The distribution of lamprey GnRH-III in brains of adult sea lampreys (Petromyzon marinus).

In the sea lamprey, Petromyzon marinus, two forms of GnRH, lamprey GnRH-I and -III, have been demonstrated to be neurohormones regulating the pituitary-gonadal axis. The objective of the present study was to determine the distribution of lamprey GnRH-III in the brains of adult sea lampreys and to compare it to the distribution of lamprey GnRH-I. For this purpose, two kinds of immunostaining were employed: one was a single immunostaining by one of two GnRH antibodies using two successive sections; the other was double immunostaining of a single section. A dense accumulation of neuronal cells immunoreactive (ir) to antisera against either lamprey GnRH-I or -III was found in the arc-shaped preoptico-anterior hypothalamic area. Additional smaller numbers of irGnRH cells were found in the periventricular zone of the posterior hypothalamus. In the above-mentioned locations, the distribution of both irGnRH-I and -III cells was intermixed and very similar, but the cells exhibiting GnRH-III immunoreactivity were distinctly different from those exhibiting GnRH-I immunoreactivity. The relative numbers of irGnRH-III cells were larger than those of irGnRH-I cells in the preoptico-anterior hypothalamic area, and more than 90% of GnRH cells in the posterior hypothalamus were irGnRH-III cells. Both irGnRH-I and -III cells projected their fibers primarily into the neurohypophysis. The relative densities of the accumulated irGnRH-III fibers were similar to those of irGnRH-I fibers in the anterior neurohypophysis but higher than those of irGnRH-I fibers in the posterior neurohypophysis. The present study provides further immunocytochemical data to the already compelling physiological evidence that indicates that both lamprey GnRH-I and -III act through the hypothalamic-pituitary-gonadal axis to modulate reproductive processes in the sea lamprey.

A brain-Hatschek's pit connection in amphioxus.

In the adult lancelet, Branchiostoma belcheri, there is a lobe of the right ventral margin of the brain that extends around the right side of the notochord and makes contact with Hatschek's pit, which also is to the right of the midline. This structural system resembles the hypothalamo-adenohypophyseal system of vertebrates and appears to make possible seasonal nervous regulation of the release of gonadotropin.

Isolation and characterization of melanotropins from lamprey pituitary glands.

Three peptides containing the melanotropin-core amino-acid sequence, YXMXHFRWG, were isolated from the pituitary glands of a modern representative of the most primitive vertebrates, the sea lamprey, Petromyzon marinus. MSH-A, a nonadecapeptide (NPELYQMNHFRWGQPPTHF), is free at both ends. MSH-B, an eicosapeptide (VQESADGYRMQHFRWGQPLP), is free at the N-terminus and amidated at the C-terminus. They differ strikingly from gnathostome MSHs in structure. The third peptide, with an apparent molecular weight of 15 kDa, was tentatively designated lamprey ACTH, based on a structural feature: the N-terminal 22-residue-MSH (SVSSPKYAMGHFRWGSPDKATI) is followed by four consecutive basic amino acids (RKRR) and a ACTH-like sequence (PVRPNTSDSPEIPDYAF--). MSH-B is 10 and 100 times more potent than alpha-MSH and MSH-A, respectively, in a frog skin assay in vitro, whereas the lamprey ACTH showed no melanotropic activity. Lamprey ACTH did, however, show corticotropic activity on the lamprey pronephric and mesonephric tissue.

The occurrence and distribution of GnRH in the brain of Atlantic hagfish, an agnatha, determined by chromatography and immunocytochemistry.

In Atlantic hagfish (Myxine glutinosa), chromatographic and immunocytochemical evidence showed that the brain contains a gonadotropin-releasing hormone (GnRH)-like molecule that is closely related to lamprey GnRH-III. The chromatographic studies (HPLC) used specific antisera directed against mammalian GnRH and lamprey GnRH-I. In addition to these antisera, other specific antisera were tested in immunocytochemical studies, including chicken GnRH-I, chicken-GnRH-II, salmon GnRH, and lamprey GnRH-III. Using a lamprey GnRH-I antiserum, an early eluting GnRH form coeluted on HPLC with lamprey GnRH-III standard and an unknown form coeluted with the chicken GnRH-II standard. Radioimmunoassay of brain tissue detected GnRH immunoreactivity only when the lamprey GnRH-I antibody was used. A GnRH-like immunoreactivity was also obtained immunocytochemically in the neurohypophysis with the use of antisera against chicken GnRH-II, salmon GnRH, lamprey GnRH-I, and lamprey GnRH-III. These studies indicate that, contrary to earlier reports, hagfish do have a GnRH-like molecule that is more closely related, in terms of immunological determinants, to lamprey GnRH-III than to other currently known vertebrate GnRH molecules.

A novel glycoprotein in the olfactory and pituitary systems of larval and adult lampreys.

A novel homodimeric glycoprotein was isolated and characterized from the pituitaries of adult sea lampreys, Petromyzon marinus, modern representatives of the earliest vertebrates. The monomer consists of 121 amino-acid residues in a sequence that has no resemblance to any known pituitary hormone. Whereas this protein is localized in most cells of the rostral pars distalis of adult lampreys, we have chosen to name it, nasohypophysial factor (NHF), because it first appears in the olfactory system of developing larval lampreys. Not only may NHF be a new pituitary hormone but a useful probe for examining the ontogenetic and phylogenetic relationships of the pituitary and olfactory systems in vertebrates.

Diffusion between the neurohypophysis and the adenohypophysis of lampreys, Petromyzon marinus.

Horseradish peroxidase (HRP), a protein that can be visualized by appropriate histochemical procedures, was injected into the third ventricle of the brain of adult lampreys, Petromyzon marinus. Within 5 to 15 min HRP had passed through the neurohypophysis, which forms the floor of the third ventricle. It appeared at this time to have diffused throughout the connective tissue separating the adenohypophysial follicles from the neurohypophysis and from each other. This observation would indicate that it is possible for neurosecretory peptides like gonadotropin-releasing hormone to diffuse from the brain (neurohypophysis) to the adenohypophysis and thus regulate its secretory activity in lampreys.

Detection of Met-enkephalin and Leu-enkephalin in the brain of the hagfish, Eptatretus stouti, and the lamprey, Petromyzon marinus.

Acid extracts of the brain of the pacific hagfish, Eptatretus stouti, and the marine lamprey, Petromyzon marinus, were each fractionated by gel filtration chromatography and aliquots of column fractions were screened with radioimmunoassays (RIAs) specific for pro-dynorphin-related end products and for pro-enkephalin-related end products. Only pro-enkephalin-related immunoreactive forms were detected. The enkephalin-sized immunoreactive material, isolated for each species, was separately fractionated by reverse-phase high-performance liquid chromatography (HPLC). Aliquots of column fractions were screened with RIAs specific for Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Phe, and Met-enkephalin-Arg-Gly-Leu. In the hagfish brain, immunoreactive forms with the same retention times as synthetic Met-enkephalin and Leu-enkephalin were detected in a ratio of approximately 2:1. In addition, an immunoreactive form was detected with the Met-enkephalin-Arg-Phe-specific RIA. This form had the same chromatographic properties as synthetic Met-enkephalin-Arg-Phe. Analyses with the Met-enkephalin-Arg-Gly-Leu RIA were negative. HPLC analysis of the lamprey enkephalin-related material revealed the presence of authentic Met-enkaphalin and Leu-enkephalin in a molar ratio of 3:1. C-terminally extended forms of Met-enkaphalin were not detected in the lamprey extracts. Collectively these observations indicate that pro-enkephalin-related opioid peptides are present in the brain of cyclostomes.

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.

Metabolic effects of salmon glucagon and glucagon-like peptide in coho and chinook salmon.

Different doses of glucagon and glucagon-like peptide (GLP) isolated from coho salmon, Oncorhynchus kisutch were tested in vivo and in vitro on juvenile coho and chinook (O. tshawytscha) salmon. Results obtained suggest an involvement of these peptides in the regulation of plasma glucose, plasma fatty acids, liver glycogen, and the hepatic enzymes: glycogen phosphorylase, pyruvate kinase, triacylglycerol lipase, and glucose-6-phosphate dehydrogenase. Metabolic effects were more enhanced in summer than either in spring or in autumn. GLP was less effective than glucagon in stimulating glycogenolysis in vivo. Salmon glucagon, especially in low concentrations, was generally more potent metabolically than mammalian (porcine/bovine) glucagon. The interaction between glucagon-family peptides and insulin seems to be different from the one described in mammals: glucagon and GLP either lowered plasma circulating levels of insulin or showed no effect. Only at the time of parr-smolt transformation did GLP slightly elevate plasma insulin levels in coho salmon.

Colocalization of glucagon-like peptide and glucagon immunoreactivities in pancreatic islets and intestine of salmonids.

Pancreatic islets of salmon contain at least two peptides of the glucagon family: 29-amino acid glucagon and 31-amino acid glucagon-like peptide (GLP). Both peptides were recently isolated from the pancreatic islets of coho salmon and sequenced (Plisetskaya et al. 1986). Antibodies generated against these two peptides and against human glucagon were used as immunocytochemical probes to investigate whether glucagon and GLP are processed in the same, or in different cell types in the pancreatic islets and the gut of salmon. Two salmonid species, rainbow trout and coho salmon, were studied. All islet A-cells in the two species were immunoreactive toward both anti-salmon (s)-glucagon and anti-s-GLP. Similar colocalization of glucagon and GLP immunoreactivites was found in open-type endocrine cells in mucosae of the small intestine (including the pyloric coecae) and the large intestine close to the vent of rainbow trout. None of the antibodies stained mucosal cells of the body of the stomach. These results suggest that in the pancreas and the gut of salmonid fish the same cells produce both glucagon and GLP. These peptides are most likely the products of a single gene coding for the preproglucagon sequence.

Different cellular distributions of two somatostatins in brain and pancreas of salmonids, and their associations with insulin- and glucagon-secreting cells.

Invariant somatostatin-14 (SST-14) and somatostatin-25 (SST-25), isolated from coho salmon pancreas (Plisetskaya et al., 1986a) are likely coded by two distinct somatostatin genes. The present study was undertaken to investigate whether these genes are expressed in the same or in different cell types in the pancreatic islets and in the brain of two salmonids: rainbow trout and coho salmon. Antibodies generated against SST-14, mammalian (m) SST-28(1-14), salmon (s) SST-25, salmon insulin, and salmon glucagon were used as immunocytochemical probes. Two distinct cell types containing SSTs were revealed in the pancreas of both salmonid species: one cell type immunoreactive to both SST-14 and mSST-28(1-14) and the other cell type immunoreactive only to sSST-25. The SST-14/mSST-28(1-14)-positive cells were limited to the more central parts of the islets, in apposition to the insulin-positive cells: sSST-25-positive cells were located more peripherally and were associated topographically with the glucagon-positive cells. In contrast to the pancreas, neurons in the neurohypophysis and hypothalamus of the rainbow trout and coho salmon contained only SST-14-like and mSST-28(1-14)-like immunoreactivities, while immunoreactivity to sSST-25 was completely absent. These results suggest that differentiation in the pancreas and brain of salmonid fishes results in cell types in which SST genes are separately expressed. The close topographical association of sSST-25 with glucagon cells, and of SST-14 with insulin cells, in the pancreatic islets implies yet unknown functional regulatory relationships that require detailed study.

Effects of somatostatin-25 and urotensin II on lipid and carbohydrate metabolism of coho salmon, Oncorhynchus kisutch.

Salmon (Oncorhynchus kisutch) somatostatin (sSS; 4 or 8 ng/g body wt) or synthetic Gillichthys urotensin II (UII; 2 or 4 ng/g body wt) were injected intraperitoneally into juvenile freshwater coho salmon. Both sSS and UII caused a dose-dependent increase in plasma free fatty acids (FFA) which diminished with time. sSS induced an initial (1 hr) transient hyperglycemia. By contrast, UII tended to induce hypoglycemia, this effect being significant 5 hr after injection of the higher dose. Both sSS and UII depressed plasma insulin titers 1 hr after injection. By 3 hr, the sSS-associated insulin depression was no longer observed. UII treatment induced a hyperinsulinemia which was present 3 and 5 hr after peptide administration. Although no decreases in liver total lipid concentration or in mesenteric fat total tissue mass were observed, lipolytic enzyme activity within each depot was significantly enhanced by both peptides. Neither sSS nor UII altered 3H2O incorporation into fatty acids or neutral lipids. However, enhanced lipogenesis, particularly by UII, was indicated by increased NADPH production resulting from glucose-6-phosphate dehydrogenase activity. Both sSS and UII enhanced glucose mobilization, as indicated by decreased liver glycogen content and increased liver glucose-6-phosphatase activity. UII, but not sSS, stimulated glycogen synthetase activity. These results suggest that both sSS and UII stimulate hyperlipidemia by enhancing depot lipase activity and that although both factors are potentially gluconeogenetic, sSS seems to be glycogenolytic and hyperglycemic, whereas UII may channel glucose to FFA synthesis.

Pituitary thyrotropin and gonadotropin of coho salmon (Oncorhynchus kisutch): separation by chromatofocusing.

Thyrotropin (TSH) and gonadotropin (GTH) were isolated from adult female coho salmon pituitary glands. After final extraction in acidic alcohol and precipitation in 85% ethanol, proteins were fractionated using gel filtration chromatography and chromatofocusing. Homologous bioassay systems were used to monitor bioactivity during the purification procedures. TSH activity was measured in vivo in coho salmon parr. GTH (steroidogenic) activity was determined in vitro using cultures of adult coho salmon ovarian follicles. Using these procedures, TSH and GTH activities were separated. TSH activity eluted as one major peak at pH 6.3 whereas GTH activity eluted as five major peaks at pH's 5.4, 5.0, 4.7, 4.3, and after 1.0 M NaCl on chromatofocusing. Molecular weights of the TSH and GTHs were estimated by gel filtration chromatography as 35 and 40 kDa, respectively. Like other vertebrate TSHs and gonadotropins, the coho salmon TSH and GTHs appeared to consist of two subunits. Coho salmon TSH and bovine TSH (bTSH) were equipotent in the TSH bioassay. The five coho salmon GTHs exhibited similar potencies in stimulating ovarian estradiol synthesis in vitro. Further biochemical analysis and tests for other gonadotropic activities are warranted to determine if these five GTHs are isoforms of one GTH or if they can be distinguished functionally in other GTH bioassays. Sufficient quantities of coho salmon TSH were isolated in this study for future studies of the hypothalamic-pituitary-thyroid axis in fish.

Characterization of coho salmon (Oncorhynchus kisutch) islet somatostatins.

Three different somatostatins have been isolated from the pancreatic islet tissue of the coho salmon (Oncorhynchus kisutch) by gel filtration and HPLC. Two of these peptides contain 14 amino acids and the larger third peptide consists of 25 amino acids. The sequence of the salmon SST-25 is Ser-Val-Asp-Asn-Leu-Pro-Pro-Arg-Glu-Arg-Lys-Ala-Gly -Cys-Lys-Asn-Phe-Tyr-Trp-Lys-Gly-Phe-Thr-Ser-Cys. The sequence of the salmon SST-14-I is Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys. The other small somatostatin (SST-14-II) which was not sequenced has an amino acid composition identical to the C-terminal 14 amino acids of the SST-25 and it is probably derived from this larger form. Evidence for low levels of a somatostatin containing 28 amino acids is also presented. This SST-28 appears to be an N-terminal extended precursor of SST-25 or a peptide derived via alternative processing of a common preprosomatostatin. Injected into juvenile salmon, SST-25 caused a decline in circulating levels of plasma insulin, depletion of liver glycogen, and activation of lipolytic pathways. Juvenile salmon treated with anti-SST-25 serum revealed elevated levels of plasma insulin as well as an increase of the glycogen content of the liver.

Occurrence and distribution of substance P-related immunoreactivity in the brain of adult lampreys, Petromyzon marinus and Entosphenus tridentatus.

The occurrence and distribution of substance P (SP)-related immunoreactivity were examined in the adult brains of two species of lampreys, Petromyzon marinus and Entosphenus tridentatus, by using the PAP technique and three different anti-SP sera (anti-SP#1, anti-SP#2, and anti-SP#3). In both species of lampreys, anti-SP#1 and anti-SP#2 yielded positive reactions in the brain, while there was no, or slight immunoreaction there to anti-SP#3. The positive reactions toward anti-SP sera in the lamprey brain were not eliminated, or insufficiently reduced by preabsorption with SP, but they were completely abolished by preabsorption with eledoisin-related peptide. Thus, SP-positive material in the lamprey brain is more closely related, in terms of immunological determinants, to eledoisin than to SP. In Petromyzon, SP-positive perikarya were found in the ventrolateral telencephalon and the ventral hypothalamus, whereas in Entosphenus they were found in the ventral thalamus, tegmentum motoricum mesencephali (two locations), and rostral rhombencephalon, as well as in the above-mentioned two regions. Nevertheless, the distributions of SP-positive fibers in the regions of the brain other than the neurohypophysis were very similar between the two species: SP-positive fibers were found at many locations of the brain, and were especially rich in the periventricular subependymal zone of the ventral telencephalon and in the diencephalon, preoptic area, hypothalamus, and interpeduncular nucleus. In Petromyzon, a heavy accumulation of positive fibers was observed in the rostral part of the anterior neurohypophysis, whereas in Entosphenus no such fibers were observed there. These results clearly suggest the presence of a neuronal system of unknown function containing a SP-related peptide in the brain of lampreys.

Isolation and structures of coho salmon (Oncorhynchus kisutch) glucagon and glucagon-like peptide.

Glucagon and glucagon-like peptide (GLP) containing 31 amino acids have been isolated from the principal islet of coho salmon (Oncorhynchus kisutch) by gel filtration of acid alcohol extracts followed by HPLC, and the complete amino acid sequence of both peptides has been determined. Salmon glucagon is a simple 29 residue peptide differing at 3 positions when compared to catfish glucagon and at 8 positions when compared to porcine glucagon. Salmon GLP differs at 6 positions when compared with the N-terminal 31 amino acids of the 34 amino acid catfish GLP. Both coho salmon glucagon and GLP cross-react weakly in our mammalian glucagon radioimmunoassay and therefore this technique could not be used to determine tissue content. Glucagon and GLP isolated amounted to 156 micrograms/g and 350 micrograms/g wet tissue, respectively.