Modification of morphology and function of integument mitochondria-rich cells in tilapia larvae (Oreochromis mossambicus) acclimated to ambient chloride levels.

Similar to those of the gills of adults, three types of mitochondria-rich (MR) cells with different morphologies of apical surfaces (wavy convex, shallow basin, and deep hole) were identified on the integument of freshwater-acclimated tilapia larvae (Oreochromis mossambicus). The object of this study is to test the hypothesis that these subtype cells may represent MR cells equipped with variable efficiencies in Cl(-) uptake. Larvae acclimated to low-Cl(-) =0.001-0.007 mM) water developed higher densities of MR cells than those acclimated to high-Cl(-) =7.3-7.9 mM) water. The percentage of wavy-convex-type cells in total MR cells was higher in low-Cl(-)-acclimated larvae than in high-Cl(-)-acclimated larvae, which displayed only deep-hole type. In addition, Cl(-) influx rates of whole larva measured with (36)Cl(-) showed a coincident correlation with MR cell densities, that is, low-Cl(-) larvae displayed higher Cl(-) influx rates than did high-Cl(-) larva, suggesting that tilapia larvae develop a higher density of MR cells with larger apical surfaces (wavy-convex type) to boost Cl(-) uptake in Cl(-)-deficient water. The distinct types of apical surfaces may represent different phases of MR cells that possess different efficiencies of Cl(-) uptake. Increased apical membrane surface areas of MR cells may provide larvae with rapid regulation of Cl(-) before new MR cells differentiate.     

Ambient salinity modulates the expression of sodium pumps in branchial mitochondria-rich cells of Mozambique tilapia,Oreochromis mossambicus

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.     

Stimulation of Cl- uptake and morphological changes in gill mitochondria-rich cells in freshwater tilapia (Oreochromis mossambicus)

The purpose of the present article is to examine the relationships between ion uptakes and morphologies of gill mitochondria-rich (MR) cells in freshwater tilapia. Tilapia were acclimated to three different artificial freshwaters (high Na [10 mM], high Cl [7.5 mM]; high Na, low Cl [0.02-0.07 mM], and low Na [0.5 mM], low Cl) for 1 wk, and then morphological measurements of gill MR cells were made and ion influxes were determined. The number and the apical size of wavy-convex MR cells positively associated with the level of Cl(-) influx. Conversely, Na(+) influx showed no positive correlation with the morphologies of MR cells. The dominant MR cell type in tilapia gills changed from deep-hole to wavy-convex within 6 h after acute transfer from a high-Cl(-) to a low-Cl(-) environment. Deep-hole MR cells became dominant 24-96 h after acute transfer from a low-Cl(-) to a high-Cl(-) environment. We conclude that wavy-convex MR cells associate with Cl(-) uptake but not Na(+) uptake, and the rapid formation of wavy-convex MR cells reflects the timely stimulation of Cl(-) uptake to recover the homeostasis of internal Cl(-) levels on acute challenge with low environmental Cl(-).     

Biochemical characterization of isolated branchial mitochondria-rich cells of Oreochromis mossambicus acclimated to fresh water or hyperhaline sea water

Mitochondria-rich cells have been separated from other epithelial cells of tilapia (Oreochromis mossambicus) gills by density gradient centrifugation on Percoll. During centrifugation two main bands of cells formed. The viability of the cells in both bands was high (>90%). In one band, 45–47% of the total cell number was mitochondria-rich cells. The other band contained at least 80% pavement cells, representing the majority of other gill epithelial cell types. A comparison of the activities of four enzymes involved in major metabolic and ion regulatory functions was made between these two different fractions of cells. Furthermore, the separation of gill epithelial cells and determination of enzymatic activity was carried out in tilapia after the fish were acclimated to fresh water or hyperhaline sea water (60 mg·ml^-1 S) to gain an indication of the relative contribution of mitochondria-rich cells and pavement cells to both NaCl excretion and absorption. Regardless of acclimation salinity, the activities of Na⁺/K⁺-ATPase, glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were significantly higher in mitochondria-rich cells than in pavement cells. However, tilapia acclimated to hyperhaline sea water possessed significantly lower carbonic anhydrase activity in mitochondria-rich cells than in pavement cells. In contrast, no significant difference of carbonic anhydrase activity was observed between the two cell fractions in tilapia acclimated to fresh water.Abbreviations ATPase adenosine triphosphatase - CA carbonic anhydrase - DASPMI dimethylaminostyrylmethylpyridinium iodine - FW fresh-water - GIDH glutamate dehydrogenase - G6PDH glucose-6-phosphate dehydrogenase - HSW hyperhaline sea water (60 mg·ml^-1) - MR cells, mitochondria-rich cells - S salinity     

Fine structure of the branchial epithelium in the teleost Oreochromis niloticus

We have studied the gill epithelium of Oreochromis niloticus using transmission electron microscopy with the particular interested relationship between cell morphology and osmotic, immunoregulatory, or other non‐regulatory functions of the gill. Pavement cells covered the filament epithelium and lamellae of gills, with filament pavement cells showing distinct features from lamellar pavement cells. The superficial layer of the filament epithelium was formed by osmoregulatory elements, the columnar mitochondria‐rich, mucous and support cells, as well as by their precursors. Light mitochondria‐rich cells were located next to lamellae. They exhibited an apical crypt with microvilli and horizontal small dense rod‐like vesicles, sealed by tight junctions to pavement cells. Dark mitochondria‐rich cells had long dense rod‐like vesicles and a small apical opening sealed by tight junctions to pavement cells. The deep layer of the filament epithelium was formed by a network of undifferentiated cells, containing neuroepithelial and myoepithelial cells, macrophage and eosinophil‐like cells and their precursors, as well as precursors of mucous cells. The lateral‐basal surface was coated by myoepithelial cells and a basal lamina. The lamellar blood lacunae was lined by pillar cells and surrounded by a basal lamina and pericytes. The data presented here support the existence of two distinct types of pavement cells, mitochondria‐rich cells, and mitochondria‐rich cells precursors, a structural role for support cells, a common origin for pavement cells and support cells, a paracrine function for neuroepithelial cells in the superficial layer, and the control of the lamellar capillary base by endocrine and contractile cells. Data further suggest that the filament superficial layer is involved in gill osmoregulation, that may interact, through pale mitochondria‐rich cells, with the deep layer and lamellae, whereas the deep layer, through immune and neuroendocrine systems, acts in the regeneration and defense of the tissue. J. Morphol. 2010. © 2010 Wiley‐Liss, Inc.     

The function of mitochondria-rich cells (chloride cells) in teleost gills

Summary The CCs are the site of Cl^– transport in teleosts in sea water. The gills of freshwater teleosts contain at least two types of mitochondria-rich cell, the type and the type (Pisam and Rambourg, 1991). During seawater acclimation, the cells vanish and the cells are transformed and proliferate, and accessory cells appear in addition. This gives rise to the question of the function of cells in fish living in fresh water.According to the studies reviewed here, although they deal only with extrabranchial epithelia, the majority of evidence indicates that CCs (or MRCs) function as sites of active Ca²⁺ transport in freshwater teleosts. Moreover, some experimental results suggest that CCs are the Cl^– uptake site in freshwater teleosts. The main problem in characterizing the CC function is that they have not yet been adequately described from the biochemical standpoint. This applies particularly to their metabolic pattern and the composition of their apical and basolateral membranes, including their integrated proteins and cell-cell junctions.Experiments with organ tissue cultures such as gill organ cultures from Oncorhynchus mykiss (McCormick and Bern, 1989) and opercular membrane cultures from Oreochromis mossambicus (McCormick, 1990) will almost certainly yield important results. Primary cell cultures of CCs would be even better for characterizing CCs. Such a cell culture of rainbow trout respiratory cells has already been established (Pärt et al., 1993).     

Salinity effects on cytometrical parameters of the kidney in the euryhaline teleost Oreochromis mossambicus Peters

Study of the effects of environmental salinity on the glomerular areas and the nuclear areas of the renal tubules in tilapia showed significant differences ( P < 0.001 or 0.05) between seawater- and freshwater-adapted fish. Upon seawater adaptation, there were decreases in the glomerular areas and the nuclear areas in the main segments of the nephron: glomerular, −24.1%; 1st proximal, −17.1%; 2nd proximal, −21.5%; distal, −9.1%; collecting, −26.5%. These cytometrical changes are discussed in relation to the different osmoregulatory functions of euryhaline teleosts adapted to sea water and to fresh water.     

Neuropeptides in the intestine of two teleost species (Oreochromis mossambicus,Carassius auratus): Localization and electrophysiological effects on the epithelium

Summary The presence of bioactive peptides in the gut and their possible electrophysiological effects on the intestinal epithelium were studied in two teleost species, the tilapia (Oreochromis mossambicus) and the goldfish (Carassius auratus). Vasoactive intestinal polypeptide-like immunoreactive nerve fibres were found beneath the intestinal epithelium of both species. Galanin-, metenkephalin-and calcitonin gene-related peptide-like immunoreactive nerve fibres were found exclusively in the mucosa of the tilapia. Both species had vasoactive intestinal polypeptide-, enkephalin- or neuropeptide Y-like immunoreactive endocrine cells; calcitonin gene-related peptide-like immunoreactive endocrine cells were additionally found in the tilapia. Somatostatin- and dopamine--hydroxylase-like immunoreactivities were not observed. Nerve cell bodies in the myenteric plexus of both species showed immunoreactivity for calcitonin gene-related peptide-, vasoactive intestinal polypeptide-, and galanin-like peptide. Enkephalin-like immunoreactive nerve cell bodies were present in the tilapia only. None of the peptides had a pronounced electrogenic effect. However, calcitonin gene-related peptide added to stripped intestinal epithelium of the tilapia, reduced the ion selectivity, and addition of galanin increased the ion selectivity. In goldfish intestine, both galanin and calcitonin gene-related peptide were without effect. Enkephalin counteracted the serotonin-induced reduction of the ion selectivity of the goldfish intestinal epithelium, but had no effect on the tilapia epithelium. In both species, vasoactive intestinal polypeptide reduced the ion selectivity of the intestinal epithelium, and neuropeptide Y induced an increase of the ion selectivity. Somatostatin showed no effect on the epithelial ion selectivity of either species. Tetrodotoxin did not inhibit the effects of the peptides studied. The changes in ion selectivity suggest that the enterocytes may be under the regulatory control of these peptides.     

Morphological changes of integumental chloride cells to ambient cadmium during the early development of the teleost, Oreochromis mossambicus

Synopsis Newly-hatched embryos of Oreochromis mossambicus were reared in freshwater and treated with 0 (control), 50 (low level) or 200 (high level) ppb cadmium for 4 days. Changes in the numbers and dimensions of chloride cell apical crypts on the skin of the free embryos were examined daily using scanning electron microscopy. The apical crypts of the chloride cells were rarely observed on the skin of the embryo trunk, and unevenly distributed on the surface of the yolksac. Two days after hatching, the chloride cells of the free embryos exposed to 50 ppb Cd were more active than those of the other two groups. Compared with the control group, the maximum dimensions of the developing apical crypts were stimulated by 50 ppb Cd and inhibited by 200 ppb Cd. The results indicated that the development of chloride cells in tilapia free embryos was provoked by low level Cd exposure and stunted by high level Cd exposure, suggesting the existence of structure/function relationships in which the activation of chloride cells may be related to the ionoregulatory mechanism in adaptation to Cd exposure.     

Immunocytochemical identification of prolactin cells in the pituitary gland of tilapia larvae (Oreochromis mossambicus: Teleostei)

Summary Using an antiserum to highly purified chum salmon prolactin, prolactin cells were identified in the putative rostral pars distalis of newly hatched tilapia larvae (Oreochromis mossambicus) by the immunogold method for the electron microscope. In the putative rostral pars distalis, some cells had another kind of secretory granule which was much less numerous, much smaller in size, and without immunoreactivity to salmon prolactin antiserum. Controls incubated with salmon prolactin-preabsorbed antiserum or normal serum showed no immunoreactive cells, confirming the specificity of the antiserum. The possible role of prolactin in the osmoregulation of tilapia larvae is discussed.     

Mitochondrion-rich cells in the embryonic yolk sac of Oreochromis mossambicus

By removing a small amount of yolk, tilapia embryos were dechorionated successfully as early as 30 h after fertilization. Using DASPEI, a mitochondrion-specific fluorescent stain, we were able to determine the first appearance of the mitochondrion rich cells on the surface of the yolk sac 26 h after fertilization ( c. 2 h after the beginning of gastrula stage). However, with scanning electron microscopy examination, no apical crypt could be found until 48 h after fertilization.     

Ca+2 transport across intestinal brush border membranes of the cichlid teleost Oreochromis mossambicus

Summary Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use of magnesium precipitation and differential centrifugation. The membrane preparation was enriched 17-fold in alkaline phosphatase. The membranes were 99% right-side-out oriented as indicated by the unmasking of latent glyceraldehyde-3-phosphate dehydrogenase and acetylcholine esterase activity by detergent treatment. The transport of Ca⁺² in brush border membrane vesicles was analyzed. A saturable and a nonsaturable component in the uptake of Ca⁺² was resolved. The saturable component is characterized by a K _m much lower than the Ca⁺² concentrations predicted to occur in the intestinal lumen. The nonsaturable component displays a Ca⁺² permeability too high to be explained by simple diffusion. We discuss the role of the saturable component as the rate-limiting step in transmembrane Ca⁺² movement, and suggest that the nonsaturable component reflects a transport mechanism operating well below its level of saturation.The authors wish to thank Tom Spanings for his superb organization of fish husbandry, and Maarten de Jong (Dept. of Physiology, Faculty of Medicine, University of Nijmegen) for making the automated stopped-flow apparatus available to us.     

The volume of mitochondria-rich cells of frog skin epithelium

Summary The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog,Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.     

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.     

Degeneration and death,by apoptosis and necrosis,of the pavement and chloride cells in the gills of the teleost Oreochromis mossambicus

Summary Degeneration and death of branchial epithelial cells were studied in an African cichlid fish. In both freshwater and seawater fish the superficially located pavement cells are sloughed off at the end of their lifecycle. This process is preceded by degeneration via a process of cytoplasmic shrinkage and condensation related to apoptotic (physiologically controlled) cell death. The chloride cells are pleomorphic, i.e., accessory, mature, and degenerating cells. Degeneration of chloride cells mainly occurs by apoptosis. Degenerating cells show shrinkage and densification of cytoplasm and nuclei, and swelling of the tubular system; these cells are then separated from the ambient water by pavement cells. They are finally phagocytosed and digested by macrophages. Apoptosis of chloride cells, but not of pavement cells, is greatly stimulated when the fish are in seawater; this reflects an increase in cellular turnover of the chloride cells. Accidental cell death (necrosis) of pavement cells or chloride cells is rarely observed in fully adapted freshwater and seawater fish. Its incidence increases in the first few days following transfer of fish from fresh water to seawater.     

Distribution of chloride cells in teleost larvae

Distribution and density of the chloride cells in the newly hatched larvae of teleosts vary depending on species and environmental salinity at hatching. In the euryhaline freshwater ayu (Plecoglossus altivelis), chloride cells are concentrated in the skin posterior to the pectoral fins and gradually decrease in number toward the head and tail. In the stenohaline sea water flounder (Kareius bicoloratus), most chloride cells are localized at the inner membrane of gill chambers and in the skin near the openings of gill chambers, but only a few cells appear in the skin of the yolk sac. In the stenohaline freshwater carp (Cyprinus carpio), only a few small chloride cells are scattered in the body skin. The density and abundance of chloride cells appears to be correlated with the different requirements for osmoregulation in teleost larvae.     

Localization of chloride conductance to mitochondria-rich cells in frog skin epithelium

Summary Cell volume determinations and electrophysiological measurements have been made in an attempt to determine if mitochondria-rich (MR) cells are localized pathways for conductive movements of Cl across frog skin epithelium. Determinations of cell volume with video microscope techniques during transepithelial passage of current showed that most MR cells swell when the tissue is voltage clamped to serosa-positive voltages. Voltage-induced cell swelling was eliminated when Cl was removed from the mucosal bath solution. Using a modified vibrating probe technique, it was possible to electrically localize a conductance specifically to some MR cells in some tissues. These data are evidence supporting the idea that MR cells are pathways for conductive movements of Cl through frog skin epithelium.     

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.     

Regulation of drinking rate in euryhaline tilapia larvae (Oreochromis mossambicus) during salinity challenges

Euryhaline tilapia larvae are capable of adapting to environmental salinity changes even when transferred from freshwater (FW) to seawater (SW) or vice versa. In this study, the water balance of developing tilapia larvae (Oreochromis mossambicus) adapted to FW or SW was compared, and the short-term regulation of drinking rate of the larvae during salinity adaptation was also examined. Following development, wet weight and water content of both SW- and FW-adapted larvae increased gradually, while the dry weight of both group larvae showed a slow but significant decline. On the other hand, the drinking rate of SW-adapted larvae was four- to ninefold higher than that of FW-adapted larvae from day 2 to day 5 after hatching. During acute salinity challenges, tilapia larvae reacted profoundly in drinking rate, that is, increased or decreased drinking rate within several hours while facing hypertonic or hypotonic challenges, to maintain their constancy of body fluid. This rapid regulation in water balance upon salinity challenges may be critical for the development and survival of developing larvae.