive ferric chloride
reaction of the immature capsule an^* positive diazo and argentaffin staining
of the 'prokeratin' granules, both indicate the presence of a polyphenol which
could participate in tanning the protein of the capsule.
Portions of inner and outer layers of a fresh, partly formed capsule were
incubated with /-tyrosine solution at pH 8-o for 12 hours at 370 C. to test for
the presence of polyphenol oxidase. Controls were set up with potassium
cyanide added to the solution. The dark outer portion of the capsule became
very dark brown, and the solution brown; the inner white portion slightly
brown with some slight browning of the solution. In both controls there was
no change in colour. These results indicate the presence of a polyphenol
oxidase which might serve to oxidize the polyphenol present in the capsule to
a quinone capable of tanning the material.
Conclusion
From these results it would seem to be incorrect to describe the protein of
the egg-capsule of selachians as a keratin, since it differs considerably in its
behaviour from the true keratins, and it is better regarded as a sclerotin in
Brown—Egg-capsule Proteins of Selachians and Trout
487
Pryor's sense (1940). It is interesting that this vertebrate protein the secretion
of which has much in common with the secretion of invertebrate structural
proteins should also have chemical affinities with invertebrate structural
proteins. The presence of disulphide bonds indicates also a relation to the
keratins but important disulphide bonds are also present in such invertebrate
structures as the cuticle and hooks of cestodes (Crusz, 1948) and in the carapace of Limulus (Lafon, 1943).
Regarding the mechanism of orientation of the fibrils in successive laminae
and the change of orientation through 900, nothing can be said with certainty.
The transverse lips (b in fig. 2, B) of the shell-gland would seem fitted to lay
down fibres transverse to the long axis of the egg-capsule and the pores
(d in fig. 2, B) of the gland fibres oriented at 900 to these; but the cause of
alternate orientation in successive layers is still obscure.
THE CHORION OF TROUT EGGS
The eggs of salmon and trout are enclosed in a tough membrane, the
chorion, which is analogous to the capsule of selachian eggs, but in the salmon
and trout is secreted by cells in the ovary and not by a gland of the oviduct.
Young and Inman (1938) studied the chemistry of the chorion of Salmo
salar. They found that it was extremely resistant to normal solvents in the
cold, but dissolved in 1% sodium hydroxide solution and hydrochloric acid at
ioo° C. Trypsin had no effect, but pepsin digested the chorion. The aminoacid content was also determined by these workers, as follows:
Salmo salar
Total N .
Arginine .
Histidine .
Lysine
Tyrosine .
Tryptophane
Cystine
Glucosamine
%
%
15-20-15-32
5 7 2 - 5-8S
1 23- 1 28
3'54- 3 47
5-12- 5 ' i 2
1-42- 1-42
1-79- 1-89
1 04
Human hair
(Block, 193S)
^
14-9
8-o
o-6
2-5
2-9
0-7
147
A few swelling experiments were made on the chorion of trout eggs (S.
irideus). Boiling distilled water caused an anisometric contraction and
thickening of the chorion, indicating the presence of weak electrovalent
linkages, hydrolysed by boiling water, and predominant orientation of protein
chains in the plane of the chorion with some preferred orientation with respect
to axes in the membrane. Neither sodium hydroxide solution alone nor with
sodium sulphide after prolonged treatment in the cold dissolves the chorion,
but in both cases strips of the chorion contracted in one direction and elongated in the other, becoming very fragile and easily broken on handling. These
dimensional changes again suggest the presence in the chorion of fibrillar
488
Brown—Egg-capsule Proteins of Selachians and Trout
proteins oriented in the plane of the chorion with some preferred orientation.
Calcium cyanate, lithium cyanate, and hydrochloric acid all cause the chorion
to contract slightly and become rubbery. Sodium hypochlorite solution was
the only reagent, besides boiling acids and alkalis, that dissolved the chorion.
Sections of the chorion, stained for —SH and —S—S— groups by the
technique of Chevremont and Frederic (1943), indicate the presence of
—S—S— groups in the chorion. Diazo and argentaffin staining for polyphenols gave negative results.
Conclusion
The material of the chorion differs in chemical behaviour from that of <xand feather-keratins. Young and Inman showed that the amino-acid content
also differs considerably from that of true keratins; and it is not a quinonetanned protein, as is the material of the selachian egg-capsule. Block (1938)
classifies it as a pseudokeratin which suggests that it has at least some keratin
characteristics, but its mode of secretion and its chemical behaviour are more
allied to invertebrate cuticular materials, though its exact nature has not yet
been determined.
Part of this work was done at the Marine Biological Laboratory, Plymouth,
to the Director and Staff of which I am most grateful for the facilities given
me. I am particularly grateful to Mr. G. A. Steven for advice on the obtaining,
handling, and identification of material. I am also indebted to Professor J. F.
Danielli for much advice about histochemical techniques and to Dr. L. E. R.
Picken who gave invaluable suggestions, criticism, and advice through all
stages of the work.
REFERENCES
BLOCK, R. J., 1938. Cold Spring Harb. Monog., 6, 79.
BROWN, C. H., 1950. Quart. J. micr. Sci., 91, 331.
1952. Ibid., 93, 481.
CHEVREMONT, M-, and FREDERIC, J., 1943. Arch. Biol. Paris, 54, 589.
CLARK, R. S., 1922. J. mar. biol. Ass. U.K., 12, 577.
CRUSZ, H., 1948. J. Helminth., 22, 179.
DANIELLI, J. F., 1947. Symposia of the Society for experimental Biology, No. 1. Nucleic acid.
Cambridge (University Press).
FAURE-FHEMIET, E., 1938. Arch. Anat. micr., 34, 23.
and BAUDOUY, C. T., 1938. Bull. Soc. Chim. Biol., 20, 14.
and GARRAULT, H., 1938. Ibid., 20, 24.
FILHOL, J., and GARRAULT, H., 1938. Arch. Anat. micr., 34, 105.
KRUKENBERG, C. F. W., 1885. Mitt. zool. Stat. Neapel., 6, 283.
LAFON, M., 1943. Bull. Inst. Oceanogr. Monaco, No. 850.
LISON, L., 1936. Histodiimie animate. Paris (Gauthier-Villars).
PRYOR, M. G. M., 1940. Proc. Roy. Soc. B, 128, 378.
RUDALL, K. M., 1946. Society of Dyers and Colourists. Fibrous proteins. 15. Proceedings of a
symposium held at the University of Leeds, May 1946.
YOUNG, G. E., & INMAN, W. R., 1938. J. biol. Chem., 124, 189.