Immunocytochemical detection of mitochondria-rich cells in the brood pouch epithelium of the pipefish, Syngnathus schlegeli : structural comparison with mitochondria-rich cells in the gills and larval epidermis

The brood pouch of the male pipefish (Syngnathus schlegeli) is a ventral organ located on the tail, with the anterior region closely associated with the genital pore. The embryos in the pouch are attached to highly vascularized placenta-like tissue which seals the pouch folds from inside during incubation. The epithelium of the placenta-like tissue consists of mitochondria-rich cells (MRCs) and pavement cells. Differences in MRC morphology in the brood pouch epithelium, the gills and the larval epidermis of the pipefish were examined by light and electron microscopy. Transmission electron microscopy revealed that the MRCs in the brood pouch and the gills shared common characteristics: the presence of numerous mitochondria packed among a well-developed tubular system and the close association of the basal parts with the capillaries running underneath the epithelia. The size of the apical opening of the elongate, flask-shaped brood pouch MRC was about one-tenth that of the apical pit of the gill MRC. The gill and larval epidermal MRCs formed a multicellular complex, in contrast to solitary brood pouch MRCs. The brood pouch MRCs were intensively stained by immunocytochemistry with an antiserum specific for Na⁺,K⁺-ATPase. The Na⁺ concentrations in the brood pouch were maintained near those in the serum rather than seawater during incubation. We conclude that the brood pouch MRCs function as an ion-transporting cell, absorbing ions from the brood pouch lumen, perhaps to protect the embryos from the hyperosmotic environment.     

Morphological responses of mitochondria-rich cells to hypersaline environment in the Australian mudskipper, Periophthalmus minutus

A population of the Australian mudskipper, Periophthalmus minutus, was found to inhabit mudflat that remained uncovered by tide for more than 20 days in some neap tides. During these prolonged emersion periods, P. minutus retreated into burrows containing little water, with a highest recorded salinity of 84 ± 7.4 psu (practical salinity unit). To explore the mechanical basis for this salinity tolerance in P. minutus, we determined the densities of mitochondria-rich cells (MRCs) in the inner and outer opercula and the pectoral fin skin, in comparison with P. novaeguineaensis, from an adjacent lower intertidal habitat, and studied morphological responses of MRCs to exposure to freshwater (FW), and 100% (34-35 psu) and 200% seawater (SW). Periophthalmus minutus showed a higher density of MRCs in the inner operculum (3365 ± 821 cells mm(-2)) than in the pectoral fin skin (1428 ± 161) or the outer operculum (1100 ± 986), all of which were higher than the MRC densities in p. novaeguineaensis. No mortality occurred in 100% or 200% SW, but half of the fish died within four days in FW. Neither 200% SW nor FW exposure affected MRC density. Transfer to 200% SW doubled MRC size after 9-14 days with no change in the proportion of MRCs with apical pits or plasma sodium concentration. In contrast, transfer to FW resulted in a rapid closing of pits and a significant reduction in plasma sodium concentration. These results suggest that P. minutus has evolved morphological and physiological mechanisms to withstand hypersaline conditions that they may encounter in their habitat.     

Ultrastructure and development of the gills in<Emphasis Type="Italic"> Rana dalmatina</Emphasis> (Amphibia,Anura)

The appearance and the modification of the gill apparatus in Rana dalmatina tadpoles have been described in the different phases of larval development. The morphology and ultrastructure have been studied using light microscopy and both scanning and transmission electron microscopy. The organization of the gills during the initial phases of development (external gills or transient gills) brings to mind the characteristics of Urodela larvae in which the gills appear to consist of three tufts of filaments supported by the gill arches III, IV and V. The cellular composition of the transient gills appears to be extremely simple and the presence of specialized cells is not noted. Basal cells, pavement cells and ciliated cells form the thin mono- or bilayered epithelium. In the persistent gills (or internal gills) of the R. dalmatina tadpole (Ortons larval type 4) the gill arches carry four rows of gill tufts branching out to the ventral region. Meanwhile, from the dorsal portion of the arch the gill filters present an axial portion from which there is much branching out, which confers a characteristic appearance on this part of the gills. The cellular composition of the gill tufts and of the filters is different: in the gill tufts basal cells, pavement cells, ciliated cells, cubic cells and mitochondria-rich cells (MRCs) have been recognized, while in the gill filters the last cellular type does not appear. The MRC has highly variable forms and dimensions and is characterized by the presence of numerous mitochondria in the cytoplasm. Often the MRCs manifest themselves grouped together, in groups of three or more. The pavement cells and the cubic cells demonstrate identical ultrastructural characteristics and have an external surface area characterized by the presence of short superficial microridges and numerous vacuoles in the apical cytoplasm.     

Salinity-dependent changes in Na+/K+-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia

The Mozambique tilapia (Oreochromis mossambicus) is prone to osmoregulatory disturbances when faced with fluctuating ambient temperatures. To investigate the underlying causes of this phenomenon, freshwater (FW)- and seawater (SW)-acclimated tilapia were transferred to 15, 25, or 35°C for 2 weeks, and along with typically used indicators of osmoregulatory status [plasma osmolality and branchial and intestinal specific Na⁺, K⁺-ATPase (NKA) activity], we used tissue microarrays (TMA) and laser-scanning cytometry (LSC) to characterize the effects of temperature acclimation. Tissue microarrays were stained with fluorescently labeled anti-Na⁺, K⁺-ATPase antibodies that allowed for the quantification of NKA abundance per unit area within individual branchial mitochondria-rich cells (MRCs) as well as sections of renal tissue. Mitochondria-rich cell counts and estimates of size were carried out for each treatment by the detection of DASPMI fluorescence. The combined analyses showed that SW fish have larger but fewer MRCs that contain more NKA per unit area. After a 2-week acclimation to 15°C tilapia experienced osmotic imbalances in both FW and SW that were likely due to low NKA activity. SW-acclimated fish compensated for the low activity by increasing MRC size and subsequently the concentration of NKA within MRCs. Although there were no signs of osmotic stress in FW-acclimated tilapia at 25°C, there was an increased NKA capacity that was most likely mediated by a higher MRC count. We conclude on the basis of the different responses to temperature acclimation that salinity-induced changes in the NKA concentration of MRCs alter thermal tolerance limits of tilapia.     

Functional plasticity of mitochondrion-rich cells in the skin of euryhaline medaka larvae (Oryzias latipes) subjected to salinity changes

A noninvasive technique, the scanning ion-selective electrode technique (SIET) was applied to measure Na(+) and Cl(-) transport by the yolk-sac skin and individual mitochondrion-rich cells (MRCs) in intact medaka larvae (Oryzias latipes). In seawater (SW)-acclimated larvae, significant outward Na(+) and Cl(-) gradients were measured at the yolk-sac surface, indicating secretions of Na(+) and Cl(-) from the yolk-sac skin. With Na(+) pump immunostaining and microscopic observation, two groups of MRCs were identified on the yolk-sac skin of SW-larvae. These were single MRCs (s-MRCs), which do not have an accompanying accessory cell (AC), and multicellular complex MRCs (mc-MRCs), which usually consist of an MRC and an accompanying AC. The percentage of mc-MRC was ～60% in 30 parts per thousand of SW, and it decreased with the decrease of external salinity. By serial SIET probing over the surface of the MRCs and adjacent keratinocytes (KCs), significant outward fluxes of Na(+) and Cl(-) were detected at the apical opening (membrane) of mc-MRCs, whereas only outward Cl(-) flux, but not Na(+) flux, was detected at s-MRCs. Treatment with 100 μM ouabain or bumetanide effectively blocked the Na(+) and Cl(-) secretion. Following freshwater (FW) to SW transfer, Na(+) and Cl(-) secretions by the yolk-sac skin were fully developed in 5 h and 2 h, respectively. In contrast, both Na(+) and Cl(-) secretions downregulated rapidly after SW to FW transfer. Sequential probing at individual MRCs found that Na(+) and Cl(-) secretions declined dramatically after SW to FW transfer and Na(+)/Cl(-) uptake was detected at the same s-MRCs and mc-MRCs after 5 h. This study provides evidence demonstrating that ACs are required for Na(+) excretion and MRCs possess a functional plasticity in changing from a Na(+)/Cl(-)-secreting cell to a Na(+)/Cl(-)-absorbing cell.     

Effects of salinity on chloride cells in the euryhaline cyprinodontid fish Rivulus marmoratus

Summary The ultrastructure and density of chloride cells in the gill, opercular epithelium, and opercular skin of the euryhaline self-fertilizing fish Rivulus marmoratus (Cyprinodontidae) were studied with electron and fluorescence microscopy. R. marmoratus raised from birth in 1, 50, 100, and 200% seawater were compared. Chloride cells from fish raised in each of the four salinities exhibited an invaginated pit structure at the apical crypt. Multicellular complexes were present in the 1% seawater group and in those fish raised in higher salinities where elaborate interdigitations were seen between cells. Chloride cells from gills of fish raised in 200% seawater had a significantly higher percentage of their cytoplasmic volume composed of mitochondria than did those from fish raised in 1% seawater (69.9% vs 37.4%). The opercular skin and opercular epithelium had the same density of chloride cells (4.2×10⁴-4.5×10⁴ chloride cells/cm²), and this number did not vary significantly with increased salinity. The opercular skin thus appears far more responsive to environmental salinity than the opercular epithelium. Chloride cells from the opercular epithelium of fish raised in 200% seawater were found to be 39% larger than those from fish raised in 1% seawater, whereas the chloride cells from the opercular skin of the 200% seawater group were 107% larger than those from the 1% seawater group.     

Gills of the medaka (Oryzias latipes): A scanning electron microscopy study

The general morphology and surface ultrastructure of the gills of adult and larvae medaka (Oryzias latipes) were studied in freshwater and seawater using scanning electron microscopy. The gills of all examined fish were structurally similar to those of other teleosts and consisted of four pairs of arches supporting (i) filaments bearing lamellae and (ii) rakers containing taste buds. Three cell types, specifically pavement cells, mitochondria‐rich cells (MRCs), and mucous cells, constituted the surface layer of the gill epithelium. Several distinctive characteristics of medaka gills were noted, including the presence of regularly distributed outgrowth on the lamellae, enlarged filament tips, the absence of microridges in most pavement cells in the filament and lamellae and the presence of MRCs in the arch at the filament base. A rapid mode of development was recorded in the gills of larval fish. At hatching, the larvae already had four arches with rudimentary filaments, rakers, and taste buds. The rudimentary lamellae appeared within 2 days after hatching. These results suggest the early involvement of larval gills in respiratory and osmoregulation activities. The responses of the macrostructures and microstructures of gills to seawater acclimation were similar in larvae and adult fish and included modification of the apical surface of MRCs, confirming the importance of these cells in osmoregulation. The potential roles of these peculiarities of the macrostructures and microstructures of medaka gills in the major functions of this organ, such as respiration and osmoregulation, are discussed.     

Intracellular voltage recordings in the opercular epithelium of Fundus heteroclitus

Opercular epithelial cells of Fundus heteroclitus were investigated using conventional microelectrodes. The area of interest was the cells lining the inside of the opercular epithelium closest to the gill arches, an area with a high density of chloride cells. Only one cell type could be discerned from the values of 60 opercular cells measured with the opercular epithelium in open circuit conditions. A mean apical voltage of -18.0 +/- 0.6 mV was observed with intracellular values ranging from -10 to -30 mV. The predicted intracellular chloride content was 59 mM/liter. Apical fractional resistance (faR) was 0.78 +/- 0.02. The intracellular potential measurements were typically difficult to maintain for extended periods (longer than 3 min). The opercular cells depolarize with serosal isoproterenol treatment (10(-6) M) corresponding to the increase in opercular transepithelial potential. The opercular cell apical fR decreased with isoproterenol treatment. These data indicate the observed opercular cells were involved in opercular chloride transport.     

Identification and immunocytochemical localization of two different carbonic anhydrase isoenzymes in teleostean fish erythrocytes and gill epithelia

Carbonic anhydrase was purified from the gills (CAB) of the rainbow trout Salmo gairdneri and from erythrocytes (CAE) of the fresh water carp Cyprinus carpio. The purification of the isozymes was confirmed by SDS acrylamide gel electrophoresis. Antibodies against the purified CAB and CAE were then raised in rabbits. Specificity was verified by immunoblotting. No cross-reaction was found between them, using the immunodot technique. CAB antiserum was used to specifically localize gill CA in the trout. Immunoperoxidase labelling revealed a concentration of enzyme on the apical region of the outer layer of the gill epithelial cells. The inner layer of the epithelium was only weakly positive. Results obtained using the immuno-gold technique confirmed the immunoperoxidase labelling: there was a concentration of label in the apical regions of chloride cells. In mucous cells, only the mucous granules were labelled. In the lamellae, the label was distributed in the apical part of the pavement cells. The villi and microplicae were strongly positive. CAE antiserum stained the red blood cells. The discrepancy between histochemical localization in the gill or in the opercular skin of killifish and our present immunolocalization was discussed. It was concluded that the most typical localization of CA is on the apical surface of the lamellar epithelium lying in contact with the environment. The result suggests that one of the main roles of gill CA may be to facilitate the diffusion of CO2 from blood to water.     

Skin cultured with or without an NaCl gradient and aldosterone influence epidermal fibronectin localizations

Fibronectin (FN) localizations in the epidermal cells of the frog Rana esculenta were detected in isolated ventral skin fragments 4 day-cultured with or without an NaCl transepithelial gradient and aldosterone. Without the gradient, few mitochondria-rich cells (MRCs) were FN-detected. Stratum germinativum and spinosum cells also contained fibronectin. With the gradient, numerous MRCs were detected. Below them, in the stratum germinativum, clear spaces were recognized. Aldosterone with or without the gradient modified the above effects: in both cases, many MRC contained fibronectin. It was interesting to note that, for each type of culture, stratum germinativum cells were dramatically FN-detected.     

Localization of a Band 3-related protein in the mitochondria-rich cells of amphibian skin epithelium

Based on immunoblotting procedure, the isolated epithelium of amphibian skin was found to contain a 180 kDa protein which cross-reacts with a polyclonal antiserum raised against human erythrocyte Band 3. Immunoperoxidase and immunofluorescence staining techniques indicated that the Band 3-related protein was localized in the mitochondria-rich cells (MRC) of this epithelium, with characteristic apical labelling pattern. Our findings show that the putative apical anion exchanger of the MRC is immunologically related to the band 3 multigenic family, which catalyzes Cl-HCO₃ ^? transmembranous exchange. It thus suggests a molecular basis for the role played by these cells in the transepithelial Cl pathway and acid-base regulation.     

Tissue kinetics,ion transport,and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm² (D_MRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO₃ (Ag) or methylene blue (MB). Part of the increased D_MRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (K_b) decreased and cell loss by moulting (K_d) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in D_MRC was accompanied by a 3-fold increase in Cl^- current (I_Cl). At day-2 there was a transient reduction in the I_Cl per MRC. H⁺ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased I_Cl and H⁺ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl^- (but not H⁺) transport capacity, was restored at day-7. We conclude that increased D_MRC following exposure to low external Cl^-, rather than being due to an increased K_b, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where morphological differentiation precedes functional differentiation.Parts of this study have been presented at the 32th International Congress of Physiological Sciences, 1–6 August, 1993, Glasgow, Scotland, and the 19th meeting of the European Study Group for Cell Proliferation, 5–9 October, 1993, Bruges, Belgium     

Morphological and morphometrical changes in chloride cells of the gills of Pimephales promelas after chronic exposure to acid water

Summary Fathead minnows, Pimephales promelas, were exposed for 129 days to Lake Superior water acidified with sulfuric acid by means of a flow-through toxicant injection system. The effects of chronic acid stress (pH 6.5, 6.0, 5.5, 5.0) on gill histology were examined. Most of the histological effects were seen at pH 5.5 and 5.0 and were confined primarily to changes in numbers, distribution, and morphology of chloride cells. At low pH levels there tend to be more chloride cells in the gill epithelium and an increased percentage of these cells in the secondary lamellae. In contrast to normal chloride cells, chloride cells from fish exposed to low pH frequently had apical pits while some had bulbous apical evaginations. The occurrence of structural changes in chloride cells during exposure to acid water suggests that chloride cells may be involved in acclimation to acid stress.     

Effects of Low pH acute exposure on survival and gill morphology in Triturus italicus larvae

We examined, from a morphological and ultrastructural point of view, the gill epithelium of Triturus italicus, both in basal conditions and after acute exposure to low pH. Our analysis of gill morphology began with the aim of determining lethal pH levels; we found that the pH value at which 50% of mortality occurs (LC50) is 4.0. We then investigated the effects of the larvae's exposure to a critical value of pH (pH 4.5). No change was observed in the gill during the first 24 hr. After 48 hr, a cellular response was evident and the gills appeared covered with a dense mucous layer. Observations that were carried out by light microscopy (LM) and electron microscopy, both scanning (SEM) and transmission (TEM), showed considerable histological and ultrastructural changes. As regards the principal filament, the alterations resulted in the presence of an external keratinized layer. More changes affected the mitochondria-rich cells (MRCs) in both a qualitative and quantitative way; this cell type shows a wide surface and seems to protrude more than the near pavement cells that, in normal conditions, partially cover the MRCs, thus reducing their external surface. The microvilli were extremely lengthened and often anatomized each other. Changes in the secondary filament affected the thickness of the epithelium, which appeared considerably smaller in the gills of the newts exposed to acid stress. The ciliated cells appeared to be more numerous than in the control specimens and the MRCs showed a widening of the apical surface.     

Apical membrane area of rabbit urinary bladder increases by fusion of intracellular vesicles: An electrophysiological study

Summary Mammalian urinary bladder undergoes, in a 24-hour period, a series of slow fillings and rapid emptying. In part the bladder epithelium accommodates volume increase by stretching the cells so as to eliminate microscopic folds. In this paper we present evidence that once the cells have achieved a smooth apical surface, further cell stretching causes an insertion of cytoplasmic vesicles resulting in an even greater apical surface area per cell and an enhanced storage capacity for the bladder. Vesicle insertion was stimulated by application of a hydrostatic pressure gradient which caused the epithelium to bow into the serosal solution. Using capacitance as a direct and nondestructive measure of area we found that stretching caused a 22% increase in area. Removal of the stretch caused area to return to within 8% of control. An alternate method for vesicle insertion was swelling the cells by reducing mucosal and serosal osmolarity. This perturbation resulted in a 74% increase in area over a 70-min period. Returning to control solutions caused area to decrease as a single exponential with an 11-min time constant. A microtubule blocking agent (colchicine) dit not inhibit the capacitance increase induced by hypoosmotic solutions, but did cause an increase in capacitance in the absence of a decreased osmolarity. Microfilament disrupting agent (cytochalasin B, C, B.) inhibited any significant change in capacitance after osmotic challenge. Treatment of bladders during swelling with C.B. and subsequent return, to control solutions increased the time constant of the recovery to control values (22 min). The Na⁺-transporting ability of the vesicles was determined and found to be greater than that of the apical membrane. Aldosterone increased the transport ability of the vesicles. We conclude that some constituent of urine causes a loss of apical membrane permeability. Using electrophysiological methods we estimated that the area of cytoplasmic vesicles is some 3.3 times that of the apical membrane area. We discuss these results in a general model for vesicle translocation in mammalian urinary bladder.     

Identification and immunocytochemical localization of two different carbonic anhydrase isoenzymes in teleostean fish erythrocytes and gill epithelia

Summary Carbonic anhydrase was purified from the gills (CAB) of the rainbow trout Salmo gairdneri and from erythrocytes (CAE) of the fresh water carp Cyprinus carpio. The purification of the isozymes was confirmed by SDS acrylamide gel electrophoresis. Antibodies against the purified CAB and CAE were then raised in rabbits. Specificity was verified by immunoblotting. No cross-reaction was found between them, using the immunodot technique. CAB antiserum was used to specifically localize gill CA in the trout. Immunoperoxidase labelling revealed a concentration of enzyme on the apical region of the outer layer of the gill epithelial cells. The inner layer of the epithelium was only weakly positive. Results obtained using the immuno-gold technique confirmed the immunoperoxidase labelling: there was a concentration of label in the apical regions of chloride cells. In mucous cells, only the mucous granules were labelled. In the lamellae, the label was distributed in the apical part of the pavement cells. The villi and microplicae were strongly positive. CAE antiserum stained the red blood cells. The discrepancy between histochemical localization in the gill or in the opercular skin of killifish and our present immunolocalization was discussed.It was concluded that the most typical localization of CA is on the apical surface of the lamellar epithelium lying in contact with the environment. The result suggests that one of the main roles of gill CA may be to facilitate the diffusion of CO₂ from blood to water.     

Effects of cortisol on gill chloride cell morphology and ionic uptake in the freshwater trout,Salmo gairdneri

Summary Daily intramuscular injection of cortisol (4 mg kg^–1 body weight) in rainbow trout,Salmo gairdneri, for 10 days caused significant increases in the number and individual apical surface area of gill chloride cells per mm² of filament epithelium. Concomitantly, whole body influxes of sodium (Na⁺) and chloride (Cl^–) increased. Acute (3 h) intra-arterial infusion of cortisol did not affect whole body Na⁺ or Cl^– influx. A significant correlation was observed between both Na⁺ and Cl^– influxes and the fractional apical surface area of filament chloride cells in control, sham (saline-injected) and experimental (cortisol-injected) fish. The chloride cells displayed similar ultrastructural modifications in trout undergoing cortisol treatment as in trout transferred to ion-deficient water. These findings suggest the existence of structure/function relationships in which branchial chloride cell morphology is an important determinant of Na⁺ and Cl^– transport capacity. We conclude that chronic cortisol treatment enhances whole body Na⁺ and Cl^– influxes by promoting proliferation of branchial chloride cells. The results of correlation analysis indicate that the chloride cell is an important site of NaCl uptake in freshwater rainbow trout.     

Gap junctions are non-randomly distributed inDrosophila wing discs

Summary The distribution of gap junctions in mature larvalDrosophila melanogaster wing discs was analyzed by means of quantitative electron microscopy. Gap junctions are non-randomly distributed in the proximal-distal disc axis and in the apical-basal cell axis of the epithelium. In the epithelial cells, the surface density, number and length of gap junctions are greatest in the apical cell region and distal disc region. The average gap junction surface density is 0.0572 m^–1 and 2.77% of the lateral cell surface is composed of gap junctions. In the adepithelial cells, the gap junction surface density is 0.0005 m^–1 and 0.06% of the cell surface is composed of gap junctions. No gap junctions were observed between epithelial cells and adepithelial cells. The absolute area of gap junctions was estimated in a proximal-distal strip of cells in the disc and is considerably less in the folded regions of the epithelium compared to the flat notum and wing pouch regions. The results are discussed with respect to pattern formation and growth control in imaginal discs.     

Active chloride cell density in some tissues of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) larvae during the early development stage

In this study, active chloride cell density in some tissues (gill arch epithelium, skin, and yolk-sac membrane) of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) larvae during the early development stage was investigated using a vital fluorescence staining technique. It was found that the numbers of active chloride cells were very variable, depending on the tissue and age of the larvae. Active chloride cells were most abundant in the skin and yolk-sac membrane, but less so in the gill arch epithelium of newly hatched larvae. With larval age, the density of active chloride cells in the gill epithelium increased, while that in the skin and yolk-sac membrane decreased.