Sternal Epithelium Research Articles

The cationic composition and pH in the moulting fluid of Porcellio scaber (Crustacea, Isopoda) during calcium carbonate deposit formation and resorption

Before moulting, terrestrial isopods resorb calcium carbonate (CaCO(3)) from the posterior cuticle and store it in sternal deposits. These consist mainly of amorphous calcium carbonate (ACC) spherules that develop within the ecdysial space between the anterior sternal epithelium and the old cuticle. Ions that occur in the moulting fluid, including those required for mineral deposition, are transported from the hemolymph into the ecdysial space by the anterior sternal epithelial cells. The cationic composition of the moulting fluid probably affects mineral deposition and may provide information on the ion-transport activity of the sternal epithelial cells. This study presents the concentrations of inorganic cations within the moulting fluid of the anterior sternites during the late premoult and intramoult stages. The most abundant cation is Na(+) followed by Mg(2+), Ca(2+) and K(+). The concentrations of these ions do not change significantly between the stages whereas the mean pH changed from 8.2 to 6.9 units between mineral deposition in late premoult, and resorption in intramoult, respectively. Measurements of the transepithelial potential show that there is little driving force for passive movements of calcium across the anterior sternal epithelium. The results suggest a possible role of magnesium ions in ACC formation, and a contribution of pH changes to CaCO(3) precipitation and dissolution.

Microscopical and functional aspects of calcium-transport and deposition in terrestrial isopods

Terrestrial isopods (Crustacea) are excellent model organisms to study epithelial calcium-transport and the regulation of biomineralization processes. They molt frequently and resorb cuticular CaCO 3 before the molt to prevent excessive loss of Ca 2+ ions when the old cuticle is shed. The resorbed mineral is stored in CaCO 3 deposits within the ecdysial gap of the first four anterior sternites. After the molt, the deposits are quickly resorbed to mineralise the posterior part of the new cuticle. The deposits contain numerous small spherules composed of an organic matrix and amorphous CaCO 3, which has a high solubility and, therefore, facilitates quick mobilization of Ca 2+ and HCO 3 - ions. During the formation and resorption of the deposits large amounts of Ca 2+, HCO 3 - and H + are transported across the anterior sternal epithelial cells. Within the last years, various light and electron microscopical techniques have been used to characterize the CaCO 3 deposits and the cellular mechanisms involved in biomineralization. The work on the CaCO 3 deposits includes studies on the ultrastructure of the deposits, the sequence of events during deposit formation and dissolution, and the mineral composition of the sternal deposits. The differentiation of the anterior sternal epithelial cells and the mechanisms of epithelial ion transport required for the mineralization and demineralisation of the deposits was studied using various analytical light and electron microscopical techniques including polarized light microscopy, immunocytochemistry, electron microprobe analysis, electron energy loss spectroscopy and electron spectroscopic imaging. Comparative analysis of deposit morphology and the differentiation of the sternal epithelia provide information on the evolution of CaCO 3 deposit formation in relation to the degree of adaptation to terrestrial environments.

Expression of Ca 2+-ATPase and Na +/Ca 2+-exchanger is upregulated during epithelial Ca 2+ transport in hypodermal cells of the isopod Porcellio scaber

It is thought that a plasma membrane Ca 2+-transport ATPase (PMCA) and a Na +/Ca 2+-exchange (NCE) mechanism are involved in epithelial Ca 2+ transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca 2+-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca 2+-transporting condition to the stages of CaCO 3 deposit formation and degradation. During Ca 2+-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.

Analysis of Ca(2+) uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber.

In terrestrial isopods, large amounts of Ca(2+) are transported across anterior sternal epithelial cells during moult-related deposition and resorption of CaCO(3) deposits. Because of its toxicity and function as a second messenger, resting cytosolic Ca(2+) levels must be maintained below critical concentrations during epithelial Ca(2+) transport, raising the possibility that organelles play a role during Ca(2+) transit. We therefore studied the uptake of Ca(2+) into Ca(2+)-sequestering organelles by monitoring the formation of birefringent calcium oxalate crystals in permeabilised anterior and posterior sternal epithelium cells of Porcellio scaber during Ca(2+)-transporting and non-transporting stages of the moulting cycle using polarised-light microscopy. The results indicate ATP-dependent uptake of Ca(2+) into organelles. Half-maximal crystal growth at a Ca(2+) activity, a(Ca), of 0.4 micromol l(-1) and blockade by cyclopiazonic acid suggest Ca(2+) uptake into the smooth endoplasmic reticulum by the smooth endoplasmic reticulum Ca(2+)-ATPase. Analytical electron microscopical techniques support this interpretation by revealing the accumulation of Ca(2+)-containing crystals in smooth membranous intracellular compartments. A comparison of different moulting stages demonstrated a virtual lack of crystal formation in the early premoult stage and a significant fivefold increase between mid premoult and the Ca(2+)-transporting stages of late premoult and intramoult. These results suggest a contribution of the smooth endoplasmic reticulum as a transient Ca(2+) store during intracellular Ca(2+) transit.

X-ray microprobe analysis of epithelial calcium transport

The sternal epithelium of Porcellio scaber was used as a novel model to study the subcellular elemental distribution in control and Ca 2+-transporting stages in situ. The anterior sternal epithelium (ASE) is specialized for transport of cuticular Ca to sternal CaCO 3 deposits during premolt, and from these deposits during intramolt. The less specialized posterior sternal epithelium transports Ca 2+ to and from the cuticle. In the ASE cells basal [Na], [Cl], and [Mg] are higher than in the apical side. The basal [Na] increases from 105 to 173 mmol/kg dry mass between control and Ca 2+-transporting stages, accompanied by a decrease in [Cl] and [K]. The [Mg] increases, suggesting transepithelial Mg 2+-transport. Cytosolic [Ca] varied insignificantly between 4.5 and 5.7 mmol/kg dry mass, however, the number of Ca hot-spots with concentrations between 15 and 50 mmol/kg dry mass increased during transport. Mitochondrial [Ca] decreased in the ASE from 3.3 in the control to 1.0 in the late premolt and to 2.0 mmol/kg dry mass in the intramolt stage. The results suggest Na +-dependent mechanisms for transcellular Ca 2+-transport and the presence of Ca 2+-binding proteins. Organelles, probably the smooth endoplasmic reticulum, sequester Ca 2+ during intracellular Ca 2+-transport. A role of mitochondria as a storage site for cuticular Ca is excluded.

Morphometric analysis of the calcium-transporting sternal epithelial cells of the terrestrial isopods Ligia oceanica, Ligidium hypnorum, and Porcellio scaber during molt

Isopods shed first the posterior and then the anterior half of the body. Before molt, most terrestrial species resorb CaCO 3 from the posterior mineralized cuticle. The mineral is stored in anterior sternal deposits, which are used to calcify the new posterior cuticle after molt. For Porcellio scaber it is known that the anterior sternal epithelium has specific structural differentiations for epithelial transport. These differentiations include the plasma membrane surface areas, and the volume fraction of the mitochondria. We analyzed the ultrastructure of the sternal epithelium and used a morphometric approach to study the variations of these parameters between species living in different terrestrial environments. In Ligidium hypnorum, which lives in moist environments, the plasma membrane surface area and volume fraction of mitochondria are much larger than in the semiterrestrial Ligia oceanica. This is in accordance with the relatively larger CaCO 3 deposits and shorter time intervals for their formation and resorption in L. hypnorum. For P. scaber, which is adapted to mesic habitats, most values are between those of L. oceanica and L. hypnorum. However, P. scaber has even larger CaCO 3 deposits which are formed and degraded within similar time intervals as in L. hypnorum. This unexpected result is considered from the standpoint of more effective mechanisms being present for epithelial ion transport.

Membrane Particle Distribution in the Sternal Epithelia of the Terrestrial Isopod Porcellio scaber Latr. (Crustacea, Oniscidea) during CaCO3 Deposit Formation and Resorption, a Freeze-Etch Analysis

The anterior sternal epithelium of terrestrial isopods transports cuticular Ca2+ to and from large sternal CaCO3 deposits. We analyzed the anterior and posterior sternal epithelium by the means of the freeze-etch technique and measured the size distribution and density of intramembrane particles (IMPs) during three different molting stages. At least three IMP size classes around 4.5, 7.7, and 9.4 nm can be distinguished on the P-face of the apical and basolateral plasma membrane. An additional size class of around 12.8 nm is restricted to the apical compartment. In the anterior sternal epithelium, the density of these large particles changes by a factor of 1.9 during the molt cycle, suggesting a role in CaCO3 formation and/or resorption. The density of the smaller IMPs rises transiently by a factor of 1.3 in the posterior sternal epithelium only. The IMP density of the basolateral plasma membrane increases significantly by a factor of 1.4 and 1.3 in the anterior and posterior sternal epithelia, respectively. The results indicate that increases in the IMP density contribute to the differentiation to an increased transport activity during the cyclic enlargements of the plasma membrane surface area in the anterior sternal epithelium.

Ultrastructural Evidence for Osmoregulatory Function of the Sternal Epithelia in Some Gammaridean Amphipods

The ion-permeabilities and the ultrastructure of the sternal epithelia were studied in 7 species of gammaridean amphipods. Evidence has been obtained that the sterna can be divided into 2 categories: ion-transporting and non-ion-transporting types. The sterna of Sternomoera japonica, Grandidierella japonica, Melita setiflagella, and Corophium uenoi, belonging to the former category, are covered by ion-permeable cuticular layers lined with a thick epithelium, which is characterized by both a shallow apical infolding system (AIS) and a well-developed basolateral infolding system (BIS). The AIS is composed of frequent lamellar infoldings without mitochondrial association originating from the apical sides of the epithelia and involves virtually every part of the apical cell membrane, forming elaborate intercellular channels in the subcuticular space. In contrast, the BIS consists of deep infoldings of the basolateral cell membranes and extensive interdigitations between epithelial cells, which are both associated with a large number of mitochondria. This BIS involves the greater part of the cell membrane and constitutes a complicated, giant labyrinth of the intercellular spaces above the basal lamina. The AIS and BIS never communicate in the sternal epithelia, though they sometimes interpenetrate. On the other hand, the sterna of Jesogammarus jesoensis, Jesogammarus hinumensis, and Haustorioides sp., belonging to the latter category, are composed of an ion-impermeable, thick cuticular layer and a thin epithelium characterized by a paucity of mitochondria and complete lack of the BIS and AIS, which are common to those of the ordinary epithelium of the body surface. The results suggest a rather widespread occurrence in amphipods of a variety of extrabranchial ion-permeable areas of the body surface. They also suggest that the sterna lined with thick, specialized epithelia, together with the gills, are involved in the active transport of electrolytes in osmoregulation as well as respiration.

Immunocytochemical localization of Na+,K(+)-ATPase in the calcium-transporting sternal epithelium of the terrestrial isopod Porcellio scaber L. (Crustacea).

Terrestrial isopods store large amounts of calcium carbonate between the epithelium and the old cuticle of the first four anterior sternites before molt. During the formation of these sternal CaCO3 deposits, large amounts of calcium are transported across the anterior sternal epithelium from the base to the apical side of the integument, and in the reverse direction during resorption of the deposit. A monoclonal antibody against the avian alpha-subunit of Na+,K(+)-ATPase was used to localize Na+,K(+)-ATPase in the anterior and the posterior sternal epithelium of Porcellio scaber. Semithin cryosections 0.5 micron thick were used for immunofluorescence microscopy and ultrathin cryosections for immunogold electron microscopy. The Na+,K(+)-ATPase was localized in the basolateral plasma membrane of the posterior and anterior sternal epithelium. The apical plasma membrane, including cytoplasmic extensions into the newly secreted cuticle, was virtually devoid of the enzyme. This pattern of immunolocalization was not affected by the direction of transepithelial calcium transport associated with the deposition and resorption phases of the molt cycle.

Ultrastructural changes of the anterior and posterior sternal integument of the terrestrial isopod Porcellio scaber Latr. ( Crustacea) during the moult cycle

The ultrastructure of the anterior (ASE) and posterior sternal epithelium (PSE) was investigated during the biphasic moult cycle. During early premoult the sternal epithelial cells increase in size, accumulate huge amounts of glycogen, and increase the abundance of cellular organelles. CaCO 3 deposit formation begins before the secretion of the epicuticle in the anterior sternal integument and continues through the secretion of the first exocuticular layers. The deposition of cuticle is delayed in the ASE until the CaCO 3 deposit is fully resorbed between the anterior and posterior moult. The development of the interstitial network (IN), which enormously increases the basolateral compartment of the plasma membrane, starts at the beginning of the exocuticle deposition. During CaCO 3 deposit formation and resorption the volume fraction of the mitochondria is much larger in the ASE than in the PSE, although the cuticle is secreted faster in the posterior integument. The results suggest that the exocuticular and epicuticular layers are permeable to calcium and probably also carbonate ions; that the IN is required during late premoult, when CaCO 3 deposition is accelerated, but not during early CaCO 3 deposition; and that active mechanisms contribute to transepithelial ion transport during CaCO 3 deposit formation and resorption.

ULTRASTRUCTURAL EVIDENCE FOR OSMOREGULATORY FUNCTION OF THE STERNAL EPITHELIA IN SOME GAMMARIDEAN AMPHIPODS

ABSTRACT The ion-permeabilities and the ultrastructure of the sternal epithelia were studied in 7 species of gammaridean amphipods. Evidence has been obtained that the sterna can be divided into 2 categories: ion-transporting and non-ion-transporting types. The sterna of Sternomoera japonica, Grandidierella japonica, Melita setiflagella, and Corophium uenoi, belonging to the former category, are covered by ion-permeable cuticular layers lined with a thick epithelium, which is characterized by both a shallow apical infolding system (AIS) and a well-developed basolateral infolding system (BIS). The AIS is composed of frequent lamellar infoldings without mitochondrial association originating from the apical sides of the epithelia and involves virtually every part of the apical cell membrane, forming elaborate intercellular channels in the subcuticular space. In contrast, the BIS consists of deep infoldings of the basolateral cell membranes and extensive interdigitations between epithelial cells, which are both associated with a large number of mitochondria. This BIS involves the greater part of the cell membrane and constitutes a complicated, giant labyrinth of the intercellular spaces above the basal lamina. The AIS and BIS never communicate in the sternal epithelia, though they sometimes interpenetrate. On the other hand, the sterna of Jesogammarus jesoensis, Jesogammarus hinumensis, and Haustorioides sp., belonging to the latter category, are composed of an ion-impermeable, thick cuticular layer and a thin epithelium characterized by a paucity of mitochondria and complete lack of the BIS and AIS, which are common to those of the ordinary epithelium of the body surface. The results suggest a rather widespread occurrence in amphipods of a variety of extrabranchial ion-permeable areas of the body surface. They also suggest that the sterna lined with thick, specialized epithelia, together with the gills, are involved in the active transport of electrolytes in osmoregulation as well as respiration.

Ultrastructural evidence for transepithelial calcium transport in the anterior sternal epithelium of the terrestrial isopod Porcellio scaber (Crustacea) during the formation and resorption of CaCO 3 deposits

Before the molt, terrestrial isopods store large amounts of calcium carbonate between the epithelium and the old cuticle of the first four anterior sternites. In order to test whether the anterior sternal epithelium has specific structural differentiations indicative of transepithelial ion transport, the anterior sternal epithelium and, as a control, the posterior sternal epithelium were studied using electron-microscopical techniques. During the formation of calcium carbonate deposits, the basolateral plasma membrane of the anterior sternal epithelium forms an elaborate interconnected network of interstitial dilations and channels. Numerous osmiophilic granules occur within this basolateral intercellular network during resorption of the calcium carbonate deposits. Electron energy-loss spectroscopy of the osmiophilic granules indicates that they contain calcium. During the resorption of the calcium carbonate deposits, the apical plasma membrane of the anterior sternal epithelium has many subcuticular folds. An interstitial network, osmiophilic granules, and apical, subcuticular folds do not occur in the posterior sternal epithelium. Taken together, these structural features are indicative of transepithelial ion transport and are probably necessary for the formation and resorption of the anterior sternal calcium carbonate deposits.

Pereopodal disk: a new type of extrabranchial ion-transporting organ in an estuarine amphipod, Melita setiflagella (crustacea)

A discoid organ, ‘pereopodal disk (PD)’, was found on the medial surface of the basipodite of each pereopod, except the third and the fourth, in an estuarine amphipod, Melita setiflagella. The silver methods showed that PD is an extrabranchial ion-permeable area of the body surface. The ultrastructural study revealed that PD is covered by a thin and soft cuticle layer suggesting high permeability to gases and ions, and is composed of a thick, transporting-type epithelium. This epithelium is characterized by deep basal infolding systems (BIS) of cell membranes exceeding two-thirds of the epithelial thickness and complicated interdigitations between adjacent epithelial cells, both associated with many mitochondria. Apical infolding systems (AIS) are shallow and not accompanied by any mitochondria. These characteristics resemble those of the sternal epithelia and form a striking contrast in the polarity of the infoldings to the gill epithelia, which are characterized by a well-developed AIS and sparse BIS. The results suggest that this unique organ may be involved in the active transport of electrolytes to maintain constant osmotic pressures of the body fluids under widely fluctuating salinities of the estuarine environments.

The ultrastructure of the sternal gills forming a striking contrast with the coxal gills in a fresh-water amphipod (crustacea)

Sternomoera yezoensis has specialized sterna with 21 sternal gills in addition to six pairs of coxal gills. Despite a common high permeability to chloride ions, the cpithelia of these two kinds of gills are diametrically opposed in the polarity of the cell membranemitochondria complex. The coxal gill epithelium (4–6 μm thick) is characterized by a welldeveloped AIS (apical infolding system) associated with a huge number of large mitochondria. The AIS exceeds two-thirds of the epithelial thickness and forms a highly sophisticated, subcuticular labyrinth. On the contrary, the sternal gill epithelium, an extension of the sternal epithelium proper, is extremely thick (10–15 μm) and is characterized by a very deep BIS (basolateral infolding system) associated with numerous slender mitochondria. The BIS reaches nine-tenths of the epithelial thickness and forms a giant, baso-lateral labyrinth. Shallower, less elaborate AIS and BIS without mitochondrial association originate from the opposite sides of these epithelia. Although AIS and BIS interpenetrate in the sternal gill epithelium, they never communicate. The results indicate that in addition to the coxal gills, the sterna with Ihe sternal gills function as transporting as well as respiratory organs, though the functional difference between these two kinds of gills remains to be elucidated.