First steps of otolith formation of the zebrafish: role of glycogen?

The first steps of otolith formation were studied by electron microscopy in zebrafish embryos at different postfertilization (PF) time intervals. Between 19 and 22 h PF, the otic cavity contains glycogen particles derived by an apocrine process from the apical portions of the epithelial cells of the inner ear. The particles are arranged in parallel arrays, then in pseudocrystalloid structures, and finally in concentric arrays to form dense clusters referred to as "spherules". At 23 h PF, a group of "globules", consisting of modified aggregated "spherules" surrounded by several free "spherules", forms the nascent otolith. At 30 h PF, fused globules form a roughly spherical otolith. Spherules undergoing their process of modification and aggregation, are located in its central part, and constitute the so-called "nucleus". At 50 h PF, the otolith is a flattened hemisphere. It is made up of fused globules surrounded by two concentric layers whose organization is similar to that observed in the otolith of the adult fish. At this stage, calcium may be detected in the otolith except in its nucleus. We suggest that glycogen molecules found in the nascent otolith might allow the insertion of molecules such as glycoproteins (collagens) which are known to fix calcium. As a result, glycogen might play a key role in initiating the formation of otoliths and possibly that of other calcified tissues.

Biological functions of trout pavement-like gill cells in primary culture on solid support: pH(i) regulation, cell volume regulation and xenobiotic biotransformation.

This review presents results obtained on rainbow trout gill cells in primary culture on solid support. Ultrastructural analysis showed that cultured gill cells displayed features of pavement cells in situ. Several biological functions have been investigated on these cultured cells. First, it was shown that their intracellular pH at rest and after acidosis is regulated by a Na+/H+ exchanger. Second, gill cells in primary culture can regulate their volume after a cell swelling. Intracellular calcium appears to be involved in this regulation. The effects of different xenobiotics on the capacity of gill cells to regulate their volume are presented. Third, cultured pavement cells contain biotransformation enzymes to metabolize xenobiotics. All these results demonstrate that gill cells in primary culture on solid support represent a promising in vitro model for the study of pavement cells physiology. In conclusion, applications of this culture are discussed and compared with the permeable filter method, together with the limitations and prospects of this in vitro model on solid support.

Effects of medium hypertonicity on water permeability in the mammalian rectum: ultrastructural and molecular correlates.

Minute-by-minute net water fluxes (Jw) were measured across the isolated rectal epithelium in rats and rabbits. Five minutes after a serosal (but not mucosal) hypertonic challenge (plus 200 mosmol/l) a significant increase in the basal Jw was recorded in both species [deltaJw, microl min(-1) cm(-2): 0.40+/-0.06 (rats); 0.45+/-0.10 (rabbits)]. At the same time, most epithelial cells shrank markedly while the intercellular spaces were wide open (electron microscopy studies). In freeze-fracture studies multi-strand tight-junction structures (only slightly modified by serosal hypertonicity in rabbits) were observed in control conditions. No structural changes were observed after mucosal hypertonicity (both in rats and rabbits). Immunohistochemical studies showed the expression of aquaporin 3 (AQP3) at the basolateral membrane of epithelial cells in the rat. A first conclusion is that the epithelium of the mammalian rectum is a highly polarized, aquaporin-3-containing, water permeability structure. The Jw increase induced by serosal hypertonicity was sensitive to mercurial agents in both species and no changes in unidirectional [14C]mannitol fluxes (Ps) or transepithelial resistance (RT) were observed during this Jw increase. These observations suggest a transcellular route for the osmotically induced increase in water fluxes. In the rabbit rectum the initial Jw response, associated with serosal hypertonicity, was a transient one. It was followed by a second, slow and HgCl2-sensitive Jw increase (a transient peak in paracellular mannitol permeability was also observed). A second conclusion is that serosal hypertonicity induces an increase in transcellular water permeability in both rat and rabbit rectum.

Chronology of the appearance of beta, A, and alpha mitochondria-rich cells in the gill epithelium during ontogenesis of the brown trout (Salmo trutta).

Three types of mitochondria-rich (MR) cells, the alpha, beta, and accessory cells, are observed in the gill epithelium of juvenile and adult freshwater teleosts. In addition to numerous mitochondria, their cytoplasm contains a network of membranous tubules, the tubular system, connected to the laterobasal plasma membrane. Because they are believed to play a role in ionic regulation, it is of interest to examine the order of appearance and the ultrastructural characteristics of such cells during the embryogenesis and larval life of the brown trout. Gills of embryos and fry maintained in freshwater were thus removed at different stages and prepared for transmission and scanning electron microscopic examination. One week before hatching, cells resembling the beta cells of juvenile and adult teleosts appeared first among the epithelial cells located at the base of the filaments in the gills of the brown trout larva. In addition to their tubular system, they contained numerous and large apical structures seemingly originating from the Golgi apparatus. At approximately hatching time, small pear-shaped cells were seen to be closely apposed to the lateral side of the beta cells; they were usually devoid of apical structures and were considered to be accessory cells. After yolk sac resorption, additional cells, the alpha cells, were present along the lamellae. In contrast to the beta cells, they only exhibited poorly developed apical structures. The possible role of these three types of MR cells in osmoregulation during fish development is discussed.

Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line.

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.

Ultrastructural study of the saccular epithelium of the inner ear of two teleosts, oncorhynchus mykiss and psetta maxima

The secretory cells and ionocytes of the saccular epithelium of the inner ear of trout (Oncorhynchus mykiss) and turbot (Psetta maxima) have been studied by electron microscopy. In these species, the saccular epithelium may be subdivided into four zones: the "macula", the "meshwork area", the "patches area", and the "intermediate area". In addition to the sensory "hair cells" and their supporting cells, the macula contains, at its periphery, "granular cells" that have the ultrastructural characteristics of secretory cells. The "meshwork area" around the macula contains large ionocytes endowed with pseudopods, many mitochondria, and three intracytoplasmic membrane systems (endoplasmic reticulum, tubular, and vesicular systems). The patches area, located at some distance from the macula, consists of groups of small mitochondria-rich ionocytes characterized by infoldings of their lateral plasma membrane. In the intermediate area, the size and organelle-content of cells decrease from the meshwork area to the patches area. There is no significant difference in cell composition or structure of the saccular epithelium between the trout and the turbot. The secreting cells might be involved in secretion of endolymph and formation of the otolith, whereas the ionocytes probably regulate the ionic composition of the endolymph.

Water pathways across a reconstituted epithelial barrier formed by Caco-2 cells: effects of medium hypertonicity.

Caco-2 cells, originated in a human colonic cancer, are currently used as model systems to study transepithelial transports. To further characterize their water permeability properties, clone P1 Caco-2 cells were cultured on permeable supports. At confluence, the transepithelial net water movement (Jw), mannitol permeability (Ps), and electrical resistance (R) were simultaneously measured. The observed results were correlated with transmission and freeze-fracture electron microscopy studies and compared with those obtained, in similar experimental conditions, in a typical mammalian epithelial barrier: the rabbit rectum. When the serosal solution was made hypertonic (50 mM polyethylene glycol-PEG), the spontaneously observed secretory Jw rapidly reversed, became absorptive and then stabilized. Simultaneously, the R values dropped and Ps went up. In the case of the rabbit rectal epithelium, a similar treatment did not elicit significant changes in the water permeability during the first 20 min following the osmotic challenge while there was a significant increase in the transepithelial resistance. After exposure to serosal hypertonicity, several morphological modifications developed in the Caco-2 cells: Localized dilations in the intercellular spaces and vacuoles in the cytoplasm appeared. Nevertheless, most cells remained in contact and no evidence of cell shrinking was observed. Simultaneously, the tight-junction structure was more or less disorganized. The filament network lost its sharpness and "omega" figures appeared, bordering the intercellular spaces. In some cases the tight-junction network was completely disrupted. In the case of the rabbit rectum the structural modifications were completely different: Serosal hypertonicity rapidly induced cell shrinking and the opening of the intercellular spaces, with no noticeable change in the tight-junction structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical structures of "mitochondria-rich" alpha and beta cells in euryhaline fish gill: their behaviour in various living conditions.

BACKGROUND: One of the characteristic features of the two types (alpha and beta) of "mitochondria-rich" (chloride) cells in the gill epithelium of freshwater fishes is the presence in their apical region of tubulovesicular structures. A further analysis of the ultrastructural features of these apical elements as well as that of their modifications under various living conditions should help to understand better the respective rôle of both alpha and beta cells in these conditions. METHODS: Atlantic salmon (Salmo salar) maintained in fresh water as well as tilapia (Oreochromis niloticus) maintained either in fresh water or in deionized water or in 20% saltwater were examined. Measurements of surface areas of apical structures in the various living conditions were also performed. RESULTS: In the alpha cells of freshwater fishes, the apical structures consisted of isolated vesicles containing a filamentous material resembling that coating the apical surface. They were closely related to the apical plasma membrane and did not penetrate the region containing the tubular system. When fishes were transferred to deionized water, the number of the apical membrane folds increased significantly, as did the number and size of apical structures which became elongated. In saltwater-adapted fishes, the apical structures showed a tendency to collapse and took the appearance of flattened and slightly curved elements. These observations tended to indicate that in alpha cells the apical structures were extensions of the apical plasma membrane and thereby might be implicated in sodium uptake when fishes are placed in fresh or deionized water and in chloride excretion when they are transferred to salt water. In beta cells, the apical structures were usually separated from the apical plasma membrane by a zone rich in cytoskeleton elements. They penetrated deeply into the supranuclear region, where they intermingled with the elements of the tubular system. They consisted mainly of tubular elements that contained a material resembling that present in the trans tubular Golgi network from which they might originate. The apical structures remained unaltered in beta cells whatever the medium (fresh or deionized water) in which the fish was placed. CONCLUSIONS: The alpha cells which are usually thought to be mainly involved in chloride excretion when fishes are transferred into seawater might also be implicated in sodium uptake in freshwater living conditions. The rôle of beta cells, in contrast, still remains to be established.

Effects of growth hormone on gill chloride cells in juvenile Atlantic salmon (Salmo salar).

Experiments were performed to investigate the effects of ovine growth hormone (oGH) on both the ultrastructural features of chloride cells and the ability of gills to extrude Na+ after transfer into seawater. February presmolts and June parrs of the Atlantic salmon (Salmo salar) were implanted with oGH. In such animals, spontaneously showing a poor ability to adapt themselves to seawater life, GH significantly increased gill Na(+)-K(+)-adenosinetriphosphatase activity as well as gill sodium efflux into seawater. When examined by electron microscope, two types of chloride cells (alpha- and beta-types) were identified in control parrs and presmolts. GH treatment induced an increase in size and number of alpha-cells that displayed an extensive tubular system, while the beta-cells, thought to be specific to freshwater life, decreased in number. There was, concomitantly, an increase in number of accessory cells associated with the apical portion of the alpha-cells and, as a result, the formation of extensive shallow junctions between these cell types. Such functional and ultrastructural modifications that mimicked those naturally occurring during the last steps of the smoltification strongly suggest that GH stimulates the differentiation of freshwater chloride cells toward a seawater type.

Effects of prolactin on alpha and beta chloride cells in the gill epithelium of the saltwater adapted tilapia "Oreochromis niloticus".

Tilapia (Oreochromis niloticus), 21 g average body weight, were divided into two groups. A group was maintained in fresh water, whereas another group was adapted for 2 weeks to 20% salt water. Among the latter, fishes were injected every 2 days for a week with tilapia prolactin (ti-PRL I). Gills were prepared for electron microscopy in order to determine the types and surface areas of chloride cells in each experimental condition. Two types of chloride cells, the alpha and beta cells were easily distinguished on the basis of their location and ultrastructural features in the gills of freshwater fishes, while only one type of cell, the saltwater alpha cells presumably derived from the transformation of the freshwater alpha cells, were encountered in saltwater adapted animals. After PRL injection of saltwater adapted fishes, small chloride cells, which displayed ultrastructural features similar to those of beta cells in freshwater tilapia, reappeared in interlamellar regions of the gills. In the same experimental conditions, the voluminous saltwater alpha cells showed a tendency to resume ultrastructural features more characteristic of the freshwater alpha cells from which they were derived. These observations tend to indicate that prolactin behaves as a "freshwater adapting hormone" and that beta cells are specifically involved in fish adaptation to freshwater living conditions.

Conversion of a rabbit proximal convoluted tubule (PCT) into a cell monolayer: ultrastructural study of cell dedifferentiation and redifferentiation.

The evolution of a primary culture of kidney proximal convoluted tubule (PCT) cells was followed step by step from the plating time of an isolated tubule to the 39th day of culture. During the first 48 h, the structural remodeling of PCT, leading to the formation of a cell monolayer without cell division, is accompanied by intracytoplasmic changes indicating cell dedifferentiation. Numerous autophagic vacuoles are observed inside the cells, and the ultrastructural features characteristic of in situ PCT cells are progressively lost. Despite these drastic modifications, cell polarity, as observed by immunocytochemical detection of the leucine aminopeptidase, remains unaltered. Starting at 48 h, the peripheral cells divide, and the culture proliferates in a centrifugal direction while newly formed cells differentiate. From 6 days onwards, glycogen granules, never encountered in in situ PCT cells, appear in cultured cells and progressively accumulate. At the optimal stage of the culture (12-17 days old), cells somewhat resemble PCT cells, but their apical brush borders remain rudimentary, and basal cytoplasmic interdigitations surrounding densely packed mitochondria are poorly developed. Subsequently, the cells become overloaded with glycogen and lipid inclusions and resemble degenerating cells.

Effects of low temperature on the formation of secretion granules in the Golgi apparatus of granular cells of the frog urinary bladder.

The formation of secretion granules has been studied in the Golgi apparatus of granular epithelial cells of frog urinary bladders maintained at room temperature or cooled at 4 degrees C for various lengths of time. In control animals, the Golgi apparatus was composed of the following stacked elements: subjacent to the cis-element made up of anastomosed tubules, two elements in the mid-compartment consisted of flattened saccules interconnected by tubules. On the trans-face, two or three sacculo-tubular elements were slightly dilated by an electron dense granular material. In the trans-Golgi elements, this material was segregated into dilatations of various sizes and shapes which are continuous with flattened portions devoid of stained material. In the trans-Golgi region, free irregular progranules, seemingly formed by rupture of the trans-most Golgi elements. In granular cells examined after 4 h at 4 degrees C, all Golgi compartments were affected by the low temperature. The cis-half portion of the Golgi apparatus consisted mainly of anastomosed membranous tubules and the cis-element was no longer recognizable. The trans-compartment was reduced to a few flattened saccules with progranules hardly visible on their trans-aspect. At later time intervals, there was a progressive reconstitution of the cis-zone while saccular elements started to pile up in the trans-compartment. At 24 h, the trans-compartment comprised six to eight saccular elements which showed irregular dilatations filled with granular material separated by large flattened portions. These various observations were interpreted as indicating that the trans-compartment was a dynamic structure undergoing continuous renewal.

Ultrastructural features of mitochondria-rich cells in stenohaline freshwater and seawater fishes.

In order to elucidate the functional significance of accessory cells in freshwater fishes, such as the rainbow trout, which displays a poor adaptability to seawater life, a search for such cells was performed in two stenohaline freshwater fishes: the loach and the gudgeon. Accessory cells were never encountered in these species; but, in contrast, two types of chloride cells were observed consistently that strikingly resembled the alpha- and beta-cells previously described in the guppy, a freshwater-adapted euryhaline fish. The alpha-cell, a pale and elongated chloride cell, was located at the base of the secondary lamellae in close contact with the arterioarterial pillar capillary. Darker, ovoid chloride cells resembling the beta-cell were found exclusively in the interlamellar region of the primary epithelium facing the central venous sinous. The latter cells frequently formed multicellular complexes linked together by deep, narrow, apical junctions. In another experiment, a stenohaline seawater fish, the turbot, was adapted to diluted 5% saltwater and to fresh water. In seawater, the gill epithelium contained only one type of chloride cell, always associated with accessory cells. Due to numerous cytoplasmic interdigitations between the accessory cells and the apical portion of the chloride cell, there was a noticeable increase in the length of the shallow apical junction, sealing off the intercellular space between the two cell types. In 5% saltwater, there was a decrease in the number of these interdigitations and a concomitant decrease in the length of the shallow apical junction. In fresh water, chloride cells were partially or completely separated from the outside medium by modified accessory cells. It is thus concluded that accessory cells are found exclusively in fish living in seawater or preadapted to seawater and that they probably are involved in the formation and modulation of paracellular pathways for ionic excretion. In contrast, the respective roles of the two types of chloride cells observed in freshwater fishes are still to be determined.

Accessory cells in the gill epithelium of the freshwater rainbow trout Salmo gairdneri.

Two types of mitochondria-rich cells were identified in the gill epithelium of the freshwater-adapted rainbow trout, Salmo gairdneri, after selective impregnation of their tubular system with reduced osmium. A first type consisted of large cells with a poorly developed and loosely anastomosed tubular system; thus, that resembled the chloride cells commonly encountered in the gill epithelium of freshwater-adapted euryhaline fishes. A second type comprised smaller cells with an extensively developed and tightly anastomosed tubular system. These never reached the basal lamina of the gill epithelium and were adjacent to chloride cells, to which they were linked by shallow apical junctions (100-200 nm); thus, they resembled accessory cells, which are currently found in the gill epithelium of seawater-adapted fishes but are usually lacking in freshwater living fishes. Transfer of the freshwater-adapted trout into seawater induced the proliferation of the tubular system in the chloride cells and the formation of lateral plasma membrane interdigitations between accessory cells and the apical portion of the chloride cells. The length of the apical junction sealing off this extended intercellular space was reduced to 20-50 nm. The tubular system of the accessory cells was not modified. The extension of the tubular system in the chloride cells of the seawater-adapted fishes indicated that, as in most euryhaline fishes, these cells have a role in the adaptation of the rainbow trout to seawater. In contrast, the function of the presumptive accessory cells in freshwater trout remains to be established.

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.

Two types of chloride cells in the gill epithelium of a freshwater-adapted euryhaline fish: Lebistes reticulatus; their modifications during adaptation to saltwater.

Two types of chloride cells were identified in the gill epithelium of freshwater-adapted guppies. One type, referred to as an "alpha-chloride cell," was a pale, elongated cell located at the base of the secondary lamella in close contact with the arterioarterial pillar capillaries. In its cytoplasm, membranous tubules in continuity with its basolateral plasma membrane formed an extended tridimensional network. The vesiculotubular system (Pisam: Anat. Rec. 200:401-414, 1981) consisted of a few tubules and vesicles located next to the apical plasma membrane. A second type, referred to as a "beta-chloride cell," was a darker, ovoid cell located in the interlamellar region of the primary epithelium facing the central venous sinus. Membranous tubules in continuity with the basolateral plasma membrane were unevenly distributed in the cytoplasm. A prominent vesiculotubular system composed of numerous vesicles and tubules was found between the Golgi apparatus and the apical surface. During seawater adaptation, the alpha-chloride cells increased in size and progressively transformed into characteristic "seawater alpha-chloride cells" with a well-developed, regular, tight tubular network and numerous vesicles and tubules of the vesiculotubular system accumulated below the apical pit. The beta-chloride cells underwent a progressive degeneration and disappeared. Thus, in freshwater-adapted guppies, there are two types of chloride cells, alpha and beta, respectively, related to the arterial and the venous vessels, whereas in seawater-adapted fishes, a single type of cell, the alpha-chloride cell, was related to both the arterial and venous channels.

The role of microtubules and microfilaments in the hydrosmotic response to antidiuretic hormone.

To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.

Two anatomical pathways for the renewal of surface glycoproteins in chloride cells of fish gills.

The cytoplasm of chloride cells found in the epithelium lining the gills of guppies (Lebistes reticulatus) contains, in addition to the Golgi apparatus and cisternae of endoplasmic reticulum, two distinct membranous components, the vesiculotubular and the tubular systems. While the latter is connected to the laterobasal plasma membrane, the former, made up of small vesicles and short membranous tubules, is seen mainly between the Golgi apparatus and the apical cavity which invaginates the apex of the cell. The role of these two systems in the transport of glycoproteins from the Golgi apparatus to the cell surface was investigated in fishes maintained in fresh and salt water, injected with 3H-fucose, and sacrificed at various intervals thereafter (10 and 30 min; 2.5, 8, 15.5, 24, and 48 hours). The distribution of the label was analyzed by quantitative radioautography in sections examined with the light and electron microscopes. The light microscopic data suggested that the label incorporated in the supranuclear region, where the Golgi apparatus is located, migrated toward the apical and the laterobasal regions of the chloride cells. The relative concentration of the tracer over the various components of the cytoplasm of these cells was calculated from data collected on electron microscope radioautographs at various intervals after 3H-fucose injection. The curves obtained supported the view that glycoproteins synthesized in the Golgi apparatus were transported to the apical surface via the vesiculotubular system, and to the laterobasal membrane via the tubular system.

Membranous systems in the "chloride cell" of teleostean fish gill; their modifications in response to the salinity of the environment.

The membranous systems of the "chloride cell" were studied in teleostean fish gills stained in ferrocyanide-reduced osmium (Karnovsky, '71). Three distinct systems were observed: (1) the tubular system, densely stained with ferrocyanide-reduced osmium, was made up of anastomosed tubules opening in the latero basal intercellular space; (2) the endoplasmic reticulum, faintly stained, and continuous with the nuclear envelope; (3) the vesiculotubular system, the staining of which was intermediate between those of both previously cited systems, was made up of vesicles and short tubules. These membranous systems underwent modifications according to the salinity of the exterior medium: (1) the tubular system formed a broad and loose network in fresh water adapted fishes; in salt water, the meshes of the network became small, tight, and regular thus increasing the cell surface area. (2) the endoplasmic reticulum, which in fresh water, consisted of dilated cisternae often studded with ribosomes; in salt water, it developed in a network of anastomosed smooth sheets interdigitated with the tubular system. (3) the vesiculotubular system seems to be also more developed in salt water than in fresh water-adapted fishes.

Polysaccharidic material in chloride cell of teleostean gill: modifications according to salinity.

The gill epithelium is known to be implicated in the hydromineral regulation of teleosts, especially owing to its "chloride cells." We have examined the polysaccharides of chloride cells from euryhaline teleosts adapted to fresh- or saltwater. The use of periodic acid-chromic acid-silver methenamine, colloidal thorium, or radioautography after incorporation of [3H]glucosamine has shown that chloride cells are characterized by a high concentration of polysaccharides in their apical region (at the level of the vesiculotubular system) and by a special polysaccharidic cell coat. The polysaccharide molecules originate from the Golgi area; by 12 h they accumulate within the vesiculotubular system and are released in the apical cavity of the cell within 24 h. In fresh- and saltwater-adapted fish, the localization of polysaccharidic material in chloride cells is basically the same. However, in saltwater-adapted fish, the amount and turnover of the polysaccharide molecules are clearly increased.