Cell and Tissue Research
Cell TissueRes. 196, 249-261 (1979)
9 by Springer-Verlag 1979
New Observations on the Fine Structure of the Liver in Goldfish (Carassius auratus) * Waykin Nopanitaya, Johnny L. Carson, Joe W. Grisham and John G. Aghajanian Department of Pathology,Schoolof Medicine,Universityof North Carolinaat ChapelHill, Chapel Hill, North Carolina, USA
Summary. A re-examination of goldfish liver was made through the use of SEM of fractured samples and TEM of ultrathin-sections and freeze-etch replicas. Several new hepatic fine structures described in the present study are morphologically similar to those reported previously in many higher vertebrates including mammals. Hepatic sinusoids of goldfish contain fenestrations which are arranged into sieve plates. Although the hepatic plates are made up of two layers of hepatocytes, the parenchymal cells of goldfish liver are morphologically similar to mammalian hepatocytes, particularly with respect to the sinusoidal surfaces which are studded with numerous microvilli. The intercellular surfaces of hepatocytes have both nexus and desmosomal junctions, similar to those found in various epithelial cells of higher vertebrates, as cell attachments and communication loci. Tight junctions are found mainly between the openings of the intracellular bile canaliculi and the intralobular bile ductules which are situated in the center of the bicellular hepatic plate. Key words: Liver - Goldfish - Ultrastructure. In their comparative study of the anatomy of liver in vertebrates, Elias and Bengelsdorf (1952) pointed out that investigations on the morphology of the liver of goldfish would be of interest from a phylogenetic aspect. Their light-microscopic studies showed that the liver of this species is composed mainly of hepatic plates that are two ceils in thickness while the hepatic plates of several other non-fish species were primarily one-cell thick. David (1961) and Yamamoto (1962, 1965), the first investigators to examine the fine structure of goldfish liver, directed their attention mainly to the unique intercellular and intracellular biliary passages and some fine structural features of the hepatocytes. These early ultrastructural findings are of interest when compared to intrahepatic features of vertebrates. Several aspects of hepatic fine structure, however, remain to be investigated fully. In particular, the ultrastructural details of various other hepatic cells remain to be demonstrated. This report, through the use of SEM and TEM with the application Dr. WaykinNopanitaya,Departmentof Pathology,School of Medicine, Universityof North Carolina at Chapel Hill, Chapel Hill, N.C. 27514, USA * Supported in part by Grants ~/ GM92and ES07017
Send offprint requests to:
0302-766X/79/0196/0249/$02.60
250
w. Nopanitaya et al.
o f b o t h c o n v e n t i o n a l u l t r a t h i n sectioning a n d freeze-etch techniques, elucidates some i n t r a h e p a t i c fine structures o f the goldfish t h a t have n o t been e x a m i n e d previously by electron m i c r o s c o p y .
Materials and Methods Goldfish (Carassiusauratus),measuring 6-8 cm in total length, were killed by decapitation immediately after being taken from the aquarium. All internal organs were quickly removed and rinsed in physiological saline prior to immersion in fixative in which the hepatic tissues were carefully dissected out under the dissecting microscope. Tissues for TEM and SEM studies were fixed in either 4% paraformaldehyde or 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 2-4h at 4~C, followed by post-fixation in 2 % OsO4 in 0.1 M phosphate buffer (pH 7.3) for 1 h at room temperature, and then processed further for TEM and SEM by conventional methods. The critical-point dried SEM samples were manually fractured prior to mounting and Au-Pd coating. Tissues used for obtaining freeze-etched replicas were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 2-4 h at 4~C. They were cut into small pieces (1.5 x 1.5 x 3 mm), immersed in a solution of 25 % glycerol in 0.9 % NaC1 for 2 h at 4~ after which they were rapidly frozen, and transferred to Balzers' apparatus in which they were freeze-etched and C-Pt replicated according to the method described by Moor (1966). Ultrathin sections and freeze-etch replicas were examined with a Zeiss EM 10A TEM equipped with a goniometer stage, using an accelerating voltage of 80 KV. The samples for SEM were viewed and photographed with an ETEC U2 Autoscan operating at 20 KV.
Results Since some features o f the fine structure o f the h e p a t o c y t e s a n d the biliary passages in goldfish have previously been d o c u m e n t e d ( Y a m a m o t o , 1965), the descriptive m o r p h o l o g y detailed in the present investigation pertains specifically to new u l t r a s t r u c t u r a l findings.
Hepatic Parenchymal Arrangement and Segmentation The a r r a n g e m e n t o f the h e p a t i c p a r e n c h y m a l cells in goldfish differs f r o m t h a t o f m a m m a l i a n h e p a t i c lobules in the absence o f connective-tissue septae t h a t delineate the h e p a t i c cell mass. A t low m a g n i f i c a t i o n , S E M o f a f r a c t u r e d s a m p l e discloses an a p p a r e n t p a r e n c h y m a l s e g m e n t a t i o n resulting f r o m the i n t e r d i g i t a t i o n o f h e p a t i c b l o o d vessels (Fig. 1). S E M studies in c o n j u n c t i o n with the light m i c r o s c o p e reveal t h a t the p o r t a l vessels a n d the h e p a t i c veins p e n e t r a t e p e r p e n d i c u l a r l y into the m a s s o f p a r e n c h y m a l cells, dividing it a n d causing it to resemble a n " h e p a t i c acinus".
Hepatic Sinusoids and Sinusoidal Endothelial Cells H e p a t i c sinusoids are tube-like channels averaging f r o m 5 to 12 ~tm in diameter. E n d o t h e l i a l cells lining the sinusoids are heavily fenestrated b y p o r e s ranging f r o m 0.051xm to 0.21xm in d i a m e t e r (Figs. 2 4 ) in g r o u p s o r clusters o f f r o m 15 to 35
Fig. 1. A low magnification SEM micrograph of fractured hepatic tissue of goldfish. Portal (p) and hepatic (hv) veins randomly penetrate the hepatic cell masses, x 190 Fig. 2. A freeze-etch replica of goldfish sinusoidal endothelial cells. Groups of fenestrae (arrows) are evident on the endothelial cells, x 13,000
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Fig. 3. Endothelial fenestrations are clearly visible under the SEM at high magnification, x 35,500 Fig.4. Endothelial fenestrae, as seen in transversely sectioned endothelium, are surrounded by endothelial cytoplasm containing numerous microfilaments. • 20,500
Fig. 5. Both filament-rich cell (Fc) and Ito cell (arrow) containing lipid inclusion (L) are found in the perisinusoidal space of Disse in goldfish, x 15,300 Fig. 6. A bicellular hepatic plate contains a bile ductule (BD) which is located between the hepatocytes (h~ and h2) forming the hepatic plate, x 7000
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Fig.7. The hepatocyte surface facing the sinusoid contains numerous microvilli(My), whereas the smooth intercellular surface (Is) is studded by only a few cytoplasmicprojections (arrow heads). • 22,000
(Figs. 2, 3). TEM of thin sections and SEM of the fenestrations show no evidence of a membraneous diaphragm. The cytoplasm bordering the pores contain numerous fine filaments that are closely associated with the plasma membrane which surrounds the endothelial pores (Fig. 4). Subendothelial basal lamina and perisinusoidal collagen fibers are not evident in the sinusoids. A careful examination of the perisinusoidal spaces of Disse reveals two distinct types of perisinusoidal cells. One type is very abundant and possesses numerous cytoplasmic microfilaments; the other is found less frequently and contains prominent, large lipid inclusions in the cytoplasm (Fig. 5).
Hepatic Plates and Hepatocytes Hepatic plates in goldfish are formed by two closely apposed layers of hepatocytes (Fig. 6) which are bordered laterally on each side by sinusoids. The cells of the hepatic plates are barrel-shaped with the largest diameter being at the level of the nucleus and the smallest diameters being at the tapered ends of the cell toward the sinusoid and toward the center of the plate.
Fig.8. A gap junction, as revealed by TEM of a thin section, is commonly found between adjoining hepatocytes, x 139,400 Fig. 9. A replica of the hepatocyte surface. It contains both small and large gap junctions (arrow heads). A pinocytotic vesicle (arrow) is also visible in this micrograph. • 68,800 Fig. 10. A desmosomal junction is occasionally seen between adjacent hepatocytes, x 101,100
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SEM observations of the three-dimensional features of the hepatocytes reveal that each cell has at least two distinct surfaces: a sinusoidal surface and an intercellular surface (Fig. 7). The sinusoidal surface, the surface of hepatocytes bordering the sinusoidal endothelium, is covered with numerous microviUi (15-20 microvilli/lxm 2) which project into the space of Disse. The microvilli are relatively uniform in size (ca. 0.1 ~tm wide by 0.6 Ixm long) and are randomly oriented. The intercellular surface of the hepatocyte is smooth and possesses a few short cytoplasmic projections measuring 0.1 ~tm wide by 0.3 Ixm long (Fig. 7). TEM observations of ultrathin sections and freeze-etch replicas reveal additional features of the intercellular surface of the goldfish hepatocytes. These include pinocytotic invaginations and the loci of intercellular attachment and communication (Figs. 8, 9). Cell-to-cell attachment may be either by a gap junction (nexus) (Figs. 8, 9) or a desmosomal junction (Fig. 10), the former being more numerous than the latter. The appearance of the gap junction of goldfish hepatocytes is similar to the typical gap junction seen in various cells of vertebrates (Staehelin and Hull, 1978). In freeze-etch replicas, the gap junction appears as a plaque-like structure which varies considerably in size. Small gap junction plaques, which are circular in outline and measure 0.08-0.1 ~tm in diameter, and large irregularly shaped gap junctions, ranging from 0.5-1.0 ~tm wide by 1.0-2.0 ~tm long, are randomly distributed on the intercellular surface of the hepatocytes (Fig. 9). The ultrastructure of the organelles of goldfish hepatocytes, as seen with the TEM, is similar to that of higher vertebrates. Glycogen particles in rosetteconfigurations are abundant (Figs. 12, 13) and are the most prominent feature of the hepatocytes. These cells contain a single, spherical nucleus, the bounding membrane of which contains randomly distributed nuclear pores which are about 90 nm in diameter (Fig. 11). The Golgi complex of each hepatocyte is consistently found in close proximity to the "intercellular bile canaliculus" described by Yamamoto (1965) (Fig. 12).
Intrahepatic Plate Biliary Pathway In each hepatocyte, the intracellular bile canaliculus opens into the lumen of the adjacent terminal ductule cells through the hepatocyte surface. This results in a direct lumenal communication between the hepatocyte and the biliary duct ceils. At this transition zone, the terminal ductule cell openings are in contact with one another at tight junctions and from a common biliary passage (Fig. 12). The lumen of an individual ductule cell is formed by infoldings of the plasma membrane which are sealed by tight junctions (Fig. 13). SEM of a favorably fractured sample clearly elucidates the three-dimensional relationship between the hepatocytes and the biliary ductule cells in hepatic plates (Figs. 6, 14). In particular, the extracellular openings of the hepatocyte intracellular bile canaliculus and of the biliary ductule cells are easily recognizable (Fig. 14). They are randomly located on the lateral surface of their respective cell type. The bile canalicular opening of the hepatocyte is cup-shaped, 1 gm in diameter, with its luminal surface covered by microvilli averaging 0.08 gm wide by 0.6gm long (Figs. 12, 13). This structure represents a mold of the similar cup-shaped openings
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Fig. 11. The nucleus of this goldfish hepatocyte has several nuclear pores, x 25,600 Fig. 12. A TEM micrograph illustrating the common biliary pathway in goldfish. An intracellular bile canaliculus of the hepatocyte (BC) opens directly into the lumen of the bile ductule cell (BDC). The hepatocyte is held to its adjacent bile ductule cell by tight junctions. Numerous glycogen particles are seen throughout the hepatocyte cytoplasm. G Golgi complex, x 25,600
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Fig. 13. A TEM micrograph demonstrating tight junctions (arrows) between the glycogen-rich hepatocyte (/-/) and the terminal bile ductule formed by a single filament-rich bile ductule cell (BDC). Also note that the cytoplasmic portions of this bile ductule cell (BDC) are joined together by tight junctions (arrow heads) thereby forming a ductular lumen (L). x 19,400 Fig. 14. A SEM micrograph showing the biliary openings (arrow heads) along the lateral cytoplasm of the bile ductule cell (BDC) and the hepatocyte (H). x 7900
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seen laterally on the ductule cell surface or at the terminal branch of the biliary ductule. The ductule cells forming the biliary passages are spindle-shaped and have thin, elongated cytoplasmic processes which extend from the bulging cell body (Fig. 14). The intercellular surface of the bile ductule cells generally is smooth, having only a few short cytoplasmic projections similar to those found on the hepatocyte intercellular surface.
Discussion
The present investigation has not only provided new ultrastructural information on goldfish hepatic cells but also has extended our knowledge of some previously described hepatic fine structures. In particular, the morphology of the hepatic acinus, the unique bicellular hepatic plates, the sinusoidal endothelial fenestrations, and the attachment complexes of the hepatocytes and their associated bile ductules are elucidated in greater detail than in earlier studies. These studies demonstrate that the hepatic parenchymal arrangement in goldfish is similar to that in other classes of higher vertebrates, especially the hepatic acinus as described by Rappaport et al. (1973). The hepatic sinusoids, which radiate from the major blood vessels of the hepatic acini and allow the blood to percolate through the parenchyma, are also similar in appearance to those reported in a variety of vertebrate species including mammals. In particular, the endothelial cells, which are highly fenestrated by small pores, are very similar to those observed in small mammals (Grisham et al., 1975; Itoshima et al., 1974; Motta, 1975; Motta and Porter, 1974; Muto, 1975; Nopanitaya and Grisham, 1975). Groups or clusters of pores are arranged in a manner comparable to the "sieve plates" found in the sinusoidal endothelium of rat and mouse (Nopanitaya and Grisham, 1975; Orci et al., 1971 ; Wisse, 1970). Large, solitary fenestrations, which are occasionally seen in rat and mouse endothelial cells and suspected to be artifacts resulting from fixation by perfusion (Ogawa et al., 1973; Wisse, 1970), are not evident in goldfish liver fixed by immersion. Whether the large type of fenestrations can be induced by fixation by perfusion or by other adverse conditions is not known. The presence of cytoplasmic microfilaments in close association with the plasma membrane which forms the endothelial pores has been described in rat by Wisse (1970) who indicated that the diameter of the endothelial pores may be changed as a result of the ability of these actin-like filaments to contract. The absence of a subendothelial basal lamina, which has been noted in most species of vertebrates (Ito, 1973; Wisse, 1970) including goldfish (Yamamoto, 1962, 1965), further suggests that the exchange of materials between the blood and the hepatocytes in goldfish is made freely through the endothelial pores. The bicellular nature of the hepatic plates of the goldfish, as described in the light-microscopic studies of Elias and Bengelsdorf (1952), is clearly demonstrated by SEM. This singular characteristic is very striking in the Osteichthyes (bony fish) (Elias and Bengelsdorf, 1952) and Amphibia (Haar and Hightower, 1976), whereas it is not evident in healthy livers of mammalian species. Bicellular hepatic plates, do occur, however, in mammals suffering from a pathological condition such as
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cirrhosis (Nopanitaya et al., 1976). The functional differences between unicellular and bicellular hepatic plates remain to be investigated. The manner in which the biliary passages associate with the hepatic plate has been described in an earlier T E M study by Yamamoto (1965) who noted the characteristics of the intercellular bile canaliculus and its relationship to the intralobular ductule cells of the biliary system. Our SEM findings not only confirm these earlier observations but also provide a three-dimensional view of the intralobular biliary passages of the goldfish. In goldfish the biliary system within the hepatic plate is formed by short intraceUular bile canaliculi which drain laterally into the lumen of bile ductules and form the intralobular biliary passage, whereas in amphibians and mammals the entire biliary system of the hepatic plates is made up of intercellular canaliculi which form a groove on the surface of two or more adjacent hepatocytes (Compagno and Grisham, 1974; Grisham et al., 1975; Haar and Hightower, 1975; Motta and Fumagalli, 1975; Nopanitaya and Grisham, 1975). It is of interest to consider the morphological differences between the hepatocytes of goldfish and those of other vertebrates that have been studied with SEM, i.e. rat, mouse, rabbit, and man. The hepatocytes of these species are morphologically similar to one another but are significantly different in appearance from the goldfish hepatocytes. All mammalian hepatocytes are sharply angulated and polyhedral in shape (Grisham et al., 1975; Nopanitaya and Grisham, 1975), whereas hepatocytes of goldfish are barrel-shaped. The number of microvilli on the sinusoidal surfaces in mammalian species (20-60 microvilli/~tm 2) (Grisham et al., 1975) is much greater than that observed in goldfish (15-20 microvilli/~tm2), and the surface area containing microvilli is much more extensive on mammalian cells. A meaningful explanation of these species differences should be sought. It is now clear that goldfish hepatocytes possess intercellular attachments which were not reported in earlier studies. In the present investigation, desmosomes and gap junctions are observed between adjacent liver cells. Our observations also suggest that, in goldfish liver, cell-to-cell communication takes place through these juntional complexes, particularly through the gap junctions, which are similar to those of various epithelial cells in higher vertebrates (Staehelin and Hull, 1978). References
Compagno,J., Grisham,J.W.: Scanningelectronmicroscopyof extrahepaticbiliaryobstruction.Arch. Path. 97, 348-351 (1974) Elias, H., Bengelsdorf,H.: The structureof the liverof vertebrates.ActaAnat. (Basel)15, 297-337(1952) Grisham, J.W., Nopanitaya, W., Compaguo, J., Nagel, A.E.H.: Scanning electron microscopy of normal rat liver: The surfacestructure of its cellsand tissuecomponents.Am. J. Anat. 144,295-322 (1975) Haar, J.L., Hightower,J.A.: A light and electronmicroscopicinvestigationof the hepaticparenchymaof the adult newt, Notophthalmus viridescens. Anat. Rec. 185, 313-324 (1976) Ito, T.: Recentadvancesin the study on the finestructureof the hepaticsinusoidalwall; a review.Gunna Rep. Med. Sci. 6, 119-163 (1973) Itoshima, T., Kobayashi,T., Shimada,Y., Murakami,T.: Fenestratedendotheliumof the liversinusoids of the guinea pig as revealedby scanning electron microscopy.Arch. Histol. Jpn. 37, 15-24 (1974) Moor, H.: Use of freeze-etchingin the study of biologicalultrastructure. Int. Rev. Exp. Pathol. 5, 179216 (1966)
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Motta, P.: A scanning electron microscopy study of the rat liver sinusoid endothelial and Kupffer cells. Cell Tissue Res. 164, 371-385 (1975) Motta, P., Fumagalli, G.: Structure of rat bile canaliculi as revealed by scanning electron microscopy. Anat. Rec. 182, 499-531 (1975) Motta, P., Porter, K.R.: Structure of rat liver sinusoids and associated tissue spaces as revealed by scanning electron microscopy. Cell Tissue Res. 148, 111-125 (1974) Muto, M.: A scanning electron microscopic study on endothelial cells and Kupffer cells in rat liver sinusoids. Arch. Histol. Jpn. 37, 369-386 (1975) Nopanitaya, W., Grisham, J.W. : Scanning electron microscopy of mouse intrahepatic structures. Exp. Mol. Pathol. 23, 441-458 (1975) Nopanitaya, W., Grisham, J,W., Carson, J.L., Dotson, M.M.: Surface features of cirrhotic liver. Virchows Arch. [Pathol. Anat.] 372, 97-108 (1976) Ogawa, K.T., Minase, T., Enomoto, K., Onoe, T. : Ultrastructure of fenestrated cells in the sinusoidal wall of rat liver after perfusion fixation. Tohoku J. Exp. Med. 110, 89-101 (1973) Orci, L.A., Matter, A., Rouiller, C.: A comparative study of freeze-etch replicas and thin sections of rat liver. J. Ultrastruct. Res. 35, 1-19 (1971) Rappaport, A.M.: The microcirculatory hepatic unit. Microvasc. Res. 6, 212-228 (1973) Staehelin, L.A., Hull, B.E. :Junctions between living cells. Sci. Am. 238, 140-153 (1978) Wisse, E.: An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids. J. Ultrastruct. Res. 31, 125-150 (1970) Yamamoto, T. : Some observations on the fine structure of the terminal biliary passages in the goldfish liver. Anat. Rec. 142, 293 (1962) Yamamoto, T.: Some observations on the fine structure of the intrahepatic biliary passages in goldfish (Carassius auratus). Z. Zellforsch. 65, 319-330 (1965) Accepted November 5, 1978
Cell TissueRes. 196, 249-261 (1979)
9 by Springer-Verlag 1979
New Observations on the Fine Structure of the Liver in Goldfish (Carassius auratus) * Waykin Nopanitaya, Johnny L. Carson, Joe W. Grisham and John G. Aghajanian Department of Pathology,Schoolof Medicine,Universityof North Carolinaat ChapelHill, Chapel Hill, North Carolina, USA
Summary. A re-examination of goldfish liver was made through the use of SEM of fractured samples and TEM of ultrathin-sections and freeze-etch replicas. Several new hepatic fine structures described in the present study are morphologically similar to those reported previously in many higher vertebrates including mammals. Hepatic sinusoids of goldfish contain fenestrations which are arranged into sieve plates. Although the hepatic plates are made up of two layers of hepatocytes, the parenchymal cells of goldfish liver are morphologically similar to mammalian hepatocytes, particularly with respect to the sinusoidal surfaces which are studded with numerous microvilli. The intercellular surfaces of hepatocytes have both nexus and desmosomal junctions, similar to those found in various epithelial cells of higher vertebrates, as cell attachments and communication loci. Tight junctions are found mainly between the openings of the intracellular bile canaliculi and the intralobular bile ductules which are situated in the center of the bicellular hepatic plate. Key words: Liver - Goldfish - Ultrastructure. In their comparative study of the anatomy of liver in vertebrates, Elias and Bengelsdorf (1952) pointed out that investigations on the morphology of the liver of goldfish would be of interest from a phylogenetic aspect. Their light-microscopic studies showed that the liver of this species is composed mainly of hepatic plates that are two ceils in thickness while the hepatic plates of several other non-fish species were primarily one-cell thick. David (1961) and Yamamoto (1962, 1965), the first investigators to examine the fine structure of goldfish liver, directed their attention mainly to the unique intercellular and intracellular biliary passages and some fine structural features of the hepatocytes. These early ultrastructural findings are of interest when compared to intrahepatic features of vertebrates. Several aspects of hepatic fine structure, however, remain to be investigated fully. In particular, the ultrastructural details of various other hepatic cells remain to be demonstrated. This report, through the use of SEM and TEM with the application Dr. WaykinNopanitaya,Departmentof Pathology,School of Medicine, Universityof North Carolina at Chapel Hill, Chapel Hill, N.C. 27514, USA * Supported in part by Grants ~/ GM92and ES07017
Send offprint requests to:
0302-766X/79/0196/0249/$02.60
250
w. Nopanitaya et al.
o f b o t h c o n v e n t i o n a l u l t r a t h i n sectioning a n d freeze-etch techniques, elucidates some i n t r a h e p a t i c fine structures o f the goldfish t h a t have n o t been e x a m i n e d previously by electron m i c r o s c o p y .
Materials and Methods Goldfish (Carassiusauratus),measuring 6-8 cm in total length, were killed by decapitation immediately after being taken from the aquarium. All internal organs were quickly removed and rinsed in physiological saline prior to immersion in fixative in which the hepatic tissues were carefully dissected out under the dissecting microscope. Tissues for TEM and SEM studies were fixed in either 4% paraformaldehyde or 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 2-4h at 4~C, followed by post-fixation in 2 % OsO4 in 0.1 M phosphate buffer (pH 7.3) for 1 h at room temperature, and then processed further for TEM and SEM by conventional methods. The critical-point dried SEM samples were manually fractured prior to mounting and Au-Pd coating. Tissues used for obtaining freeze-etched replicas were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 2-4 h at 4~C. They were cut into small pieces (1.5 x 1.5 x 3 mm), immersed in a solution of 25 % glycerol in 0.9 % NaC1 for 2 h at 4~ after which they were rapidly frozen, and transferred to Balzers' apparatus in which they were freeze-etched and C-Pt replicated according to the method described by Moor (1966). Ultrathin sections and freeze-etch replicas were examined with a Zeiss EM 10A TEM equipped with a goniometer stage, using an accelerating voltage of 80 KV. The samples for SEM were viewed and photographed with an ETEC U2 Autoscan operating at 20 KV.
Results Since some features o f the fine structure o f the h e p a t o c y t e s a n d the biliary passages in goldfish have previously been d o c u m e n t e d ( Y a m a m o t o , 1965), the descriptive m o r p h o l o g y detailed in the present investigation pertains specifically to new u l t r a s t r u c t u r a l findings.
Hepatic Parenchymal Arrangement and Segmentation The a r r a n g e m e n t o f the h e p a t i c p a r e n c h y m a l cells in goldfish differs f r o m t h a t o f m a m m a l i a n h e p a t i c lobules in the absence o f connective-tissue septae t h a t delineate the h e p a t i c cell mass. A t low m a g n i f i c a t i o n , S E M o f a f r a c t u r e d s a m p l e discloses an a p p a r e n t p a r e n c h y m a l s e g m e n t a t i o n resulting f r o m the i n t e r d i g i t a t i o n o f h e p a t i c b l o o d vessels (Fig. 1). S E M studies in c o n j u n c t i o n with the light m i c r o s c o p e reveal t h a t the p o r t a l vessels a n d the h e p a t i c veins p e n e t r a t e p e r p e n d i c u l a r l y into the m a s s o f p a r e n c h y m a l cells, dividing it a n d causing it to resemble a n " h e p a t i c acinus".
Hepatic Sinusoids and Sinusoidal Endothelial Cells H e p a t i c sinusoids are tube-like channels averaging f r o m 5 to 12 ~tm in diameter. E n d o t h e l i a l cells lining the sinusoids are heavily fenestrated b y p o r e s ranging f r o m 0.051xm to 0.21xm in d i a m e t e r (Figs. 2 4 ) in g r o u p s o r clusters o f f r o m 15 to 35
Fig. 1. A low magnification SEM micrograph of fractured hepatic tissue of goldfish. Portal (p) and hepatic (hv) veins randomly penetrate the hepatic cell masses, x 190 Fig. 2. A freeze-etch replica of goldfish sinusoidal endothelial cells. Groups of fenestrae (arrows) are evident on the endothelial cells, x 13,000
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Fig. 3. Endothelial fenestrations are clearly visible under the SEM at high magnification, x 35,500 Fig.4. Endothelial fenestrae, as seen in transversely sectioned endothelium, are surrounded by endothelial cytoplasm containing numerous microfilaments. • 20,500
Fig. 5. Both filament-rich cell (Fc) and Ito cell (arrow) containing lipid inclusion (L) are found in the perisinusoidal space of Disse in goldfish, x 15,300 Fig. 6. A bicellular hepatic plate contains a bile ductule (BD) which is located between the hepatocytes (h~ and h2) forming the hepatic plate, x 7000
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Fig.7. The hepatocyte surface facing the sinusoid contains numerous microvilli(My), whereas the smooth intercellular surface (Is) is studded by only a few cytoplasmicprojections (arrow heads). • 22,000
(Figs. 2, 3). TEM of thin sections and SEM of the fenestrations show no evidence of a membraneous diaphragm. The cytoplasm bordering the pores contain numerous fine filaments that are closely associated with the plasma membrane which surrounds the endothelial pores (Fig. 4). Subendothelial basal lamina and perisinusoidal collagen fibers are not evident in the sinusoids. A careful examination of the perisinusoidal spaces of Disse reveals two distinct types of perisinusoidal cells. One type is very abundant and possesses numerous cytoplasmic microfilaments; the other is found less frequently and contains prominent, large lipid inclusions in the cytoplasm (Fig. 5).
Hepatic Plates and Hepatocytes Hepatic plates in goldfish are formed by two closely apposed layers of hepatocytes (Fig. 6) which are bordered laterally on each side by sinusoids. The cells of the hepatic plates are barrel-shaped with the largest diameter being at the level of the nucleus and the smallest diameters being at the tapered ends of the cell toward the sinusoid and toward the center of the plate.
Fig.8. A gap junction, as revealed by TEM of a thin section, is commonly found between adjoining hepatocytes, x 139,400 Fig. 9. A replica of the hepatocyte surface. It contains both small and large gap junctions (arrow heads). A pinocytotic vesicle (arrow) is also visible in this micrograph. • 68,800 Fig. 10. A desmosomal junction is occasionally seen between adjacent hepatocytes, x 101,100
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SEM observations of the three-dimensional features of the hepatocytes reveal that each cell has at least two distinct surfaces: a sinusoidal surface and an intercellular surface (Fig. 7). The sinusoidal surface, the surface of hepatocytes bordering the sinusoidal endothelium, is covered with numerous microviUi (15-20 microvilli/lxm 2) which project into the space of Disse. The microvilli are relatively uniform in size (ca. 0.1 ~tm wide by 0.6 Ixm long) and are randomly oriented. The intercellular surface of the hepatocyte is smooth and possesses a few short cytoplasmic projections measuring 0.1 ~tm wide by 0.3 Ixm long (Fig. 7). TEM observations of ultrathin sections and freeze-etch replicas reveal additional features of the intercellular surface of the goldfish hepatocytes. These include pinocytotic invaginations and the loci of intercellular attachment and communication (Figs. 8, 9). Cell-to-cell attachment may be either by a gap junction (nexus) (Figs. 8, 9) or a desmosomal junction (Fig. 10), the former being more numerous than the latter. The appearance of the gap junction of goldfish hepatocytes is similar to the typical gap junction seen in various cells of vertebrates (Staehelin and Hull, 1978). In freeze-etch replicas, the gap junction appears as a plaque-like structure which varies considerably in size. Small gap junction plaques, which are circular in outline and measure 0.08-0.1 ~tm in diameter, and large irregularly shaped gap junctions, ranging from 0.5-1.0 ~tm wide by 1.0-2.0 ~tm long, are randomly distributed on the intercellular surface of the hepatocytes (Fig. 9). The ultrastructure of the organelles of goldfish hepatocytes, as seen with the TEM, is similar to that of higher vertebrates. Glycogen particles in rosetteconfigurations are abundant (Figs. 12, 13) and are the most prominent feature of the hepatocytes. These cells contain a single, spherical nucleus, the bounding membrane of which contains randomly distributed nuclear pores which are about 90 nm in diameter (Fig. 11). The Golgi complex of each hepatocyte is consistently found in close proximity to the "intercellular bile canaliculus" described by Yamamoto (1965) (Fig. 12).
Intrahepatic Plate Biliary Pathway In each hepatocyte, the intracellular bile canaliculus opens into the lumen of the adjacent terminal ductule cells through the hepatocyte surface. This results in a direct lumenal communication between the hepatocyte and the biliary duct ceils. At this transition zone, the terminal ductule cell openings are in contact with one another at tight junctions and from a common biliary passage (Fig. 12). The lumen of an individual ductule cell is formed by infoldings of the plasma membrane which are sealed by tight junctions (Fig. 13). SEM of a favorably fractured sample clearly elucidates the three-dimensional relationship between the hepatocytes and the biliary ductule cells in hepatic plates (Figs. 6, 14). In particular, the extracellular openings of the hepatocyte intracellular bile canaliculus and of the biliary ductule cells are easily recognizable (Fig. 14). They are randomly located on the lateral surface of their respective cell type. The bile canalicular opening of the hepatocyte is cup-shaped, 1 gm in diameter, with its luminal surface covered by microvilli averaging 0.08 gm wide by 0.6gm long (Figs. 12, 13). This structure represents a mold of the similar cup-shaped openings
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Fig. 11. The nucleus of this goldfish hepatocyte has several nuclear pores, x 25,600 Fig. 12. A TEM micrograph illustrating the common biliary pathway in goldfish. An intracellular bile canaliculus of the hepatocyte (BC) opens directly into the lumen of the bile ductule cell (BDC). The hepatocyte is held to its adjacent bile ductule cell by tight junctions. Numerous glycogen particles are seen throughout the hepatocyte cytoplasm. G Golgi complex, x 25,600
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Fig. 13. A TEM micrograph demonstrating tight junctions (arrows) between the glycogen-rich hepatocyte (/-/) and the terminal bile ductule formed by a single filament-rich bile ductule cell (BDC). Also note that the cytoplasmic portions of this bile ductule cell (BDC) are joined together by tight junctions (arrow heads) thereby forming a ductular lumen (L). x 19,400 Fig. 14. A SEM micrograph showing the biliary openings (arrow heads) along the lateral cytoplasm of the bile ductule cell (BDC) and the hepatocyte (H). x 7900
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seen laterally on the ductule cell surface or at the terminal branch of the biliary ductule. The ductule cells forming the biliary passages are spindle-shaped and have thin, elongated cytoplasmic processes which extend from the bulging cell body (Fig. 14). The intercellular surface of the bile ductule cells generally is smooth, having only a few short cytoplasmic projections similar to those found on the hepatocyte intercellular surface.
Discussion
The present investigation has not only provided new ultrastructural information on goldfish hepatic cells but also has extended our knowledge of some previously described hepatic fine structures. In particular, the morphology of the hepatic acinus, the unique bicellular hepatic plates, the sinusoidal endothelial fenestrations, and the attachment complexes of the hepatocytes and their associated bile ductules are elucidated in greater detail than in earlier studies. These studies demonstrate that the hepatic parenchymal arrangement in goldfish is similar to that in other classes of higher vertebrates, especially the hepatic acinus as described by Rappaport et al. (1973). The hepatic sinusoids, which radiate from the major blood vessels of the hepatic acini and allow the blood to percolate through the parenchyma, are also similar in appearance to those reported in a variety of vertebrate species including mammals. In particular, the endothelial cells, which are highly fenestrated by small pores, are very similar to those observed in small mammals (Grisham et al., 1975; Itoshima et al., 1974; Motta, 1975; Motta and Porter, 1974; Muto, 1975; Nopanitaya and Grisham, 1975). Groups or clusters of pores are arranged in a manner comparable to the "sieve plates" found in the sinusoidal endothelium of rat and mouse (Nopanitaya and Grisham, 1975; Orci et al., 1971 ; Wisse, 1970). Large, solitary fenestrations, which are occasionally seen in rat and mouse endothelial cells and suspected to be artifacts resulting from fixation by perfusion (Ogawa et al., 1973; Wisse, 1970), are not evident in goldfish liver fixed by immersion. Whether the large type of fenestrations can be induced by fixation by perfusion or by other adverse conditions is not known. The presence of cytoplasmic microfilaments in close association with the plasma membrane which forms the endothelial pores has been described in rat by Wisse (1970) who indicated that the diameter of the endothelial pores may be changed as a result of the ability of these actin-like filaments to contract. The absence of a subendothelial basal lamina, which has been noted in most species of vertebrates (Ito, 1973; Wisse, 1970) including goldfish (Yamamoto, 1962, 1965), further suggests that the exchange of materials between the blood and the hepatocytes in goldfish is made freely through the endothelial pores. The bicellular nature of the hepatic plates of the goldfish, as described in the light-microscopic studies of Elias and Bengelsdorf (1952), is clearly demonstrated by SEM. This singular characteristic is very striking in the Osteichthyes (bony fish) (Elias and Bengelsdorf, 1952) and Amphibia (Haar and Hightower, 1976), whereas it is not evident in healthy livers of mammalian species. Bicellular hepatic plates, do occur, however, in mammals suffering from a pathological condition such as
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cirrhosis (Nopanitaya et al., 1976). The functional differences between unicellular and bicellular hepatic plates remain to be investigated. The manner in which the biliary passages associate with the hepatic plate has been described in an earlier T E M study by Yamamoto (1965) who noted the characteristics of the intercellular bile canaliculus and its relationship to the intralobular ductule cells of the biliary system. Our SEM findings not only confirm these earlier observations but also provide a three-dimensional view of the intralobular biliary passages of the goldfish. In goldfish the biliary system within the hepatic plate is formed by short intraceUular bile canaliculi which drain laterally into the lumen of bile ductules and form the intralobular biliary passage, whereas in amphibians and mammals the entire biliary system of the hepatic plates is made up of intercellular canaliculi which form a groove on the surface of two or more adjacent hepatocytes (Compagno and Grisham, 1974; Grisham et al., 1975; Haar and Hightower, 1975; Motta and Fumagalli, 1975; Nopanitaya and Grisham, 1975). It is of interest to consider the morphological differences between the hepatocytes of goldfish and those of other vertebrates that have been studied with SEM, i.e. rat, mouse, rabbit, and man. The hepatocytes of these species are morphologically similar to one another but are significantly different in appearance from the goldfish hepatocytes. All mammalian hepatocytes are sharply angulated and polyhedral in shape (Grisham et al., 1975; Nopanitaya and Grisham, 1975), whereas hepatocytes of goldfish are barrel-shaped. The number of microvilli on the sinusoidal surfaces in mammalian species (20-60 microvilli/~tm 2) (Grisham et al., 1975) is much greater than that observed in goldfish (15-20 microvilli/~tm2), and the surface area containing microvilli is much more extensive on mammalian cells. A meaningful explanation of these species differences should be sought. It is now clear that goldfish hepatocytes possess intercellular attachments which were not reported in earlier studies. In the present investigation, desmosomes and gap junctions are observed between adjacent liver cells. Our observations also suggest that, in goldfish liver, cell-to-cell communication takes place through these juntional complexes, particularly through the gap junctions, which are similar to those of various epithelial cells in higher vertebrates (Staehelin and Hull, 1978). References
Compagno,J., Grisham,J.W.: Scanningelectronmicroscopyof extrahepaticbiliaryobstruction.Arch. Path. 97, 348-351 (1974) Elias, H., Bengelsdorf,H.: The structureof the liverof vertebrates.ActaAnat. (Basel)15, 297-337(1952) Grisham, J.W., Nopanitaya, W., Compaguo, J., Nagel, A.E.H.: Scanning electron microscopy of normal rat liver: The surfacestructure of its cellsand tissuecomponents.Am. J. Anat. 144,295-322 (1975) Haar, J.L., Hightower,J.A.: A light and electronmicroscopicinvestigationof the hepaticparenchymaof the adult newt, Notophthalmus viridescens. Anat. Rec. 185, 313-324 (1976) Ito, T.: Recentadvancesin the study on the finestructureof the hepaticsinusoidalwall; a review.Gunna Rep. Med. Sci. 6, 119-163 (1973) Itoshima, T., Kobayashi,T., Shimada,Y., Murakami,T.: Fenestratedendotheliumof the liversinusoids of the guinea pig as revealedby scanning electron microscopy.Arch. Histol. Jpn. 37, 15-24 (1974) Moor, H.: Use of freeze-etchingin the study of biologicalultrastructure. Int. Rev. Exp. Pathol. 5, 179216 (1966)
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Motta, P.: A scanning electron microscopy study of the rat liver sinusoid endothelial and Kupffer cells. Cell Tissue Res. 164, 371-385 (1975) Motta, P., Fumagalli, G.: Structure of rat bile canaliculi as revealed by scanning electron microscopy. Anat. Rec. 182, 499-531 (1975) Motta, P., Porter, K.R.: Structure of rat liver sinusoids and associated tissue spaces as revealed by scanning electron microscopy. Cell Tissue Res. 148, 111-125 (1974) Muto, M.: A scanning electron microscopic study on endothelial cells and Kupffer cells in rat liver sinusoids. Arch. Histol. Jpn. 37, 369-386 (1975) Nopanitaya, W., Grisham, J.W. : Scanning electron microscopy of mouse intrahepatic structures. Exp. Mol. Pathol. 23, 441-458 (1975) Nopanitaya, W., Grisham, J,W., Carson, J.L., Dotson, M.M.: Surface features of cirrhotic liver. Virchows Arch. [Pathol. Anat.] 372, 97-108 (1976) Ogawa, K.T., Minase, T., Enomoto, K., Onoe, T. : Ultrastructure of fenestrated cells in the sinusoidal wall of rat liver after perfusion fixation. Tohoku J. Exp. Med. 110, 89-101 (1973) Orci, L.A., Matter, A., Rouiller, C.: A comparative study of freeze-etch replicas and thin sections of rat liver. J. Ultrastruct. Res. 35, 1-19 (1971) Rappaport, A.M.: The microcirculatory hepatic unit. Microvasc. Res. 6, 212-228 (1973) Staehelin, L.A., Hull, B.E. :Junctions between living cells. Sci. Am. 238, 140-153 (1978) Wisse, E.: An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids. J. Ultrastruct. Res. 31, 125-150 (1970) Yamamoto, T. : Some observations on the fine structure of the terminal biliary passages in the goldfish liver. Anat. Rec. 142, 293 (1962) Yamamoto, T.: Some observations on the fine structure of the intrahepatic biliary passages in goldfish (Carassius auratus). Z. Zellforsch. 65, 319-330 (1965) Accepted November 5, 1978
