Endocrine Control of Calcium Metabolism in Teleosts

It is evident that fishes regulate their serum calcium efficientlybut that endocrine systems involved may be different from thosein tetrapods. A functional parathyroid gland has not yet beendemonstrated in fishes. The majority of evidence indicates thatcalcitonin has little or no effect on fish calcium regulation.Instead, the corpuscles of Stannius and the pituitary glandare necessary for maintaining fish serum calcium levels. Inthe killifish, Fundulus heteroclitus, the removal of the corpusclesproduces hypercalcemia in sea water but not in artificial seawater deficient in calcium. Transplants of the corpuscles orthe administration of corpuscle homogenate corrects the increasein calcium. On the other hand, hypophysectomy elicits hypocalcemiaunder calcium deficient conditions but not in calcium rich seawater. Replacement therapy with pituitary homogenate or hypophysialtransplant prevents the fall in calcium. It is postulated thatthe hypocalcemic corpuscles of Stannius and the hypercalcemicpituitary gland enable the euryhaline killifish to regulateits serum calcium levels in high calcium sea water and low calciumfresh water, respectively.     

Hypocalcemic effects of bovine parathyroid hormone (1-34) and Stannius corpuscle homogenates in teleost fish adapted to low-calcium water

Injections of bovine parathyroid hormone (PTH 1-34) and homogenates of corpuscles of Stannius produce hypocalcemia in male killifish and tilapia adapted to calcium-deficient seawater or fresh water, respectively. In fish from water with normal calcium concentrations no effects are noticeable. These results suggest similarity in bioactivity between PTH, the hypercalcemic hormone of terrestrial vertebrates, and the hypocalcemic factor of the corpuscules of Stannius in teleost fish.     

A second cell type in stannius bodies of two euryhaline teleost species

In the corpuscles of Stannius of sticklebacks and eels two cell types are described of presumably endocrine nature. The predominating type, comparable to the cells observed in other species, responds to variation in calcium content of the medium and possibly produces a hypocalcemic hormone. The second cell type is unreactive to calcium. Since it is more active in freshwater than in seawater specimens, this cell type is possibly involved in ionic regulation. It was not found in two seawater teleost species.     

Evidence of hypocalcemic factor from corpuscles of Stannius of the teleost Heteropneustes fossilis (Bloch)

The administration of acid extract of corpuscles of Stannius has no effect on the plasma calcium level, in the normal fish, whereas the same dose shows typical hypocalcemic response in hypercalcemic fish, H. fossilis. It is concluded that acid extract of corpuscles of Stannius is effective only in hypercalcemic condition.     

Intraspecific variation in gene expression after seawater transfer in gills of the euryhaline killifish Fundulus heteroclitus

Previous research has suggested that northern populations of the euryhaline killifish (Fundulus heteroclitus) are better adapted to freshwater environments than their southern counterparts. In this study, we examined whether this adaptation has come at an ionoregulatory cost in seawater, by comparing published data for northern killifish to newly acquired data on the molecular responses of southern killifish to seawater transfer. After abrupt transfer from brackish water (10 per thousand) to seawater, Na,K-ATPase activity, Na,K-ATPase alpha(1a) mRNA expression, and NKCC1 mRNA expression increased 1 and 4 days after transfer in the gills of southern fish (by 2-3-fold), but increased at 1 day and not 4 days after transfer in northern fish. Small increases in mRNA expression were observed in both populations at 14 days. CFTR expression also increased in southern and northern fish at 1 and 4 days into seawater, and was also elevated at 14 days in northern fish. Because fish from both southern and northern populations maintained plasma Na(+) and Cl(-) balance after seawater transfer, the differences in activity and expression could not have been caused by differences in plasma ion levels. Instead, some other regulatory factor may account for the differences in expression between populations. This study shows that freshwater adaptation in northern populations of killifish has not necessarily come at a significant ionoregulatory cost in seawater, but has altered the molecular responses of their gills to seawater transfer compared to southern killifish.     

The relationship between the ionic composition of the environment and the secretory activity of the endocrine cell types of stannius corpuscles in the teleost Gasterosteus aculeatus

Summary The corpuscles of Stannius of threespined sticklebacks contain two glandular cell types of presumed endocrine nature. To elucidate the function of both cell types the secretory activity of the cells was studied in fully adapted seawater and freshwater fishes and in specimens transferred from sea water to fresh water or adapted to media of various ionic composition. The secretory activity was established, in tissue sections and freeze-etch replicas, by estimating the volume of the nuclei, the density of the nuclear pores, and the frequency of exocytotic phenomena.The type-1 cells, ultrastructurally comparable to the predominant or only cell type described in many other teleosts, are more active in sea water than in fresh water. The activity of the type-2 cells, whose ultrastructural appearance is known only for salmonids and eels, is higher in fresh water. Transfer of seawater fishes to fresh water results in reduction of type-1 cells and activation of type-2 cells. The factors responsible for these changes were analyzed by exposure of fishes to solutions of various salts in fresh water and to artificial sea water with a reduced content of one of its components. The high activity of type-1 cells in sea water proved to be related to the high calcium content of this medium. These cells probably produce a substance comparable to hypocalcin, the endocrine factor isolated from the Stannius corpuscles of some other teleost species. The high activity of type-2 cells in fresh water appeared to be connected with the low sodium and potassium levels of this medium. Type-2 cells possibly produce a hitherto unknown hormone involved in the control of sodium and/or potassium metabolism.The technical assistance of Miss C. Mein and Mr. J. Zagers is gratefully acknowledged     

Fundulus heteroclitus acutely transferred from seawater to high salinity require few adjustments to intestinal transport associated with osmoregulation

The common killifish, Fundulus heteroclitus, has historically been a favorite organism for the study of euryhalinity in teleost fish. Despite the species' large range of salinity tolerance, studies of osmoregulation in high salinity are rare, with most previous studies focused on fish transferred between freshwater and seawater. Similarly, while branchial transport properties have been studied extensively, there are relatively few studies investigating the role of the intestine in osmoregulation in killifish. This study sought to characterize the fluid and ion transport occurring in the intestinal tract of killifish adapted to seawater, and furthermore to investigate the adjustments that occur to these mechanisms following acute transfer to high salinity (70ppt). In vivo samples of blood plasma and intestinal fluids of seawater-acclimated killifish indicated absorption of Na(+), Cl(-), and water, the relative impermeability of the intestine to Mg(2+) and SO(4)(2-), and active secretion of HCO(3)(-) into the intestinal lumen. The details of these processes were investigated further using in vitro techniques of isolated intestinal sac preparations and an Ussing chamber pH-stat titration system. However, these methods were discovered to be of limited utility under physiologically relevant conditions due to tissue deterioration. Results that could be validly interpreted suggested that there are few changes to intestinal transport following transfer to high salinity, and that adjustments to epithelial permeability occur in the first 24h post-transfer.     

Effects of calcium and phosphate on the corpuscles of Stannius of the teleost fish,Oreochromis mossambicus

Summary Removal of the corpuscles of Stannius (CS) in Oreochromis mossambicus leads to hypercalcemia and hypophosphatemia. The effects on CS size and ultrastructure of different calcium and phosphate concentrations of the ambient water and of the food were investigated. A six-fold increase of the calcium concentration of the water leads to a four-fold increase in CS volume; this is mainly caused by an increase in the size and number of the type-1 cells. The effect of external calcium is most probably mediated by the calcium concentration of the blood plasma. Plasma ionic calcium may be the relevant factor. Changes in the calcium concentration of the food had no effect on the CS. Similarly, hyperphosphatemia or hypophosphatemia induced by high phosphate concentrations of the water or the food, or by a phosphate-deficient diet, had no noticeable effect on the CS. The results support the hypothesis that the type-1 cells produce the hypocalcemic factor of the CS. There is no evidence for the production by the CS of an endocrine factor involved in the control of phosphate metabolism.     

A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F.heteroclitus

The killifish,Fundulus heteroclitus, is a euryhalineteleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at amolecular level, a 5.7-kilobase cDNA homologous to human cysticfibrosis transmembrane conductance regulator (hCFTR) was isolated froma gill cDNA library from SW-adapted killifish. This cDNA encodes aprotein product (kfCFTR) that is 59% identical to hCFTR,the most divergent form of CFTR characterized to date. Expression ofkfCFTR in Xenopus oocytes generatedadenosine 3',5'-cyclic monophosphate-activated,Cl-selective currentssimilar to those generated by hCFTR. In SW-adapted killifish,kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increasedseveralfold, suggesting a role for kfCFTR in salinity adaptation. Undersimilar conditions, plasma Na⁺levels rose significantly after 8 h and then fell, although it is notknown whether these changes are directly responsible for the changes inkfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.

     

Stannius corpuscles and plasma calcium levels during the reproductive cycle in the cichlid teleost fish,Oreochromis mossambicus

Summary The corpuscles of Stannius (CS) of the cichlid Oreochromis mossambicus (formerly Sarotherodon mossambicus) were studied in relation to sexual maturation and plasma calcium levels. After sexual maturation, the CS are enlarged in female fish, because of an increase in size and number of the type-1 cells. During the ovarian cycle, the size of the CS increases in parallel with the growth of the ovaries. Concurrently, plasma total calcium increases markedly until spawning. This increase is mainly accounted for by calcium bound to proteins (vitellogenins), but the ultrafiltrable calcium fraction is also slightly higher than in males. Ovariectomy is followed by a reduction in the size of the CS, mainly a result of involution of the type-1 cells, and by a reduction in plasma calcium to levels typical for males. Gonadectomy in males does not affect size or ultrastructure of the CS, or plasma calcium levels. Since the type-1 cells of the CS are the presumptive source of a hypocalcemic hormone, we conclude that activation of the CS during the female reproductive cycle is a response to elevated calcium levels that accompany ovarian maturation. We suggest that the CS respond in particular to the elevated ultrafiltrable or ionic calcium levels.     

Vascularization,innervation, and ultrastructure of the endocrine cell types of stannius corpuscles in the teleost Gasterosteus aculeatus

The blood circulation of the Stannius corpuscles, like that of the kidneys to which the corpuscles are attached, represents a portal system. The corpuscles receive blood from the dorsal caudal vein and from a vein coming from the hypaxial musculature. They are drained by veins which enter the caudal parts of the kidneys and therefore endocrine substances released by the corpuscles pass through the kidneys before they enter the general body circulation. The corpuscles are penetrated by sympathetic nerves coming from a small subvertebral ganglion. It is likely that these nerves innervate the muscular coat around the blood vessels. The muscular coat surrounding the renal blood vessels, the collecting tubules and part of the ureters, is innervated by nerves from the same ganglion. The secretory activity of the gland cells appears to be controlled by blood borne factors, because neither synaptic contacts with these cells, nor gap junctions among the cells, have been found in thin sections and freeze-etch replicas of the corpuscles. The corpuscles contain two cell types, both presumed to have endocrine function. Histochemical and ultrastructural data indicate that the gland cells produce glycoproteins. It is likely that the contents of the secretory granules are released by exocytosis. One cell type is structurally similar to the cells described in many other teleosts and thought to be engaged in the synthesis of a hypocalcemic hormone. The ultrastructure of the second cell type resembles cells described only in other migratory species: salmonids and eels. It may be involved in the control of monovalent ions.     

Differential Activity of Stanniocalcin in Male and Female Fresh Water Teleost Mastacembelus armatus (Lacepede) during Gonadal Maturation

The present study was carried out to analyze the differences in the activity of hormone stanniocalcin (STC) between male and female fishes of Mastacembelus armatus during their gonadal cycle. A large variation in nuclear diameter of cells of corpuscles of Stannius (CS) were recorded in relation to testicular cycle as well as ovarian cycle which indicates that the cellular activity varied with different phases of reproductive cycle in both male and female fish. Similar changes in nuclear diameter of CS cells were also observed after 17alpha-methyltestosterone administration in males and 17 β-estradiol administrations in females. A positive correlation was observed between plasma STC levels, gonadosomatic index (GSI) and the sex steroids in both sexes, suggesting that STC has a role in the processes involved in gonadal development. In addition females showed remarkable changes in plasma calcium level during gonadal cycle while no such change for males were observed. In females the plasma calcium level estimated during different phases of reproductive cycle indicates positive correlation between plasma level of calcium and gonad growth. Thus hyperactivity of CS cells was noted in both male and female fishes during gonadal cycle along with the differences in the activity of STC as well. In female it may act as hypocalcemic factor and bring the level of calcium to normal which increases during preparatory and pre spawning phases to fulfill the increased demand of calcium for vitellogenesis. However data of male fishes indicated that plasma STC concentration varied widely during gonadal cycle but showed no consistent relationship to plasma calcium level.     

The Calcium-sensitive Cells of the Pars Intermedia in the Eel

In the pituitary, the PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia appear less active in seawater (SW)- than in freshwater (FW)-adapted eels. The kinetics of their response during adaptation to SW or readaptation to FW was investigated. Morphometric studies show that transfer to SW induces a rapid nuclear atrophy which accentuates in eels kept for several weeks in SW. Readaptation to FW stimulated the Ca-s cells after 2–10 days; after 1 or 2 months, the cells tend to be similar to those of eels kept in FW. Plasma calcium decreases slightly but significantly in SW eels. The response of the Ca-s cells is not modified by an ovine prolactin treatment inducing hypercalcemia, hypernatremia and stimulation of the corpuscles of Stannius. Minor changes occurring in the MSH cells remain difficult to interpret; the short stimulation during readaptation to FW may be related to a stress effect and/or to release of other peptides present in the MSH cells of fish.     

Extracellular calcium-sensing receptors in fishes

The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca(2+)](o)) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca(2+) and Mg(2+) concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca(2+) sensing related both to organismal calcium homeostasis and to intercellular Ca(2+) signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.     

Cellular localization of somatolactin in the pars intermedia of some teleost fishes

Summary We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish pars intermedia.     

Extracellular calcium-sensing receptors in fishes.

The extracellular calcium-sensing receptor (CaSR) in fishes, like the CaSRs of tetrapod vertebrates, is a dimeric seven transmembrane, G protein-coupled receptor. The receptor is expressed on the plasma membranes of a variety of tissues and cells where it functions as a sensor of extracellular calcium concentration ([Ca(2+)](o)) in the physiological range. In the context of systemic calcium homeostasis, CaSR expressed in endocrine tissues that secrete calciotropic and other hormones (pituitary gland and corpuscles of Stannius) may play a central role in global integrative signaling, whereas receptor expressed in ion-transporting tissues (kidney, intestine, gills, and elasmobranch rectal gland) may have local direct effects on monovalent and divalent ion transport that are independent of endocrine signaling. In fishes, specifically, CaSR expression at the body surface (at the gills and olfactory tissues, for example) may permit direct sensing of environmental Ca(2+) and Mg(2+) concentrations, especially in the marine environment. Additionally, CaSRs may have other widespread and diverse roles in extracellular Ca(2+) sensing related both to organismal calcium homeostasis and to intercellular Ca(2+) signaling. As a consequence of the broad spectrum of recognized ligands, including polyvalent cations and amino acids, and of binding site shielding by monovalent cations, additional receptor functionalities related to salinity and nutrient detection are proposed for CaSRs. CaSR expression in the gastrointestinal tract may be multifunctional as a sensor for polyvalent cations and amino acids. Structural and phylogenetic analyses reveal strongly conserved features among CaSRs, and suggest that calcium sensing by mammalian parathyroid gland-type CaSR proteins may be restricted to chordates. Comparative functional and genomic studies that include piscine CaSRs can be useful model systems for testing existing hypotheses regarding receptor function, and will shed light on the evolutionary developmental history of calcium homeostasis in the vertebrates.     

Improvement in abnormal secretion of thyrotropin and gonadotropin after restoration of serum calcium is pseudohypoparathyroidism

A 25 yr-old woman patient was admitted because of convulsion. The diagnosis of pseudohypoparathyroidism was made on the basis of typical stigmata, lowered serum calcium, increased serum phosphorus and parathyroid hormone levels, and defective response in urinary excretion of cyclic AMP and phosphorus to exogenous parathyroid extract. Endocrine studies performed in the hypocalcemic state revealed several abnormalities of the pituitary gland such as an exaggerated response of TSH to TRH, high basal levels and exaggerated responses of LH/FSH and a blunted GH response to arginine-HCl, while there was no clinical evidence of hypothyroidism and hypogonadism. These abnormalities of anterior pituitary function were normalized after the restoration of normocalcemia by using 1 alpha-hydroxy-cholecalciferol. These results suggest that some endocrine abnormalities observed in pseudohypoparathyroidism might be functional and reversible disorders secondary to hypocalcemia rather than genetic ones.     

Morpholino gene knockdown in adult Fundulus heteroclitus: role of SGK1 in seawater acclimation

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism.     

Inhibition of whole body Ca2+ uptake in fresh water teleosts, Channa punctatus and Cyprinus carpio in response to salmon calcitonin

In many fish, ultimobranchial-derived calcitonin (CT) has been shown to be a potent hypocalcemic regulator. But an equal number of studies failed to show any correlation between CT and plasma calcium levels. Again, in fish, where CT has been shown to function as a hypocalcemic hormone, the way this is brought about is not well known. While the regulatory role of many hormones e.g., stanniocalcin, pituitary-derived prolactin and cortisol on gill calcium (Ca2+) transport (GCAT) has been well established, very few studies have been done to examine the effects of CT on GCAT in fish. In the present study we examined the effects of synthetic salmon calcitonin (sCT) in vivo on GCAT in two distinctly different species of fresh water teleost, Channa punctatus (partially air breathing) and Cyprinus carpio (fully gill breathing). Whole body calcium uptake, a measure of GCAT, was lower in the partial air breathing fish. We found that salmon CT had significant inhibitory effect on GCAT in both the fish species, kept either in normal tap water or low-calcium water. Fish, kept in high-calcium water, showed little response. In parallel studies we also observed that inhibition of GCAT was correlated with simultaneous changes in plasma calcium levels in response to exogenous administration of sCT. The present findings therefore suggest that CT in fresh water teleosts regulate its hypocalcemic action through inhibition of GCAT.     

Endocrine Organs of a Parasitic Male Deep-Sea Angler-Fish,Edriolychnus schmidti

The parasitic male deep-sea angler-fish Edriolychnus schmidti Regan 1925, which was described by Regan and Trewavas 1932, was reinvestigated with respect to the endocrine organs. Although the original microscopical sections are not in a good condition, it could be concluded that those organs which are involved in regulation of various internal environmental factors, as well as those involved in adaption to external factors, appear poorly developed or reduced. Mechanisms regulating reproductive activities seem to be well developed; a pronounced tuberis region is seen in the otherwise reduced brain and the peculiar pituitary gland appears to contain secretory active cells.