Caudal Neurosecretory System Research Articles

Immunohistochemical Distribution of Urotensins I and II in the Central Nervous System of the Senegal Bichir, Polypterus senegalus

Abstract The distribution of the urotensin I (UI)- and II (UII)-like immunoreactants in the central nervous system of the primitive bony fish Polypterus senegalus was studied by means of immunohistochemistry. Noticeably, UI-positive perikarya were present chiefly in the brain stem and the entire spinal cord with the exception of the caudalmost spinal cord segments, and their axons terminated on the wall of the adjacent blood vessels. In the cranial portion, the positive cells were found in the nucleus preopticus, the caudal diencephalon and the medulla oblongata. In contrast, the UII-immunoreactive fibers were distributed in the terminal part of the spinal cord, anterior to the 15th-to-the last vertebra; and a few UII-immunopositive perikarya occurred only in the caudalmost 5 to 6 segments. The most caudal region of the spinal cord showed a simplified or reduced neurohemal structure, where only UII-immunoreactive fibers were found. Accordingly, this area may represent the caudal neurosecretory system of t...

Development of the caudal neurosecretory system of the chum salmon, Oncorhynchus keta, as revealed by immunohistochemistry for urotensins I and II

In order to make an immunohistochemical analysis of the development of the caudal neurosecretory system of the chum salmon, Oncorhynchus keta, we employed the peroxidase-anti-peroxidase technique using antisera specific for urotensins (U) I and II on artificially reared embryos, larvae, and juveniles of this species. Immunoreactivities for UI and UII were first demonstrated in the embryo immediately before hatching, showing labeled perikarya and fibers in the most caudal region of the spinal cord where the presumptive caudal neurosecretory system is located. However, distinct differentiation of the histological neurohemal organ had not yet begun in the embryo. Immunoreactive perikarya and fibers gradually increased in number, and an elaborate urophysis comparable to that of adults was demonstrated in the larvae about 5 months after hatching. At this stage, weak immunoreactivity against UI was detected in the neurohypophysis.

Isolation and primary structure of urotensin II from the brain of a tetrapod, the frog Rana ridibunda

A peptide related to urotensin II has been isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the peptide was established as Ala-Gly-Asn-Leu-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val and this sequence was confirmed by chemical synthesis. Frog urotensin II contains an additional amino acid residue compared with fish urotensin II peptides but the structure of the cyclic region of the molecule has been fully conserved. The data show that urotensin II is not confined to the caudal neurosecretory system of fish but is present in the central nervous system of a tetrapod.

Isolation and amino acid sequence of urotensin II from the sturgeon Acipenser ruthenus

Urotensin II (UII) peptides have previously been isolated from the urophysis (the neurohemal organ of the caudal neurosecretory system) of several teleost fish, and the UII amino acid sequences have been determined. Chondrostean fish, such as the Acipenseridae (sturgeon), though without a distinct urophysis, also have a caudal neurosecretory system, which has been indicated by bioassay and immunological evidence to contain UII-like peptides. In the present studies, we investigated by UII radioimmunoassay the UII-like peptides in the spinal cord of three Acipenser species, and isolated and sequenced UII from one of them. As expected, much more UII immunoreactivity (UII-IR) was found in caudal than in anterior spinal cord extracts. In addition, caudal extracts from A. ruthenus were found to contain much more UII-IR (whether determined on a UII-IR/weight or UII-IR/fish basis) than those from the larger A. stellatus and A. guldenstadti. UII was therefore isolated from A. ruthenus and its amino acid sequence was shown to be H-Gly-Ser-Thr-Ser-Glu- Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH . This sequence is identical at positions 6–11 (the disulfide ring) with the known teleost UII peptides, and has acidic and hydrophobic amino acids at positions 5 and 12, respectively, as do the teleost UII peptides. Overall sequence identity with the various forms of teleost UII was 58–83%.

Increased urotensin I and II immunoreactivity in the urophysis of Gillichthys mirabilis transferred to low salinity water

The function of the fish caudal neurosecretory system is uncertain, but a role in osmoregulation has been suggested by many investigators. Our objective was to determine if acclimation to water of different salinity has an effect on immunoreactive patterns and staining intensities of the two caudal neuropeptides, urotensins I (UI) and II (UII), in Gillichthys mirabilis. Five fish, originally maintained in seawater, were transferred to deionized fresh water (FW), and five were transferred to new seawater (SW). After 24 hr spinal cords were removed and fixed, FW and SW spinal cords were paired in blocks to receive identical treatment, and cryostat sections were double immunostained for both peptides using a double sequential immunofluorescence procedure. FW spinal cords exhibited increased staining intensities for both UI and UII in their urophyses (the neurohemal organ) compared to the SW spinal cords. The magnitude of intensity difference appeared greater for UI than for UII. In addition, the FW urophyses had more loci displaying intense, perivascular UI immunoreactivity than the SW urophyses. Thus, it appears that environmental salinity has an effect on the urophysial content of UI and UII in this euryhaline fish. The increased immunoreactivity in FW fish could reflect increased synthesis and storage, decreased release of the stored peptides, or decreased peptide degradation.

Endothelin-Like Immunoreactivity in the Nervous System of Invertebrates and Fish

There have been no reports on the distribution of immunoreactive endothelin (ir-ET) in lower vertebrates and invertebrates, except for our previous studies on the nereid Neanthes diversicolor and the earthworm Eisenia foetida. In the present study, we found ET-like immunoreactivity in five species of invertebrates and two species of fish with antiserum against synthetic endothelin-1 (ET-1). Immunoreactive perikarya and nerve fibers were observed in the central nervous system of the slug Limax marginatus, the freshwater snail Indoplanorbis exustus, and the mussel Mytilus edulis in mollusks, the field cricket Gryllus bimaculatus in insects, and the tube tunicate Ciona intestinalis in protochordates. In the medaka, Oryzias latipes, ir-ET was found in the hypothalamoneurohypophysial system, the caudal neurosecretory system, the gill, and the kidney. Immunoreactive cells were also found in the mucous gland of the slug and in the adenohypophysis of the lamprey, Lampetra japonica. The wide distribution of ET-like substances in invertebrates and fish provides evidence for the case that ET found in mammals has a long evolutionary history.

Salinity tolerance and its seasonal variation in the flounder, Platichthys flesus

1. Following transfer from 50% SW the increases in plasma chloride concentration and osmolality in SW compared to FW adapted fish were less marked in winter than in summer. 2. Urine chloride concentration and osmolality were higher and urine volume lower in SW than in FW adapted animals throughout the year. Urinary bladder volume was significantly restricted during winter by the presence of greatly enlarged gonads, particularly in females. This was associated with a reduced urine osmolality in FW and SW adapted animals in winter. 3. Activity of the caudal neurosecretory system varied in accordance with seasonal migration and change in reproductive status of the flounder.

The caudal neurosecretory system of Platichthys flesus: General morphology and responses to altered salinity

1. The general morphology of the caudal neurosecretory system of the flounder is described. 2. Immunocytochemical staining and quantification of peptides within the spinal cord, associated with the last eight pre-terminal vertebrae, indicate spatial separation of urotensin II (UII) and corticotropin-releasing factor (CRF)/urotensin I (UI) producing cells. 3. Quantification of urophysial peptides indicates that tissue levels are elevated in seawater (SW) compared with freshwater (FW) adapted fish.

Immunohistochemical demonstration of urotensins I and II in the caudal neurosecretory system of the Japanese charr,Salvelinus leucomaenis, retained in sea water

This paper is concerned with part of the role and function of the caudal neurosecretory system of the charr,Salvelinus leucomaenis, studied by immunohistochemistry. In order to elucidate the different histologic changes, we examined the immunoreactivities of urotenisn I (UI) and urotensin II (UII) in 3 experimental groups: the feral (river) fish, the fresh-water aquarium-, and sea water aquarium-retained fish. Coexistence of UI and UII was demonstrated in most of the smaller and larger neurons distributed in and near the urophyseal system of all 3 groups. However, some of the larger neurons were immunoreactive only to a single hormone, UI or UII. Merely a few neurons indicated no reactivity for either UI or UII. No such clearcut differences were encountered immunohistochemically in the 3 groups. Neuronal and urophysial immuno-reactivity to UI of feral and fresh-water-retained fish was slightly stronger than that of sea water-retained fish. Moreover, in sea water-retained fish, the intensity of immunoreactivity for UI was variable, and the number of neurons positive for UII only was somewhat larger than that in feral and fresh-water-retained fish. A series of UII-positive cerebrospinal fluid (CSF)-contacting neurons were seen in the ependymal and subependymal layers ventral to the central canal of the spinal cord in every group. These CSF-contacting neurons might constitute another neurosecretory system aside from the ordinary caudal neurosecretory system equipped with urophysis. In contrast to the hypothalamohypophysial neurosecretory system, the caudal neurosecretory system did not show any significant changes among the 3 groups. This suggests that urotensins I and II have no essential role in osmoregulation of the charr.

Steroidogenic role of the caudal neurosecretory system in the flounder, Platichthys flesus

Transfer of flounders from seawater (SW) to fresh water (FW) resulted in a small reduction in circulating cortisol levels and urophysial protein storage. Transfer of flounders from FW to SW resulted in a larger increase in plasma cortisol and specific urophysial protein storage. Over the first 4 days after transfer from FW to SW there was a positive correlation between the observed changes in urophysial urotensin I (UI) content and plasma cortisol. This apparent steroidogenic effect of UI was supported by the increases in plasma cortisol observed following iv injection of crude flounder urophysial gland extract and synthetic Catostomus commersoni UI. The study supports a contribution of the caudal neurosecretory system to the control of interrenal steroidogenesis as part of the integrated osmoregulatory physiology of euryhaline species like the flounder.

Differential effects of low pH and aluminium on the caudal neurosecretory system of the brook trout, Salvelinus fontinalis

Brook trout were subjected to soft water at pH 6·5, 5·5 or 5·0 without aluminium added, or to water at pH 5·5 with 200,300 or 500 μg Al I‐1 added. The response of the caudal neurosecretory system to low pH or aluminium was evaluated after one week by measuring the urotensin I and urotensin II concentrations in the urophysis by radioimmunoassay, and by morphometric analysis of the caudal neurosecretory cells. A positive correlation was found between urotensin I concentrations and acidity, and a negative correlation was found between urotensin II concentrations and total aluminium in the water. Morphometric indices (cell size and proportion of lobed nuclei in the caudal neurosecretory cells) suggested increased synthetic activity in the caudal neurosecretory cells of fish at pH 5·5 compared to pH 6·5.

Sauvagine/urotensin I-like immunoreactivity in the caudal neurosecretory system of a seawater fish Diplodus sargus L. in normal and hyposmotic milieu

We report the presence of sauvagine/urotensin I-like immunoreactive (SV/UI-LI) elements in the caudal neurosecretory system of a teleost ( Diplodus sargus L.) collected from aquaria tanks of the Aquaculture Center (Talassographic Institut of CNR) of Messina or maintained in an hyposmotic milieu for different periods. In normal specimens, SV/UI-LI material was recognizable in discrete or little amounts both in Dahlgren cell cytoplasm and in their axons that reach the urophysis. On the contrary, the specimens transferred in an hyposmotic milieu showed a fast and dramatic increase of immunoreactivity mainly in neurohemal endings of the urophysis. This suggests a physiological role of caudal neurosecretory products on osmoregulatory mechanisms.

In situ hybridization demonstrating coexpression of urotensins I, II-α, and II-γ in the caudal neurosecretory neurons of the carp, Cyprinus carpio

In order to examine the coexpression of different urotensins in the same caudal neurosecretory neurons, in situ hybridization with synthetic oligonucleotide probes specific for mRNAs for urotensins I, II-α, and II-γ (UI, UII-α, and UII-γ) was applied to the caudal neurosecretory system of the carp, Cyprinus carpio, together with immunohistochemistry. Almost all identifiable caudal neurosecretory neurons were labeled with the oligonucleotide and cDNA probes for UI mRNA. Further, any two of the probes for UI, UII-α, and UII-γ mRNAs labeled the same neurons in serial sections. These results suggest that essentially all caudal neurosecretory neurons of the carp coexpress UI, UII-α, and UII-γ. Although most neurons were densely labeled with oligonucleotide probes, only a small portion of the neurons were intensely immunoreactive. This suggests that most caudal neurosecretory neurons actively synthesize all three hormones, but that in some neurons, all or some of the hormones are rapidly transported to the urophysis, resulting in low or undetectable immunoreactivity in the perikarya.

Immunoreactive methionine-enkephalin in the caudal neurosecretory system of the carp, Cyprinus carpio.

Methionine-enkephalin (Met-enk) was detected by immunocytochemistry and radioimmunoassay in the caudal neuro-secretory system of the carp Cyprinus carpio. Some cells showing urotensin I (UI)-immunoreactivity reacted to Met-enk antiserum, but others did not. Neurons with urotensin II (UII)-immunoreactivity did not react to Met-enk antiserum; neurons with both UI and UII immunoreactivities also displayed a negative Met-enk reaction. Met-enk was detected by radioimmunoassay in the urophysis, although the content was relatively small compared with that found in other parts of the central nervous system and in the pituitary.

A double sequential immunofluorescence method demonstrating the co-localization of urotensins I and II in the caudal neurosecretory system of the teleost, Gillichthys mirabilis.

A double immunofluorescence method was devised to localize simultaneously urotensin-I (UI) and -II (UII) immunoreactivities in the caudal neurosecretory system of the goby, Gillichthys mirabilis. In a sequential fashion, sections of the posterior spinal cord and urophysis were treated with antiserum to corticotropin-releasing factor (CRF) that cross-reacts with UI, fluorescein-conjugated sheep anti-rabbit IgG, biotinylated anti-UII and rhodamine-conjugated avidin. UI and UII immunoreactivities appeared to coexist in some neurons and in most fibers and urophysial tissue; the remainder of the fibers and urophysis and the majority of neurons were immunoreactive for CRF/UI only. No convincing evidence of immunoreactivity for UII only was found. A few nonreactive cells were seen, but these may not be neurosecretory neurons. The two immunoreactive cell types were not segregated topographically, and the intensity of perikaryal immunofluorescence for CRF/UI was variable. To explain these results a hypothesis that all caudal neurosecretory cells may synthesize both UI and UII and that immunoreactive differences may reflect different states of cellular activity, is suggested. This sequential double immunofluorescence method offers several advantages over other techniques and is especially useful for co-localization studies when primary antisera from different species are not available.

The distribution of ‘extraurophyseal’ urotensin I-immunoreactivity in the central nervous system of Catostomus commersoni after urophysectomy

The occurrence and distribution of the fish neuropeptide urotensin I-immunoreactive (IR-UI) perikarya and fibres was investigated by peroxidase-anti-peroxidase immunohistochemistry in paraffin and vibratome sections of the central nervous system of urophysectomized and control fishes. IR-UI neurones not described previously were found in the area ventralis telencephali pars dorsalis, in the area pretectalis and in two unidentified brainstem nuclei of control and urophysectomized fishes. In urophysectomized fishes a 4-fold increase in the number of parvocellular IR-UI neurons was observed in the nucleus lateralis tuber-is, whereas no significant differences were seen in other IR-UI neuronal groups. A functional relationship between IR-UI ‘extraurophyseal’ (i.e. brain) and caudal neurosecretory systems is suggested.

Extraurophyseal distribution of urotensin II immunoreactive neuronal perikarya and their processes

The use of the unlabeled antibody enzyme method on serially adjacent sections permitted the demonstration of urotensin II (UII) and urotensin I (UI) immunoreactivities colocalized in most of the cells of the caudal neurosecretory system of Catostomus commersoni. The study of the upper regions of the central nervous system from the spinal cord anterior to the fifth preterminal vertebral region up to and including the brain stem revealed the presence of UII immunoreactivity in cerebrospinal fluid-contacting neurons, located ventral to the central canal along the entire length of the spinal cord and medulla. Beaded nerve fibers were observed projecting to the ventrolateral surface of the spinal cord and also forming a seemingly ascending immunoreactive-UII longitudinal bundle directed toward the brain. The presence of this "extraurophyseal" system of immunoreactive-UII cells and fibers suggests that the UII peptide may be released in upper regions of the central nervous system in response to stimuli conveyed via the cerebral spinal fluid. Thus, separate functions may be postulated for the urophyseal and the cerebral spinal fluid-contacting urotensin II systems.

Cloning and sequence analysis of cDNAs encoding precursors of urotensin II-alpha and -gamma

The primary structures of the precursors of urotensin II (UII)-alpha and -gamma, neuropeptide hormones of the caudal neurosecretory system of the carp, Cyprinus carpio, have been determined by analyzing the nucleotide sequences of cloned DNAs complementary to the mRNAs encoding them. A cDNA library, constructed with poly(A)+RNA in the preterminal spinal cord, was screened using 32P-labeled synthetic oligonucleotides representing all possible cDNA sequences corresponding to the pentapeptide common to all forms of carp UII (UII-alpha, -beta, and -gamma). Twenty out of 39 positive clones were analyzed with the restriction endonucleases, HindIII and PvuII, and classified into 4 groups. Nucleotide sequence analysis of 4 clones representing each group revealed that 2 clones encode the precursor of UII-alpha and the other 2 that of UII-gamma. Both precursors consist of 125 amino acid residues, and UII-alpha and -gamma exist at the carboxyl-termini preceded by Arg-Lys-Arg. The homology in both nucleotide and amino acid sequences between the precursors of UII-alpha and -gamma is more than 90%, suggesting that the genes were generated from a common ancestral gene by duplication. There is no sequence homology between the precursors of UII and urotensin I, another peptide hormone of the caudal neurosecretory system, nor between the precursors of UII and somatostatin-14. RNA transfer blot analysis indicated that mRNAs encoding the precursors of UII-alpha and -gamma are present in the spinal cord but not in the brain, intestine, liver, or kidney of the carp.(ABSTRACT TRUNCATED AT 250 WORDS)

Coexistence of urotensin I/corticotropin-releasing factor and urotensin II immunoreactivities in cells of the caudal neurosecretory system of a teleost and an elasmobranch fish

By use of the indirect immunofluorescence method in conjunction with an elution-restaining technique, cells in the caudal neurosecretory system of the big skate ( Raja binoculata) and the plainfin midshipman ( Porichthys notatus) were shown to contain both urotensin I (UI)/corticotropin-releasing factor (CRF) and urotensin II (UII) immunoreactivities. In the skate coexistence was complete, while in the midshipman some cells demonstrated immunoreactivity for only one or the other neuropeptide. The present results do not support previous suggestions that, like vasopressin and oxytocin in the hypothalamic magnocellular system, UI and UII exist in separate cellular populations in the caudal neurosecretory system. Results of these and other experiments indicate that there exists a wide range of species variation in the degree of coexistence of UI/CRF and UII immunoreactivities within caudal neurosecretory cells.

In vitro effects of somatostatin and urotensin II on prolactin and growth hormone secretion in tilapia, Oreochromis mossambicus

The control of release of two recently characterized forms of prolactin (PRL) of molecular mass 24 and 20 kDa was investigated. The rostral pars distalis of male tilapia was incubated singly in a hypotomic modified Krebs-Ringer bicarbonate medium in order to stimulate PRL release; for comparison, the proximal pars distalis containing growth hormone (GH) cells was incubated in isotonic medium with or without 1 μg/ml cortisol to stimulate GH release. The release of both PRLs and GH into the medium was measured by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by densitometry. Both somatostatin and synthetic ( Gillichthys) urotensin II, a partial somatostatin homolog and analog from the teleost caudal neurosecretory system, significantly inhibited the release of both PRLs. Somatostatin significantly inhibited GH release, but urotensin II had no significant effect.