Hydrolagus Colliei Research Articles

Long-term transportation of ratfish, Hydrolagus colliei, and tiger rockfish, Sebastes nigrocinctus

Long-term transportation of ratfish, Hydrolagus colliei, and tiger rockfish, Sebastes nigrocinctus

Innervation of the medial rectus muscle in the ratfish, Hydrolagus colliei.

Oculomotor organization in elasmobranch fish (sharks, skates, and rays) differs from that in other vertebrates in that the medial rectus muscle is innervated by contralateral rather than ipsilateral motoneurons. Distinguishing whether this innervation pattern is unique to the elasmobranchs, or is the ancestral pattern for cartilaginous fishes, requires examination of a representative of the sister group to the elasmobranchs, the holocephalans (ratfish). In the present study, the innervation pattern of the medial rectus was examined in a ratfish, Hydrolagus colliei, by using biotinylated dextran amines (BDA, 3,000 MW). Labeled cells were revealed in the contralateral oculomotor nucleus. Therefore, an innervation pattern in which the medial rectus muscle is innervated by contralateral motoneurons is the primitive condition for cartilaginous fishes.

Embryonic staging and external features of development of the Chimaeroid fish, Callorhinchus milii (Holocephali, Callorhinchidae).

The development of Callorhinchus milii, a primitive chondrichthyan fish (Subclass Holocephali) is described in detail based on a complete series of embryos from stage 17 to hatching. The external features of these specimens, in comparison with other chondrichthyan embryos, are used to establish the first staging table for any chimaeroid species. Each stage of C. milii is defined by a suite of morphological characters in addition to total length, including the number of somites, extent of external pigmentation, eye size and shape, head flexure, heart morphology, and size and shape of paired and unpaired fins. Particular attention is given to features of the gill arches and associated structures, including external gill filaments and the opercular flap. Embryos of this species also possess a transient rostral bulb, a feature unique to chimaeroids. Embryological development of Callorhinchus milii is similar to that previously described for sharks and batoids (Subclass Elasmobranchii), including the spiny dogfish, Squalus acanthias, the Japanese bullshark, Heterodontus japonicus, the lesser spotted dogfish, Scyliorhinus canicula, the frill shark, Chlamydoselachus anguineus, the guitarfish, Rhinobatus halavi, and the skate, Raja brachyura. Callorhinchus milii is also similar in overall development to another holocephalan, Hydrolagus colliei. A review of previous staging schemes confirms that early morphological development in all three major chondrichthyan lineages (sharks, batoids, and chimaeras) can be correlated using a common set of stages. A uniform staging system is provided that should prove useful in continuing ontogenetic and phylogenetic studies of this entire clade of fishes. J. Morphol. 236:25-47, 1998. © 1998 Wiley-Liss, Inc.

Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes

Immunoglobulin heavy chain (IgH) genes in representative chondrichthyan fishes (sharks and skates) consist of independently functioning clusters, containing separate variable (VH), diversity (DH), and joining (JH) region elements and constant (CH) region exons. IgH loci have been characterized in Hydrolagus colliei (spotted ratfish), a modern representative of a major independent chondrichthyan lineage. Three distinct families of IgH gene clusters were identified. The most numerous genes consist of unjoined VH-D1-D2-JH segments that correspond to the most abundant Hydrolagus spleen (cDNA) transcripts which apparently arise from a diversified gene family. In the second cluster type, VH, DH, and JH segments are germline-joined, whereas the CH exons exhibit typical organization. This gene type is found in only a few copies per haploid genome and both transmembrane and secretory transcripts have been identified. A third cluster type has been identified that consists of unjoined VH elements but lacks a typical CH1 exon, which is substituted with a second CH2-like exon. Transcripts from this third cluster type also appear to derive from a diversified gene family. Genomic D regions of the two unjoined clone types exhibit structural differences that are consistent with incorporation of recombination machinery-mediated events. Genomic library screening indicates that 90% of VH+ clones are truncated, nearly identical pseudogenes (lacking JH and CH). These studies demonstrate an early phylogenetic origin for the cluster type of gene organization and document extensive organizational diversification within an apparent single class of IgH genes.

The primary structure of a chondrichthyan protamine: a new apparent contradiction in protamine evolution.

We have determined the primary structure of protamine R3 from ratfish (Hydrolagus colliei), a species belonging to the order Chimaeriformes (an old phylogenetic line among the chondrichthyes). Protamine R3 contains 48 residues organized as follows: ARRRH SMKKK RKSVR RRKTR KNQRK RKNSL GRSFK (Q/A)HGFL KQPPR FRP. Comparison of this sequence with both protamine Z3 from Scyliorhinus canicula (a chondrichthyan) and typical protamines from bony fish generates an apparent contradiction: Two relatively close species (H. colliei and S. canicula, both chondichthyes) display different protamines, whereas species more distant in evolution (S. canicula and bony fish) contain very similar protamine molecules. We note that this is not an isolated case in the evolution of sperm nuclear basic proteins (SNBPs) and discuss the possible significance of this fact.

Hypophysial and extrahypophysial projections of the neurosecretory system of cartilaginous fishes: an immunocytochemical study using a polyclonal antibody against dogfish neurophysin.

Immunocytochemistry using antibodies against the neurohypophysial nonapeptides has given equivocal results regarding relevant aspects of the classical neurosecretory system of elasmobranchs. The lack of antibodies reacting with the elasmobranch neurophysins (Nps) has prevented the study of this neurosecretory system by Nps immunocytochemistry. This led us to purify Nps from Scyliorhinus canicula, and to use them to raise a polyclonal antibody. This antibody reacted strongly with the elasmobranch neurophysin neurons, revealing their most delicate and distant hypophysial and extrahypophysial projections. A detailed mapping of the neurosecretory system of five elasmobranch species (Etmopterus spinax, Squalus acanthias, Scyliorhinus canicula, Galeus melanostomus, Raja radiata) and one holocephalian species (Hydrolagus colliei) was performed. In elasmobranchs, the magnocellular neurophysin cells formed a distinct preoptic nucleus, whereas in Hydrolagus the immunoreactive cells were scattered. Distinct parvicellular neurophysin cells were present in the preoptic nucleus. In Raja the nucleus "O" contained parvicellular Nps-immunoreactive neurons. The findings at the pituitary level point to the possibility that neurophysin neurons, in addition to releasing nonapeptides into the systemic capillaries of the neural lobe, also participate in the regulation of the function of the rostral, medial and intermediate lobes of the adenohypophysis by a dual mechanism, i.e., a neurovascular pathway and a direct neural input. The extrahypophysial projections of the neurophysin neurons were highly developed to a degree not comparable to any other vertebrate group. The targets of these projections were located in the telencephalon, diencephalon and hindbrain. The evolutionary and functional implications of this phenomenon are discussed.

Raphe nuclei in three cartilaginous fishes, Hydrolagus colliei, Heterodontus francisci, and Squalus acanthias.

The vertebrate reticular formation, containing over 30 nuclei in mammals, is a core brainstem area with a long evolutionary history. However, not all reticular nuclei are equally old. Nuclei that are widespread among the vertebrate classes are probably ones that evolved early. We describe raphe nuclei in the reticular formation of three cartilaginous fishes that diverged from a common ancestor over 350 million years ago. These fishes are Hydrolagus colliei, a holocephalan, Squalus acanthias, a small-brained shark, and Heterodontus francisci, a large-brained shark. Nuclear identification was based on immunohistochemical localization of serotonin and leu-enkephalin, on brainstem location, and on cytoarchitectonics. Raphe nuclei are clustered in inferior and superior cell groups, but within these groups individual nuclei can be identified: raphe pallidus, raphe obscurus, and raphe magnus in the inferior group and raphe pontis, raphe dorsalis, raphe centralis superior, and raphe linearis in the superior group. Hydrolagus lacked a dorsal raphe nucleus, but the nucleus was present in the sharks. The majority of immunoreactive cells are found in the superior group, especially in raphe centralis superior, but immunoreactive cells are present from spinal cord to caudal mesencephalon. The distribution and cytoarchitectonics of serotoninergic and enkephalinergic cells are similar to each other, but raphe nuclei contain fewer enkephalinergic than serotoninergic cells. The cytoarchitectonics of immunoreactive raphe cells in cartilaginous fishes are remarkably similar to those described for raphe nuclei in mammals; however, the lack of a raphe dorsalis in Hydrolagus indicates that either it evolved later than the other raphe nuclei or it was lost in holocephalan fishes.

Analysis of the Post-translational Processing of α-MSH in the Pituitaries of the Chondrostean Fishes, Acipenser transmontanus and Polyodon spathula

Multiple forms of α-MSH were isolated from the pituitaries of the white sturgeon, Acipenser transmontanus, and the paddlefish, Polyodon spathula, following fractionation of the pituitary extracts by reversed-phase HPLC. In both species, immunoreactive forms which eluted with the same retention times as synthetic mammalian ACTH(1-13)NH 2, monoacetylated α-MSH, and diacetylated α-MSH were detected. In the pituitary of the sturgeon, N-acetylated forms of α-MSH represented 91% of the total α-MSH isolated. However, in the pituitary of the paddlefish, N-acetylated forms of α-MSH accounted for only 45% of the total α-MSH isolated. The presence of N-acetylated forms of α-MSH in the pituitaries of representatives of the two extant genera of chondrostean fish coupled with the observation that N-acetylated forms of α-MSH are present in the pituitaries of cladistian and neopterygian fishes indicates that the α-MSH specific N-acetylation mechanism evolved prior to the radiation of the Actinopterygii. A conspicuous feature of the chondrostean α-MSH N-acetylation reaction was the low levels of diacetylated α-MSH produced relative to the levels of monoacetylated α-MSH. Similar observations have been made for the cladistian fish, Calamoichthys calabaricus, and the cartilaginous fish, Hydrolagus colliei. However, in holostean fishes, lungfishes, and tetrapods, the diacetylated form of α-MSH is the major end product produced in the intermediate pituitary. The phylogenetic implications of these observations will be discussed.

Sperm storage in male elasmobranchs: a description and survey.

Two basic types of spermatozoan aggregates, spermatophores and spermatozeugmata, found in 14 different species of sharks, one species of skate, and one species of chimaera (holocephalan), were investigated using light and scanning electron microscopy. Spermatophores, aggregates (usually 1,000-6,000 microns in diameter and larger) of randomly clumped sperm embedded in and surrounded by an eosinophilic matrix, were found in Alopias superciliosus, Odontaspis taurus, Carcharodon carcharias, Isurus oxyrinchus, and Lamna nasus. Three types of spermatozeugmata, sperm structures without a surrounding capsule or matrix, are described. The first, clumps of 60-200 sperm unbound in a supporting matrix, are found in Squalus acanthias and Hydrolagus colliei. In the second type, single-layered spheres are formed of sperm clumps with the sperm heads bound in a common supporting matrix. These are found in Carcharhinus limbatus and Carcharhinus plumbeus. The third type of spermatozeugmata are large multilayered, compound structures formed by the accretion of several single-layered aggregates. These multilayered structures characteristically are found in Carcharhinus falciformis, C. limbatus, Carcharhinus obscurus, C. plumbeus, Carcharhinus porosus, Prionace glauca, Rhizoprionodon terraenovae, and Sphyrna lewini. Sperm aggregates of all types are stored between the septa and in the lumen of the terminal ampulla of the epididymis. In their various forms they are the final product of the mature male elasmobranch reproductive tract. In a male with mature claspers, the presence of sperm aggregates is a more reliable indicator of maturity and sexual activity than is clasper condition alone.

Identification of gonadotropin-releasing hormone and associated binding substances in the blood serum of a holocephalan ( Hydrolagus colliei)

The identity of the gonadotropin-releasing hormone (GnRH) form and the presence of GnRH-binding substances in the blood serum of the holocephalan, spotted ratfish ( Hydrolagus colliei), were investigated. The GnRH-like peptides in the serum were identified on the basis of relative hydrophobicity using reverse-phase HPLC. [His 5,Trp 7,Tyr 8]GnRH (chicken GnRH-II) was the only GnRH form detected in the serum. It has been previously shown to be the only GnRH form in the brain of this species. The presence of GnRH-binding substances was inferred by anomalous HPLC elution of GnRH, ultrafiltration behavior, and by the direct binding of iodinated GnRH analogues by blood serum components. The mean GnRH concentration in the extracted blood serum was 125 ± 11 pg ml −1 ( n = 5) in males and 64 ± 48 pg ml −1 ( n = 4) and 155 ± 26 ( n = 4) in two separate groups of females. Measurement of GnRH in the blood serum is complicated by the presence of GnRH-binding substances, which may cause the coprecipitation of GnRH during extraction with organic solvents. The high concentration of GnRH and the presence of GnRH-binding substances suggest that systemic blood is the route by which GnRH reaches the gonadotropes and/or that GnRH may have a hormonal role in H. colliei.

Chemical Identification of the Mammalian Oxytocin in a Holocephalian Fish, the Ratfish ( Hydrolagus colliei)

The neurohypophysial hormones of the ratfish ( Hydrolagus colliei), a species belonging to the subclass Holocephali of cartilaginous fishes, have been investigated. An oxytocin-like hormone has been isolated from acetone-desiccated pituitary glands by using successively molecular sieving and high-pressure liquid chromatography. The peptide has been identified as oxytocin by coelution with synthetic oxytocin in HPLC, amino acid sequencing, mass spectrometry, and C-terminal sequencing through carboxypeptidase Y. Vasotocin may be present in a very small amount. Cartilaginous fishes appear to display a great diversity in their oxytocin-like hormones since five different peptides have been identified in rays and sharks that belong to the second subclass Selachii.

Sperm-Specific Basic Proteins in the Holocephalan Fish Hydrolagus colliei (Chondrichthyes, Chimaeriformes) and Comparison with Protamines from an Elasmobranch.

Seven basic proteins can be isolated from sperm nuclei of the holocephalan ratfish Hydrolagus colliei. Two of these proteins (R3 and m0) are devoid of cysteine, whereas five of them (R1, R2, m1, m2, and m3) contain low levels of this amino acid residue. The proteins R1, R2, and R3 are major ones in the sperm nuclei of H. colliei, and they are analogous to basic proteins Z1, Z2, and Z3 (scylliorhinines) from the sperm of the elasmobranch Scyliorhinus canicula. However, taking into account the partial sequence of R3 protein and the number of cysteines in R1 and R2, these proteins do not seem to be homologous to the scylliorhinines. A comparison of sperm basic proteins between H. colliei (a holocephalan) and S. canicula (an elasmobranch) suggests a remarkable divergence of these proteins from a common ancestral pattern during the evolution of Chondrichthyes.

Ampullary sense organs, peripheral, central and behavioral electroreception in chimeras (Hydrolagus, Holocephali, Chondrichthyes).

Ampullary sense organs are distributed in groups over the head of Hydrolagus colliei with their pores in clusters and innervated by the buccal, hyomandibular and superficial ophthalmic branches of the anterior lateral line nerve. The ampullae contain ciliated sense cells in an alveolate-shaped epithelium, which communicates to the surface through a jelly-filled tube. The sense cells synapse at their bases with the afferent nerve fibers that terminate in the dorsal nucleus of the anterior lateral line lobe of the medulla. The anatomy and ultrastructure support the homology with the ampullae of Lorenzini of elasmobranchs. Single units recorded from the buccal branch of the anterior lateral line nerve are either lateral line or ampullary in character, the former being sensitive only to mechanical stimuli, the latter to both mechanical and to weak electric stimuli. They are also distinguished by the positions of their receptive fields. The electroreceptive units are spontaneously active and are excited by a cathode placed near the opening of their pore and inhibited by an anode. Compound evoked potentials are recorded from beneath the lateral aspect of the tectum in response to weak electric fields in the bath. Each recording locus has a best position and orientation of the electric field. The electric fields are effective if their duration is longer than ca. 2 ms; longer than 10 ms makes no difference until an OFF effect becomes distinct at ca. 50 ms. The reception is tuned to low frequencies but is not sensitive to maintained current (DC). Evoked potentials summating moderate numbers of responses are clear at < 1 microV/cm. Ratfish were conditioned in a ring-shaped tank to reverse the direction of swimming when an electric field was switched ON. The stimulus was a 5 Hz square wave or the onset of a DC of 1-10 microA between a pair of electrodes on the floor of the tank. The fish responded to fields as weak as 0.2 microV/cm. A specialized sense modality for electroreception, similar to that in elasmobranchs and most other groups of nonteleost fishes, except for Myxini and Neopterygii (holosteans), is present in the subclass Holocephali. The notion is supported that this modality and its central as well as peripheral apparatus arose early in the evolution of vertebrates. Only two losses of the whole system need be hypothesized, on this idea, once in the ancestors of the hagfishes and once in the ancestors of the neopterygians, which include the teleosts. Some orders of teleosts then evolved a new system of electroreception independently. The ciliary receptor cells are probably primitive; microvillar sense cells evolved independently.

Immunohistochemical localization of serotoninergic, enkephalinergic, and catecholaminergic cells in the brainstem and diencephalon of a cartilaginous fish, hydrolagus colliei

We localized serotonin (5-HT), leu-enkephalin (LENK), and tyrosine hydroxylase (TH) immunoreactive cells in the brain of a holocephalian, Hydrolagus colliei, by use of antibodies made in rabbit and the peroxidase-antiperoxidase technique. Only three locations contained TH+ cells, the caudal myelencephalon, the locus coeruleus, and the diencephalon. Of these locations, the diencephalon contained the most cells and the locus coeruleus the least cells. The caudal TH+ myelencephalic cells formed a single large group that spanned both the dorsal and ventral portions of the brain (A1A2). The diencephalic TH+ cells were located in the posterior tuberculum, in the ventromedial and ventrolateral thalamic nuclei, and in the inferior lobe of the hypothalamus. Hydrolagus differed from mammals and the elasmobranchs, their sister group, in that no substantia nigra (A9), ventral tegmental area (A10), or A5 cell group was found. Distribution of LENK+ and 5-HT+ cells were similar to each other; the raphe nuclei contained most of the 5-HT+ and LENK+ cells. These 5-HT+ and LENK+ cells were found at all rostrocaudal levels of the myelencephalon. The nucleus reticularis magnocellularis, reticularis paragigantocellularis lateralis, the ventral met- and mesencephalon (B7 and B9 cell groups), the hypothalamus, and the pretectal area contained additional 5-HT+ and LENK+ cells. The solitary complex contained LENK+ cells but not but 5-HT+ cells. A dorsal raphe nucleus, which is the largest 5-HT+ cell group in mammals, was absent in Hydrolagus. A dorsal raphe nucleus is present in one galeomorph shark radiation but is absent in three radiations of batoids (rays, skates, and guitarfish). Thus even within cartilaginous fish, there are differences in the distribution of neurochemicals and possibly nuclei within their brains.

Primary Structure of gonadotropin-releasing hormone from the brain of a holocephalan (ratfish: Hydrolagus colliei)

Gonadotropin-releasing hormone (GnRH) in the ratfish brain has been isolated and purified using reverse-phase high performance liquid chromatography. Amino acid composition and sequence analysis indicate that the primary structure is pGlu-HisTrpSerHisGlyTrpTyrProGly-NH 2. The presence of the amino terminal pyroglutamic acid has been confirmed by degradation studies with pyroglutamyl aminopeptidase. The amidated carboxy terminus and molecular weight were confirmed using mass spectrometry. Moreover, sequence comparison and coelution studies with one of the synthetic forms of GnRH (chicken GnRH II) indicate that the ratfish and chicken GnRH II molecules are identical. This represents the first sequence data of a GnRH molecule from a cartilaginous fish (class: Chondrichthyes). It is argued that the ratfish GnRH molecule has been retained for over 400 million years of evolution and is expressed in most vertebrate classes.

Ser 5]-somatostatin-14: Isolation from the pancreas of a holocephalan fish, the Pacific ratfish ( Hydrolagus colliei)

The holocephalan fishes were the first class of vertebrate in evolution to develop a pancreatic gland with both endocrine and exocrine parenchyma. An extract of the pancreas of one such fish, the Pacific ratfish ( Hydrolagus colliei) contained somatostatin-like immuno-reactivity (141 pmol/g wet wt), measured with an antiserum raised against mammalian somatostatin-14. Automated Edman degradation and fast atom bombardment-mass spectrometry established the primary structure of the major molecular form as Ala-Gly-Cys-Lys- Ser-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys. A minor component of somatostatin-like immunoreactivity, constituting 8% of the total, was of approximate molecular weight 6000. Thus, in the ratfish pancreas prosomatostatin-I is processed predominantly to somatostatin-14, as in the mammalian pancreas, but the resulting tetradecapeptide contains the substitution Ser for Asn at position 5.

Fine structural observations on the process of spermiation in the holocephalan fish Hydrolagus colliei.

The process of spermiation in the ratfish Hydrolagus colliei is described and compared with that in mammals and amphibians. Spermiation in this species involves prior fluid space expansion both within the apical parts of the Sertoli cytoplasm and in the spaces between Sertoli cells and spermatids. The apical ends of Sertoli cells fragment, including the parts immediately around the spermatid acrosomes. Intercellular material between the spermatid tips and the Sertoli cells dissolves. Concurrently an opening from the seminiferous follicle into the efferent ductule is made by means of changes in cell shape and separation of Sertoli cells and efferent ductule cells that adhere to each other up to the time of sperm release.

Biological activity of a bombesin-like peptide extracted from the intestine of the ratfish, Hydrolagus colliei

1. Ratfish ( Hydrolagus colliei) intestines were boiled in water to inactivate proteases and then treated with cold 4° trifluoroacetic acid to extract bombesin-like peptides. 2. The extract was fractionated in several steps using reverse-phase and ion exchange HPLC, and bombesin-like immunoreactive peptides were detected by radioimmunoassay using an antiserum specific for the bioactive C-terminal region of bombesin. 3. A highly purified bombesin-like peptide-containing fraction stimulated amylase release in a dose-responsive fashion from rat pancreatic acini; the dose-response curve was parallel to a bombesin standard, and the ratfish peptide stimulated the same maximal rate of amylase secretion as the bombesin standard. 4. A potent, highly selective bombesin receptor antagonist completely abolished the stimulation of amylase release caused by the ratfish peptide, demonstrating the specificity of the response. 5. Estimates of the bombesin-like peptide concentration of this fraction by radioimmunoassay and by bioassay were nearly identical, indicating that ratfish bombesin is very similar biologically and antigenically to frog skin bombesin.

Differential fate of mitochondria during spermiogenesis in the ratfish Hydrolagus

During spermiogenesis in the ratfish Hydrolagus colliei, spermatid mitochondria follow one of three fates. Some are retained as functional spermatozoan mitochondria clustered in the midpiece, some are enclosed in large vacuoles and phagocytosed by Sertoli cells, and the remaining mitochondria are segregated to the remnant cytoplasm. In the latter site rhodamine 123 fluoresence studies show that the mitochondrial derivatives, although much altered morphologically, retain the electrochemical gradient characteristic of the inner mitochondrial membrane. Some mitochondrial derivatives show activity even after the remnant cytoplasm is cast off from the spermatozoon.

The origin of the mammalian form of GnRH in primitive fishes

The presence of neuroendocrine hormones in extant agnathan fishes suggests that a method of control involving these hormones was operating 500-600 million years ago in emerging vertebrates. Data on a limited number of species show that several members of the GnRH family of peptides may have arisen in non-teleost fishes. Lamprey (Petromyzon marinus) GnRH has a unique composition and has not been detected in other vertebrates. It is not yet clear whether the chicken II GnRH-like molecule arose in cartilaginous fishes, but a chromatographically and immunologically similar molecule is found in dogfish (Squalus acanthias) and ratfish (Hydrolagus colliei). Finally, a mammalian GnRH-like molecule is detected in three primitive bony fish: sturgeon (Acipenser transmontanus), reed fish (Calamoichthys calabaricus), and alligator gar (Lepidosteus spatula). Minor forms are also present, but are not yet characterized. Clearly, the basic structure of GnRH peptides was established in primitive fish. In contrast, at least three other identified forms of GnRH have been detected in teleosts or tetrapods: Salmon I, catfish I, and chicken I GnRH. Evidence for the presence of members of the GnRH family and the neurohypophysial hormone family in primitive fishes argues for the importance of neuroendocrine control throughout the history of vertebrates.