Fine structure of the liver in the larval lamprey, Petromyzon marinus

J. Anat. (1980), 131, 3, pp. 501-519 499With 16 figuresPrinted in Great Britain

Fine structure of the liver in the larval lamprey, Petromyzonmarinus L.; bile ducts and gall bladder*

E. W. SIDON,t W. D. PEEKt, J. H. YOUSONtAND M. M. FISHERt

tDepartment of Zoology, Scarborough College, University of Toronto,West Hill, Ontario MlC 1A4 Canada and,

tDepartment ofPathology, University of Toronto, Toronto, Ontario M5S 1L5 Canada

(Accepted 11 March 1980)

INTRODUCTION

The liver of the lamprey is unique because, although it contains a system of bileducts and an intrahepatic gall bladder during the larval stage of the life cycle, thebiliary system is no longer present in the liver of adults (Bertolini, 1965; Sterling,Meranze, Winsten & Krieger, 1967; De Vos, Wolf-Peeters & Desmet, 1973; Shin,1977). Light microscopic observations have shown that the events leading to thedegeneration of the entire apparatus for bile transport and storage during meta-morphosis in lampreys closely resemble those of human biliary atresia (Youson &Sidon, 1978). We wish to document this process by electron microscopy, but therehas been no description of ultrastructural characteristics of bile ducts and the gallbladder, prior to the onset of degeneration, in any species of lamprey. The purposeof the present investigation is to describe these structures in the liver of larvae of thesea lamprey, Petromyzon marinus, and to compare them with those of othervertebrates. A study of the hepatocytes of this species was recently conducted (Peek,Sidon, Youson & Fisher, 1979).

MATERIALS AND METHODS

Larval sea lampreys, Petromyzon marinus, were collected, maintained in thelaboratory, and anaesthetized prior to fixation procedures as previously described(Ooi & Youson, 1977; Peek et al. 1979). For routine electron microscopy, liverswere chopped into small blocks and fixed in a solution of ice-cold (4 °C) 2%glutaraldehyde in phosphate buffer (Millonig, 1961) at pH 7-3 for 2 hours. Thetissues were then rinsed in buffer and post-fixed for 2 hours in 1% OS04 in thesame buffer. The tissues were then dehydrated in ethanols to propylene oxide andembedded in Epon-Araldite (Mollenhauer, 1964). Silver-gold sections were cuton glass knives using a Reichert OMU-2 ultramicrotome, mounted on uncoatedcopper grids, and double stained with saturated uranyl acetate (Watson, 1958) andlead citrate (Reynolds, 1963). All grids were examined using a Zeiss EM-9S electronmicroscope.For freeze-fracture, livers were chopped into small blocks and fixed for 1 hour in a

solution of ice-cold (4 °C) 2 % glutaraldehyde in 0-1 M sodium cacodylate at pH 7T3.Following fixation, tissues were rinsed in buffer, soaked in 30 % glycerol in 0OI M

* Reprint requests to Dr J. H. Youson.

0021-8782/80/2828-8270 $02.00 © 1980 Anat. Soc. G.B. & I

500 E. W. SIDON AND OTHERS

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Fig. 1. The wall of the gall bladder (Gb) is composed of columnar epithelial cells (E), smoothmuscle fibres (S), and connective tissue (CT) while bile ducts (BD) are lined with cuboidal cells.Bile canaliculi (B) are surrounded by hepatocytes which are adjacent to hepatic sinusoids (U).x 315.

sodium cacodylate (pH 7 3) overnight in the refrigerator and fractured in a Balzer'sfreeze-etch unit (Balzer's, Liechtenstein) according to the method of Shivers &Brightman (1976). Replicas were mounted on uncoated copper grids and examinedin a Siemens 102 electron microscope operating at 80 kV.

RESULTS

The general structure of the liver of larval P. marinus has been described (Peeket al. 1979) as a true tubular gland, its tubular nature being derived from thearrangement of hepatocytes about bile canaliculi. These were drained by bile ducts,which were ramified throughout the hepatic parenchyma and finally united to forma single, large common bile duct. The common bile duct exited from the liverdorsally, and joined with the anterior intestine. The gall bladder was intrahepatic(Fig. 1).

Fig. 2. Cells of a bile duct lie flat on a thin basal lamina (BL) and are separated by wide butirregular intercellular spaces (ICS). Numerous apical microvilli (Mv) extend from the apicalsurface into the lumen (L) while smaller numbers (arrows) protrude into the lateral intercellularspaces. The cytoplasm contains numerous mitochondria (M), a Golgi complex (G), and a fewsecondary lysosomes (Ly). Intracytoplasmic cistemae (C) appear as vacuolar structures in thelateral, peripheral cytoplasm. The apparent presence of cistemae in the apical cytoplasm is dueto the plane of section. N, nucleus. x 6300.

Fig. 3. A cross section of a cell from a bile duct demonstrates the complex system of peripheralcisternae (C). Cistemae appear as vacuoles and may show continuity (arrow) with the inter-cellular space (ICS). The irregular lateral surface of the cell bears microvilli (Mv) which projectinto the intercellular space. The cytoplasm contains numerous mitochondria (M), a large Golgicomplex (G), and a few single cistemae of rough endoplasmic reticulum (R). x 15000.

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Bile ductsThe lumina of the bile ducts were of variable diameter, ranging from 4 0 ,tm where

they drained bile canaliculi to 35 0 ,um at their point of junction with the commonbile duct. They were surrounded by a thin layer of fibrous connective tissue and,frequently, were closely associated with sinusoids. The cells of the bile ducts formeda simple cuboidal to low columnar epithelium, 2-0-13-5 ,um in height. Aggregationsof cilia projected into the lumen of large branches of the common bile duct, but wereabsent in smaller ducts. In other respects the main ultrastructural features of allbile duct cells were similar, regardless of the size of the duct.The apical surface of cells of the bile ducts bore large numbers of long (1 6,um),

densely packed microvilli (Fig. 2) which projected into the lumina. The microvilliextended from a layer of filamentous ectoplasm and usually contained a core ofmicroffilaments arranged parallel to their long axes. Wide intercellular spacesseparated the cells of the bile ducts along their lateral margins (Figs. 2-5). Thesespaces were up to 2-22tm in width and of similar dimension in both thin sectionedand freeze-fractured material, indicating that their size is not likely to be a result ofsome aspect of routine tissue preparation such as dehydration. The lateral inter-cellular spaces were usually closed basally (Fig. 2). The lateral margins of the cellswere irregular in outline (Figs. 2, 3) and bore moderate numbers of microvilli whichprojected up to 0-8 ,tm into the intercellular space (Figs. 2-5).The most prominent surface specialization of the bile duct cells was revealed, in

its true nature, only through the use of freeze-fracture. Thin sections of the cellsrevealed large numbers of apparently empty, 'vacuolar' or 'vesicular' structures inthe peripheral cytoplasm (Figs. 2, 3). These structures were round to elongate andfrequently of irregular outline, showing constricted and dilated regions. Rarely,they appeared to be confluent with the lateral intercellular space. Freeze-fracture,however, revealed that these 'vacuoles' and/or 'vesicles' actually represented profilesof sectioned 'cisternae' which formed a complex, peripheral network within thecells (Figs. 4, 5). These cisternae, as had been suggested in thin section, were irregularin shape, with tubular and broad, saccular regions appearing with approximatelyequal frequency. Adjacent saccular areas were often joined by highly constrictedsegments. In addition to revealing the true cisternal nature of the peripheral'vacuoles' and 'vesicles', freeze-fracture showed that these structures represented atrue surface specialization. The lateral plasma membranes of the cells of the bileducts were perforated by numerous pores, approximately 90 0 nm in diameter andrandomly distributed over the lateral surface (Fig. 5). Freeze-fracture replicas, inwhich the plane of fracture crossed through the cytoplasm immediately beforeentering the lateral plasma membrane, revealed that the lateral pores actuallyrepresented openings to the surface of the otherwise intracytoplasmic cisternae

Fig. 4. A replica of a freeze-fractured bile duct shows portions of two cells separated by a widelateral intercellular space (ICS) and containing a cytoplasmic projection (PR). The nuclearenvelope is perforated by nuclear pores (NP) and an elongate mitochondrion (M) is shown.Peripherally, intracytoplasmic cisternae (C) form an anastomosing (arrows) network. The areawithin the enclosure is enlarged in the inset. The large arrowheads in the lower right corners ofthese and subsequent freeze-fracture micrographs indicate the direction of platinum shadowing.Mv, apical microvilli. x 35000. Inset. Intracytoplasmic cisterna (C) opens into the intercellularspace (ICS) via pores (P) in the lateral plasma membrane (LM). PR, cytoplasmic projection.x 66000.

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Fine structure of lamprey liver(Figs. 4, 5, insets). Although the pores were numerous, their numbers were pro-portionately low when compared with the volume of cytoplasm occupied by thecisternae and their relative surface area. There did not appear to be any intra-membranous specialization or surface coat unique to the cisternal membranes.

Infoldings and microvilli were absent from the basal surfaces of the cells of thebile ducts, which lay flat on a thin basal lamina (Fig. 2). Occasionally, pinocytoticvesicles were present in close association with the basal plasma membrane. Intra-cytoplasmic cisternae were not observed to open to basal surfaces.The cells of the bile ducts were joined, apically, by junctional complexes consisting

of well-developed zonulae occludentes surmounting zonulae adhaerentes. Laterally,small desmosomes were common but communicating (gap) junctions were notobserved. The zonulae occludentes, as observed in freeze-fracture replicas, weresomewhat variable in their apicobasal depth, ranging from 90 0 to 200-0 nm. Theyappeared as a series of highly interconnected ridges on P-face replicas and assimilarly arranged grooves on E-face replicas. The degree of interconnection ofridges or grooves was such that a honeycomb-like meshwork was generated (Figs.5-7). There was a degree of variability in the relative continuity of the P-face ridgescomposing the zonulae occludentes. Areas in which long, continuous ridges werepresent alternated with regions in which the ridges were composed of rows ofparticles and bars separated by narrow gaps (Fig. 6). Below the zonula occludens,the zonula adhaerens was marked, in freeze-fracture, by the presence of varyingnumbers of 20-0 nm particles on E-face replicas (Fig. 7). There was no apparentdifferentiation marking the junction on P-face replicas.The cytoplasmic matrix was of uniform electron density and was differentiated

into a thin, but well-defined, filamentous ectoplasm and a central, organelle-containing endoplasm. Nuclei were large, spherical to ovoid, and positioned nearthe basal surface of duct cells. The nuclear envelope was perforated by moderatenumbers of nuclear pores (Fig. 4) and its cytoplasmic surface was studded withribosomes. Mitochondria were abundant and in thin sectioned tissue (Figs. 2, 3,8-10) they ranged from round to elongate in profile and in freeze-fracture they werefrequently seen with their long axes parallel to the apicobasal plane of the cells(Fig. 4). The mitochondrial matrix was similar in electron density to that of thecytoplasmic matrix and contained varying numbers of intramitochondrial granules.Cristae were abundant and plate-like, frequently spanning the entire width of themitochondria. The moderately large Golgi complex(Figs. 3, 9) ofeach cell was usuallyin the form of two or more stacks of narrow saccules surrounded by numerousvesicles and was generally located in the supranuclear cytoplasm. Rough endoplasmicreticulum was sparsely distributed and single cistemae of rough endoplasmicreticulum frequently appeared to separate the apical ectoplasm from the endoplasmbeneath. Clusters of irregularly shaped, secondary lysosomes appeared in most bileduct cells, usually in the supranuclear cytoplasm (Fig. 10). These containedamorphous and particulate matter of variable electron density. The cytoplasm also

Fig. 5. A P-face replica from a freeze-fractured bile duct shows that the lateral plasma membraneis perforated by numerous pores (P) and bears the bases of numerous microvilli (arrows) whichhave been broken away during fracturing. An apical zonula occludens (ZO)forms a band acrossthe membrane face. The apical surface of the cell bears numerous microvilli (Mv) and inter-cellular spaces (ICS) are wide. x 35000. Inset. A saccular, intracytoplasmic cisterna (C) opensto the intercellular space (ICS) via a pore (P). Note the branched nature of the cisterna (arrows).x 99000.

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Fine structure of lamprey livercontained large numbers of microfilaments, frequently arranged in bundles (Fig. 8),numerous microtubules, and scattered deposits of glycogen in the form of 20-0 nmparticles.

Gall bladderThe gall bladder was intrahepatic and was located anteriorly. It was linked to the

common bile duct by a short cystic duct. A thick layer of fibrous connective tissuesurrounded the gall bladder which was generally up to 205-0 1am in diameter (Fig. 1).Blood vessels and smooth muscle fibres were scattered throughout the connectivetissue (Fig. 1). The simple epithelium was composed of columnar cells (Fig. 11),about 19 ,um in height, lying on a thin basal lamina. Numerous microvilli up to1-3 ,am in length extended from the apical surface into the lumen. They usuallycontained a core of microfilaments which arose from an unusually thick layer offilamentous apical ectoplasm. This layer, which was entirely devoid of organelles,was clearly visible in both thin sectioned (Figs. 11, 12) and freeze-fractured (Fig. 13)tissue. It was relatively uniform in thickness and was continuous with a well defined,though thinner, layer of lateral (Fig. 12) and basal ectoplasm. As in the cells of thebile ducts, the ground cytoplasm was dense and afforded limited contrast in thinsection tissue. Organelles, with the exception of mitochondria, were sparse andconsisted of a moderately large, supranuclear Golgi complex, a few isolated cisternaeof rough endoplasmic reticulum (Fig. 12), scattered tubules of smooth endoplasmicreticulum and a few dense bodies. Mitochondria were numerous, round to elongatein shape, and evenly distributed throughout the cytoplasm. The cytoplasm alsocontained very large pools of glycogen in the form of closely packed arrays of40 0 nm particles (Figs. 11, 12), and numerous microtubules and microfilaments.

In addition to the apical microvilli, the cells bore extensive lateral folds whichextended into the intercellular spaces (Figs. 11, 12), greatly increasing the lateralsurface area. Peripheral cisternae and pores, as observed in the cells of the bile duct,were absent from the cells of the gall bladder. The intercellular spaces were ofvariable width, both in thin sectioned and freeze-fractured preparations, but werenarrower than those observed in bile ducts. They were separated from the lumen ofthe gall bladder by junctional complexes consisting of zonulae occludentes andadhaerentes. In freeze-fracture, the structure of the zonulae occludentes was similarto that seen in the bile ducts. Junctional depth (apicobasal), however, was moreuniform, approximating 160-0 nm. P-face ridges and E-face grooves formed closelymeshed, anastomosing networks (Figs. 14, 15). P-face ridges were usually dis-continuous and formed of rows of bars and particles (Figs. 14, 15). As in the bile

Fig. 6. A portion of a replica of a freeze-fractured bile duct shows the complex meshworkformed by P-face ridges of a zonula occludens (ZO). Towards the right of the micrograph theP-face ridges are composed of short bars and particles separate narrow gaps (arrows). Mv,microvilli. x 54000.Fig. 7. A portion of a replica of a freeze-fractured bile duct shows the complex meshwork ofE-face grooves (EG) and P-face ridges (PR) forming the zonula occludens. The region of thezonula adhaerens (ZA) is marked by the presence of numerous large particles (arrows) on theE-face. x 48 000.Fig. 8. The basal region of a cell from a bile duct lies on a thin basal lamina (BL) and lacksinfolds. The cytoplasm contains numerous mitochondria (M), bundles of microfilaments (Mf),and a thin basal layer of filamentous ectoplasm (Ec). C, intracytoplasmic cisternae; Gl, glycogen;N, nucleus. x 33 000.

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Fine structure of lamprey liverducts, the region of the zonula adhaerens was marked, in freeze-fracture, by thepresence of 9'6 nm E-face particles but this feature was much less constant in thegall bladder. Laterally, the cells were joined by desmosomes. Communicatingjunctions were not observed.

DISCUSSION

Features generally considered to be characteristic of transporting epitheliainclude elaborately folded cell surfaces, well developed cell junctions, and numerousmitochondria. Extensive amplification of cellular surfaces results in the formationof complex, extracellular channels which, in combination with cell junctions, arebelieved to constitute the mechanism for transepithelial transport of fluids (Berridge& Oschman, 1972). One of the most widely accepted models for isosmotic absorptionof fluid is based on mammalian gall bladder (Kaye, Wheeler, Whitlock & Lane, 1966;Tormey & Diamond, 1967). In the present study fine structural observations of thebile ducts and gall bladder of the larval sea lamprey reveal features suggestive ofthis function.

Bile ductsThe bile ducts of most vertebrates are composed of a transporting epithelium

which is involved in secretion and absorption of water and bicarbonate ions(Goldfarb, Singer & Popper, 1963; Wheeler, 1969; Forker, 1977). The apical surfacesof the cells of biliary epithelia bear numerous microvilli, which are reflective of theirabsorptive nature, and the lateral surfaces are plicated due to the presence ofirregular folds and microvillus-like projections (Marinozzi, Muto, Correr & Motta,1977). Hollander & Schaffner (1968) suggested that the ion pumps, which are anecessary component of the fluid-transporting mechanism (Diamond & Tormey,1966), are most probably located along the lateral borders of the cells. Further, ithas been demonstrated that the width of lateral intercellular spaces in bile ducts isvariable, reflecting the rate of transepithelial flow of water and electrolytes (Yamada,1969a).Judged by their structural features, it is apparent that the cells of the bile ducts of

the larval lamprey are probably active in fluid transport. The cellular apices arethickly populated with microvilli, suggesting a high rate of uptake, and intercellularspaces are wide, indicating that flow rates are high (Yamada, 1969a). The functionallength of the intercellular channel is increased, to a moderate degree, by the presenceof lateral microvilli. These intercellular spaces may be more than simply sites of cellattachment and ionic flux. In some organisms (Schaffner & Popper, 1961; Steiner

Fig. 9. A portion of a cell from a bile duct shows a Golgi complex (G), a few mitochondria (M),scattered deposits of glycogen (Gl), a portion of a nucleus (N) and sections of peripheral, intra-cytoplasmic cistemae (C). x 21 300.Fig. 10. The apical cytoplasm from a cell from a bile duct contains numerous secondarylysosomes (Ly), scattered glycogen (GI) deposits and a few single cisternae of rough endoplasmicreticulum (R). x 29200.Fig. 11. The cells of the gall bladder bear numerous apical microvilli (Mv) and lateral folds(LF). They lie on a thin basal lamina (BL) and are separated by complex intercellular spaces(ICS). Below the basal lamina there is a thick layer of fibrous connective tissue (CT). The cells arejoined, apically, by junctional complexes (J) and contain a thick apical layer of filamentousectoplasm (Ec). Nuclei (N), large deposits of glycogen (GI), mitochondria (M), and a few lipiddroplets (LD), from which the lipid has been extracted, are present. x 5000.

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Fine structure of lamprey liver& Carruthers, 1961) they represent regions where biliary canaliculi can drain neigh-bouring hepatocytes directly through the walls of ductules. Interdigitating plicationsbetween ductular cells of hagfishes probably confer a degree of elasticity to thetubular wall (Mugnaini & Harboe, 1967). The characteristic arrangement of lateralcell membranes within ductule cells of larval P. marinus could offer additionalelasticity in a similar manner. This property would represent a functional advantagein the regulation of biliary pressure.The presence of vacuole-like cisternae at the periphery of the cells of the bile ducts

in larval P. marinus represents a distinguishing feature and one which has notpreviously been reported in any transporting epithelium under normal conditions.The cisternae form a complex, three dimensional network of intracytoplasmicchannels (Fig. 16), which are continuous with the lateral intercellular spaces viapores. It must be noted here that the term pore is used in the sense of a surfaceopening leading into a space which is located below the surface. In this respect it isdissimilar, for example, to a nuclear pore, which simply represents a 'window' in thenuclear envelope. The continuity of the cisternae with the intercellular spacesindicates that the cisternal lumina actually represent extracellular space and, hence,the cisternae must be regarded as a surface specialization. In this respect theyresemble the smooth tubules present in the chloride cells of gills of young, adult P.marinus (Peek & Youson, 1979). The relative paucity of pores, when compared withthe volume of cytoplasm occupied by the cisternae, indicates that the cisternallumina, although structurally extracellular, may be regarded as a functionallyseparate compartment from the intercellular space. Perhaps they act as sites ofsequestration of water and electrolytes, thus enhancing the absorptive capacity ofthe epithelium. Future studies, to measure levels of membrane-bound, Na-K-APTase, are required in order to confirm the role of the cisternal membranes inion transport, since this enzyme is usually associated with transfer of electrolytesacross membranes (Kaye et al. 1966; Bonting, 1970). Vacuoles, similar to thecisternae observed in the present study, have been reported in hepatocytes of ratliver perfused with chlorpromazine (Hruban, Tavoloni, Reed & Boyer, 1978). Inthis case, satellite vacuoles communicate with large, irregular vacuoles next tosinusoids but there is no demonstrated continuity with extracellular spaces. Ac-cording to these authors, the formation of intracellular vacuoles may be a con-sequence of an alteration in membrane flow. This causes interference with thenormal process of membrane internalization following fusion of pinocytotic vesicleswith the plasma membrane. Excessive limiting membrane, which is normally removedby membrane internalization, is left instead to form networks of vacuoles. It is alsoconceivable that the cisternae in duct cells of lampreys act as depots for largemolecules or metabolites of epithelial cells in an intercellular drainage system, since

Fig. 12. Portions of cells from the gall bladder exhibit a thick apical layer of filamentousectoplasm (Ec) and thinner lateral layers, which extend into lateral folds (LF). The cells arejoined, apically, by a junctional complex (J), and laterally, by a desmosome (D). The cytoplasmcontains large pools of glycogen (GI), numerous microtubules (Mt), isolated cistemae of roughendoplasmic reticulum (R), and a supranuclear Golgi complex (G). ICS, intercellular space.x 18900.Fig. 13. A portion of freeze-fractured gall bladder shows the thick apical layer of ectoplasm(Ec), which is devoid of organelles. The plane of fracture has exposed a region of P-facemembrane showing a segment of a zonula occludens (ZO). x 64000.

511

E. W. SIDON AND OTHERS512

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Fine structure of lamprey liverintercellular spaces in bile ductules of humans represent outlets for metabolicproducts of the cells (Sternlieb, 1965).

It has been demonstrated that there is a direct relationship between the morphol-ogy of the zonula occludens, as seen in freeze-fracture, and the relative degree ofpermeability of the junction (Claude & Goodenough, 1973). The zonulae occludentesof bile ducts in the present study were of variable depth and complexity withfrequent gaps in the P-face ridges. Such a morphology is consistent with that of a'leaky' epithelium (Claude & Goodenough, 1973) and leads to the suggestion,therefore, that the zonula occludens of lamprey bile ducts forms an incompletebarrier against electrolytes. Undoubtedly some regions of the junction, where depthand complexity are great, are physiologically 'tight', but since relative tightness ofthe junction is the sum of all of the local permeabilities (Claude & Goodenough,1973), it seems logical to suggest that, overall, the junction is 'leaky'. This observationsupports the findings of Fromter & Diamond (1972) which show that the junctionsbetween cells of many epithelia form the main route of passive ion permeation. Inview of the apparent leakiness of the junction, it is attractive to speculate that thecisternae of the bile duct cells may provide a mechanism whereby absorption ofwater and electrolytes from the ductular lumina is accomplished while minimizingbackward diffusion through the zonula occludens.The relative paucity of organelles in the cells of the bile ducts of larval P. marinus

indicates that these cells are less active metabolically and synthetically than theadjacent hepatocytes (Peek et al. 1979). Similar observations have been reported forbiliary ductules of humans (Sternlieb, 1965) and the common bile duct of rats(Yamada, 1969b). In lampreys, particularly significant differences are noted in thesparse quantities of rough and smooth endoplasmic reticulum and ribosomes.Mitochondria are numerous in the duct cells, apparently indicative of energy require-ments of the cells in active transport of ions.

Gall bladderThe gall bladder of larval P. marinus is completely enclosed within the liver and it

differs from the bile ducts by its large size. One distinguishing feature of gall bladdermucosal cells is the appearance of large pools of glycogen in the form of closelypacked arrays of particles. Although large deposits of glycogen can be seen in gallbladders of some mammals, such as monkeys (Rhodin, 1974), this cytoplasmicinclusion is less prominent in other mammalian species (Luciano, 1972; Koga, 1973).

Fig. 14. A replica of freeze-fractured bile duct shows the discontinuous, but highly inter-connected, meshwork formed by P-face ridges of the zonula occludens (ZO). Note gaps in P-faceridges (small arrows) and fragments of E-face attached to P-face (large arrows). x 75000.Fig. 15. A replica of freeze-fractured bile duct shows the highly interconnected meshworkformed by P-face ridges (PR) and E-face grooves (EG) of the zonula occludens. The area of thezonula adhaerens (ZA) is marked by the presence of a few irregular particles (arrows) on theE-face. x 78000.Fig. 16. A diagrammatic representation of a cell from a bile duct is shown. The top of the cellhas been removed revealing a number of intracytoplasmic cisternae which have been cut incross section (C(cs)). Below this, to the left, the plasma membrane is intact, revealing numerouspores (P) and microvilli (Mv). To the right, the plasma membrane has been removed revealingthe complex network of intracytoplasmic cistemae en face (C(ef)). These are highly inter-connected and exhibit constricted and dilated areas. They are continuous with the plasmamembrane (which has been removed) via pores (P). Other cytoplasmic details have been omittedfor the sake of clarity.

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E. W. SIDON AND OTHERS

Cell-to-cell variations in glycogen content could be attributed to relative functionalstates of individual mucosal epithelia.Another characteristic of gall bladder cells in lampreys as well as mammals (Kaye

et al. 1966) is the extensive band of filamentous ectoplasm along their apical surfaceswhich is free of organelles. The property of contractility has been attributed tomicrofilaments in the cytoplasm (Fawcett, 1966) and it may be speculated that theyparticipate in the regulation of biliary pressure, especially since the gall bladder ofP.marinus contains a sparse distribution of muscle fibres. A gall bladder is absent inrats (Jones & Spring-Mill, 1977) and bundles of microfilaments as well as micro-tubules have been reported in its common bile duct epithelium (Yamada, 1969b;Riches, 1972). Filaments and microtubules may also participate in water absorption(Behnke, 1964) or produce a method of defence against the corrosive effects of bile(Riches, 1972). In this latter context, dense bodies observed in the present studyappear lysosomal in character and could participate in digestion of bile factors.They have been reported in gall bladder epithelia of the rabbit (Yamada, 1974)and the mouse (Yamada, 1968) and possibly represent sites of enzymic degradationof pinocytosed bile products (Yamada, 1974) which could reduce toxic effects ofthese substances on mucosal epithelia.

In fish, rabbit and man, gall bladder cells are water transporting epithelia thatalter the concentration of bile by active removal of sodium and three anions:chloride, bromide, and bicarbonate, along with water (White, Sarles & Benhamou,1977). Fluids and electrolytes might then be transported into intercellular spaceswhich act as cell compartments for creation of a standing osmotic gradient (Kayeet al. 1966). These lateral intercellular spaces are of variable width depending uponthe rate of fluid movement (Johnson, McMinn & Birchenough, 1962; Hayward,1962; Kaye et al. 1966; Tormey & Diamond, 1967) and the lateral plasma membranesform complex interdigitations with adjacent cells (Kaye et al. 1966; Tormey &Diamond, 1967). In these transporting epithelia, the numerous mitochondria tendto be concentrated in the supranuclear cytoplasm (Berridge & Oschman, 1972). Incontrast, the smaller numbers of mitochondria and the narrow intercellular channelsin the gall bladder of larval P. marinus suggest that the epithelium in this vertebratespecies is not as well equipped for transport. However, the condition as describedmay be subject to variability depending upon the particular physiological state of theorgan.As was the case in the bile ducts of larval P. marinus, the zonulae occludentes of

the gall bladder also appear to be 'leaky'. This is not unusual since the epithelium ofgall bladders of mammals and amphibians (Fromter & Diamond, 1972; Claude &Goodenough, 1973) and fish (Fromter & Diamond, 1972) have been shown to havelow transepithelial electrical resistance, and, thus, are leaky to ions. In spite of thisleakiness, Van Os & Slegers (1973) have demonstrated that even though the zonulaoccludens may be the main route of passive transport across the epithelium, the bulkof osmotic flow across rabbit gall bladder is through, rather than between, the cells.The possible effect of junctional leakiness in the gall bladder of larval P. marinuscannot yet be determined since little is known about the functional role of theepithelium. Definitive conclusions can be provided only after further physiologicalstudies.

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SUMMARY

The purpose of the present investigation was to describe ultrastructural charac-teristics of hepatic bile ducts and the gall bladder in larvae of the sea lamprey,Petromyzon marinus, using freeze-fracture replicas as well as ultrathin sections.Comparison of these structures with those of other vertebrates was necessary toprovide a basis in future studies for characterization of biliary degeneration duringmetamorphosis.The bile ducts were composed of a simple cuboidal to columnar epithelium with

the cells separated by wide lateral intercellular spaces and containing a prominentbrush border. Vacuole-like intracytoplasmic cisternae formed a peripheral networkwithin the cells and were confluent with intercellular spaces at the site of numerouspores in the lateral plasma membranes. The cells were joined apically by welldeveloped zonulae occludentes surmounting zonulae adhaerentes. The zonulaeoccludentes, as observed in freeze-fracture replicas, appeared as a honeycomb-likemeshwork. Frequent gaps in P-face ridges suggested a 'leaky' epithelium.The cytoplasm of bile duct cells contained few organelles except for large numbers

of mitochondria; many microfilaments were present. The ultrastructural features ofthese cells reflected an epithelium specialized for absorption and transport and theywere similar to cells of the bile ducts in other vertebrates.The general organization of epithelium in the gall bladder resembled that of bile

ducts, but intercellular spaces were narrower, peripheral pores and cisternae wereabsent laterally, and the brush border was less extensively developed at the apicalsurface. The cytoplasm also contained large pools of glycogen and numerous micro-filaments were situated in the apical ectoplasm. The overall appearance of the gallbladder of lampreys suggested that the epithelium was less specialized compared tothe water transporting organs of other vertebrate species.

This investigation was supported by a grant from the J. P. Bickell Foundation toJ. H. Y. and M. M. F. and Grant A5945 from the Natural Sciences and EngineeringResearch Council of Canada to J. H. Y. The authors thank Dr R. R. Shivers,Department of Zoology, University of Western Ontario, London, Canada, for hisprovision of freeze-fracture facilities.

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Fine structure of the liver in the larval lamprey, Petromyzon marinus