J. Anal. (I1989), 164, pp. 19-39 19With 8 figures

Printed in Great Britain

A microscopic study of the lung of Testudo graeca (Chelonia)

L. M. PASTOR, J. BALLESTA, M. T. CASTELLS, R. PEREZ-TOMAS,J. A. MARIN AND J. F. MADRID

Department of Cell Biology, Section of Histology and General Embryology,Medical School, University of Murcia, Murcia, Spain

(Accepted 3 August 1988)

INTRODUCTION

The ultrastructure of the mammalian lung has been widely studied. Thus, thereexist numerous reports on the ultrastructural characteristics of the intrapulmonaryairways, area of gas-exchange and pulmonary interstitium of mammals (Meyrik &Reid, 1970; Weibel, 1973; Breeze & Wheeldon, 1977; Breeze & Turk, 1984; Gail &Lenfant, 1983).

In reptiles, the ultrastructure of the area of gas-exchange is well known, there beingseveral works concerned with lizards, snakes and turtles (Okada et al. 1962; Klika,Tesik & Nedved, 1976; Meban, 1977, 1978a, b; Welsch & Muller, 1980; Bartels &Welsch, 1983, 1984). However, studies on the intrapulmonary airways and pulmonaryinterstitium of reptiles are very scarce (Klemm, Gatz, Westfall & Fedde, 1979; Luchtel& Kardong, 1981; Solomon & Purton, 1984) and no reports exist on the lung of theterrestrial tortoise of the important super-family Testudinoidea (Gibe, 1970).On the other hand, two of the most recent research fields on lung histology are the

localisation ofendocrine cells and the histochemical characterisation ofcarbohydrates.These studies have been mainly carried out in mammals (DiAugustine & Sonstegard,1984; Spicer, Schulte & Thomopoulos, 1983); thus, no information exists on lowervertebrates, including reptiles.

In the present work, a variety of morphological techniques, including carbohydratehistochemistry, lectin histochemistry, immunocytochemistry and conventional lightand electron microscopy, are applied to study the lung of the Mediterranean tortoise,Testudo graeca.

MATERIALS AND METHODS

Light microscopyThe animals were killed by injecting an overdose of sodium pentobarbitone into the

peritoneal cavity.Lungs from adult specimens of Testudo graeca in non-hibernating (n = 4) and

hibernating (n = 4) periods were fixed either by instillation through the trachea or byimmersion. The fixatives used were Bouin's fluid or 10% formalin. The samples wereroutinely processed and embedded in paraffin. The sections, 5,tm thick, weresubjected to carbohydrate conventional histochemistry (Table 1) and lectin histo-chemistry.

Basically, after endogenous peroxidase blocking with 0-3 % hydrogen peroxidesections were incubated for 1 hours at room temperature in horseradish peroxidase-conjugated lectins (Sigma, St Louis, MO) at the appropriate dilution (Table 2). Afterwashing in TBS, peroxidase was developed in TBS containing 0-05% 3,3-

L. M. PASTOR AND OTHERS

diaminobenzidine tetrahydrochloride and 0 3 % hydrogen peroxide. When developed,the sections were dehydrated, cleared and mounted in DPX. Controls for lectinstaining included (a) substitution of lectin-HRP conjugates for TBS, (b) exposure toeach lectin-HRP conjugate in the presence of a 0-1 M concentration of thecorresponding inhibitory sugar (Table 2).

Immunocytochemistry was performed according to the PAP method (Sternberger,1979) using rabbit antibodies to serotonin (1: 10000). Specific controls included (1)preabsorption of the antibodies with the corresponding antigen, (2) normal serumfrom a non-immunised rabbit as first layer.

Electron microscopyBefore fixation for light microscopy, small pieces of the lungs were immersed in

2-5 % glutaraldehyde, postfixed in 1 % osmium tetroxide, dehydrated in acetone andembedded in Epon 812. Ultrathin sections were cut using a Reichert-Imy Ultracutultramicrotome and stained with uranyl acetate and lead citrate. Electron microscopywas performed with a Zeiss EM/10 cR. For scanning electron microscopy (SEM),after washing in buffer and postfixation, the specimens were dehydrated in acetone,critical-point dried, mounted on aluminium stubs, sputtered with gold and studiedwith a JEOL-T300 scanning electron microscope.

RESULTS

Light microscopyThe lungs of Testudo graeca were subdivided into several compartments each

opening independently into a single short intrapulmonary bronchus which terminatedin a wide, tabicated chamber. The faviform parenchyma of the lung was formed bythree orders of trabeculae: primary, secondary and tertiary (Fig. 1 a). Intertrabecularsepta ran between these trabeculae. Tertiary trabeculae led into a faveolus whichusually extended to the pleural surface of the lung.The intrapulmonary bronchus was covered by a pseudostratified epithelium

composed of ciliated, mucous and basal cells (bronchial-type epithelium). In thelamina propria of the bronchus, patches of hyaline cartilage could be observed (Fig.1 b).The luminal surface of the primary trabeculae was covered by a bronchial-type

epithelium (Fig. 1 c). The abluminal surface was lined by two types of epithelial cells:squamous cells with a thick central region which contained the nucleus and thinnerperipheral sheets of cytoplasm (Type I pneumonocytes) and cuboidal cells showingcytoplasmic granules (Type II pneumonocytes). This is the typical epithelium of thegas-exchange area.The luminal surface of the secondary trabeculae showed a progressive loss of

bronchial-type epithelium which was substituted by gas-exchange epithelium (Fig.1 d). The abluminal surface of the secondary trabeculae was completely covered bygas-exchange epithelium. In the tertiary trabeculae, both the luminal and abluminalsurfaces were covered by gas-exchange epithelium (Fig. 1 e). Intertrabecular septawere also lined by this epithelium (Fig. 11).An extensive superficial capillary network was observed deep to the epithelium of

the trabeculae (Fig. 1 d-J). Smooth muscle bands, connective tissue, fibroblasts andpigment cells were visible in the central core of all trabeculae (Fig. 1 a, c-e). In theprimary trabeculae, elastic fibres could also be seen. The axis of the intertrabecularsepta contained fibroblasts, immune cells and a few smooth muscle fibres. Large nerve

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Fig. 1 (a-f). (a) Primary, secondary and tertiary trabeculae (T1, T2, T3) of the lung of Testudo graeca.Note intertrabecular septa (IS) between the trabeculae. H & E. x 150. (b) Pseudostratified epitheliumof an intrapulmonary bronchus. Basal, ciliated and mucous cells are present, H and E. x 400.(c) Pseudostratified epithelium of a primary trabecula. Note bundles of smooth muscle (SM) andpigment cells (arrows) in the central core of the trabeculae. Toluidine blue. x 600. (d) Secondarytrabeculae showing a mixture of gas-exchange and ciliated pseudostratified epithelia. Toluidine blue.x 600. (e) Tertiary trabeculae lined by typical gas-exchange epithelium. Note a central core of smoothmuscle (SM) bands and the superficial capillary network. Toluidine blue. x 800. (f) Intertrabecularsepta covered by gas-exchange epithelium. Toluidine blue. x 400.

Lung of Testudo graeca 23

Table 3. Conventional mucin histochemistry results

AB ABPAS Am-PAS pH 2-5 pH 1 AB-PAS HID HID-AB

Mucous cells 2P 2P 2B 2B 3BP 2N 3NB

Ciliated cellSurface coat IP IP lB lB lBP IN INBCilia

Surface of PnmI -/IP -/lP -/lB -/lB -lBP -/IN -/INBSurface of PnmII -/1P -/lP -/lB -/lB -lBP -/IN -1/NB

Key to symbols in Table: P, PAS-positive; B, AB-positive; N, HID-positive; BP, a mixture of AB- and PAS-positive mucins, with AB-positive mucins predominating; NB, a mixture of HID- and AB-positive mucins,with HID-positive mucins predominating; -, negative. Symbols separated by / indicate a mixture of cells withdifferent content. Numerical values from 1 to 3 correspond to increasing intensity of staining.

Table 4. Lectin histochemistry results

Con-A WGA DBA PNA SBA LTA UEA-I

Mucous cells -/+ + + + + + + - -

Ciliated cellsSurface coat + + + + - - -

Cilia + + ++ + - -

Pneumonocytes ICytoplasmic -+ + + + -+ ++ - -

processesCell body

Pneumonocytes IISurface cell + + + + + -/+ +Cytoplasm + + + + - +

Endothelial cell + + -/+ + - -/ -Interstitial + + + -/+ - - - -/+collagen fibres

Staining intensity:-, negative; +, weak; + +, moderate, + ++, strong. Symbols separated by / indicatea mixture of cells with different reactivity.

trunks containing nerve fibres were also observed within the lung. Intrapulmonarynerve ganglia were observed in the trabeculae and intertrabecular septa (Fig. 2a).

Conventional histochemical techniques for mucosubstances detected both sialo-and sulpho-mucins in three regions within the lung: mucous cells, surface coat ofciliated cells and surface mucous layer of pneumonocytes (Table 3; Fig. 2b).Mucous cells had a strong affinity for Con-A, WGA, DBA, PNA and SBA (see

Table 2 for abbreviations used). The surface coat of the ciliated cells and the ciliathemselves showed a similar lectin binding pattern except for PNA (Table 4).Cytoplasmic processes of Type I pneumonocytes stained intensely with all lectinsexcept LTA and UEA-I, whereas the cell body was unreactive to the lectins. Thecytoplasm of Type II pneumonocytes stained with Con-A, WGA, DBA and SBA.Interstitial collagenous fibres bound to Con-A, WGA, DBA and UEA-I andendothelial cells of capillaries reacted with Con-A, WGA, DBA and SBA (Table 4;Fig. 2c-e).

24 L. M. PASTOR AND OTHERS

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Fig. 2(a-f). (a) Intrapulmonary ganglia. Note ganglion cell bodies. H and E. x 400. (b) Primarytrabeculae. Positive reaction for sialosulpho-mucins is located in both the surface coat of the ciliatedcells (arrow) and the mucous cells (double arrow). HID-AB. x 400. (c) Reactivity to SBA is located inthe cilia, surface coat of ciliated cells, mucous cells and surface coat of Type I pneumonocytes. x 300.(d) The surface coat of Type I pneumonocytes appears strongly labelled by WGA. x 150. (e) Boththe surface coat of Type I pneumonocytes and the endothelial cells of capillaries reacted with DBA.x 800. (J) Secondary trabeculae. Cluster of serotonin-immunoreactive cells. PAP technique. x 150.

Serotonin-immunoreactive cells were observed as solitary cells or forming groups,called neuroepithelial bodies (Fig. 2]), along the bronchial-type epithelium of theintrapulmonary bronchus and primary and secondary trabeculae. Occasionally,serotonin-containing cells were also found in the respiratory exchange epithelium.Neuroepithelial bodies were formed by groups of serotonin-immunoreactive cellslocated basally in the epithelium and covered by ciliated and mucous cells. Solitarycells presented a pyramidal shape although a clear contact with the lumen was notobserved.

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Scanning electron microscopyThe lung of Testudo graeca showed a multicameral structure. The bronchi, luminal

zone of the primary trabeculae and some areas of the secondary trabeculae werecovered by ciliated and microvillous cells. The gas-exchange areas were lined by twocell types: squamous cells showing short microvilli and marked cell margins (Type Ipneumonocytes) (Fig. 3 a, b) and small cuboidal cells exhibiting long apical microvilli(Type II pneumonocytes).

Transmission electron microscopyBronchial-type epitheliumThe bronchial-type epithelium covering the intrapulmonary bronchi and some areas

of primary and secondary trabeculae consisted of mucous, ciliated, basal andendocrine cells (Fig. 3c). Mucous cells showed characteristic secretory granules ofvariable electron density in the apical region. Ciliated cells had abundant cilia withtheir corresponding basal bodies, prominent nucleoli and numerous mitochondria.Basal cells did not show contacts with the lumen. The cytoplasm of these cellscontained abundant filaments. Endocrine cells were found either as solitary cells orforming neuroepithelial bodies (Figs. 3c, 4). Morphology of the endocrine cells wassimilar in both cases: low electron-dense cells presenting dense-cored granules in thebasal region. The diameter of the granules varied between 120 and 140 nm. EndocrinecelIs of the neuroepithelial bodies were covered by a single layer of ciliated and mucouscells. No contacts with the lumen were observed (Fig. 4a). A different cell type,characterised by a highly electron-dense cytoplasm and a heterochromatic nucleus,was also seen within the neuroepithelial bodies (Fig. 4a). Around the neuroepithelialbodies, nerve terminals containing clear vesicles (mean diameter S0 nm) were detected(Fig. 4a).

Gas-exchange epitheliumThe epithelium of the respiratory exchange area can be further characterised

ultrastructurally as Type I pneumonocytes, Type II pneumonocytes and undiffer-entiated cells. Free faveolar macrophages were also observed.Type I pneumonocytes were characterised by a heterochromatic nucleus surrounded

by a thin layer of cytoplasm where few organelles could be seen. The remainder of thecell consisted of thin cytoplasmic processes which extended over the surface capillarynetwork (Fig. 5a). These cytoplasmic processes contained abundant pinocytoticvesicles and microfilaments and formed tight junctions and desmosomes withprocesses of other Type I pneumonocytes. The luminal surface of the cell showed amoderate number of short microvilli which, occasionally, possessed a granularmaterial on the surface. The basal lamina of Type I pneumonocytes frequentlyappeared to be fused with that of the capillaries. A subpopulation of Type Ipneumonocytes contained abundant cytoplasmic granules of mucous appearancesimilar to those found in the mucous cells of the bronchial-type epithelium (Fig.Sb).Type II pneumonocytes had a cuboidal shape. The free luminal surface of the cell

had numerous short microvilli. The cytoplasm contained abundant mitochondria,segments of rough endoplasmic reticulum, centrioles and lipid droplets in the basalportion of the cells (Fig 5c). As already described in Type I pneumonocytes, apopulation of Type II pneumonocytes presented numerous mucous granules in the

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Fig. 3 (a-c). (a) Secondary trabeculae. Zone of transition between gas-exchange (G) and bronchial-type (B) epithelium. x 1500. (b) Secondary trabeculae. Zone of transition. Surface of ciliated andsquamous (Type I pneumonocytes) cells. x 3000. (c) Pseudostratified epithelium of a primarytrabecula. Basal (B), ciliated (C) and mucous (Ml) cells are present. x 4000. Inset shows a solitaryendocrine cell within the epithelium. x 6300.

Fig. 4(a-b). (a) Neuroepithelial body of the lung of Testudo graeca. Endocrine cells (E) are basallylocated and mainly covered by ciliated cells. x 3000. Upper inset: nerve terminal containing clearvesicles in close contact with an endocrine cell (E). x 20000. Lower inset: clear (C) and dense (D)endocrine cells. x 4500. (b) Solitary endocrine cell in the gas-exchange area of a primary trabecula.PnmI, Type I pneumonocyte. x 18000. Inset shows a low magnification of the endocrine cell. Notethe close proximity to a blood vessel. x 1800.

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Lung of Testudo graeca 29apical portion of the cytoplasm (Fig. 5d). These cells were usually detected near thebronchial-type epithelium. Typical Type II pneumonocytes (without mucous granules)were predominantly observed in the faveoli.The main ultrastructural characteristic of all Type II pneumonocytes was the

presence of lamellar bodies (Fig. Sc, d), the structure of which was similar to that ofmammals. In Type II pneumonocytes with mucous granules, a merocrine secretion ofboth lamellar bodies and mucous granules was observed.

Several precursor structures of lamellar bodies were observed within Type IIpneumonocytes: (a) Lipid droplets and mitochondria presenting lamellate inclusions(Fig. Se, f), (b) segments of rough endoplasmic reticulum (RER) with lamellateinclusions (Fig. 5g) and (c) multivesicular bodies showing a progressive differentiationtowards lamellar bodies (Fig. 5h-j). These three structures were frequently associatedwith mature lamellar bodies.

Undifferentiated cells were frequently found in the transition between bronchial-type and respiratory exchange-type epithelia. Undifferentiated cells were characterisedby abundant segments of RER, short cytoplasmic flanges and a few mucous granulessimilar to those observed in some Type I and II pneumonocytes (Fig. 6a).

Occasionally, free macrophages showing numerous lysosomes and short cyto-plasmic protrusions (pseudopodia) were observed in thb faveolar lumen (Fig. 6b).

HibernationDuring the hibernation period, morphological alterations were observed in

pneumonocytes; namely an intense vacuolisation of the RER of Type II pneumono-cytes and an increase in the electron density of the cytoplasm of Type I pneumonocytes(Fig. 6c).

InterstitiumThe central core of all trabeculae was composed of a band of smooth muscle fibres.

Interspersed between the muscle fibres, nerve terminals containing clear (50 nm meandiameter) and dense-cored (100 nm mean diameter) vesicles were observed (Fig.7 a). Large cells containing numerous highly electron-dense granules were observed inthe connective tissue which surrounded the muscle fibres (Fig. 7 b, d). These cells werealso seen close to nerves and blood vessels. Other cell types detected in the interstitiumwere: fibrocytes (Fig. 7 c), pericytes, lymphocytes, leucocytes and macrophages.Collagenous fibres were numerous (Fig. 7 d) while elastic fibres were mainly seeninterspersed between the smooth muscle fibres.

Endothelial cells of the interstitial blood vessels, including the capillary network,showed abundant pinocytotic vesicles and Weibel-Palade bodies (Fig. 8 a). Theadventitia of the blood vessels possessed nerve terminals containing numerous clearvesicles.The relationship between epithelial cells and fibrocytes in the area of gas-exchange

Fig. 5 (a-i). (a) Type I pneumonocyte. Note the cell body and the thin cytoplasmic processes. x 4000.(b) Type I pneumonocyte showing granules of mucous appearance. x 12000. (c) Type IIpneumonocytes showing abundant lamellar bodies. x 12000. (d) Type II pneumonocytes containinglamellar bodies and granules of mucous appearance (arrows). x 8000. (e) Lipid droplets associatedwith lamellate material. x 25000. (J) Lamellate inclusion within a mitochondrion (arrow). x 15 500.(g) Osmiophilic material within cisternae of rough endoplasmic reticulum (arrows). x 30000. (h)Clear (arrow) and dense (double arrow) multivesicular bodies. x 40000. (i) Lamellate inclusionswithin dense multivesicular bodies. x 45000. (j) Mature lamellar bodies containing small vesicles(arrows). x 40000.

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Lung of Testudo graeca 31

was mediated by thin cytoplasmic processes of Type I and II pneumonocytes directedtowards the cell membrane of the fibrocytes, but in no instance did they form clearjunctions with these cells (Fig. 8b).

Lamellate inclusions similar to those present in the laminar bodies were observed inthe endothelial cells, the pulmonary interstitium and the capillary lumen (Fig. 8c).

Bundles of nerve fibres containing clear and dense-cored vesicles were also observedin the interstitium (Fig. 8 d, e). Intrapulmonary ganglion cells had a regular shape, anda centrally located nucleus showing prominent nucleoli. The cytoplasm presented afew organelles and abundant free ribosomes (Fig. 8J).

DISCUSSION

Gross anatomyThe lung of Testudo graeca showed a multicameral structure with three orders of

trabeculae. The faviform parenchyma presented a honeycomb-like appearance. Thegeneral structure of the lung was more complex than that of other Lacertidae andOphidia (Klemm et al. 1979; Luchtel & Kardong, 1981) although the intrapulmonarybronchi were not well developed as occurs in Chelonia mydas (Solomon & Purton,1984).

Intrapulmonary airwaysIn Testudo graeca, the structure and histochemical reactivity of the mucous cells of

the lung were similar to those of the mucous cells of the extrapulmonary airways(Pastor et al. 1987). Mucous cells have also been described in the intrapulmonaryairways of Chelonia mydas (Solomon & Purton, 1984); however, this cell type wasdifferent to the secretory cells of the extra- and intrapulmonary airways of someOphidia and Lacertidae (Klemm et al. 1979; Luchtel & Kardong, 1981; Tesik, 1984;Pastor et al. 1988). Histochemically, the mucous cells contained sialosulpho-mucins.Lectin histochemistry revealed the presence of GalNac, Gal and GlcNac. These resultswere similar to those previously reported in the mucous cells of the extrapulmonaryairways of Testudo graeca (Pastor et al. 1987), indicating that the mucin content of themucous cells is similar throughout the respiratory airways. No fucose residues weredetected in Testudo graeca; however, in mammals, abundant fucose groups have beendescribed in human bronchi and in extrapulmonary airways of rodents (Mazzuca,Hermitte, Lafitte & Roussel, 1982; Geleff, Bock & Stockinger, 1986).

Basal cells have been classically considered as the stem cells of the epithelia althoughrecent works suggest that mucous, intermediate and serous cells also have aregenerative potential (Evans, Shami, Cabral-Anderson & Dekker, 1986). Thedecrease of basal cells observed in the secondary trabeculae of Testudo graeca may becompared with the decrease of these cells in the terminal zones of mammalian airways(Breeze & Wheeldon, 1977).

Ciliated cells showed a similar morphology to that of mammals (Breeze &Wheeldon, 1977). It is important to note the presence of an apical surface coatcontaining sialosulpho-mucins, Fuc, GlcNac, GalNac and Man. A carbohydrate-rich

Fig. 6(a-c). (a) Undifferentiated cell containing granules of mucous appearance. x 14000.(b) Faveolar macrophage showing abundant lysosomes and short pseudopodia. x 8000. (c) Gas-exchange area of hibernating Testudo graeca. Note the marked vacuolisation of the roughendoplasmic reticulum of Type II pneumonocyte (PnmIl) and the electron density of Type Ipneumonocytes (PnmI). x 4200.

Fig. 7 (a-d). (a) Nerve terminals in a bundle of smooth muscle fibres. x 20000. Inset: detail of thenerve terminals showing clear (arrow) and dense-cored (double arrow) vesicles. x 45 000. (b) Pigmentcell of the lung. x 7000. (c) Intertrabecular septum. F, Fibrocyte; PnmI, Type I pneumonocyte;PnmII, Type II pneumonocyte. x 2000. (d) Collagenous fibres and pigment cells in the interstitiumof the lung. x 6500.

Lung of Testudo graeca 33surface coat has also been described in ciliated cells ofmammals by means of light andelectron microscopy (Spicer, Mochizuki, Setser & Martinez, 1980; Geleff et al. 1986).A function of minimising friction or adhesion between the crowded cilia has beensuggested for this surface coat (Spicer et al. 1983).Both solitary and grouped (neuroepithelial bodies) endocrine cells of the lung of

Testudo graeca were immunocytochemically and ultrastructurally similar to those ofthe extrapulmonary airways of this tortoise (Pastor et al. 1987). The ultrastructureof the Chelonian neuropithelial bodies has been previously described in Pseudemysscripta elegans (Scheuermann, De Groodt-Lasseel, Stilman & Meisters, 1983). In thisspecies, the neuroepithelial bodies were covered by Type I pneumonocytes; however,in Testudo graeca, as in amphibians, the neuroepithelial bodies were lined by bothmucous and ciliated cells (Rogers & Haller, 1978; Wasano & Yamamoto, 1978).

The blood-air barrierThe epithelium of the respiratory region consisted of four types of pneumonocytes:

typical Type I and II pneumonocytes and Type I and II pneumonocytes with mucousgranules. Previous studies have described the exchange-type epithelium of reptiles ascomposed either by two cell types (typical Type I and II pneumonocytes) (Meban,1977, 1978 a, b; Klemm et al. 1979; Solomon & Purton, 1984) or by three cell types(typical Type I and II pneumonocyte and Type II pneumonocyte showing mucous orserous granules or pneumonocytes similar to mammalian Clara cells) (Welsch &Muller, 1980; Luchtel & Kardong, 1981; Scheuermann & De Groodt-Lasseel, 1982).Therefore, the present results in Testudo graeca show two cell types not previouslydescribed in vertebrates, Type I pneumonocytes with mucous granules andundifferentiated cells with mucous granules but lacking lamellar bodies. These findingssuggest the existence of a plasticity in the epithelium, all cell types described beinginterrelated by processes of cell differentiation.Four possible formation pathways of the lamellar bodies of Testudo graeca have

been found: from multivesicular bodies, mitochondria, lipid droplets and roughendoplasmic reticulum. Similar findings have been reported in mammals, birds andamphibians but not in reptiles. The osmiophilic material observed in the RER issimilar to the 'bar-like' structures described in other veterbrates (Shimura, Aoki, Sato& Takishima, 1980; Pastor, Ballesta & Lopez, 1985). These structures have beenassociated with the origin of the lamellar bodies and with modifications in thesynthesis of the surfactant (Vincent & Nadeau, 1987). The origin of lamellar bodiesfrom lipid droplets and mitochondria has been described in some amphibian and avianspecies (Matsumura, 1984; Lopez, Sesma, Diaz de Rada & Villaro, 1986). Theformation of lamellar bodies from multivesicular bodies is similar to that described inmammals but different from amphibians (Sorokin, 1966; Goniakowska-Witalinska,1986).The luminal surface of the pneumonocytes contained sialosulpho-mucins and

reacted with all lectins except LTA and UEA-I. A similar finding has been reportedin mammals, suggesting a rich carbohydrate content in the glycocalyx of the cells ofthe gas-exchange region (Atwal & Brown, 1980; Meban, 1984, 1986 a, b; Faraggiana,Villari, Jagirdar & Patil, 1986). This surface coating, as occurs in mammals, maytherefore operate as a water trap and help to maintain the aqueous subphase of thesurfactant complex. By this mechanism it could also protect the delicate alveolarepithelium from desiccation (Meban, 1984).The capillaries of the lung of Testudo graeca bound strongly to Con-A, WGA and

DBA and weakly to SBA but no staining was observed with LTA and UEA-I,

34 L. M. PASTOR AND OTHERS

Lung of Testudo graeca 35indicating the presence of abundant Man and GlcNac residues. The capillaries of thelungs of higher vertebrates have been demonstrated to contain sialic acid and Gal and,only in the human lung, Man and Fuc residues have been detected (Alroy, Goyal &Skutelsky, 1987). Differences may be related to the important function of theglycocalyx in the endothelial cells described recently in mammals (Ryan, Ryan &Crutchley, 1985).

Putative macrophages have been observed in the faveoli of Testudo graeca. Alveolarmacrophages are common cells in the mammalian lung where they play importantroles in several lung diseases (Bowden, 1987). Free macrophages have so far not beendescribed in the non-mammalian lung, probably because they represent a very smallcell population in normal conditions being markedly increased in pathologicalsituations.

HibernationHibernating specimens of Testudo graeca showed a cytoplasmic vacuolisation,

mainly referred to the RER of pneumonocytes II, as well as an increased electrondensity of Type I pneumonocytes. Similar findings have been reported in the liver andkidney of this species (Zuasti, Ferrer, Ballesta & Pastor, 1986, 1987; Ferrer et al. 1987).These phenomena are probably related to a reduction of extracellular fluid duringhibernation and a massive intake of water into the cells (Lawrence & Hawkey,1986).

The lung interstitiumThe trabeculae contained abundant smooth muscle fibres. Nerve terminals showing

vesicles of probably cholinergic and peptidergic nature were observed in closeassociation with the muscle bundles and the adventitia of blood vessels. These nerveterminals are similar to those described in other vertebrates (Burnstock, 1986). Theabsence of typical adrenergic vesicles, as occurs in Chelodina longicollis, may supportthe hypothesis that the reptilian lung is poorly supplied by adrenergic nerves (Smith& Satchell, 1987). Previous works have reported the absence of substance P-immunoreactive nerve fibres in the repitilian lung (Smith & Satchell, 1987); however,the presence of large, dense-cored vesicles, typical of peptidergic nerves, in the lung ofTestudo graeca suggests the existence of regulatory peptides within the nerveterminals.The Weibel-Palade bodies found in the endothelial cells were similar to those found

in mammals and Anura (Weibel & Palade, 1964; Goniakowska-Witalinska, 1986).These bodies may play an important role in some pulmonary diseases; thus, theexistence of histamine within Weibel-Palade bodies has recently been described insome anurans (Fujimoto et al. 1984).Pigment cells had the typical structure of melanophores (Bagnara & Hadley, 1969).

Abundant melanophores have been described in different organs of reptiles (skin,mesentery, skeletal muscle and blood vessels) but not, hitherto, in the lung (Zamorano

Fig. 8 (a-f). (a) Artery of the lung of Testudo graeca. The endothelial cells contain abundantWeibel-Palade bodies. The internal elastic lamina and numerous smooth muscle cells are also shown.x 5000. (b) Type II pneumonocyte showing short basal processes projecting towards the basal laminawhich appears to be continuous. A portion of a fibrocyte is seen immediately deep to the basallamina. x 8000. (c) Lamellate material in a faveola and within a capillary (arrow). x 4500.(d) Unmyelinated nerve fibres in the lung interstitium. Sch, Schwann cell. x 12 000. (e) Detail ofa nerveterminal of the interstitium containing clear and dense-cored (arrows) vesicles. x 45 000. (/) Ganglioncell showing a large nucleus with a prominent nucleolus and abundant cytoplasmic free ribosomes.x 6000.

L. M. PASTOR AND OTHERS

et al. 1977; Gopalakrishnakone, 1986). In the lung of Testudo graeca, as described inother zones, melanophores were associated with blood vessels, nerves or smoothmuscle fibres or were dispersed in the connective tissue. It has been suggested thatmelanophores play an important role in the thermal regulation of reptiles(Gopalakrishnakone, 1986).Although numerous fibrocytes were observed in the interstitium, no myofibroblasts

were detected. This is in contrast with the occurrence of myofibroblasts in theinterstitium of the mammalian lung where this cell type represents 50% of allinterstitial cells (Martin, Normand, Cornu & Le Bouffant, 1977).

Lamellate inclusions have been observed within both the endothelial cells and thecapillary lumen of Testudo graeca. In mammals, this finding has been associated withthe renewal of the surfactant (Kalina & Young, 1980).

All types of pneumonocytes showed cytoplasmic processes projecting towards theinterstitium, being more abundant in Type II pneumonocytes. Although theseprocesses were close to the interstitial fibrocytes, no discontinuity of the basal laminawas detected. Similar processes have been described in Type II penumonocytes ofmammals (Vaccaro & Brody, 1981; Pastor et al. 1985). Occasionally, these processespassed through the basal lamina and came into contact with the fibrocytes,representing a mechanism of epithelial-mesenchymal interaction allowing some kindof regulation of the pneumonocytes by the fibroblasts (Adamson & King, 1986).

In conclusion, the lung of Testudo graeca (Chelonia) showed a structure morecomplex than that of Lacertidae and Ophidia presenting a large variety of cell typesin the gas-exchange area.

SUMMARY

The lung of the tortoise, Testudo graeca (Chelonia) was studied by means of lightand electron microscopy, histochemistry and immunocytochemistry.The lung showed the typical faviform structure of the reptilian lung. Three orders

of trabeculae were observed. The epithelium of primary and secondary trabeculae wascomposed of ciliated, mucous, basal and endocrine cells. Mucous cells contained sialo-and sulpho-mucins and were reactive to the lectins Con-A, WGA, DBA, PNA andSBA. Endocrine cells were observed as solitary cells or forming neuroepithelial bodies.By means of immunocytochemistry, endocrine cells were demonstrated to containserotonin.

In the gas-exchange area Types I and II pneumonocytes and undifferentiated cellswere observed. Free macrophages were detected in the faveolar lumen. The lunginterstitium contained smooth muscle cells, fibrocytes, pigment cells, myelinated andunmyelinated nerves and intrapulmonary ganglia. Nerve terminals containing clearand dense-cored vesicles were observed in the adventitia of the blood vesicles andinterspersed between the smooth muscle bands. The lung of the hibernating specimensshowed a marked vacuolisation of pneumonocytes.

In conclusion, the lung of Testudo graeca showed a complex histologicalorganisation. Marked differences from mammalian lung were found.

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