The surface epithelium of teleostean fish gills. Cellular and junctional adaptations of the chloride cell in relation to salt adaptation.

Various species of teleostean fishes were adapted to fresh or salt water and their gill surface epithelium was examined using several techniques of electron microscopy. In both fresh and salt water the branchial epithelium is mostly covered by flat respiratory cells. They are characterized by unusual outer membrane fracture faces containing intramembranous particles and pits in various stages of ordered aggregation. Freeze fracture studies showed that the tight junctions between respiratory cells are made of several interconnecting strands, probably representing high resistance junctions. The organization of intramembranous elements and the morphological characteristics of the junctions do not vary in relation to the external salinity. Towards the base of the secondary gill lamellae, the layer of respiratory cells is interrupted by mitochondria-rich cells ("chloride cells"), also linked to respiratory cells by multistranded junctions. There is a fundamental reorganization of the chloride cells associated with salt water adaptation. In salt water young adjacent chloride cells send interdigitations into preexisting chloride cells. The apex of the seawater chloride cell is therefore part of a mosaic of sister cells linked to surrounding respiratory cells by multistranded junctions. The chloride cells are linked to each other by shallow junctions made of only one strand and permeable to lanthanum. It is therefore suggested that salt water adaptation triggers a cellular reorganization of the epithelium in such a way that leaky junctions (a low resistance pathway) appear at the apex of the chloride cells. Chloride cells are characterized by an extensive tubular reticulum which is an extension of the basolateral plasma membrane. It is made of repeating units and is the site of numerous ion pumps. The presence of shallow junctions in sea water-adapted fish makes it possible for the reticulum to contact the external milieu. In contrast in the freshwater-adapted fish the chloride cell's tubular reticulum is separated by deep apical junctions from the external environment. Based on these observations we discuss how solutes could transfer across the epithelium.

Seawater teleosts: evidence for a sodium-potassium exchange in the branchial sodium-excreting pump.

The net sodium extrusion rate by the gill of the seawater-adapted euryhaline flounder is identical to the potassium influx. The excretion of sodium is blocked in K(+)-free seawater solutions. The instantaneous sodium outflux readjustment pattern of flounders transferred from seawater to solutions of various sodium chloride or potassium chloride concentrations is consistent with the hypothesis of a linkage between Na(+) outflux and K(+) influx through a common exchange carrier. External Na(+) and K(+) compete for this comnmonz carrier. It is suggested that the exchange diffusion mechanism (linkage of sodium influx and outflux) and the high internal sodium turnover rate which characterizes all seawater teleosts are the results of this competitive process. The sodium-potassium dependent adenosine triphosphatase system occurring in the gill of the seawater teleosts may play a central role in this sodium-potassium exchange pump.

M23C6 carbides and Cr2N nitrides in aged duplex stainless steel: A SEM, TEM and FIB tomography investigation.

The precipitation evolution during ageing of a 2101 lean duplex stainless steel was investigated, revealing that the precipitate type and morphology depends on the nature of the grain boundary. Triangular M23C6 carbides precipitate only at γ/δ interfaces and rod-like Cr2N nitrides precipitate at both γ/δ and δ/δ interfaces. After 15min of ageing, the M23C6 size no longer evolves, whereas that of the Cr2N continues to evolve. For Cr2N, the morphology is maintained at γ/δ interfaces, whereas percolation occurs to form a continuous layer at δ/δ interfaces. By combining 2D and 3D characterisation at the nanoscale using transmission electron microscopy (TEM) and focused ion beam (FIB) tomography, a complete description of the precipitation evolution was obtained, including the composition, crystallographic structure, orientation relationship with the matrix phases, location, morphology, size and volume fraction.

Exchange diffusion effect and euryhalinity in teleosts.

The sea water (SW)-adapted euryhaline Platichthys flesus, and the marine Serranus exchange about 50% of their internal sodium with the external sodium per hour. This rate of exchange decreases with decreasing salinity of the adaptation medium. When the flounder is transferred from SW to FW an instantaneous 90% reduction of the Na and Cl outflux is observed. About 30 min later a second, progressive, reduction occurs. The outflux reductions appear to result from two types of regulatory mechanisms reducing gill permeability and preventing excessive salt loss. The first reduction corresponds to independent "Na- and Cl-free effects" as shown by transfers to artificial media containing either Na or Cl with an impermeant co-ion. The pattern of simultaneous rapid variations of Na influx and outflux for a range of salinity changes in flounder adapted to SW, 1/2 SW, or 1/4 SW has been studied. The data are compatible with the hypothesis of an exchange diffusion mechanism characterized by a coupling of both unidirectional fluxes. The affinity of the exchange diffusion carrier for sodium has been measured (Km approximately equal to 400 mM). The delayed reduction would result from a progressive diminution of the quantity of carrier available but without modification of its affinity for sodium. When the stenohaline marine perch is transferred from SW to FW, a 40% reduction of the outflux is observed. But it is not the result of an exchange diffusion effect as it is related to the external osmolarity change and not to the NaCl concentration change. Furthermore no delayed reduction is observed after transfer into FW. This transfer is accompanied by a heavy loss of electrolytes resulting in a rapid decline of the plasma electrolyte level and death. A comparative survey of the relative importance of these regulatory mechanisms has been made.

Branchial sodium exchange in seawater-adapted Tilapia mossambica: effects of prolactin and hypophysectomy.

The effects of exogenous prolactin and hypophysectomy were tested on Na exchange across the gills of Tilapia mossambica adapted to sea-water. Exogenous prolactin produced a 75% decrease of both total Na influx and Na efflux. Total Na influx was measured simultaneously with the drinking rate. Water ingestion stopped almost completely in prolactin-treated fish. Thus branchial net excretion flux of Na was reduced 75% by prolactin. Hypophysectomy produced a 50% inhibition of Na efflux across the gill.

Transport of ions and water across the epithelium of fish gills.

The teleostean gill is characterized by an exceptionally low permeability to water. Water moves along the osmotic gradient across the gill, being gained in fresh water and lost in sea water. Coupling of water movement to solute movement has not been reported. In fresh water, the gill is the site of independent active uptake of sodium and chloride. Na+ uptake is coupled to H+ or NH4+ excretion, Cl- uptake to HCO3- excretion. Amiloride blocks sodium transport and thiocyanate inhibits the chloride pump. In sea water, sodium and chloride exchanges across the gill are about 100 times faster than in fresh water, up to 100% of the internal sodium or chloride being exchanged per hour. Chloride is actively excreted, while sodium movement may well be passive. The chloride pump is associated with a mechanism for Na/K exchange; both pump and Na/K exchange are blocked by thiocyanate and possibly by ouabain. Three enzymes are involved in the ionic pumps: carbonate dehydratase (EC 4.2.1.1; carbonic anhydrase), sodium/potassium-stimulated adenosine-triphosphatase (EC 3.6.1.3, ATPase) and anion-stimulated ATPase. Specialized cells ('chloride cells') are presumably the site of the active transport.

Microtubules in the "chloride cell" of the gill and disruptive effects of colchicine on the salt balance of the sea water adapted Mugil capito.

Electron-microscopic examination of the gill of the grey mullet, Mugil capito adapted to sea water, reveals the presence of numerous microtubules in the apical region of the mitochondria-rich cells. No microtubules are found in other types of epithelial cell. Exposure of the fish to colchicine (10(-4)M) for four hours induces a 20% increase of plasma Na and Cl. Colchicine leaves the water permeability of the gill unchanged but Cl and Na exchange fluxes are inhibited by 30-50% and the gill potential pattern is altered. Salt gain replaces salt excretion across the gill. The K dependent Na and Cl efflux components, independent of the gill potential shift produced by K, are totally inhibited by colchicine. Exposure to lumicolchicine is not followed by a significant change of these Na and Cl efflux-components and potential pattern. The possibility that microtubules intervene in the salt excretion process across the chloride cells is discussed.

Environmental salinity and sodium and chloride exchanges across the gill of Tilapia mossambica.

10 Freshwater-(FW)-adapted, one-third seawater (1/3 SW)-adapted and seawater (SW) adapted Tilapia mossambica were compared for their branchial Na+ influx and efflux as well as Cl- efflux. Na+ and Cl- effluxes were identical. Rates of effluxes were in 1/3 SW- and in SW-adapted fish 10 times and 200 times higher respectively than in FW specimens. 20 Shock due to handling and transfer to small experimental chambers induced, within 20 to 45 min., a considerable increase in Na+ efflux and a more discrete augmentation of the Na+ influx. 30 Branchial Mg++-and Na+-K+ activated ATPase activities increased significantly upon adaptation from FW to 1/3 SW. No significant increase was apparent upon adaptation from 1/3 SW to SW. 40 The trans-branchial potential observed in SW Tilapia resembled the pattern previously described in other species of teleosts.

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.

The effects of calcium and magnesium on sodium fluxes through gills of Carassius auratus, L.

1. Removal of calcium from Carassius either with chelating agents or by adaptation to deionized water increased sodium influx through the gills twofold.2. Treatment of fish with chelating agents increased sodium outflux, whereas adaptation to deionized water reduced sodium outflux. The effects on outflux do not appear to result from calcium removal.3. Addition of calcium (2-20 mM) reversed the effects on influx of chelating agents and of adaptation to deionized water.4. Magnesium (2-10 mM) was without significant effect on sodium influx, but increased the outflow in fish adapted to deionized water.

Branchial effects of epinephrine in the seawater-adapted mullet. II. Na+ and Cl- extrusion.

Injection of epinephrine into Mugil capito adapted to seawater is followed by a 40-60% inhibition of the Na and Cl effluxes. Simultaneously the Na influx is decreased by 30%, the overall result being a reduction of the net sodium extrusion rate by the gill. The change in Na influx is in part explained by a 75-80% decrease of the oral ingestion of seawater. This branchial adrenergic response is sensitive to alpha-blockade by phentolamine and tolazoline and insensitive to beta-blockade by propranolol. Both alpha-blockers are ineffective when injected alone. Propranolol injected alone mimics epinephrine while simultaneous injection of phentolamine blocks the response to propranolol. Rapid transfer experiments suggest that epinephrine inhibits the branchial Cl pump and its associated Na/K exchange mechanism. The leak pathway for these ions remains insensitive to epinephrine.

A study of the unidirectional fluxes of Na and Cl across the gills of the dogfish Scyliorhinus canicula (Chondrichthyes).

The gills of the dogfish Scyliorhinus canicula are more permeable to Cl than to Na. In sea water, influx of Na and Cl exceeded the efflux of these ions. Under these conditions the fish were slightly electronegative, by about 2 mV, to the external solution. The net accumulation of Cl could be accounted for by diffusion along the observed electrochemical gradient byt the movement of Na into the fish was more consistent with an electrically neutral active Na transport mechanism (using the Ussing flux ratio criterion). When the external pH was was changed from 7-8 to 6-9,, influxes of Na and Cl were depressed, while the effluxes were unaffected, and the fish became slightly less electronegative. In artificial solutions, in which the concentrations of Na and Cl were lowered and replaced with urea to maintain the total osmotic concentration, Na influx displayed saturation kinetics, while Na efflux increased with decreasing Na concentrations. Cl influx decreased linearly, while Cl efflux remained constant. The efflux of Cl could not be reconciled with a process of passive diffusion along any of the observed electrochemical gradients and thus could reflect the presence of an active transport mechanism.

Cellular relationship impairment in maturation arrest of human spermatogenesis: an ultrastructural study.

Ultrastructural examination of testicular biopsies from cases of maturation arrest showed that there were characteristic abnormalities of the Sertoli cell junctional connections. These abnormalities together with the meiotic failure afford an explanation for the severe oligospermia or azoospermia noted in patients with maturation arrest. Premature setting up of ectoplasmic specializations in front of early spermatids and/or spermatocytes were also observed.

Effects of epinephrine on branchial non-electrolyte permeability in rainbow trout.

Using isolated heads perfused at constant pressure, at rates close to those occurring in vivo, the permeability of the gills of the trout Salmo gairdneri to a range of solutes was measured. Under epinephrine-free conditions, butanol and water showed similar high branchial permeability coefficients. Urea, inulin and dextrans (mol. wt 3000 and 20 000) were 7-12 times less permeant, and mannitol 60-70 times less permeant than water or butanol. Epinephrine, at 10(-6) M, greatly increased the permeability of the gills to the small hydrophilic molecules, water and urea, and to the lipophilic substance, butanol, but did not affect the penetration of the large hydrophilic solutes, mannitol, inulin and dextrans. In the presence of 10(-6) M propanolol, a beta-blocker, epinephrine had no effect on the permeation of any of the test substances except that the permeability to urea decreased somewhat. The results suggest that epinephrine increases the permeability of the membranes of the branchial cells but does not affect the permeation of substances that cross the gill walls by paracellular routes or via an intracellular 'bulk-transport' mechanism. Such an action would be expected to increase the branchial transfer of oxygen.

Epinephrine effects on branchial water and urea flux in rainbow trout.

In isolated trout heads, perfused at constant pressure, epinephrine (10(-6) M) was found to double water and urea efflux but increased Ringer perfusion rate by only 33%. Drastic changes in perfusion rate (by clamping) produced smaller changes of both efflux rates. Epinephrine-stimulated increase in water and urea efflux, and perfusion rate, was blocked by propranolol (beta-blocker) but not by phentolamine (alpha-blocker). Both blockers together canceled out all epinephrine effects. Epinephrine increased water influx across isolated unperfused gill arches, the effect again being blocked by propranolol but not by phentolamine. Both blockers together canceled any epinephrine effect. We conclude that epinephrine alters branchial vascular flow and functional surface area via alpha- and beta-adrenergic receptors, but also increases branchial permeability to water and probably urea, via beta-adrenergic receptors. To test the validity of the perfused head technique, water and urea efflux rates were compared with in vivo values.

The effects of calcium on branchial sodium fluxes in the sea-water adapted eel, Anguilla anguilla, L.

1. The sodium influx through the gills of eels placed in calcium-free sea water for 15 hr was double that of controls. The effect was reversed in 1 hr by addition of calcium.2. The total sodium outflux through the gills of fish placed in calcium-free sea water for 15 hr was double that of controls. The effect was only partially reversed in 15 hr by addition of calcium.3. The passive outflux component of the total outflux was increased fourfold when calcium was removed and was restored to normal in 15 hr by addition of calcium. The active (exchange) outflux component of the total outflux was halved by calcium removal and increased above normal following calcium addition.4. The inability of calcium to restore the total outflux to normal within 15 hr in calcium-depleted fish, together with the raised plasma sodium concentration at this time, suggests that the raised outflux is caused by homoeostatic mechanisms, rather than permeability changes in the gill epithelium.

Reproductive failure and pericentric inversion in man.

Pachytene analysis was carried out on an infertile man heterozygous for a pericentric inversion of chromosome 6. The synaptic behavior of the bivalent 6 inversion was analyzed using electron microscopy in silver stained surface microspread of the inversion-bearing spermatocytes. Possible mechanisms of the sterilizing effect caused by the autosomal inversion are discussed.

Heterosynapsis in two fertile but hypoprolific boars carriers of reciprocal translocations.

The authors report here two new cases of reciprocal translocations in two fertile and hypoprolific boars. Silver stained synaptonemal complexes in surface-spread pachytene nuclei from a boar heterozygous for a reciprocal translocation, and from another one carrying two different reciprocal translocations, were analyzed by electron microscopy. In such heterozygotes, cross-shaped quadrivalent configurations are expected to form in order to allow homologous pairing. In the same boar, the lengths of the fully synapsed arms of the quadrivalent varies from one quadrivalent to the other and heterosynapsis was obvious. Heterosynapsis was also observed with asymmetrical pairing of the non-homologous partners of the quadrivalent. This heterosynapsis is assumed to be a mechanism preventing spermatocyte loss, but inducing a secondary segregational type of impairment of fertility due to foetal wastage leading to reduced prolificacy.