Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout

A magnetic cell separation technique (MACS) was developed for isolating and characterizing peanut lectin agglutinin positive (PNA(+)) cells from rainbow trout gills. Percoll density separated mitochondria-rich (MR) cells were serially labeled with PNA-FITC and an anti-FITC antibody covalently coupled to a 50-nm iron particle and then applied to a magnetic column. PNA(+) MR cells were enriched to >95% purity. Transmission electron microscopy analysis of both the PNA(+) and PNA negative (PNA(-)) fraction showed that PNA binds to MR chloride cells while the PNA(-) cell fraction is comprised of MR cells with features characteristic of pavement cells. Western blotting demonstrated that both PNA(+) and PNA(-) fractions had high levels of Na(+)-K(+)-ATPase and Sco1 expression; however, relative expression of H(+)-ATPase in PNA(+) and PNA(-) cells demonstrated that untreated fish had twofold higher H(+)-ATPase levels in PNA(-) cells relative to the PNA(+) cells. Furthermore, hypercapnic acidosis significantly increased the relative H(+)-ATPase expression on PNA(-) cells only, whereas metabolic alkalosis had no significant effect.     

Cellular and epithelial adjustments to altered salinity in the gill and opercular epithelium of a cichlid fish (Oreochromis mossambicus)

Morphological features of the gill and opercular epithelia of tilapia (Oreochromis mossambicus) have been compared in fish acclimated to either fresh water (FW) or hypersaline water (60 S) by scanning electron and fluorescence microscopy. In hyperosmoregulating, i.e., FW-acclimated, tilapia only those mitochondria-rich (MR) cells present on the filament epithelium of the gill were exposed to the external medium. After acclimation of fish to hypersaline water these cells become more numerous, hypertrophy extensively, and form apical crypts not only in the gill filament but also in the opercular epithelium. Regardless of salinity, MR cells were never found to be exposed to the external medium on the secondary lamellae. In addition, two types of pavement cells were identified having distinct morphologies, which were unaffected by salinity. The gill filaments and the inner operculum were generally found to be covered by pavement cells with microridges, whereas the secondary lamellae were covered exclusively by smooth pavement cells.     

Fine structure of the gill epithelium of the terrestrial mudskipper, Periophthalmodon schlosseri

In this paper, we describe the fine structure of the branchial epithelium of the amphibious, air-breathing mudskipper Periophthalmodon schlosseri, and relate the observed structure to functions in gas exchange, and to the elimination of sodium chloride and ammonia. Also, we describe the fine structure of the opercular epithelimicrom. The gill lamellar epithelium is thickened by the presence of large mitochondria-rich (MR) cells. These MR cells are further characterized by an extensive tubular system that is continuous with the basolateral plasma membrane and by a deep apical crypt often lined with microvilli. There are very few specialized MR accessory cells, which are associated with NaCl excretion in marine teleosts. Instead, MR cells are commonly isolated from each other laterally by flattened cells rich in intermediate filaments. These filament-rich (FR) cells are interconnected by desmosomes and have unusual canaliculi. These branchial FR cells are unique to P. schlosseri and may have a structural role. Electron-dense pavement cells rich in vesicles and large vacuous mitochondria compose the superficial layer of the epithelium. The unusual morphology of P. schlosseri's gill lamellae may be related to the animal's ability to effectively eliminate ammonia during air exposure. The inner opercular lining and parts of the leading edge of the filament have intraepithelial capillaries, which provide a more suitable gas exchange surface than the thickened lamellae with its restricted interlamellar water spaces. The arrangement of respiratory and ion exchange epithelia is opposite to that found in all other fish in which the lamellae typically function in gas exchange and the gill filament in ion regulation.     

Ion-deficient environment induces the expression of basolateral chloride channel, ClC-3-like protein, in gill mitochondrion-rich cells for chloride uptake of the tilapia Oreochromis mossambicus

Gill mitochondrion-rich (MR) cells contain different molecules to carry out functionally distinct mechanisms. To date, the putative mechanism of Cl(-) uptake through the basolateral chloride channel, however, is less understood. To clarify the Cl(-)-absorbing mechanism, this study explored the molecular and morphological alterations in branchial MR cells of tilapia acclimated to seawater (SW), freshwater (FW), and deionized water (DW). Scanning electron microscopic observations revealed that three subtypes of MR cells were exhibited in gill filament epithelia of tilapia. Furthermore, in DW-acclimated tilapia, the subtype I (ion-absorbing subtype) of MR cells predominantly occurred in gill filament as well as lamellar epithelia. Whole-mount double immunofluorescent staining revealed that branchial ClC-3-like protein and Na(+)/K(+)-ATPase (NKA), the basolateral marker of MR cells, were colocalized in tilapia. In SW-acclimated tilapia, all MR cells of gill filament epithelia exhibited faint fluorescence of ClC-3-like protein. In contrast, only some MR cells in gill filament epithelia of FW and DW tilapia expressed basolateral ClC-3-like protein; however, the fluorescence was more intense in FW and DW tilapia than in SW fish. In hyposmotic groups, the number of MR cells immunopositive for ClC-3-like protein was significantly higher in DW-exposed tilapia. Meanwhile, in gill lamellar epithelia of DW tilapia, all MR cells (subtype I) were ClC-3-like protein immunopositive. Double immunostaining of ClC-3-like protein and Na(+)/Cl(-) cotransporter (NCC) revealed that basolateral ClC-3-like protein and apical NCC were colocalized in some MR cells in FW and DW tilapia. Moreover, both mRNA and protein amounts of branchial ClC-3-like protein were significantly higher in DW-acclimated tilapia. To identify whether the expression of branchial ClC-3-like protein responded to changes in environmental [Cl(-)], tilapia were acclimated to artificial waters with normal [Na(+)]/[Cl(-)] (control), lower [Na(+)] (low Na), or lower [Cl(-)] (low Cl). Immunoblotting of crude membrane fractions for gill ClC-3-like protein showed that the protein abundance was evidently enhanced in tilapia acclimated to the low-Cl environment compared with the other groups. Our findings integrated morphological and functional classifications of ion-absorbing MR cells and indicated that ion-deficient water elevated the numbers of subtype I MR cells in both filament and lamellar epithelia of gills with positive ClC-3-like protein immunostaining and increased the expression levels of ClC-3-like protein. This study is the first to illustrate the exhibition of a basolateral chloride channel potentially responsible for Cl(-) absorption in the ion-absorbing subtype of gill MR cells of tilapia.     

Distribution and development of rodlet cells in the gills and pseudobranch of the bass, Dicentrachus labrax (L)

Rodlet cells in various stages of development were found in large numbers in the bass gill and pseudobranch. In the gill, rodlet cells were found in the epithelium at the base of the secondary lamellae and on the filament between adjacent lamellae, whilst in the pseudobranch they were found over the whole area of the secondary lamellae as well as in the filament epithelium.
During early development, rodlet cells are characterised by their amorphous cell inclusions, prominent supranuclear Golgi complex and network of granular endoplasmic reticulum. Later, with formation of a fibrous border the arrangement of the cell organelles undergoes reorganisation; the endoplasmic reticulum becomes distended, numerous vesicles appear and the mitochondria aggregate in the apical region of the cell. One of the most striking features is the development of club-shaped sacs containing electron dense cores, which are orientated towards the open apex of the cell.
Various staining properties of rodlet cells for light and electron microscopy were compared with those of mucous cells found in the same tissues. Possible functions of the cell are discussed.     

Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus)

Summary Exposure of adult brown bullheads Ictalurus nebulosus (120–450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (J _net ^Na+ ) and net chloride flux (J _net ^Cl- ) resulting largely from changes in whole body sodium influx (J _in ^Na+ ) and chloride influx (J _in ^Cl- ). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in J _in ^Cl- during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in J _in ^Na+ did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the post-hypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl^-/HCO ₃ ^- exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.     

Developmental and environmental regulation of chloride cells in young American shad, Alosa sapidissima

Location, abundance, and morphology of gill chloride cells were quantified during changes in osmoregulatory physiology accompanying early development in American shad, Alosa sapidissima. During the larval-juvenile transition of shad, gill chloride cells increased 3.5-fold in abundance coincident with gill formation, increased seawater tolerance, and increased Na(+),K(+)-ATPase activity. Chloride cells were found on both the primary filament and secondary lamellae in pre-migratory juveniles. Chloride cells on both the primary filament and secondary lamellae increased in abundance (1.5- to 2-fold) and size (2- to 2.5-fold) in juveniles held in fresh water from August 31 to December 1 (the period of downstream migration) under declining temperature. This proliferation of chloride cells was correlated with physiological changes associated with migration (decreased hyperosmoregulatory ability and increased gill Na(+),K(+)-ATPase activity). Increases in chloride cell size and number of fish in fresh water were delayed and of a lower magnitude when shad were maintained at constant temperature (24 degrees C). When juveniles were acclimated to seawater, chloride cell abundance on the primary filament did not (though size increased 1.5- to 2-fold), but cells on the secondary lamellae disappeared. Na(+),K(+)-ATPase was immunolocalized to chloride cells in both fresh water and seawater acclimated fish. The disappearance of chloride cells on the secondary lamellae upon seawater acclimation is evidence that their role is confined to fresh water. The proliferation of chloride cells in fresh water during the migratory-associated loss of hyperosmoregulatory ability is likely to be a compensatory mechanism for increasing ion uptake. J. Exp. Zool. 290:73-87, 2001.     

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.     

Gross morphology and surface ultrastructure of the gills of Odontesthes argentinensis (Actinopterygii, Atherinopsidae) from a Southwestern Atlantic coastal lagoon

Odontesthes argentinensis was collected from Mar Chiquita Coastal Lagoon, the Southernmost coastal Atlantic Lagoon of Argentina. The morphology of the gills was analyzed by scanning electron microscopy. The morphology of the superficial structures of the gill filaments and pharyngeal region of the gill arch was discussed and related to their functional aspects. The gills arches are structurally similar to those of other teleosts and bring out the osmoregulatory capacity of this species. The epithelium that covers the surface of the filaments and the pharyngeal region of the gill arch is formed by polygonal pavement cells with conspicuous microridges. These folds in the membrane are not denoted in the epithelium of the respiratory lamellae. Apical crypts of chloride cells are present on the afferent and interlamellar filament surfaces, but are absent elsewhere on the gill arch. The highest density of mucous cells is observed into the gill filament and the pharyngeal region which indicates the existence of a protective strategy of the respiratory lamellae and the pharynx. The epithelium of the gill arches and the rakers is studded with spines. There are taste buds along the whole pharyngeal region that may be associated with their participation in tasting at this zone.     

Presence of chloride cells in gill filaments and lamellae of the skate Torpedo marmorata

The morphology of chloride cells in the gill of the skate Torpedo marmorata is described from a light and an electron microscopic study. Chloride cells have been regularly observed in both the gill filament (or primary epithelium) and the lamellae (or secondary epithelium). Chloride cells located in the filament usually display convex apical regions with microvilli protruding amongst microridges of neighbouring pavement cells, whereas chloride cells in the lamellae are located between the two epithelial layers, and contact with the external milieu is via a narrow apical opening. Present observations are discussed in relation to data on the presence of chloride cells in the lamellae of marine teleost fish, and it is suggested that the occurrence of chloride cells all along the lamellae might be in some way inversely related to fish activity.     

Morphological effects of mercury exposure on windowpane flounder gills as observed by scanning electron microscopy

Windowpane flounder, Scophthalmus aquosus Mitchill, were exposed for 60 days to 5 or 10 μg 1⁻¹ mercury and gill samples were examined by scanning electron microscopy. The response of the gill epithelium was different at the two levels of mercury exposure. The number of chloride cell apical pits and gill filaments bearing 'cratered' epithelial cells increased at the 5-μg 1⁻¹ level and decreased at the higher exposure level.
Focal swellings demonstrated a dose-dependent relationship, their numbers being greatest at the higher exposure level. Marked fragmentation of pavement cell microridge patterns and swelling of the respiratory epithelial cells was evident at the 10-μg 1⁻¹ exposure level.     

Surface ultrastructural changes in the gills of sockeye salmon (teleostei: Oncorhynchus nerka) during seawater transfer: Comparison of successful and unsuccessful seawater adaptation

Sockeye salmon were transferred rapidly from freshwater to seawater and the changes in gill morphology, in particular the distribution and sizes of chloride and mucous cells on the afferent filamental surface examined. Salmon that successfully adapted to seawater were compared with salmon that did not adapt to seawater and died as a consequence of osmoregulatory failure. The number of mucus cells (density), determined from scanning electron microscopy, increased significantly after seawater challenge. A greater increase in mucus cell density occurred in the salmon that failed to adapt to seawater. Light microscopy of transverse sections of gills detected no difference in mucus cell numbers after seawater challenge. It is proposed that mucus cells that lie just beneath the gill epithelium are activated in response to the seawater challenge, and migrate and open onto the epithelium. Freshwater-adapted salmon that had low densities of chloride cells prior to the seawater challenge failed to adapt, whereas salmon that had high densities of chloride cells adapted successfully to seawater. In the latter, the density of chloride cells on the afferent surface decreased after 30 days in seawater. The apical surface of the chloride cells of freshwater-adapted sockeye were either smooth or covered with microvilli. A greater proportion of microvilli-covered chloride cells occurred in the freshwater-adapted salmon that subsequently adapted to seawater.     

Thyroid status alters gill ionic metabolism and chloride cell morphology as evidenced by scanning electron microscopy in a teleost Anabas testudineus (Bloch): short and long term in vivo study

Gill is the main organ of osmotic regulation in teleosts and chloride cells are the sites of ion transport across gill epithelium. Thyroid hormones are implicated in the regulation of osmotic balance in teleosts also. Treatment with 6-propyl thiouracil (6-PTU) inhibited the membrane bound enzyme Na+K+ ATPase in the gill while triiodothyronine (T3) injection stimulated it in a short-term in vivo study in the teleost Anabas testudineus. Na+, K+ and Ca2+ ions were also decreased in the 6-PTU treated fish and the T3 treatment increased their concentrations in the gill lamellae. The gill morphology also changed according to the thyroid status in the long term study. 6-PTU treatment altered the typical serrated morphology of the gill lamellae, while the T3 treatment reversed it. T3 injection increased the density of pavement and chloride cells as evidenced by scanning electron microscopy. The results demonstrate that physiological status of the thyroid influences gill Na+ pump activity and chloride cell morphological changes. Further, the study suggests a regulatory role of T3 on gill ions (Na+, K+ and Ca2+), Na+K+ and Ca2+ ATPase activity and the different gill cell types in A. testudineus.     

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.     

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.     

Functional characterization and localization of a gill-specific claudin isoform in Atlantic salmon

Claudins are the major determinants of paracellular epithelial permeability in multicellular organisms. In Atlantic salmon (Salmo salar L.), we previously found that mRNA expression of the abundant gill-specific claudin 30 decreases during seawater (SW) acclimation, suggesting that this claudin is associated with remodeling of the epithelium during salinity change. This study investigated localization, protein expression, and function of claudin 30. Confocal microscopy showed that claudin 30 protein was located at cell-cell interfaces in the gill filament in SW- and fresh water (FW)-acclimated salmon, with the same distribution, overall, as the tight junction protein ZO-1. Claudin 30 was located at the apical tight junction interface and in cell membranes deeper in the epithelia. Colocalization with the α-subunit of the Na(+)-K(+)-ATPase was negligible, suggesting limited association with mitochondria-rich cells. Immunoblotting of gill samples showed lower claudin 30 protein expression in SW than FW fish. Retroviral transduction of claudin 30 into Madin-Darby canine kidney cells resulted in a decreased conductance of 19%. The decreased conductance correlated with a decreased permeability of the cell monolayer to monovalent cations, whereas permeability to chloride was unaffected. Confocal microscopy revealed that claudin 30 was expressed in the lateral membrane, as well as in tight junctions of Madin-Darby canine kidney cells, thereby paralleling the findings in the native gill. This study suggests that claudin 30 functions as a cation barrier between pavement cells in the gill and also has a general role in cell-cell adhesion in deeper layers of the epithelium.     

Gill morphology in the zebrafish, Brachydanio rerio (Hamilton- Buchanan)

A light and electron microscopic study of the gills of the zebrafish, Brachydanio rerio , were made to serve as a morphological basis for future investigations. It was found that for fixation of B. rerio gills, a mixture of 1·5% gluturaldehyde and 1·5% paraformaldehyde gives a mucus-free surface. Morphometric measurements of structural components of the gill secondary lamellae were made. Observations at SEM were correlated with those made at TEM. The different cell types in the branchial epithelium were characterized. Chloride cells were mainly located in the interlamellar regions and on the afferent side of the primary lamellae. Two morphologically different chloride cells were seen. The first type communicates with the external environment through a reservoir-like lumen, which is normally absent in freshwater fishes. The second type of chloride cell has more direct contact with the ambient water, resembling chloride cells from other freshwater fishes. Another cell type with features similar to those of the rodlet cell was frequently observed. This cell is interposed between other types of cells in the epithelium, and sometimes junctional complexes were present between the rodlet cell and surrounding cells.     

波形纤维蛋白与Nup 180的体外结合

本文作者采用大肠杆菌表达的波形纤维蛋白与大鼠肝细胞分离的核孔蛋白进行体外结合实验,以分析波形纤维与核孔的关系。实验结果显示,细菌表达的波形纤维蛋白在体外能组装成10 nm纤维,在体外反应体系中加入核孔蛋白后,室温反应30 min,SDS-聚丙烯酰胺凝胶电泳及免疫印迹法检测,结果表明180 kD核孔蛋白(Nup 180)与波形纤维蛋白有亲和反应。结合免疫胶体金标记与电镜负染色方法显示,核孔蛋白结合于体外装配的10 nm波形纤维上。本文结果提示在细胞内波形纤维可能通过与Nup 180的结合锚定于核孔复合体上。     

Ultrastructural features of chloride cells in the gill epithelium of the Atlantic salmon, Salmo salar, and their modifications during smoltification

To elucidate the ultrastructural modifications of the gill epithelium during smoltification, gills of the Atlantic salmon (Salmo salar) were examined by electron microscopy at three stages of this process, which were defined as follows: "parrs" were freshwater fish that had not yet started their transformation; "freshwater smolts" were freshwater fish that were ready to enter seawater; and "seawater smolts" were smolts that had been transferred from fresh water and maintained for 4 days in seawater (35%). In the gill epithelium of parrs, there were two types of chloride cells. The large chloride cells contained deeply stained mitochondria and numerous apical, irregular, dense, membrane-bound bodies that formed 77% of the chloride cell population and were distinguished easily from small chloride cells that have distinctly paler mitochondria and no dense bodies in their apical cytoplasm. In freshwater smolts, the large chloride cells formed 95% of the chloride-cell population. In contrast to the small chloride cells that were not modified, they almost doubled in size. Their tubular system developed extensively to form a tight network with regular meshes significantly smaller than those observed in parr chloride cells. Forty percent of the large chloride cells were associated with a new type of cell, the accessory cell, to which they were bound by shallow apical junctions. Half of these accessory cells were not seen to be in contact with the external medium. In seawater smolts, 80% of the large chloride cells were associated with accessory cells. Most accessory cells reached the external medium and sent numerous cytoplasmic interdigitations within the apical portion of the adjacent chloride cells. As a result, a section through the apical portion of the chloride cells and their associated accessory cells revealed a mosaic of interlocked cell processes bound together by an extended, shallow apical junction. It was concluded that the Atlantic salmon develops in fresh water most of the ultrastructural modifications of the gill epithelium which in most euryhaline fish are triggered by exposure to seawater. The effective transfer into seawater would act only as a final stimulus to achieve some adequacy between the freshwater smolt and its new environment.