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Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.
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.
While there is considerable structural evidence that insulin-like growth factors (IGFs) share a long evolutionary history, little is known about the conservation of IGF function. In order to address this, we have made recombinant hagfish IGF, hence allowing characterization of an IGF from a representative of the primitive vertebrate class, Agnatha. The production of recombinant hagfish IGF has been complicated by a number of factors including the requirement of a longer leader peptide for fusion protein expression, reduced solubility of the protein, as well as problems in the refolding procedure. However, we were able to produce a small quantity of hagfish IGF with an N-terminal glycine addition which is biologically active. Furthermore, N-terminal amino acid sequencing and mass spectrometry confirm that we have produced hagfish IGF. In vitro assessment of recombinant hagfish IGF in cultured cells indicates that hagfish IGF indeed shares functional properties with mammalian IGFs. Thus, hagfish IGF stimulates protein synthesis in rat myoblasts, but 20- and 5-fold more peptide, respectively, is required to achieve the same half-maximal responses as with human IGF-I (hIGF-I) or IGF-II (hIGF-II). Hagfish IGF also competes for binding to the type-1 IGF receptor present both on rat myoblasts and on salmon embryo fibroblasts, though with somewhat lower affinity than either hIGF-I or hIGF-II. However, studies investigating binding to the IGF-II-specific type-2 receptor suggest that hagfish IGF may in fact be more closely related to IGF-I than to IGF-II. These results indicate that motifs important for functions associated with mammalian IGFs appear to have evolved prior to the Agnathans diverging from the main line of vertebrate evolution 550 million years ago. Accordingly, we now have functional as well as structural evidence that the IGFs have a long evolutionary history.
The hagfish is considered the most primitive vertebrate known, living or extinct. It remains an enigma whether adenohypophysial hormones similar to those of more advanced vertebrates are present in the hagfish pituitary gland or not. The present study aimed to detect immunoreactive adenohypophysial hormones in the hagfish pituitary gland, using antisera to tetrapod and fish adenohypophysial hormones as immunohistochemical probes. For this purpose, two species of hagfish, the Atlantic hagfish, Myxine glutinosa, and the Pacific hagfish, Eptatretus burgeri, were used. In both species, three different types of immunoreactive cells were detected in the adenohypophysis. (1) The first type of cells was gonadotropin (GTH)-like cells which were stained by antisera to LH-related GTHs, such as ovine LHbeta, human LHbeta, bullfrog LH, salmon LHbeta and sturgeon LHbeta in both species of hagfish. (2) The second type of cells that were detected was growth hormone (GH)/prolactin (PRL)-like cells. In M. glutinosa the cells were stained by antisera to salmon GH, salmon PRL, sturgeon GH, sturgeon PRL, blue shark GH, and lamprey GH. In E. burgeri the cells were only stained by anti-human GH and anti-sturgeon PRL. (3) The last type of cells was adrenocorticotropin (ACTH)-like cells. These cells were stained by antisera to lamprey ACTH and human beta-endorphin. In both species of hagfish, GTH-like cells were relatively abundant, and were distributed throughout the adenohypophysis, whereas GH/PRL-like and ACTH-like cells were few in number in the adenohypophysis. Based on these findings, we suggest that hagfish may have retained ancestral characteristics of key anterior pituitary hormones.
Hox genes code for transcription factors that play a major role in the development of all animal phyla. In invertebrates these genes usually occur as tightly linked cluster, with a few exceptions where the clusters have been dissolved. Only in vertebrates multiple clusters have been demonstrated which arose by duplication from a single ancestral cluster. This history of Hox cluster duplications, in particular during the early elaboration of the vertebrate body plan, is still poorly understood. In this paper we report the results of a PCR survey on genomic DNA of the pacific hagfish Eptatretus stoutii. Hagfishes are one of two clades of recent jawless fishes that are an offshoot of the early radiation of jawless vertebrates. Our data provide evidence for at least 33 distinct Hox genes in the hagfish genome, which is most compatible with the hypothesis of multiple Hox clusters. The largest number, seven, of distinct homeobox fragments could be assigned to paralog group 9, which could imply that the hagfish has more than four clusters. Quartet mapping reveals that within each paralog group the hagfish sequences are statistically more closely related to gnathostome Hox genes than with either amphioxus or lamprey genes. These results support two assumptions about the history of Hox genes: (1) The association of hagfish homeobox sequences with gnathostome sequences suggests that at least one Hox cluster duplication event happened in the stem of vertebrates, i.e., prior to the most recent common ancestor of jawed and jawless vertebrates. (2) The high number of paralog group 9 sequences in hagfish and the phylogenetic position of hagfish suggests that the hagfish lineage underwent additional independent Hox cluster/-gene duplication events.
Complementary DNAs encoding a preproinsulin-like growth factor (prepro-IGF) have been cloned from a primitive vertebrate species, the Atlantic hagfish, by using a DNA amplification strategy based on the polymerase chain reaction. A composite sequence containing a 414-nucleotide open reading frame encoding 138 amino acids and 164 nucleotides in the 3'-untranslated region was obtained. The deduced partial sequence of hagfish prepro-IGF reveals that it is organized like the mammalian prepro-IGFs with an unusually large (greater than 39-amino acid) signal peptide (initiator methionine residue is missing), 29-amino acid B, 15-amino acid C, 21-amino acid A, 10-amino acid D, and 26-amino acid E domains. All the invariant residues necessary to form the correct tertiary fold of an insulin-like molecule have been conserved in hagfish IGF. Sequence comparisons revealed that the A and B domains of hagfish IGF are equally similar to those of human IGF-I (35 out of 50 amino acids) or IGF-II (37 out of 53 amino acids). In contrast, the similarity between hagfish and mammalian pro-IGFs in the C, D, and E domains is relatively low. Northern blot analysis of RNA isolated from hagfish brain, heart, liver, skeletal muscle, and islet organ, however, indicated that hagfish IGF, like mammalian IGF-I, is expressed predominantly in the liver as a 4.2-kilobase transcript. DNA blot analysis revealed that hagfish IGF is a single copy gene. The predicted sequence of hagfish prepro-IGF thus demonstrates that the divergence of the IGF and insulin genes occurred prior to the separation of the Agnatha and that the organization and tertiary structure of IGF have been well maintained throughout 550 million years of vertebrate evolution.
Changes in gonadal morphology, gonadal estradiol, and progesterone were examined in Atlantic hagfish, Myxine glutinosa, during a period of 17 months, beginning in April, 2001. Atlantic hagfish were captured from the ocean on a monthly basis. A total of 60 hagfish were divided into three different size classes of twenty hagfish each (small 20-35 cm, medium 35-45 cm, large 45-55+cm) and transported to the University of New Hampshire for sampling. Overall, in the medium and large size hagfish, estradiol and progesterone had significantly elevated peaks in January, 2001. There were significant increases in estradiol concentrations in January, with relatively low fluctuations in levels for the rest of the sampling period. Progesterone concentrations increased significantly in January, 2002, in medium and large hagfish, and remained elevated until June and April, 2002, for the two size classes respectively. The majority of hagfish sampled were females or hermaphrodites; few true males were identified in any of the samples. The number of females with large eggs increased following the estradiol peak in January and hermaphrodites with mature sperm were identified in the July, 2002, sample. These data represent the first evidence for a seasonal reproductive cycle in M. glutinosa and only the second seasonal reproductive cycle identified in any hagfish species.
M. glutinosa is a cyclostome, living in the mud in seawater of high salinity. It probably is a stationary scavenger feeder. About 28,000 hagfish from the Gullmar Fjord were examined during a 5-year period for the occurrence of tumors. Hepatomas were found to be predominant neoplasm, observed at a frequency that decreased from 5.8% in 1972 to 2.9% in 1973 and finally to 0.6% in 1974--76. Islet cell hamartomas and frank neoplasms decreased from 0.5% in 1972 to less than 0.1% in 1973--76. Occasional subcutaneous and mesenterial neoplasms were also observed during 1972--74. In hagfish caught 12 km out in the open sea, the hepatoma incidence decreased from 2.8% in 1972 to 0.9% in 1974. Given this background, it is possible that pollution of the Gullmar Fjord by carcinogenic substances with low biodegradability has occurred until 1972, and this pollution could be of etiologic significance for these hagfish tumors. In fact, the use of PCBs became prohibited by law in Sweden in 1971--72. Severe restrictions were also introduced for the use of chlorinated pesticides, notably DDT, and associated substances (DDD, DDE). Preliminary analyses for the presence of PCBs, DDT (and its metabolites), and aflatoxins (the notorious hepatocarcinogen) were performed by gas chromatography and thin-layer chromatography. Livers (with and without neoplasms) from hagfish caught inside the threshold of the fjord contained about 5 mg/kg of wet weight of PCBs and about 0.1--0.4 mg/kg of dry weight of DDT, DDD, or DDE, whereas those from hagfish caught in the open sea had a much lower PCB concentration (about 0.2 mg/kg of wet weight). No PCBs and no chlorinated pesticides were found in analyses of the mud at the catching site. High PCB concentrations (3 mg/kg of wet weight) were, however, observed in livers from cod living in the Gullmar Fjord, and it was proposed that bony fish may be the source of hagfish liver PCBs. PCB chromatograms of hagfish livers differed from those of PCB standards and cod liver. This strange pattern, which was not seen in livers from hagfish caught in the open sea, might be explained by an unusual mode of metabolization. The assays for aflatoxins gave completely negative results.
Of the extant vertebrate animals, the hagfishes are generally considered to be the group which diverged first from the ancestral vertebrate lineage, although molecular sequence analysis has recently suggested that they form a monophyletic group with lampreys (Stock and Whitt, 1992). The circulatory system of hagfishes has features that have been described as 'primitive' (Burggren et al. 1985), but their gills are effective gas exchangers. The gills are contained within discrete muscular pouches, and the anatomy of the blood system and ventilatory ducts has an ideal countercurrent arrangement (Mallatt and Paulsen, 1986; Elger, 1987). Reite (1969) first reported effects of catecholamines and other drugs on the branchial vasculature of hagfish. Recent studies of both perfused gills in situ and of blood flow in vivo have suggested that blood flow through the gills of hagfish is under tonic control by catecholamines (Axelsson et al. 1990; Forster et al. 1992). In teleosts, several studies have shown that adrenergic control mechanisms are involved in the distribution of blood between the arterio-arterial and the arterio-venous pathways of the gill vasculature (see Nilsson, 1983). Anatomical and ultrastructural studies have demonstrated the existence of similar pathways in hagfish (Cole, 1925; Mallatt and Paulsen, 1986; Elger, 1987). The experiments reported here demonstrate that, in the hagfish gill pouch, both adrenaline and isoprenaline can increase the proportion of fluid leaving via the efferent arterial route, at the expense of the venous outflow. Hagfish (Eptatretus cirrhatus Forster) were collected off Motunau, North Canterbury, New Zealand, and held in seawater aquaria until used. The masses of the 11 animals used in these experiments ranged from 680 to 1720 g with a mean of 1140±110 g (s.e.m.). Animals were anaesthetized in a 0.4 % solution of benzocaine in sea water. The hagfish were opened ventrally to expose the gills and their blood supply. Individual gill pouches were prepared for perfusion studies.
The head of adult hagfishes (jawless craniates, Myxinoidea) of the family Eptatretidae displays a number of skin grooves of uncertain origin. These grooves have been homologized to the neuromast lines of other craniates, and they are innervated by two ganglionated cranial nerves that have been interpreted as lateral line nerves. The grooves do not, however, contain the compound receptors that are typical of a lateral line (i.e. neuromasts or electroreceptors), and both their development and function have remained enigmatic. To elucidate the embryonic origin of the grooves (which should develop from placodes if they are homologues of the lateral line system), embryos of Pacific hagfish were examined by means of three-dimensional reconstructions from serial sections. Because of the scarcity of specimens of embryonic hagfishes, only two embryos were reconstructed, but these reconstructions clearly show that a number of placodes and placodal derivatives (i.e. sensory ridges, receptor primordia, and cranial ganglia) occur in the head of embryonic eptatretid hagfishes. Some of these placodes correspond to the lens and epibranchial placodes of other craniates, but there are also three other placodes which represent possible homologues of lateral line placodes. The topology of the placodes in this latter group corresponds to the topology of the grooves of adult hagfishes, and we therefore reach three conclusions: (i) that an embryonic lateral line system is present in hagfishes; (ii) that the grooves of adult hagfishes in all probability derive from lateral line placodes; and (iii) that the presence of lateral line placodes is a primitive character of craniates.
The capillary tuft of glomeruli of the hagfish mesonephros contains both 'low'-pressure and 'high'-pressure glomerular vessels (LPGVs and HPGVs). The existence of the HPGV raised the possibility that pressure filtration could occur in the hagfish kidney when the blood pressure was sufficiently high. Therefore, measurements of glomerular capillary pressure were made in HPGVs and LPGVs whilst single glomeruli were perfused with hagfish Ringer's solution that contained the colloid Ficoll 70. Calculations of the effective colloid osmotic pressure in perfused capillaries were made; these showed that hydrostatic pressures within the HPGV were inadequate to effect pressure filtration except at high rates of perfusion. However, high rates of perfusion provoked perfusion pressures that exceeded the highest values measured in the renal blood supply of lightly anaesthetised hagfish. It was concluded that some process other than pressure filtration must account for formation of the primary urine by hagfish glomeruli. The proportion of the perfusate that became urine, the single glomerulus filtration fraction (SGFF), bore a strong positive relationship to the vascular resistance of perfused glomeruli. Both the SGFF and the vascular resistance were inversely related to the rate of perfusion except when that rate was very high. From these two observations it was concluded that at least two flow pathways exist in hagfish glomeruli: one that has a high vascular resistance and that contributes to the elaboration of the urine, and one that has a low vascular resistance and does not contribute to urine formation. The possible anatomical location of the various flow pathways through hagfish glomeruli and how they may function are discussed.
A factor present in the homogenate of the corpuscles of Stannius from a teleost, the longfin eel Anguilla dieffenbachii, showed calcitropic effects in an agnathan, the hagfish Eptatretus cirrhatus. This factor greatly increased calcium efflux from an isolated perfused gill pouch preparation and had no effect on calcium influx. Hagfish regulated plasma total calcium at a concentration which was about 50% of that of the ambient seawater. Intact hagfish had rates of calcium influx and efflux which were similar to those recorded in teleosts. Hagfish are thus similar to teleosts to the extent that they can and do maintain a plasma calcium concentration which is quite different from that of the ambient medium. They also resemble teleosts in that transepithelial calcium fluxes across the hagfish gill pouches respond to extracts of Stannius corpuscles. Hagfish, however, are quite unlike teleosts in that the Stannius corpuscle extract stimulates calcium efflux rather than inhibiting calcium influx.
Concentrations of regulatory peptides in an extract of the intestine of the cyclostome, Myxine glutinosa (Atlantic hagfish), were measured by radioimmunoassay using 12 antisera of defined regional specificity that were raised against mammalian gastrointestinal peptides. The hagfish gut contained somatostatin-, cholecystokinin/gastrin-, C-terminal substance P-, and neurokinin A-like immunoreactivity in concentrations that were 10 to 100 times less than the corresponding concentrations in the rat intestine. The hagfish gut also contained glucagon-like immunoreactivity, measured with both C- and N-terminally directed antisera, but the immunoreactivity did not dilute in parallel with the porcine glucagon standard in radio-immunoassay. No immunoreactivity was detected using antisera to calcitonin gene-related peptide, gastrin-releasing peptide, neuromedin U, neurotensin, N-terminal substance P, and vasoactive intestinal polypeptide. The somatostatin-like immunoreactivity in the hagfish gut was resolved by HPLC into components with the retention times of somatostatin-34 and somatostatin-14, previously isolated from the hagfish islet organ (relative abundance 2:1). The retention times of hagfish glucagon and of the multiple molecular forms of the tachykinin-like peptides were appreciably different from the retention times of the corresponding mammalian peptides.
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.
A homologous protein to human plasma alpha-2-macroglobulin (alpha-2-M) was purified from the blood plasma of hagfish (Eptatretus buergeri) and its structure and function were studied. The hagfish protein inhibited several proteinases and its inhibitory activity was blocked with methylamine as in the case of human alpha-2-M. The molecular weight and sedimentation coefficient of the hagfish inhibitor were 390,000 +/- 20,000 and 11.0 S, respectively, as determined by sedimentation studies. The frictional ratio calculated from these parameters was 1.75. The Stokes radius estimated from HPLC gel chromatography was 8.8-8.9 nm, which was similar to that of human alpha-2-M despite the fact that the hagfish inhibitor was only one-half as large as human alpha-2-M in molecular weight. The hagfish inhibitor was expected to be more asymmetric and/or more hydrated than the human inhibitor. The electron micrographs of the negatively stained hagfish inhibitor showed that it had an open, rectangular quaternary structure of 15 +/- 1.5 X 19 +/- 2 nm in which two semiglobular units were located at the two shorter sides with a gap of 8 +/- 1 nm in width. Each semiglobular unit had an approximate width of 5 +/- 0.5 nm. The thickness of the unit was estimated to be 3 to 3.5 nm from the result of fixed-angle shadowing experiments. Although the two semiglobular units must be connected by some structure, very little material could be seen between them. Such an open quaternary structure may explain the high frictional ratio and large Stokes radius of this protein. The structural change of the inhibitor after reaction with proteinases or methylamine could be detected by electron microscopy and gel chromatography.
Using a highly sensitive fluorimetric assay, significant levels of angiotensin I -converting enzyme-like activity (ACELA) were detected in a range of tissues (branchial heart, gill, kidney with associated vasculature and archinephric duct, liver, whole brain and gut) from the Atlantic hagfish (Myxine glutinosa). The highest ACELA occurred in heart and gill (1.8 and 1.5 nmol His-Leu min(-1) mg protein(-1), respectively). The mammalian angiotensin I-converting enzyme (ACE) inhibitor, captopril, at 10(-5) M was a potent inhibitor of the ACELA found in all hagfish tissues. Radioimmunoassay showed that immunoreactive angiotensins (251.8+/-11.8 pM) were detectable in hagfish plasma. The validity of the assay for measurement of hagfish angiotensins was indicated by the parallelism of the angiotensin II standard curve against serially diluted hagfish plasma. Measurement of immunoreactive plasma angiotensins and detection of significant levels of ACELA in a wide range of tissues gives indirect evidence for the presence of a renin-angiotensin system in hagfishes, the earliest evolved group of craniates.