- Email: [email protected]
Endocytosis of ferritin and hemoglobin by the trabecular endocardium in swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L. (Poecilidae: Teleostei)
ANNALS OF ANATOMY
Endocytosis of ferritin and hemoglobin by the trabecular endocardium in swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L. (Poecilidae: Teleostei) Ingvar Leiv Leknes Faculty of Science, Sogn og Fjordane College, Box 133, N-6851 Sogndal, Norway
Summary. The cardiac atrium and ventricle of swordtail,
take up and store large amounts of various foreign organ-
Xiphophorus helleri L. and platy, Xiphophorus maculatus ic macromolecules and particles from the blood stream L., are spongious, consisting of muscle trabeculae covered by endocardial cells. The cardiac trabecular endocardium is able to take up and store large amounts of horse-spleen ferritin and bovine hemoglobin from the blood stream. No such uptake was registered in endocardial cells lining the cardiac valves, atrio-ventricular junction and ventriculo-bulbar junction. The trabecular endocardium in these species seems to be unable to accumulate latex beads or bovine myoglobin, cytochrome C and holotransferrin from the blood stream. It is proposed that the trabecular endocardium in these species is able to clear the blood stream of some types of waste macromolecnles; i.e. this tissue may have a scavenger function. The present results indicate that the uptake of foreign ferritin in bony fish endocardium can be clearly demonstrated at the light microscopic level in deparaffined sections by means of acid ferrocyanide or Mallory solutions. A similar uptake of hemoglobin is demonstrated by means of Mallory stain.
Key words: Endocardium - Endocytosis - Ferritin - Hemoglobin - Poecilidae
Introduction Previously, it has been shown that the endocardial cells in some bony fish families are structurally specialized (Leknes 1983). Recently, several workers have found that the specialized atrial endocardium in gadoids is able to
E-mail: [email protected]
Ann Anat (2001) 183:251-254 © Urban & FischerVerlag
http:/Iwww.urbanfischer.de/journals/annanat.
(Leknes 1987; SmedsrCd et al. 1995; Dalmo et al. 1996, 1998; SCrensen et al. 1997, 1998). The main purpose of the present study has been to examine and compare the ability and capacity of the endocardium of two poecilid species, swordtail (Xiphophorus helleri) and platy (Xiphophorus maculatus), to accumulate various iron-containing proteins from the blood stream. In particular, we intend to find staining techniques which make it possible to demonstrate such an uptake at the light microscopic level.
Materials and methods 30 specimens of swordtail (X. helleri), 1-3 years old, and 30 specimens of platy (X. maculatus), 1-3 years old, kept in a well aerated aquarium at 21 °C, were injected intraperitoneally by 0.030.05 ml 10% solutions of horse-spleen ferritin, bovine hemoglobin, bovine myoglobin, bovine cytochrome C, bovine holotransferrin or 10% dyed latex beads (diameter 0.055 ~tm) solution (Sigma). The fishes were then, 18-77 h later, given an anaesthetic, chlorobutanol, and the heart was quickly excised and fixed at 4 °C in 4% formaldehyde, made up from paraformaldehyde~ a day before use, in phosphate buffer. In addition the hearts from 15 uninjected specimens, were also fixed. After washing in buffer, the tissue was dehydrated through an ethanol series, treated with xylene, embedded in paraffin wax and sectioned. Some deparaffined sections, 4 pm thick, were stained in Mayer's haemalum/eosin as described by Grimstone and Skaer (1972). Most sections, were, however, stained in a modified Mallory solution (Grimstone and Skaer 1972), or in acid ferrocyanide solution followed by solution of neutral red (Pearse 1980). The Mallory solution was made by dissolving 0.5 g phosphotunpestic acid, 1 g orange G, 0.5 g aniline blue and 1.5 g acid fuchsin in 100 ml
0940-9602101118313-251 $15.00/0
Fig. 1. a) Atrial trabeculae (T) in swordtail, 77 h after intraperitoneal injection of horse-spleen ferritin, stained with Mallory. The endocardial cells are packed with large orange-brown granules (arrows). b) Atrial trabeculum (T) in platy, 26 h after intraperitoneal injection of bovine hemoglobin, stained with Mallory. The endocardial cells are packed with blue granules. The pink structures at the left and right edges of the picture are erythrocytes, c) Ventricular (V) and atrial (A) tissue in platy, 26 h after intraperitoneal injection of horse-spleen ferritin, treated with acid ferrocyanide solution, and stained by neutral red. The trabecular endocardial layer displays a distinct blue colour, as the Fe 3+ ions of the horse-spleen ferritin accumulated by these cells, have reacted with ferrocyanide; i.e. Prussian blue has been produced (arrow). d) Atrial trabeculum (T) from c at higher magnification. The endocardial cells (arrows) are completely filled with Prussian blue; i.e. large amounts of horse-spleen ferritin are accumulated in these cells, e) Atrial trabeculum (T) with endocardial layer (arrow) in swordtail, 31 h after intraperitoneal injection of cytochrome C, stained with Mallory. f) Atrial trabeculum with endocardial layer (arrow) in swordtail, 26 h after intraperitoneal injection of myoglobin, stained with Mallory g)
252
water, in the order given. Deparaffined sections were stained at room temperature in this solution for 10 rain, washed rapidly in water, dehydrated very rapidly in 90% ethanol, and then in two or three bathes of 100% ethanol, and mounted in Mountex medium (Sigma). The ferrocyanide solution was made by dissolving 2 g potassium ferrocyanide in 100 ml 2% hydrochloric acid solution, and the neutral red solution was made by dissolving i g of this stain in 100 ml water. Deparaffined sections were treated with the ferrocyanide solution for 0.5 h, and then with the neutral red solution for 5 min. The sections were then treated as those stained in the Mallory solution.
Results Normal tissue. The cardiac tissue in X. helleri and X. maculatus was completely spongious, consisting of muscle trabeculae covered by endocardial cells. The atrial endocardial cells were usually high, up to 8 gm, whereas the corresponding ventricular cells were flat. Normally, some granules occurred, diameter about 1 gm, in the trabecular endocardial cells (Fig. i g). These granules displayed a blue colour when stained with Mallory solution, and no colour when treated with an acid ferrocyanide solution (Fig. 1 g, i). Tissue from specimens injected with horse-spleen ferritin or bovine hemoglobin. Macroscopically, the cardiac atrium often displayed a faint brown colour in swordtail and platy injected intraperitoneally with ferritin or hemoglobin. The trabecular endocardium in the atrium and the ventricle was always tightly packed with granules when the time elapsed between the injection and fixation was 18 h or more (Fig. 1 a-d). In the atrium these granules sometimes showed a diameter similar to the hight of the cells, up to 8 pm. When stained with a Mallory solution, these granules displayed a brown-orange colour in tissue from ferritin injected specimens and a blue colour in tissue from hemoglobin injected specimens (Fig. i a, b). When treated with acid ferrocyanide solution, these granules displayed a blue colour in tissue from ferritin injected specimens (Fig. 1 c, d, h). The endocardial tissue of the cardiac valves, atrio-ventricular junction and ventriculo-bulbar junction from ferritin treated or hemoglobin treated specimens displayed no stained granules when treated with Mallory or ferrocyanide solutions (Fig. 1 h). Tissue from specimens injected with bovine myoglobin, cytochrome C, holotransferrin or dyed latex beads. MacroscopicaUy, the atrium displayed a normal colour in swordtail and platy injected intraperitoneally with these
substances. Microscopically, the trabecular endocardium in these hearts displayed the same structure and colour as the corresponding control tissue when stained in Mallory solutions (Fig. 1 e-g).
Discussion Previously, it has been shown that the heart endocardial layer covering the atrial muscle trabeculae in bony-fish of the families Adrianichthyidae, Cichlidae, Gadidae, and Poecilidae consists of high cells, about 8-10 l~m, containing numerous bristlecoated vesicles (BCV) and moderately electron dense bodies (MDB), diameter 0.5-1.5 gm (Saetersdal et al. 1974; Lemanski et al. 1975; Leknes 1980, 1981, 1983; Nakamura and Shimozawa 1994). In species hitherto examined of other teleosts, the trabecular endo: cardial layer consists of attenuated cells which often seem to lack BCV and MDB, and thus have a shape and ultrastructure as generally found for these cells in vertebrates (Leknes 1983). Thus, the trabecular endocardium in the species of Adrianichthyidae, Cichlidae, Gadidae, and Poecilidae hitherto studied, may be regarded as specialized, whereas the endocardium in the other teleostean families hitherto studied appears to be unspecialized. Recently, it has been shown by electron microscopic techniques that the atrial endocardial cells in the cod, Gadus morhua, are able to take Up by pinocytosis, probably by means of the BCV-system, large amounts of various foreign organic macromolecules from the blood stream (SmedsrCd et al. 1995; Dalmo et al. 1996, 1998; Koren et al. 1997; SCrensen et al. 1997, 1998). In additior~, an in vitro pinocytotic uptake of horse-spleen ferritin, has been demonstrated in the specialized atrial endocardium of another gadoid, coalfish (Pollachius virens) by Leknes (1987). No such cardiac uptake of organic molecules and particles has, as far as we know, been reported for bony fish species with an unspecialized endocardium (Smedsrod et al. 1993). Ultrastructural studies have previously shown that horse-spleen ferritin is mainly taken up by BCV in rat intestinal and hepatic endothelial cells and swordtail atrial endocardium; i. e. this uptake is pinocytotic (De Bruyn et al. 1983; Leknes 1987). The present light microscopic study reveals that the trabecular endocardium in both the atrium and ventricle of X. helleri and X. maculatus is able to take up and store large amounts of horse-spleen ferritin and bovine hemoglobin, whereas this tissue seems not to accumulate latex beads or bovine myoglobin, cytochrome C and transfer-
Atrial trabeculum with endocardial layer (arrow) in uninjected platy, stained with Mallory. The intertrabecular erythrocytes are pink. h) Ventricular tissue from c at higher magnification. The endocardial layer (arrow) facing the lumen (L) of the atrioventricular junction contains no ferritin, in contrast to the trabecular endocardial layer, i) Atrial trabeculum (T) with endocardial layer (arrows) in uninjected platy, treated with acid ferrocyanide solution followed by neutral red. Only the endocardial nuclei are coloured. Bar is 20 gm in all pictures, except c) and i), where it is 50 gm. 253
tin. A n endocardial uptake of hemoglobin has, as far as we know, not previously been described either for teleosts or for other vertebrates. Furthermore, an uptake of ferritin in the ventricular endocardium, has, as far as we know, not previously been described for teleosts. We propose that the present uptake of horse-spleen ferritin and bovine hemoglobin in the trabecular endocardium in swordail and platy may reflects a scavenger function of this tissue in these species. Previously, it has been reported that the endocardial tissue of the cardiac valves and junctions in cichlid species appears to be structurally unspecialized (Leknes 1981, 1986). The present results indicate that these cells in swordtail and platy are not able to accumulate horsespleen ferritin or bovine hemoglobin. Thus, these cells probably may have no scavenger function in these species. The present study shows that the pinocytotic uptake of ferritin in the specialized endocardium in teleosts can be clearly demonstrated in paraffin embedded tissue, when the sections are deparaffined and stained with acid ferrocyanide or Mallory solutions. In particular, we recommend the former method; i.e. the Prussian blue method, to demonstrate the uptake of ferritin, and the Mallory m e t h o d to demonstrate the uptake of hemoglobin.
References Dalmo RA, Ingebrigtsen K, Sveinbjcrnsson B, Seljelid R (1996) Accumulation of immunomodulatory laminaran (13 (1,3)-Dglucan) in the heart, spleen and kidney of Atlantic cod, Gadus morhua L. J Fish Diseas 19:129-136 Dalmo RA, Seternes T, Arnesen SM, JCrgensen TO, B0gwald J (1998) Tissue distribution and cellular uptake of Aeromonas salmonicida lipopolysaccharide (LPS) in some marine fish species. J Fish Diseas 21:321-334 De Bruyn PPH, Cho Y, Michelson S (1983) In vivo endocytosis by bristle-coated pits of protein tracers and their intracellular transport in the endothelial cells lining the sinuses of the liver. I. The endosomal diposition. J Ultrastruct Res 85:272-289
Grimstone AV, Skaer RJ (1972) A guidebook to microscopical methods. Cambridge University Press, London Koren CWR, SveinbjCrnsson B, SmedsrCd B (1997) Isolation and culture of endocardial endothelial cells fl'om Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua). Cell Tissue Res 290:89-99 Leknes IL (1980) Ultrastructure of atrial endocardium and myocardium in three species of Gadidae (Teleostei). Cell Tissue Res 210:1-10 Leknes IL (1981) Ultrastructure of myocardial widened Z bands and endocardial cells in two teleostean species. Cell Tissue Res 218:23-28 Leknes IL (1983) Ultrastructural and ultrahistochemical studies on ventricular endocardial cells in teleosts (Pisces). J Zool 201:507-513 Leknes IL (1986) Fine structure and cytochemistry of the endothelial cells and rodlet cells in the bulbus arteriosus in species of Cichlidae (Teleostei). J Fish Biol 28:29-36 Leknes IL (1987) Endocytosis of horse-spleen ferritin by bony fish endocardiuml Acta Histochem 81:175-182 Lemanski LF, Payson Fitts E, Marx BS (1975) Fine structure of the heart in the Japanese medaka Oryzias latipes. J Ultrastruct Res 53:3%65 Nakamura H, Schimozawa A (1994) Phagocytotic cells in the fish heart. Arch Histol Cytol 57:415-425 PearseAGE (1980) Histochemistry: theoretical and applied. Churchill Livingstone, Edinburgh Saetersdal TS, Justesen NP, Krohnstad AaW (1974) Ultrastructure and innervation of the teleostean atrium. J Mol Cell Card 6:415-437 Smedsr0d B, GjCen T, Sveinbj0rnsson B, Berg T (1993) Catabolism of circulating collagen in the Atlantic salmon (Salmo salar). J Fish Biol 42:279-291 Smedsrcd B, Olsen R, Sveinbjcrnsson B (1995) Circulating collagen is catabolized by endocytosis mainly by endothelial cells of endocardium in cod (Gadus morhua). Cell Tissue Res 280: 39-48 SCrensen KK, Dahl LB, SmedsrCd B (1997) Role of endocardial endothelial cells in the turnover of hyaluronan in Atlantic cod (Gadus morhua). Cell Tissue Res 290:101-109 Scrensen KK, Melkko J, Smedsrcd B (1998) Scavenger-receptormediated endocytosis in endocardial endothelial cells of Atlantic cod Gadus morhua. J Exp Biol 201:1707-1718 Accepted November 20, 2000
254
Endocytosis of ferritin and hemoglobin by the trabecular endocardium in swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L. (Poecilidae: Teleostei) Ingvar Leiv Leknes Faculty of Science, Sogn og Fjordane College, Box 133, N-6851 Sogndal, Norway
Summary. The cardiac atrium and ventricle of swordtail,
take up and store large amounts of various foreign organ-
Xiphophorus helleri L. and platy, Xiphophorus maculatus ic macromolecules and particles from the blood stream L., are spongious, consisting of muscle trabeculae covered by endocardial cells. The cardiac trabecular endocardium is able to take up and store large amounts of horse-spleen ferritin and bovine hemoglobin from the blood stream. No such uptake was registered in endocardial cells lining the cardiac valves, atrio-ventricular junction and ventriculo-bulbar junction. The trabecular endocardium in these species seems to be unable to accumulate latex beads or bovine myoglobin, cytochrome C and holotransferrin from the blood stream. It is proposed that the trabecular endocardium in these species is able to clear the blood stream of some types of waste macromolecnles; i.e. this tissue may have a scavenger function. The present results indicate that the uptake of foreign ferritin in bony fish endocardium can be clearly demonstrated at the light microscopic level in deparaffined sections by means of acid ferrocyanide or Mallory solutions. A similar uptake of hemoglobin is demonstrated by means of Mallory stain.
Key words: Endocardium - Endocytosis - Ferritin - Hemoglobin - Poecilidae
Introduction Previously, it has been shown that the endocardial cells in some bony fish families are structurally specialized (Leknes 1983). Recently, several workers have found that the specialized atrial endocardium in gadoids is able to
E-mail: [email protected]
Ann Anat (2001) 183:251-254 © Urban & FischerVerlag
http:/Iwww.urbanfischer.de/journals/annanat.
(Leknes 1987; SmedsrCd et al. 1995; Dalmo et al. 1996, 1998; SCrensen et al. 1997, 1998). The main purpose of the present study has been to examine and compare the ability and capacity of the endocardium of two poecilid species, swordtail (Xiphophorus helleri) and platy (Xiphophorus maculatus), to accumulate various iron-containing proteins from the blood stream. In particular, we intend to find staining techniques which make it possible to demonstrate such an uptake at the light microscopic level.
Materials and methods 30 specimens of swordtail (X. helleri), 1-3 years old, and 30 specimens of platy (X. maculatus), 1-3 years old, kept in a well aerated aquarium at 21 °C, were injected intraperitoneally by 0.030.05 ml 10% solutions of horse-spleen ferritin, bovine hemoglobin, bovine myoglobin, bovine cytochrome C, bovine holotransferrin or 10% dyed latex beads (diameter 0.055 ~tm) solution (Sigma). The fishes were then, 18-77 h later, given an anaesthetic, chlorobutanol, and the heart was quickly excised and fixed at 4 °C in 4% formaldehyde, made up from paraformaldehyde~ a day before use, in phosphate buffer. In addition the hearts from 15 uninjected specimens, were also fixed. After washing in buffer, the tissue was dehydrated through an ethanol series, treated with xylene, embedded in paraffin wax and sectioned. Some deparaffined sections, 4 pm thick, were stained in Mayer's haemalum/eosin as described by Grimstone and Skaer (1972). Most sections, were, however, stained in a modified Mallory solution (Grimstone and Skaer 1972), or in acid ferrocyanide solution followed by solution of neutral red (Pearse 1980). The Mallory solution was made by dissolving 0.5 g phosphotunpestic acid, 1 g orange G, 0.5 g aniline blue and 1.5 g acid fuchsin in 100 ml
0940-9602101118313-251 $15.00/0
Fig. 1. a) Atrial trabeculae (T) in swordtail, 77 h after intraperitoneal injection of horse-spleen ferritin, stained with Mallory. The endocardial cells are packed with large orange-brown granules (arrows). b) Atrial trabeculum (T) in platy, 26 h after intraperitoneal injection of bovine hemoglobin, stained with Mallory. The endocardial cells are packed with blue granules. The pink structures at the left and right edges of the picture are erythrocytes, c) Ventricular (V) and atrial (A) tissue in platy, 26 h after intraperitoneal injection of horse-spleen ferritin, treated with acid ferrocyanide solution, and stained by neutral red. The trabecular endocardial layer displays a distinct blue colour, as the Fe 3+ ions of the horse-spleen ferritin accumulated by these cells, have reacted with ferrocyanide; i.e. Prussian blue has been produced (arrow). d) Atrial trabeculum (T) from c at higher magnification. The endocardial cells (arrows) are completely filled with Prussian blue; i.e. large amounts of horse-spleen ferritin are accumulated in these cells, e) Atrial trabeculum (T) with endocardial layer (arrow) in swordtail, 31 h after intraperitoneal injection of cytochrome C, stained with Mallory. f) Atrial trabeculum with endocardial layer (arrow) in swordtail, 26 h after intraperitoneal injection of myoglobin, stained with Mallory g)
252
water, in the order given. Deparaffined sections were stained at room temperature in this solution for 10 rain, washed rapidly in water, dehydrated very rapidly in 90% ethanol, and then in two or three bathes of 100% ethanol, and mounted in Mountex medium (Sigma). The ferrocyanide solution was made by dissolving 2 g potassium ferrocyanide in 100 ml 2% hydrochloric acid solution, and the neutral red solution was made by dissolving i g of this stain in 100 ml water. Deparaffined sections were treated with the ferrocyanide solution for 0.5 h, and then with the neutral red solution for 5 min. The sections were then treated as those stained in the Mallory solution.
Results Normal tissue. The cardiac tissue in X. helleri and X. maculatus was completely spongious, consisting of muscle trabeculae covered by endocardial cells. The atrial endocardial cells were usually high, up to 8 gm, whereas the corresponding ventricular cells were flat. Normally, some granules occurred, diameter about 1 gm, in the trabecular endocardial cells (Fig. i g). These granules displayed a blue colour when stained with Mallory solution, and no colour when treated with an acid ferrocyanide solution (Fig. 1 g, i). Tissue from specimens injected with horse-spleen ferritin or bovine hemoglobin. Macroscopically, the cardiac atrium often displayed a faint brown colour in swordtail and platy injected intraperitoneally with ferritin or hemoglobin. The trabecular endocardium in the atrium and the ventricle was always tightly packed with granules when the time elapsed between the injection and fixation was 18 h or more (Fig. 1 a-d). In the atrium these granules sometimes showed a diameter similar to the hight of the cells, up to 8 pm. When stained with a Mallory solution, these granules displayed a brown-orange colour in tissue from ferritin injected specimens and a blue colour in tissue from hemoglobin injected specimens (Fig. i a, b). When treated with acid ferrocyanide solution, these granules displayed a blue colour in tissue from ferritin injected specimens (Fig. 1 c, d, h). The endocardial tissue of the cardiac valves, atrio-ventricular junction and ventriculo-bulbar junction from ferritin treated or hemoglobin treated specimens displayed no stained granules when treated with Mallory or ferrocyanide solutions (Fig. 1 h). Tissue from specimens injected with bovine myoglobin, cytochrome C, holotransferrin or dyed latex beads. MacroscopicaUy, the atrium displayed a normal colour in swordtail and platy injected intraperitoneally with these
substances. Microscopically, the trabecular endocardium in these hearts displayed the same structure and colour as the corresponding control tissue when stained in Mallory solutions (Fig. 1 e-g).
Discussion Previously, it has been shown that the heart endocardial layer covering the atrial muscle trabeculae in bony-fish of the families Adrianichthyidae, Cichlidae, Gadidae, and Poecilidae consists of high cells, about 8-10 l~m, containing numerous bristlecoated vesicles (BCV) and moderately electron dense bodies (MDB), diameter 0.5-1.5 gm (Saetersdal et al. 1974; Lemanski et al. 1975; Leknes 1980, 1981, 1983; Nakamura and Shimozawa 1994). In species hitherto examined of other teleosts, the trabecular endo: cardial layer consists of attenuated cells which often seem to lack BCV and MDB, and thus have a shape and ultrastructure as generally found for these cells in vertebrates (Leknes 1983). Thus, the trabecular endocardium in the species of Adrianichthyidae, Cichlidae, Gadidae, and Poecilidae hitherto studied, may be regarded as specialized, whereas the endocardium in the other teleostean families hitherto studied appears to be unspecialized. Recently, it has been shown by electron microscopic techniques that the atrial endocardial cells in the cod, Gadus morhua, are able to take Up by pinocytosis, probably by means of the BCV-system, large amounts of various foreign organic macromolecules from the blood stream (SmedsrCd et al. 1995; Dalmo et al. 1996, 1998; Koren et al. 1997; SCrensen et al. 1997, 1998). In additior~, an in vitro pinocytotic uptake of horse-spleen ferritin, has been demonstrated in the specialized atrial endocardium of another gadoid, coalfish (Pollachius virens) by Leknes (1987). No such cardiac uptake of organic molecules and particles has, as far as we know, been reported for bony fish species with an unspecialized endocardium (Smedsrod et al. 1993). Ultrastructural studies have previously shown that horse-spleen ferritin is mainly taken up by BCV in rat intestinal and hepatic endothelial cells and swordtail atrial endocardium; i. e. this uptake is pinocytotic (De Bruyn et al. 1983; Leknes 1987). The present light microscopic study reveals that the trabecular endocardium in both the atrium and ventricle of X. helleri and X. maculatus is able to take up and store large amounts of horse-spleen ferritin and bovine hemoglobin, whereas this tissue seems not to accumulate latex beads or bovine myoglobin, cytochrome C and transfer-
Atrial trabeculum with endocardial layer (arrow) in uninjected platy, stained with Mallory. The intertrabecular erythrocytes are pink. h) Ventricular tissue from c at higher magnification. The endocardial layer (arrow) facing the lumen (L) of the atrioventricular junction contains no ferritin, in contrast to the trabecular endocardial layer, i) Atrial trabeculum (T) with endocardial layer (arrows) in uninjected platy, treated with acid ferrocyanide solution followed by neutral red. Only the endocardial nuclei are coloured. Bar is 20 gm in all pictures, except c) and i), where it is 50 gm. 253
tin. A n endocardial uptake of hemoglobin has, as far as we know, not previously been described either for teleosts or for other vertebrates. Furthermore, an uptake of ferritin in the ventricular endocardium, has, as far as we know, not previously been described for teleosts. We propose that the present uptake of horse-spleen ferritin and bovine hemoglobin in the trabecular endocardium in swordail and platy may reflects a scavenger function of this tissue in these species. Previously, it has been reported that the endocardial tissue of the cardiac valves and junctions in cichlid species appears to be structurally unspecialized (Leknes 1981, 1986). The present results indicate that these cells in swordtail and platy are not able to accumulate horsespleen ferritin or bovine hemoglobin. Thus, these cells probably may have no scavenger function in these species. The present study shows that the pinocytotic uptake of ferritin in the specialized endocardium in teleosts can be clearly demonstrated in paraffin embedded tissue, when the sections are deparaffined and stained with acid ferrocyanide or Mallory solutions. In particular, we recommend the former method; i.e. the Prussian blue method, to demonstrate the uptake of ferritin, and the Mallory m e t h o d to demonstrate the uptake of hemoglobin.
References Dalmo RA, Ingebrigtsen K, Sveinbjcrnsson B, Seljelid R (1996) Accumulation of immunomodulatory laminaran (13 (1,3)-Dglucan) in the heart, spleen and kidney of Atlantic cod, Gadus morhua L. J Fish Diseas 19:129-136 Dalmo RA, Seternes T, Arnesen SM, JCrgensen TO, B0gwald J (1998) Tissue distribution and cellular uptake of Aeromonas salmonicida lipopolysaccharide (LPS) in some marine fish species. J Fish Diseas 21:321-334 De Bruyn PPH, Cho Y, Michelson S (1983) In vivo endocytosis by bristle-coated pits of protein tracers and their intracellular transport in the endothelial cells lining the sinuses of the liver. I. The endosomal diposition. J Ultrastruct Res 85:272-289
Grimstone AV, Skaer RJ (1972) A guidebook to microscopical methods. Cambridge University Press, London Koren CWR, SveinbjCrnsson B, SmedsrCd B (1997) Isolation and culture of endocardial endothelial cells fl'om Atlantic salmon (Salmo salar) and Atlantic cod (Gadus morhua). Cell Tissue Res 290:89-99 Leknes IL (1980) Ultrastructure of atrial endocardium and myocardium in three species of Gadidae (Teleostei). Cell Tissue Res 210:1-10 Leknes IL (1981) Ultrastructure of myocardial widened Z bands and endocardial cells in two teleostean species. Cell Tissue Res 218:23-28 Leknes IL (1983) Ultrastructural and ultrahistochemical studies on ventricular endocardial cells in teleosts (Pisces). J Zool 201:507-513 Leknes IL (1986) Fine structure and cytochemistry of the endothelial cells and rodlet cells in the bulbus arteriosus in species of Cichlidae (Teleostei). J Fish Biol 28:29-36 Leknes IL (1987) Endocytosis of horse-spleen ferritin by bony fish endocardiuml Acta Histochem 81:175-182 Lemanski LF, Payson Fitts E, Marx BS (1975) Fine structure of the heart in the Japanese medaka Oryzias latipes. J Ultrastruct Res 53:3%65 Nakamura H, Schimozawa A (1994) Phagocytotic cells in the fish heart. Arch Histol Cytol 57:415-425 PearseAGE (1980) Histochemistry: theoretical and applied. Churchill Livingstone, Edinburgh Saetersdal TS, Justesen NP, Krohnstad AaW (1974) Ultrastructure and innervation of the teleostean atrium. J Mol Cell Card 6:415-437 Smedsr0d B, GjCen T, Sveinbj0rnsson B, Berg T (1993) Catabolism of circulating collagen in the Atlantic salmon (Salmo salar). J Fish Biol 42:279-291 Smedsrcd B, Olsen R, Sveinbjcrnsson B (1995) Circulating collagen is catabolized by endocytosis mainly by endothelial cells of endocardium in cod (Gadus morhua). Cell Tissue Res 280: 39-48 SCrensen KK, Dahl LB, SmedsrCd B (1997) Role of endocardial endothelial cells in the turnover of hyaluronan in Atlantic cod (Gadus morhua). Cell Tissue Res 290:101-109 Scrensen KK, Melkko J, Smedsrcd B (1998) Scavenger-receptormediated endocytosis in endocardial endothelial cells of Atlantic cod Gadus morhua. J Exp Biol 201:1707-1718 Accepted November 20, 2000
254