Pacific Hagfish Research Articles

Sex differentiation in the hagfish Eptatretus stouti

A detailed study was made of sex differentiation in three anteroposteriorly distributed body regions of the long gonadal fold in young Pacific hagfish ( Eptatretus stouti) from 12.4 to 45 cm in length. In general, the posterior region, adjacent to the cloaca, remained undifferentiated, and only in specimens more than 28 cm long was there development of this region into a differentiated testis. On the other hand, the anterior gonadal region of all specimens under 20 cm developed continually and progressively through stages that included cystic nests of cells derived from the germinal epithelium, acquisition of follicular investments around these cells, growth of these young oocytes and break-up of their original follicular groupings, and, finally, in larger specimens, vitellogenesis. Although the earliest oocytes formed in cyst-like nests of cells, later oocytes were derived individually directly from the germinal epithelium. In larger specimens, in which testicular development occurred in the posterior part of the gonad, the anterior gonad involuted, leaving a thin structure that lacks germ cells. There were numerous exceptions to this general description of events, indicating a certain degree of lability of determination of a particular gonadal region to either male or female differentiation. Some earlier stages of female differentiation could occur in the posterior gonadal region of a few smaller animals. Some posteriorly located ovotestes (differentiated elements of both sexes) developed in a few specimens between 16 and 21 cm in length, but were not seen in larger animals. Some testicular follicles appeared in the anterior gonadal regions of three specimens between 20 and 25 cm in length, but not in specimens larger than that. Three hermaphroditic specimens between 29 and 33 cm in length were found in which there was a well-differentiated testis posteriorly and a well-differentiated ovary (vitellogenic eggs) anteriorly. Questions of hermaphroditism, lability of sex determination, and possible epigenetic influences on these phenomena are taken up in the discussion. Since this study provides the first correlation of stages of sex differentiation with animal length, it forms the basis for experimental approaches in E. stouti to analysis of mechanisms of sex determination and differentiation.

Projections ascending from the spinal cord to the brain in petromyzontid and myxinoid agnathans

The course of projections ascending through the rostral spinal cord to nuclei in the brains of petromyzontid and myxinoid agnathans was examined with silver staining of anterograde degeneration and horseradish peroxidase histochemistry. As in jawed vertebrates, the ascending spinal projections of lampreys and hagfishes appear to be organized into two major systems, the spinal lemniscal and dorsal column pathways. The spinal lemniscal pathway, extending rostrally along the ventrolateral margin of the spinal and medullary central gray, consists of a spinoreticular and possibly a spinovestibular projection in both aganthan groups. In Pacific hagfish, spinal lemniscal fibers reach the ipsilateral mesencephalic tectum, but no spinal projection to the thalamus was evident. The spinal lemniscus of lampreys ascends to the region of the isthmus and may extend into the mesencephalic tegmentum. Anterograde and retrograde tracing methods indicate that a very small population of cells in the far rostral cord of lampreys may project to the optic tectum and diencephalon; however, spinotectal and spinothalamic projections, if present, are limited in extent. The dorsal column pathway in agnathans, consisting in part of primary spinal afferents, ascends in the dorsal funiculus of the cord. The dorsal column fibers of agnathans, like those of some other anamniotes, continue beyond the spinomedullary junction through the length of the hindbrain, possibly conveying ascending somatosensory input to the sensory nuclei of the alar medulla.

Retinofugal and Retinopetal Projections in the Pacific Hagfish, <i>Eptatretus stouti </i>(Myxinoidea) (Part 1 of 2)

The retinofugal and retinopetal connections of the Pacific hagfish were determined using the fluorescent compound DiI as an in vitro tracer. The retinofugal connections were found to be bilateral and to reach preoptic, thalamic and pretectal, as well as tectal, targets. No distinct basal optic root was found. A retinopetal projection in hagfishes is described here for the first time. It originates from two cells groups in the rostral mesencephalic tegmentum. These retinal connections in hagfishes are compared to the organization of the visual system in the second Recent group of jawless fishes, the lampreys. Both visual systems are similar in the number and location of the targets of retinal fibers. Furthermore, there is a striking similarity in the retinopetal systems. Major differences are found in the organization of the optic tract. These observations allow the identification of some cell groups not previously recognized in myxinoid brains or interpreted differently. Finally, the findings are discussed in relation to visually guided behavior in hagfishes.

Retinofugal and Retinopetal Projections in the Pacific Hagfish, <i>Eptatretus stouti </i>(Myxinoidea) (Part 2 of 2)

The retinofugal and retinopetal connections of the Pacific hagfish were determined using the fluorescent compound DiI as an <i>in vitro</i> tracer. The retinofugal connections were found to be bilateral and to reach preoptic;, thalamic and pretectal, as well as tectal, targets. No distinct basal optic root was found. A retinopetal projection in hagfishes is described here for the first time. It originates from two cell groups in the rostral mesencephalic tegmentum. These retinal connections in hagfishes are compared to the organization of the visual system in the second Recent group of jawless fishes, the lampreys. Both visual systems are similar in the number and location of the targets of retinal fibers. Furthermore, there is a striking similarity in the retinopetal systems. Major differences are found in the organization of the optic tract. These observations allow the identification of some cell groups not previously recognized in myxinoid brains or interpreted differently. Finally, the findings are discussed in relation to visually guided behavior in hagfishes.

Insulin-like immunoreactivity in the brain of two hagfishes, Eptatretus stouti and Myxine glutinosa

Immunocytochemical investigation of the brains of hagfish Myxine glutinosa and Eptatretus stouti with antisera raised against salmon insulin revealed the presence of groups of immunoreactive cells discretely localized in the mid- and hindbrain of both species. Subpopulations of these cells reacted weakly with antisera against Myxine islet insulin and equivocally with anti-bovine insulin serum. Extracts prepared from Myxine brain were subject to gel filtration and were found by radioimmunoassay to contain two forms of insulin-like material, one of large molecular weight (<66 kDa) and another smaller molecule (6 kDa). The relationship of these molecules to the insulin-related growth factor family of neurohormonal peptides is discussed and their potential function assessed in terms of a possible homology with Muller-type cells and involvement in axonal regeneration phenomena.

Origins of the descending spinal projections in petromyzontid and myxinoid agnathans.

The origins of the descending spinal pathways in sea lampreys (Petromyzon marinus), silver lampreys (Ichthyomyzon unicuspis), and Pacific hagfish (Eptatretus stouti) were identified by the retrograde transport of horseradish peroxidase (HRP) placed in the rostral spinal cord. In lampreys, the majority of HRP-labeled cells were located along the length of the brainstem reticular formation in the inferior, middle, and superior reticular nuclei of the medulla, mesencephalic tegmentum, and nucleus of the medial longitudinal fasciculus. Labeled reticular cells included the Mauthner and Müller cells. Horseradish-peroxidase-filled cells were also present in the descending trigeminal tract, intermediate and posterior octavomotor nuclei, and a diencephalic cell group, the nucleus of the posterior tubercle. As in lampreys, the reticular formation of the Pacific hagfish was the largest source of descending afferents to the spinal cord. Labeled cells were found in the dorsolateral and ventromedial reticular nuclei, the dorsal tegmentum at the juncture of the medulla and midbrain, and the nucleus of the medial longitudinal fasciculus. Additional medullary cells projecting to the cord were located in the perivagal nucleus, the central gray, and the anterior and posterior magnocellular octavolateralis nuclei. The existence of reticulospinal and possible vestibulo-, trigemino-, and solitary spinal projections in lampreys and hagfishes and the wide distribution of these pathways in jawed vertebrates suggest that they evolved in the common ancestor of gnathostomes and both groups of jawless fishes. However, descending spinal pathways from the cerebellum, red nucleus, and telencephalon appear to be gnathostome characters.

The preparation of poly(A)+mRNA from the hagfish slime gland.

The isolation of translatable poly(A)+mRNA from the slime glands of the Pacific hagfish, Eptatretus stouti, is not possible by the commonly used procedures because of the viscous slime that is formed when the contents of the glands are hydrated. This paper reports on a procedure developed to overcome this problem. Briefly, the tissue was powdered in liquid nitrogen, mixed with sodium lauroylsarcosine and proteinase K and lyophilized. The lyophilized powder was then mixed with 0.3 mm diameter glass beads, thoroughly ground and wetted with buffer and digested at 37 degrees C. The RNA from the digest was recovered by ultracentrifugation through a CsCl cushion. Further purification of the RNA was accomplished by the usual methods with slight modifications.

The sensory trigeminal tract of Pacific hagfish. Primary afferent projections and neurons of the tract nucleus.

The central projections of primary trigeminal afferents in Pacific hagfish (Eptatretus stouti) were examined by transganglionic transport of horseradish peroxidase (HRP) placed in the principal nerves of the trigeminal complex. Central to the trigeminal ganglia, labeled afferents in the ophthalmic, external and dental nerves coursed caudally in the descending trigeminal tract of the ipsilateral medulla. The trigeminal tract occupies an extensive portion of the dorsolateral medulla and is subdivided into a consistent pattern of longitudinal fascicles surrounded by narrow cellular bands. Primary trigeminal afferents were arranged somatotopically in the fascicles. Large-diameter afferents of the ophthalmic, external and dental nerves segregated into separate medial fascicles extending from the dorsomedial to the ventrolateral descending trigeminal tract. Fine-fibered afferents of the ophthalmic and external nerves formed separate dorsolateral fascicles. Additional dental afferents spread through these lateral fascicles. Labeled trigeminal fibers reached the dorsolateral funiculus of the rostral spinal cord. In addition, HRP injections of the descending trigeminal tract revealed a largely crossed projection to the mesencephalic tectum. Tectal injections of HRP retrogradely labeled neurons in the contralateral sensory trigeminal tract, confirming the existence of a tract nucleus comprised in part of neurons located in the bands of cells bordering the primary afferent fascicles.

Why do hagfish have gill "chloride cells" when they need not regulate plasma NaCl concentration?

Two enzymes implicated in branchial ion transport, Na+-K+-ATPase and carbonic anhydrase, were localized in gill ionocytes ("chloride cells") of the Pacific hagfish, Eptatretus stouti, by light microscopic histochemical techniques. In hagfish, ouabain-sensitive Na+-K+-ATPase activity was confined to apical halves of ionocytes, where most of the cytoplasmic tubular system is located. In marine teleosts, Na+-K+-ATPase was noted in chloride cells and erythrocytes. Acetazolamide and potassium cyanate sensitive carbonic anhydrase activity occurred throughout the cytoplasm and nucleus of hagfish ionocytes. Biochemical assay of hagfish gill homogenates for Na+-K+-ATPase yielded a specific activity of 3.1 μmol Pi∙mg protein−1∙h−1 at 37 °C. This resembles values we obtained for freshwater fish (Carassius auratus: 3.3 μmol Pi∙mg protein−1∙h−1; Tilapia shirana: 3.7 μmol Pi∙mg protein−1∙h−1), and is less than values we obtained for marine teleosts (Pomacentrus spp.: 13 μmol Pi∙mg protein−1∙h−1; Gillichthys mirabilis: 6.7 μmol Pi∙mg protein−1∙h−1). Hagfish resemble freshwater teleosts in many other gill features related to ion transport. The presence of carbonic anhydrase in gill ionocytes of hagfish supports the proposal that these cells function in acid–base regulation, i.e., that they exchange H+ for Na+ and [Formula: see text] for Cl−.

Cellular requirements for mixed leucocyte reactivity in the cyclostome, Eptatretus stoutii.

Unfractionated peripheral blood leucocytes from allogeneic hagfish proliferated vigorously in one-way mixed leucocyte reaction (MLR) at 20-22 degrees. Maximum responsiveness was observed after 5 days of co-culture of responder and irradiated stimulator cells. Separation of leucocytes on the basis of laser scatter parameters yielded a small leucocyte population which responded but did not stimulate in MLR and a large leucocyte population capable of stimulating but not responding. Adherent cell depletion of the total leucocyte population did not affect the ability to respond in MLR but did ablate the stimulatory capacity of hagfish leucocytes. Together with previous phenotypic studies, these results confirm the presence of lymphocyte and macrophage/accessory cell populations in hagfish peripheral leucocytes.

The Different Vascular Patterns of Slime Glands in the Hagfishes, Myxine glutinosa Linnaeus and Eptatretus stouti Lockington A Scanning Electron Microscope Study of Vascular Corrosion Casts*

AbstractThe vascular patterns of the epidermally derived slime glands of the Atlantic hagfish, Myxine glutinosa, and of the Pacific hagfish, Eptatretus stouti, have been studied by scanning electron microscopy of vascular corrosion casts. In Myxine a simple two‐dimensional vascular network sheaths the slime glands, while in Eptatretus there is also a great number of capillary loops of different lengths arising from the sheathing network and extending into the interior of the glands. These basic differences in slime glands vascular patterns are thought to reflect substantially different physiological behaviour of the slime glands in Myxine and Eptatretus.

Evolutionary development of lymphocyte heterogeneity: Leucocyte subpopulations in the pacific hagfish

Flow cytometric analysis of forward angle versus 90 degree scatter patterns of hagfish peripheral blood revealed two distinct leucocyte populations with size characteristics analogous to mammalian monocytes/granulocytes (hagfish large leucocytes) and small lymphocytes (hagfish small leucocytes). A cell population enhanced for the small leucocytes was obtained by density gradient centrifugation. Over 70% of the small leucocyte population consistently stained with a rabbit antiserum directed against polypeptide determinants on hagfish immunoglobulin, while staining of the large cell population was greatly reduced (<10%). A panel of monoclonal antibodies raised against a crude hagfish leucocyte preparation distinguished the two cell populations and revealed the existence of subpopulations of both small and large leucocytes.

The absence of an arterial pressure effect on filtration by perfused glomeruli of the hagfish, Eptatretus stouti (Lockington).

Single renal corpuscles of hagfish were perfused with a Ringer solution containing Ficoll 70 to simulate the colloid osmotic pressure of hagfish plasma. Simultaneous measurements were made of single glomerulus filtration rate (SGFR), perfusion pressure and the pressure in a vessel of the renal vasculature. The results confirm that SGFR is independent of pressure in the glomerular capillaries (PGC). The results also suggest that flow through glomeruli and SGFR are closely linked. Studies of the pressures in glomerular capillaries during periods when the perfusion rate was varied indicate that PGC reflects the area of the active capillaries and the rate of perfusion. Therefore, in the hagfish, PGC appears to be an effect of factors that cause glomerular filtration, not the main cause of that process.

Gill ultrastructure of the Pacific hagfish Eptatretus stouti.

At the gross anatomical level, hagfish gills show unusual features not seen in any other fish gills. Our study was undertaken to determine if peculiarities also characterize the microscopic anatomy and ultrastructure of hagfish gills. To the contrary, branchial respiratory lamellae of Pacific hagfish were found to resemble the lamellae of lampreys, elasmobranchs, and teleosts, often down to the finest subcellular details. As in other fish, hagfish lamellae are lined by epithelium containing pavement cells with organelles indicative of a secretory function, basal cells showing undifferentiated cell features, and branchial ionocytes. The ionocytes are identical to chloride cells of teleosts in cytostructure, distribution, and abundance. There are pillar and marginal capillaries in hagfish gill lamellae. Pillar cells contain bundles of 5-nm microfilaments, and they associate with collagen columns as in other fish. Hagfish pillar cells do exhibit odd features, however: They cluster (groups of up to nine were seen), and their extracellular collagen columns are rarer than in other fish gills (averaging only two columns per three pillar cells). Other special features of hagfish gills are the following: lipid droplets and smooth endoplasmic reticulum are well developed in all cell types; pavement cells secrete a lipomucous product (stains with periodic acid-Schiff, Alcian blue, and Sudan black B); and goblet cells are absent. The presence of "chloride cells" in hagfish is puzzling, as hagfish body fluids are iso-osmotic to seawater and there is no need to osmoregulate for sodium chloride; the ionocytes contain carbonic anhydrase, suggesting a function in acid/base regulation.

Corticotrophin-like bioactivity in the pituitary gland and brain of the pacific hagfish, Eptatretus stouti

The cytochemical bioassay for corticotrophin (ACTH) was used in an attempt to detect ACTH-like activity in the Pacific hagfish, Eptatretus stouti. Extracts of the pituitary gland and brain were active in this assay system but those of liver and skeletal muscles were not. The slopes of the dose-response lines of the pituitary extracts were less than those of the mammalian corticotrophin standard preparation but greater than those of the brain extracts. The results suggest that the corticotrophic factor in the hagfish differs from mammalian ACTH.

Immunoglobulin bearing blood leucocytes in the Pacific hagfish

The occurrence of immunoglobulin (Ig) bearing leucocytes in the blood of the Pacific hagfish, Eptatretus stoutii , was examined using a murine monoclonal antibody (45.3) and a rabbit antiserum specific for hagfish serum Ig. Binding of antibody 45.3 to hagfish leucocytes assessed by radioimmunoassay was inhibited by preincubation of antibody with purified serum Ig thus verifying the presence of cell surface Ig cross reactive with serum Ig. The monoclonal antibody identified approximately 65% of blood leucocytes as Ig+ve while the rabbit antiserum indicated 81% Ig+ve cells. Both antibody preparations failed to react specifically with cells from mouse, horned shark, tunicate or sea star; this indicates the distinctive nature of hagfish Ig. The high percentage of blood cells bearing surface Ig in the hagfish raises the possibility that lymphocyte divergence to separate B and T pathways may not have occurred in this most primitive vertebrate. Alternatively, an Ig-like specificity characteristic of both “T” and “B” lymphocytes may have been detected. In any event, a subset of Ig negative leucocytes is evident in hagfish.

Plasma cells in adult Atlantic hagfish, Myxine glutinosa.

Hagfishes, the most primitive vertebrates, are of special interest for the evolution of immune responses. Eptatretus stoutii, the Pacific hagfish, is able to mount cellular and humoral immune responses but all attempts to demonstrate in them the presence of plasma cells have failed. In the present study we demonstrate for the first time plasma cells identifiable by ultrastructural criteria in the pronephros, a primitive lymphohaemopoietic organ, of Myxine glutinosa, the Atlantic hagfish.

The hagfish slime gland thread cell. I. A unique cellular system for the study of intermediate filaments and intermediate filament-microtubule interactions.

Thread cell differentiation in the slime gland of the Pacific hagfish Eptatretus stouti has been studied using light microscopy and scanning and transmission electron microscopy. Thread cell differentiation is remarkable in that the life history of the cell is largely dedicated to the production of a single, tapered, cylindrical, highly coiled, and precisely packaged cytoplasmic thread that may attain lengths of 60 cm and diameters approaching 1.5 micron. Each tapered thread, in turn, is comprised almost entirely of large numbers of intermediate filaments (IFs) bundled in parallel. During differentiation of the thread, the IFs become progressively more tightly packed. Various numbers of microtubules (MTs) are found among the bundled IFs during differentiation of the thread but disappear during the latter stages of thread differentiation. Observations of regularly spaced dots in longitudinal bisections of developing threads, diagonal striations in tangential sections of developing threads, and circumferentially oriented, filament-like structures observed at the periphery of developing threads cut in cross section have led us to postulate a helically oriented component(s) wrapped around the periphery of the developing thread. The enormous size of the fully differentiated thread cell, its apparent singular dedication to the production of IFs, the ease of isolating and purifying the threads and IF subunits (see accompanying paper), and the unique position of the hagfish in the phylogenetic scheme of vertebrate evolution all contribute to the attractiveness of the hagfish slime gland thread cell as a potential model system for studying IF subunit synthesis, IF formation from IF subunits, aggregation of IFs into IF bundles and the interaction(s) of IFs and MTs.

A study of triiodothyronine-catecholamine interactions: their effect on plasma fatty acids in pacific hagfish Eptatretus stouti

1. 1. Plasma circulating levels of fatty acids (PFA) were measured in intact and in triiodothyronine (T 3)-treated Pacific hagfish Eptatretus stouti. Intraperitoneal injection of both pharmacological (200 ng/g) and physiological (1 ng/g) doses of T 3 caused an elevation of PFA 24 and 48 hr after treatment. The implantation of capsules containing the antithyroid compound, 6-propylthiouracil (6-PTU) decreased PFA levels. 2. 2. Twenty-four hr after T 3-injection the norepinephrine (NE) content of plasma declined, whereas epinephrine (E) titres increased. Forty-eight hr after T 3-treatment the mean titres of dopamine (D) and NE were lower than in the control group of hagfish. 3. 3. The treatment of hagfish with E in either pharmacological (2–3 μg/g) or physiological (2–3 ng/g) doses did not cause any elevation of PFA during the 72 hr following injection. In the same experiment the high dose of E induced long-lasting hyperglycemia, whereas the low dose had no substantial effect on glycemic levels. 4. 4. The data provide evidence that in hagfish, unlike in higher animals, the observed lipolytic effect of administered T 3 is not mediated through the potentiation of the fat mobilizing action of catecholamines.

Fractionation of hagfish slime gland secretions: partial characterization of the mucous vesicle fraction.

This paper deals with the collection, fractionation and partial characterization of the slime gland secretion of the Pacific hagfish (Eptatretus stouti) with emphasis on the mucous fraction. Secretions were collected by electrical stimulation of the glands of anesthetized hagfish and, using three different methods, separated into three fractions: 1) the thread cells, 2) the mucous vesicles of the mucous cells, and 3) the soluble fraction. The methods take advantage of the stabilization of the thread cells and mucous vesicles by ammonium sulfate and sodium citrate.