Some observations on the collagen fibrils of the egg capsule of the dogfish, Scyliorhinus canicula

The egg capsule of the dogfish Scyliorhinus canicula is a collagenous material with a laminated, plywood (orthogonal) construction. The collagen fibrils which constitute the bulk of the egg capsule wall have a unique, highly ordered structure (Knight and Hunt, 1974; 1976, 1986; Gathercole et al., 1993) which is thought to represent a smectic A liquid crystalline phase (Knight et al., 1993). The egg capsule is extremely strong and chemically inert (Hunt, 1985). It is stored, secreted and formed by the nidamental gland (Rusaou?n 1976, 1990 a, b; Knight and Feng, 1992). During intracellular storage, secretion and fibrillogenesis, the dogfish egg capsule collagen appears to pass through a remarkable series of textures within a lyotropic liquid crystalline phase diagram (Knight et al., 1993). In the present communication, further observations on the ultrastructure of the collagen fibrils and their arrangement within the laminae of the fully-formed egg capsule are reported. The effect of tilting ultrathin sections of fibrils in the goniometer stage of a transmission electron microscope are described, demonstrating that the crystalline lattice within the fibril appeared twisted more or less regularly into a long pitch helix. Other observations indicated that some of the fibrils were in turn twisted round one another to form fibres which therefore had a coiled-coil structure. The fibres are arranged parallel to one another in the laminae which are stacked to give an orthogonal plywood construction. The effects of staining fibrils with cuprolinic blue and with tannic acid are reported. Reduction in the water content of the fibrils before fixation appeared to move some of the fibrils through the part of the lyotropic phase transition diagram converting them from smectic A to smectic C. Finally, evidence is presented that the fibrils shrank, but remarkably, still retained a longitudinally-ordered but modified, molecular arrangement even after boiling in water for periods of up to 10 min. These observations are discussed in relation to other collagens.     

The effect of PH on fibrillogenesis of collagen in the egg capsule of the dogfish, Scyliorhinus canicula

The collagen of the egg capsule of the dogfish, Scyliorhinus canicula is stored and secreted by the secretory cells of the D-zone of the nidamental gland (Rusaou?n-Innocent, 1990b). The collagen appears to pass through several morphologically distinct textures during storage, secretion and fibril formation which may represent different lyotropic liquid crystalline phases (Knight et al., 1993). In the present communication we report evidence that a fall in hydrogen ion concentration induces fibrillogenesis during the secretion of the dogfish egg capsule. In an attempt to understand the factors involved in collagen assembly, we investigated the effects of subjecting isolated collagen storage granules in vitro to solutions ranging in pH from 2-11 and Na(+), K(+), Ca(++), Mg(++), Zn(++) and Cu(++) ions at concentrations varying from 0.01-0.5 M. From pH 2 to pH 4 most granules appeared completely amorphous; from pH 5 to pH 7 granules showed the following previously reported liquid crystalline textures: isotropic, lamellar, micellar, hexagonal columnar, transversely banded twisted nematic, and unbanded twisted nematic. At pH 8 granules showed both the hexagonal columnar phase (phase IV) and small quantities of the final fibrillar phase together with a previously undescribed texture. The latter texture, which we refer to as phase VII, had a D period (17.5 nm) half that of the lamellar texture (phase II) and the final egg capsule fibrils (phase VI). From pH 9 to pH 11, only the final fibrillar texture (phase VI) together with small quantities of the new texture (phase VII) were present. Na(+), K(+), Ca(++), Mg(++), Zn(++) and Cu(++) ions did not appear to have an observable effect on the phases found in isolated granules at pH 7.0. The role of pH in collagen storage and fibrillogenesis was confirmed by direct estimation of the pH in vivo using vital staining with neutral red, a range of pH indicators applied to unfixed cryostat sections and direct measurements of the pH of the jelly within the egg capsule. The implications of these findings for the mechanism of collagen storage and fibrillogenesis in the dogfish egg capsule and other collagenous systems are discussed.     

Secretion and stabilization of the layers of the egg capsule of the dogfish Scyuorhinus canicula

The egg capsule of the dogfish is a unique, collagcnous, layered structure secreted by the nidamental gland which has nine, remarkably discrete, transverse zones of tubular glands. The present paper traces the origin of the four layers of the capsule to particular zones within the gland. Evidence is presented for the existence of DOPA, DOPA oxidase. protein(s) rich in tyrosyl residues and a peroxidase within the same storage granules within the secretory cells of the C and E zones. It is suggested that these interact when secreted to cross-link the inner and outer surfaces of the egg capsule. Evidence is presented that the middle layer which forms the bulk of the thickness of the egg capsule and has the highest collagen content may be partly stabilized by the peroxidation of tyrosyl residues. The mechanical significance of crosslinking in this system and the possible cytological mechanisms involved in the secretion of the tanning agents and enzymes are discussed.     

The effect of progressive hypoxia on respiration in the dogfish (scyliorhinus canicula) at different seasonal temperatures.

1. Dogfish were acclimated to 7, 12 or 17 degrees C and exposed to progressive hypoxia at the temperature to which they had been acclimated. During normoxia, the Q10 values for oxygen uptake, heart rate, cardiac output and respiratory frequency over the full 10 degrees C range were: 2.1, 2.1, 2.1 and 2.5 respectively. Increased acclimation temperature had no effect on cardiac stroke volume or systemic vascular resistance, although there was a decrease in branchial vascular resistance, pHa and pHv. 2. Progressive hypoxia had no effect on heart rate or oxygen uptake at 7 degrees C, whereas at 12 degrees C and 17 degrees C there was bradycardia, and a reduction in O2 uptake, with the critical oxygen tension for both variables being higher at the higher temperature. Cardiac stroke volume increased during hypoxia at each temperature, such that cardiac output did not change significantly at 12 and 17 degrees C. Neither pHa nor pHv changed significantly during hypoxia at any of the three temperatures. 3. The influence of acclimation temperatures on experimental results from poikilotherms is pointed out. Previously-published results show quantitative differences. 4. The significance of the present results with respect to the functioning and location of oxygen receptors is discussed. It is argued that as the metabolic demand and critical oxygen tension of the whole animal are increased at high acclimation temperatures the same must be the case with the oxygen receptor. This would raise the stimulation threshold and could account for the bradycardia seen during hypoxia becoming manifest at higher values of PI,O2, Pa,O2 and Pv,O2 as the acclimation temperature is raised.     

The developmental expression of foxl2 in the dogfish Scyliorhinus canicula

The FoxL2 genes are a subfamily of the Fox (forkhead box) gene family. FOXL2 is mutated in the disorder Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome (BPES), which is characterized by eyelid malformations, and Premature Ovarian Failure (POF). In the mouse expression is seen in the perioptic mesenchyme, developing eyelids, ovary and pituitary. We have isolated a foxl2 cDNA from the dogfish Scyliorhinus canicula (also known as the lesser spotted catshark), allowing the characterisation of this gene's sequence and expression from a lineage that diverged early in the evolution of gnathostomes. Molecular phylogenetic analysis strongly grouped this sequence with the gnathostomes within the FoxL2 subfamily. We demonstrate the early expression of Scyliorhinus canicula foxl2 in the mandibular head mesoderm and later in continuous populations of mandibular arch cells and mandibular head mesenchyme cells around the developing pituitary. As development proceeds expression decreases in the mesenchyme of the head but is seen in the mesenchyme around the eye and later in the developing eyelids. Additionally expression is seen in regions of pharyngeal arch mesoderm and in ectoderm from which gill buds will form. This expression is maintained in the developing and elongating gill buds. Thus, S. canicula foxl2 is a marker for the mandibular mesoderm and gill buds and its expression is conserved in the perioptic mesenchyme, developing eyelids and pituitary.     

Interaction of link protein with collagen

Link protein (Mr = 42,000) is an integral component of cartilage as well as of some noncartilagenous tissues. In cartilage, it forms a macromolecular complex with cartilage proteoglycan and hyaluronic acid, but its function in other tissues is unknown. We provide evidence here that the link protein of cartilage binds well to native collagen types I and III. The binding occurs only if both link protein and collagen are native. The binding of link protein to collagen type fibrils is higher than to monomeric collagen. Link protein binding to collagen fibrils is saturable and occurs at molar ratio of collagen to link protein of 7-13:1. These data suggest that the link protein binds to collagen and that the binding requires the collagen to be in its native triple helical structure. This interaction may play a role in collagen fibril formation.     

Partially systematic molecular packing in the hexagonal columnar phase of dogfish egg case collagen.

The collagen that forms the egg case of the dogfish Scyliorhinus canicula is stored in bulk in the female nidamental glands. Here the collagen molecules are thought to undergo a series of distinct pH-dependent liquid crystalline aggregation phase changes before assembling into the final arrangement encountered in the mature egg case. One liquid crystalline phase is hexagonal with the centres of two adjacent hexagons about 36 nm apart. We have collected tilt series of the hexagonal phase from plastic sections of the nidamental gland and have produced a three-dimensional reconstruction of the collagen arrangement of this phase. The reconstruction features axial columns of protein density lying regularly on the vertices of hexagonal cells of edge length 21 nm. Each column is connected to three nearest neighbours by irregular sheets of protein, but there appear to be preferred molecular directions at about 40 degrees to 50 degrees to the columns. The reconstruction has been interpreted in terms of known interactions of this collagen in other assemblies.     

Characterization of a chymosin-like pepsin from the dogfish Scyliorhinus canicula

1. Pepsin II extracted from the gastric mucosa of Scyliorhinus canicula has been characterized and compared to calf chymosin. 2. The kcat and Km of the dogfish enzyme for the synthetic hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe have been determined. The kcat/Km ratio is close to that of calf chymosin. Its milk-clotting efficiency is however 21-fold lower than that of calf chymosin. 3. The proteolytic activity against haemoglobin is optimal at pH 2.5. It clots the milk up to pH 6.8. 4. The dogfish pepsin II shows relatively better activity at low temperatures than calf chymosin.     

Metal accumulation and metallothionein induction in the spotted dogfish Scyliorhinus canicula

Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10 µM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Cu (635 µg/L), and Ag (1079 µg/L and two additional exposures at 10 µg/L and 1 µg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.     

Studies on the reticulo-endothelial system of the dogfish,Scyliorhinus canicula

Summary Clearance and subsequent localisation of a range of materials, including colloidal carbon, latex beads, sheep erythrocytes, bacteria and dextran were followed in the lesser spotted dogfish, Scyliorhinus canicula. It was found that two populations of peripheral blood leucocytes — monocytes and thrombocytes, but not granulocytes — were involved in clearance of the circulation. In the case of carbon, this material was cleared from the plasma after 12 h, and both the colloid-containing thrombocytes and monocytes disappeared from circulation by 8 weeks post injection. Upon injection of some of the materials, and particularly bacteria, a settling out of monocytes containing phagocytosed material was seen in the secondary lamellae and cavernous bodies of the gills. Large clumps of monocytes were found in the gills as early as 30 min post injection and these increased in size for up to one week, after which they gradually dispersed. The lining cells of the cavernous body, known as CB cells, were also responsible for the sequestration of carbon, latex beads and probably erythrocytes, but dextran and bacteria were not internalised. The origin, functions and phylogenetic significance of the CB cells are discussed.     

Tachykinin-like immunoreactivity in the sinus venosus of the dogfish (Scyliorhinus canicula)

The sinus venosus of the elasmobranch heart is characterized by the presence of large bundles of unmyelinated nerve fibres that bulge into the cardiac lumen, below the endocardium. In the dogfish (Scyliorhinus canicula), these fibres contain numerous dense-core membrane-bounded granules of about 200 nm in diameter. Most intramural ganglion cells of the sinus venosus also show densely packed granules similar to those found in the subendocardial fibres. We have observed strong substance-P-like immunoreactivity in the large fibre bundles and in the perikarya of the ganglion cells. Preabsorption of the antisera with fragment 7–11 of substance P has shown that the antisera recognize the tachykinin canonic sequence. Our findings suggest that an undetermined tachykinin is secreted in the elasmobranch heart, and that it is probably released into the blood stream in the context of a little-known neuroendocrine system.     

Immunohistochemical mapping of galanin-like immunoreactivity in the brain of the dogfish Scyliorhinus canicula.

The distribution of galanin-like immunoreactivity in the brain of the dogfish Scyliorhinus canicula was investigated using the indirect immunofluorescence technique. In the telencephalon, positive cells and fibers were located in the mid-caudal part of the area superficialis basalis, the n. septi caudoventralis and in the n. interstitialis commissurae anterioris. Most of the galanin-containing neurons observed in the hypothalamus were located in the magnocellular preoptic nucleus. Positive perikarya were also found in the n. lobi lateralis hypothalami and in the n. lateralis tuberis. A dense network of positive nerve processes was noted in the caudal part of the median eminence. In the dorso-caudal part of the diencephalon numerous immunoreactive neurons were seen in the recessus posterioris. A large bundle of galanin-containing fibers, which divided in two branches, was observed in the basal midbrain tegmentum. The widespread distribution of galanin-like material suggests that, in the dogfish, galanin may be involved in various brain functions including neuroendocrine regulations.     

Structural studies on the thymus of the dogfish, Scyliorhinus canicula L.

Histological and ultrastructural studies of the thymus of the dogfish, Scyliorhinus canicula L., revealed paired lobular masses above the first two gill arches of embryonic and newly hatched dogfish, involuting at approximately 3 weeks. It did not have defined cortex and medullary regions and ultrastructural investigations showed the tissue to be composed mainly of a range of different sized lymphocytes within a connective tissue capsule and reticular epithelial cell framework. Lymphoblasts often in mitosis were also present being more electron lucent and larger than lymphocytes. Macrophages contained cellular debris and frequently whole small lymphocytes. A cell with cytoplasmic granules was occasionally observed.     

Proteomic analysis of the spermatogonial stem cell compartment in dogfish Scyliorhinus canicula L

In the dogfish (Scyliorhinus canicula L.) the testicular germinative zone (GZ), composed of large isolated spermatogonia surrounded by elongating pre-Sertoli cells, is located between the albuginea and the ventrolateral intratesticular vessel. During the spermatogenic wave, cysts radiate in maturational order forming distinct testicular zones. In this study, soluble proteins of the GZ and of the zone containing cysts with spermatocytes were separated by two-dimensional electrophoresis. Gel images were matched and then evaluated for GZ-specific proteins. From the1400 protein spots identified, 680 were found to be apparently specific to this zone. Using MALDI-TOF/TOF mass spectrometry, de novo sequences were obtained for 33 proteins out of the 169 selected for identification by mass spectrometry, but only 16 of these 169 proteins were identified. One of them, proteasome subunit alpha-6, was analyzed further by immunohistochemistry. This study demonstrates the utility of the dogfish as a model for proteome analysis of the spermatogonial stem cell niche, even if it remains restricted by the lack of genomic data available on Elasmobranchs.     

X-ray diffraction analysis of the 3D organization of collagen fibrils in the wall of the dogfish egg case

The wall of the egg case of the dogfish,Scyliorhinus canicula, contains a network-forming collagen assembled into a regular three-dimensional (3D) structure. It accomplishes supportive, protective and filtering functions for the embryo contained within it. The collagen molecules in the egg case are organized into a body-centred unit cell of dimensions (mean ± s.d.) (11.6 ± 1.0) nm X (11.6 ± 1.0) nm X (81.6 ± 3.2) nm, which belongs to the I422 space group. At a higher hierarchical level, the collagen molecules assemble into parallel arrays of fibrils, ca. 100 nm in diameter, which aggregate to form laminae ca. 0.5 μm thick. These laminae are organized into a plywood-like structure and account for 98% of the thickness of the wall of the egg case. X-ray diffraction patterns of the wall of the egg case were taken along mutually perpendicular directions, one being perpendicular to the surface of the egg case. Three different kinds of diffraction pattern were observed. One of the patterns was characteristic of an X-ray direction perpendicular to the laminae in the egg case (along the x-direction). The two other patterns were obtained with the X-rays directed parallel to the plane of the laminae, either along the capsule long axis (z) or perpendicular to this (y). These two patterns were observed interchangeably in either of the x- or y-directions depending on the specimen. The diffraction patterns were analysed and interpreted taking into consideration the 3D electron microscope data of the egg case. The results confirm and extend previous findings from transmission electron microscopy and low-angle X-ray diffraction and they suggest that there is only one major type of ordered collagen arrangement in the wall of the egg case.     

The functional anatomy of the gills of the dogfish (Scyliorhinus canicula)

Vascular casting techniques combined with scanning electron microscopy of fixed tissue have been used to describe the vascular anatomy of the gills of the dogfish ( Scyliorhinus canicula ). Two distinct blood pathways were revealed: a 'respiratory' blood pathway which conveys deoxygenated blood from the heart to the gas exchange surface of the gills and then carries oxygenated blood away to the systemic circulation, and a 'non-respiratory' blood pathway which interconnects with the respiratory blood pathway and which diverts blood into the venous drainage from the gills.
The physiological function of this complex circulation in the gills of elasmobranchs is discussed in terms of both the osmotic constraints upon the animal, and the possible control of blood flow.