Ultrastructural studies on the conjunctiva in Agamid Lizards

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Ultrastructural studies on the conjunctiva in Agamid Lizards Uwe Hiller Institute of Anatomy, University of Miinster, Vesaliusweg2-4, 48149 Miinster, Germany

Summary. The conjunctivae of lizards with free movable eyelids have been studied by means of SEM and TEM. This study was restricted to the agamid species, which are characterized by large and extensively mobile eyelids. The conjunctival surface pattern can be divided into the centrally positioned A zone coinciding largely with the tarsus, and the surrounding B zone, which includes the fornix conjunctivae. The A zone is characterized by pronounced epithelial projections or even lamellated formations, which include in some cases the lamina propria. The conjunctival epithelium is stratified and composed of columnar or cuboidal cells. The great variety of cell protrusions is characteristic of all the species studied. Furthermore, even adjacent cells demonstrate great differences in electrondensity and in the condition of the cell organelles. This can be traced back to secretory activity which could be demonstrated following different modes. The structure of the underlying lamina propria conjunctivae is identical for both the A and B zones which contain mainly collageneous connective tissue, arranged in the deeper regions in a plywood-like manner. Besides smooth muscle cells and nervous tissue, wandering cells such as lymphoid and plasma cells could also always be observed, sometimes even penetrating the conjunctival epithelium near the conjunctival sac. In the B-zone the luminal protrusions of the conjunctival epithelium diminish. In the direction of the fornix, conjunctival goblet cells occur increasingly, forming in some cases epithelial mucous glands. Thus, the agamid lizards studied demonstrate, in comparison with mammalian conjunctiva, a great morphological range of cellular formations and conjunctival components, which can be used for further systematic, functional and ecophysiological studies. Key words: Conjunctiva phology - Reptiles

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Eyelids -

Ann Anat (1995) 177: 553-562 Gustav Fischer Verlag lena

Functional mor-

Introduction Light microscopic studies of the vertebrate eyelids have been carried out almost since the invention of the light microscope (survey by Reich 1875; Virchow 1911). For the very first time, Kolmer (1925/26) described the saurian conjunctival epithelium of the nictitating membrane. As in this study, the author also found in birds the socalled feathered epithelium (Kolmer 1923 / 24), indicating the numerous cellular protrusions of the epithelium. This is provided with scattered goblet cells, previously described in lizards by Leydig (1872). The earliest TEM studies of vertebrate conjunctival epithelium were published by Bielek (1976), who investigated the avian nictitating membrane. Schramm et al. (1985) studied the avian conjunctiva by means of SEM and like Kolmer (1923/24) described a feathered epithelium. Schramm et al. (1986) traced back similar structures on the bulbar conjunctiva of the nictitating membrane in an agamid lizard (Physignathus sp.). Preliminary combined TEM / SEM studies of various lizards (Hiller and Dieterich 1986· Hitler and Dieterich 1989) led to the result that aga~id lizards demonstrate a highly differentiated palpebral conjunctiva. In continuation of that work, this paper is concerned with closer studies on the functional morphology of the agamid conjunctiva.

Material and methods The use of living agamid lizards in this study was carried out in accordance with the Bundesnaturschutzgesetz and the Bundesartenschutzverordnung (version of 01.01. 1987). The sampling of tissue was licensed by the Regierungspriisident Miinster in 25 . 02. 1988. Additionally, 70070 alcohol-fixed specimens from the collection of the Museum Konig (Bonn, Germany) as well as specimens of other collections were used (see table 1), but these are all restricted ex-

clusively to SEM-observations. Preliminary comparative studies have demonstrated to what extent collection material may be used in SEM regarding cellular damage (Hiller and Dieterich 1989). SEM pictures, which were made from collection material are marked with an asterisk. The following animals were examined: Table 1. List of agamid lizards used in this study. Species

Origin

Fixation

Agama aculeata Agama agama Agama anchieta Agama impalearis Agama impalearis Ctenophorus nuchalis Physignathus cocincinus

Bonn ZFMK 18382 Commercial dealer Bonn ZFMK 32815 Bonn ZFMK 44173 Commercial dealer Senckenberg Museum, Ffm. Zoo Frankfurt, Germany

Alc. CA. imm. Alc. Alc. CA. perf Jroz.lAlc. CA. perf

Abbreviations: Alc. first fixed in 70% alcohol; Jroz. deep frozen material at about - 18°C; CA . Glutaraldehyde-fixed; imm. fixed by immersion; perf fixed by perfusion; ZFMK Registration number Forschungsmuseum Konig, Bonn (Germany) EM-preparation: The lizards were anesthetized with Inactin® i. p. (0.05 ml/g body weigth) and perfused via the cannulated left ventricle of the heart with phosphate buffered 2.3070 glutaraldehyde solution (pH 7.4/ 400 mOsm at 4 °C) and about 45 mm Hg pressure following a brief rinsing with Haemaccel® (Fischer and Dieterich 1985). The immersion fixation with the glutaraldehyde solution also led to satisfactory results. In all cases the solution mentioned was also used for postfixation (2 h, 4°C). One complete eyelid was excised for TEM, the other one for SEM. Subsequently the samples were dehydrated by alcohol and en-bloc-contrasted by 0.5% uranyl acetate and 1% phosphotungstic acid (Wohlfahrt-Bottermann 1957) and embedded in Epon. For the light microscopic control

semi thin-sections were made (LKB Pyramitome 11 8(0) and stained with methylene blue-azur II (Richardson et at. 1960). The ultrathinsections (Porter-Blum Ultra-Microtome MT-2B, Sorvall) were stained with 5"70 uranyl acetate in water followed by lead citrate, 5 min in each solution, and examined with a Siemens Elmiskop I A electron microscope or a Philips CM 10. SEM preparation: For SEM the specimens were fixed as described above for TEM so far as the fresh samples were concerned. These and the specimens from collections were subsequently dehydrated by graded series of alcohol and critical-point dried in liquid CO2 (Polaron CP-Dryer E 5100) and mounted with contact glue (Gocke) on SEM-stubs. Finally they were sputtercoated (Polaron E 5100) with Au/ Pd and examined mainly in a LeitzAMR 1200 B, also PHILIPS XL 20 (Figs. 2 b, 3 d), and JEOLIKontron 6400 F (Fig. 7 c). Terminology: The description of the ocular adnexa is that found in Rohen (1964) and Winckels (1914). The authors distinguish between the temporal from the nasal (= rostral) palpebral region so as to keep these terms for both humans and other vertebrates. The central part that represents the greatest area of each eyelid is called the tarsus palpebrae (superior and inferior), covered by the tunica conjunctiva or conjunctiva (palpebrae). The conjunctiva runs into the fornix conjunctivae on the cornea as the conjunctiva corneae. For morphological orientation the eyelids can be subdivided in the both the central A zone in the proximity of the Iidgap and the surrounding B zone, including the fornix conjunctivae (see Fig. 1 a).

Results The conjunctival epithelium of the tarsal region (A zone) The centrally positioned A zone forms the boundary of the lidgap elliptically and can be differentiated by its topo-

a Fig. 1. a) Schematic drawing of reptilian eyelids demonstrating the conjunctival surface which can be subdivided into a central A zone followed by the B zone including the fornix conjunctivae. Both regions surround the palpebral fissure concentrically; RA rostral angle, TA temporal angle of the eyelids; DL platelike disk of underlying connective tissue. b) Agama anchieta*, survey of the palpebral conjunctiva; OL / Bz upper eyelid I B zone; OL / Az upper eyelid I A zone; dotted line transition between A- and B zone; UL lower eyelid (A zone); arrow rostral angle of the eyelids; DL: platelike disk. SEM. x 28

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graphy from the adjacent B zone. This turns into the peripheral conjunctiva of the fornix conjunctivae with its transition to the conjunctiva corneae. As in other reptiles, the agamids likewise demonstrate in the lower eyelid a platelike disc of dense connective tissue (see Figs. 1 a, 1 b) covered by conjunctival epithelium (Hiller, in preparation). The A zone is marked off from the B zone by a distinct type of lamella (Fig. 1 b), which is formed by both conjunctiva and lamina propria. The second type is formed by the epithelium only and can sometimes be found in the adjacent B zone. Only in Agama acu/eata, are both types of lamellae restricted to the A zone completely. In the central area of the upper eyelid the free borders of the lamellae are flattened. Here also the flanks of lamellae are in touch with the bulbus, forming a corresponding concavity (Fig. 2a). This is also true for the apical regions of single protruding cells, demonstrating reduced surface structures. However, in peripheral and deeper regions, as in gaps between lamellae or prominent epithelial cells, the surface morphology remains unchanged and will be described in detail below. For most parts of the peripheral lamellar regions, single cell rows with few cells give the free margin of the lamellae a comb-like appearance that can vary to a large extent (Fig.2b). In the rostral and temporal regions there is a lamellar transition of the upper and lower eyelid, maintaining without changing the general morphology. Only in some cases are their number and size reduced and some of the lamellae continue from the rostral corner to the corneal fornix of the membrana nictitans, forming epithelial bulges (see Fig. 1 b). The cells of the stratified conjunctival epithelium of the A zone are not only characterized by microvilli and microridges but also by extremely variable protrusions. They may even consist of several small cells (Fig. 2c). The length of apical protrusions and similar cell formations can go up to 2.2 Ilm and have a diameter of 0.6 Ilm (Fig. 3 a), whereas in some cases they appear rather short and thick. Sometimes they arise from prominent cell apices and additionally they can be ramified in a featherlike manner (Fig. 3 a, inset). Besides the cell protrusions, the morphology of the apical cell membranes also varies considerably, even in adjacent cells. Thus the cells usually demonstrate pores of varying density and diameter or scattered areas of fissured apical cell regions (Fig. 3 b).

Fig. 2. a) Ctenosaurus nuchalis*, flattened rim of a lamella (A zone) which is in touch with the ocular surface; La edge of lamella without any protruding cells; R interlamellar space. SEM. x 810 b) Agama aculeata*, laminary protruded conjunctival cells (Lp) at the border of a lamella (A zone); Dm discharged mucoid material. SEM. x850 c)Agama anchieta*, A zone; line of a few little cells (0) forming long protrusions; Z individually protruding cells with short ramifications (arrowheads); inset: great protrusion consisting of

both a pedunculated cell and an apically situated smaller cell. SEM. x 1.250 (inset: x 660)

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Moreover, especially in the lower eyelid the epithelial cells demonstrate marginal crests or grooves created by zonulae occludentes indicating the course of the epithelial cell borders (see Figs. 3 a, 6 a).

ed cell organelles are discharged within membrane-coated blebs (Fig. 4 b). Before discharging, individual droplets or groups of them may fuse to become greater vacuoles before secretion (Fig. 4c). This is true especially in the areas of the free margins of lamellae. As a result of the discharging processes the superficial cells lining the conjunctival sac seem to be porous (see Fig. 3 b). Because of the preceding preparatory steps, the secretory products deposited as part of the mucous layer are to a large extent wiped off (see Nichols et al. 1985). In this way only little homogeneous material of few particles of cellular debris can be found in both SEM and TEM.

Intraepithelial and related intracellular structures Generally, the conjunctival epithelium of the A zone is simple or two-layered, and the luminal layer consists of columnar or cuboidal cells. The great difference of electron density between adjacent cells is striking (Fig. 4a). The clear ones contain a clear nucleus and swollen mitochondria, and also a few - apparently empty - vesicles, which occur in the apical cell region. This cell type alternates more or less frequently with dark cells, containing a dark nucleus and numerous (secretory) vesicles and normally shaped mitochondria. They also demonstrate features like extremely enlarged clear vesicles, dilated intercellular clefts and reduced plasma extensions. Especially in degenerating cells, the intercellular spaces enlarge to intercellular canaliculi, often provided with a clear matrix. Although sometimes cell debris or free cells can also be identified, using the space as an intercellular pathway.

Lamina propria conjunctivae: connective tissue, muscle cells, nervous tissue and vessels

Intraepithelial secretory cells Apart from goblet cells, which occur mainly in the peripheral regions of the eyelid, numerous epithelial cells of the A zone release secretory products into the conjunctival sac. The content of the vacuoles or droplets varies extremely from a morphological point of view. Sometimes even reduc-

The course of the basal membrane between the lamina propria and epithelium depends on the region concerned. In the A zone the course is smooth and there are both few hemidesmosomal contacts and short anchoring filaments. The connective tissue proper mainly consists of collageneous fibres varying greatly in their abundance. They often course as bundles with the superficial fibres mostly parallel to the margin of the eyelid. Preferably in deeper regions of the lamina propria, they change their direction periodically in layers by an angle of about 90° in a plywoodlike manner (Fig. 4d). In Physignathus cocincinus, the collagen fibrils involved demonstrate a diameter of 62 nm and cross striations at 54 nm intervals. The cell protrusions of the slender fibrocytes are pointed more or less in the same direction as the collagen fibres. In addition to the ground substance, they can be observed in all

Fig. 3. a) Agama anchieta*, A zone; elongated slender protrusion without any ramifications; arrows: crests between adjacent cells; inset: featherlike ramifying protrusions. SEM. x3.200 (inset: x2.700) b) Agama acu/eata, conjunctival region showing, after discharge of mucoid blebs, reduced apical cell portions (B) which border on naked areas (N), sometimes provided with pores (PO); Me: mucoid material. SEM. x2.300

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Fig. 4. Intraepithelial and intracellular conjunctival and subconjunctival structures; a) Physignathus cocincinus, rim of conjunctival lamella demonstrating nearby clear (L), intermediate (l), and dark (D) epithelial cells; arrowheads intercellular clefts surrounding dark cells containing clear vesicles. TEM. x3.200 b) Physignathus cocincinus, secretory bleb (SB) containing reduced cell organelles pouring through the apical cell membrane. TEM. x 7.400 c) Agama impaiearis, intermediate cell type with secretory vesicle (S) and confluent ones (Sc); D: dark cells; arrowheads: intercellular crest. TEM. x3.400 d) Physignathus cocincinus, lamina propria conjunctivae provided with collagenous fibers arranged in a plywood manner at angles of about 90 E: part of an epithelial cell; F: fibrocyte; NF: nerve fibre. TEM. x24.000 0

;

regions of the lamina propria. Furthermore, in this region both unfenestrated capillaries and capillaries with distinctly fenestrated endothelium occur, creating luminal marginal folds. The pores in Physignathus cocincinus are about 100 nm in diameter and at least partially closed by slender diaphragms (Fig. 5 a, inset). Nervous tissue can be found scattered all over the lamina propria, as is also true for myelinated nerve fibres of varying calibre, which frequently course together with nonmyelinated

fibres. The latter also occur near smooth muscle cells, which are likewise scattered all over the lamina propria. They are embedded in collagen fibres and/or amorphous ground substance. Sometimes the unmyelinated fibres show synaptic formations ("synapses en distance") near to smooth muscle cells (Fig. 5 b). Nevertheless, neither here nor in the epithelium proper could free nerve endings or correlated structures be observed. These results, which refer to the lamina propria, can also be applied to the peripheral B zone.

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Wandering cells and pigment cells of the lamina propria Mobile wandering cells occur in both the epithelium and lamina propria of the A and the B zone. This cell population is composed mainly of mast cells, lymphoid cells, plasma cells and macrophages. Within both the collagen tissue of the lamina propria and the conjunctival epithelium, lymphocytes can be found aggregated with plasma cells. Like the latter, lymphocytes also invade the epithelium individually via the lamina propria and appear preferably near the basement membrane. Thus in some cases lymphoid cell aggregations can be observed in both epithelium and lamina propria (Fig. 5 c). Macrophages occur mostly near vascular structures. These are characterized by a polymorphic morphology and abundant micropinocytotic vesicles (see Fig. 5 a). They are also able to enter the epithelium without any essential change in morphology.

The conjunctival epithelium of the B zone As in the central A zone, the conjunctiva of the surrounding B zone (see Fig. 1 a) is lined by a stratified squamous epithelium, composed of two or three layers. The lamina propria conjunctivae is likewise equipped as already demonstrated in that of the B zone. Usually the surface pattern of the A zone flattens in the direction of the adjacent B zone, which can be observed particularly in species with pronounced lamellae (Fig.6a). However, in cases without any lamellae the cell protrusions also diminish increasingly and the epithelium turns into the more or less smooth surfaced B zone which includes the fornix conjunctivae. Here the surface of the luminal cells is covered weakly by irregularly distributed slender cell processes, microplicae or microvilli. Thus the marginal elevations as described above for the A zone or even small grooves between adjacent cells can be identified, representing the apical cell borders.

Goblet cells Goblet cells are restricted mainly to the B zone. Their density increases in the direction of the fornix conjunctivae. This cell type can be identified in SEM as apical pores after secre-

Fig.S. a) Physignathus eocincinus, fenestrated capillary (Ca) of the lamina propria conjunctivae, associated with a macrophage (Ma) and lymphocyte (Ly); NE: nucleus of endothelial cell; NF: nerve fibre; arrowheads: basal membrane between conjunctiva and lamina propria; inset: fenestrated endothelium of capillary; TEM. x 7.000 (inset: x 28.000) b)Agama impalearis, myoneuronal contact (arrow) between smooth muscle cell (SM) and unmyelinated nerve fiber (NF); CC: collagenous fibers of connective tissue. TEM. xl 0.300 c) Physignathus eocincinus, lymphoid cell aggregations (LY) , partly associated with a plasma cell (PL) situated between epithelial cells (E); Me: mast cell; arrowheads: epithelial basal lamina. TEM. x 3.700

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Fig. 6. a) Agama impalearis*, transition from the A zone (Az, right) to the B zone (Bz, left) with minor epithelial formations, marked by dotted line; in the B zone the cell borders are visible clearly (arrows). SEM. x 1.200 b) Agama impa/earis, common great porus (PO) of goblet cell populations with mucoid material (MC) pouring onto the epithelial surface. SEM. x2.600

tion of the mucus. The release of the secretory products takes place after liquefaction of the secretory granules through a pore onto the conjunctival surface. Its diameter differs within a wide range, depending on the number of goblet cells involved (Fig. 6b). The secretory material consists mainly of a fine filamentous and/or granule containing matrix and reduced cell organelles, which vary greatly in number. The secretory masses may, in spite of the preceding treatment of the tissue, exceed the height of the epithelium proper. Normally, at least some residual rests still remain. As a rule, the mucoid droplets of the goblet cells do not undergo such liquefaction before extruding, which is also true for the secretory cells of the A zone. Their membranes are preserved until a short time before they are released individually, or in groups composed of the numerous granules themselves. The granules can also mature at different rates, which leads to a mosaic-like pattern of the cell content (Fig. 7 a). In course of time, the initially electron-dense aspect of the goblet cell turns to an electron-lucent one, which may be characteristic for all secreting goblet cells. The secretory product can be also found in SEM scattered all over the epithelial surface (see Figs. 2 b, 3 b, 6 b). In Agama impalearis, the density of the goblet cells increases exceedingly, forming an epithelial mucous gland. Obviously groups of the goblet cells mature at the same rate, so that in mature parts this glandular region can be identified by numerous pores and gaps (Fig. 7b). In SEM the mucus can be traced back as a filamentous, sometimes also granule-containing material, which occasionally can be observed flowing together on the conjunctival surface. The junctional complex of the goblet cells described is developed in a similar way to that in the other epithelial cells, but is less pronounced. So the number of desmosomes of the lateral interdigitating cell protrusions is decreased, as is the development of the intermediate filaments.

A further type of cell with secretory activity can be found in both the A- and the surrounding B zones. Here the cells secrete their whole apical portion as spherical blebs which occur simultaneously in different regions. Their diameter varies to some extent, thus in Agama agama from 2 to 5 11m. After release the secretory droplets liquefy and may flow together as described above for the goblet cells. In Agama aculeata the secretory product can be traced back on the apical cell regions as uniform secretory blebs (Fig. 7 c). Special properties of the conjunctival epithelium and lamina propria of the disc-region in the lower eyelid will be published elsewhere.

Discussion The conjunctival outer morphology of the agamid species reported here is characterized mainly by the occurrence of epithelial folds running parallel to the margins of the eyelids. These folds are present in all the agamid species studied in a more or less pronounced manner and include the lamina propria conjunctivae. In this way it is possible to divide the ocular side of the agamid eyelid into a lamellated A zone and a smooth B zone, in which goblet cells are predominantly situated. Winckels (1914) distinguished different palpebral regions by means of the goblet cells in Voeltzkowia mira as did Oelrich (1956) in Ctenosaura pectinata. The first mention of reptilian conjunctival lamellae, however, was published by Duke-Elder (1958) in crocodiles. Also in mammalian vertebrates the subdivision of the conjunctiva is preferably based on the goblet cell density (for instance, Parsons 1904; Weingeist 1973; Rohen and Steuhl 1982; Weyrauch 1983; Steuhl and Rohen 1984). Conjunctival lamellae have so far been described in lizards (Hiller and Dieterich 1986) and crocodiles (Duke-Elder 1958) only.

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Fig. 7. Secretory cells of the B zone. a) Physignathus cocincinus, secretory droplets of a mature goblet cell filled with numerous granules; with increasing maturity the dark regions within the droplets are reduced. TEM. x 21 .000 b) Agama impalearis*, mature epithelial mucous gland, consisting of numerous epithelial pores of goblet cell populations; Me: mucoid material. SEM. x 315 c) Agama anchieta*, secretory cells found in both A zone and B zone; SB: apically extruded spherical secretory blebs (freeze fracture); arrows: apical junctional cell complex. SEM. x3.500

Also the polymorphism of cell protrusions and the related release of secretory material surpass those of domestic ruminants (Weyrauch 1983) and man (Greiner et al. 1980), which is accompanied by the separation of cell portions, as for instance, proliferated protrusions and joined by a mostly prominent terminal web system. The functional significance of the lamellae can be regarded as an equipment for removing invading particles by gathering them in the interlamellar clefts. Because of the congruence between the partially broadened rims of the lamellae and the surface of the bulbus on the other side, even very small particles my be swept off. Subsequently they will be transported, together with cell debris which originates mainly from separated cell protrusions and degenerating cells, to the rostral palpebral corner by means of the tear flow or/and the nictitating membrane. Here it can be often observed deposited as dried material. Because of the close contact of the free distal surface, the cell protrusions are reduced or at least flattened in this region only. This "physiological adaptation" was traced back experimentally by Podhoranyi et al. 1968. The related regeneration of conjunctival cells could be demonstrated in the conjunctiva of various mammals (Stichting et al. 1966; Binder 1952; Hanna and O'Brien 1960). According to Hazlett et al. (1980) the occurrence of electron light and -dark cells, like the morphology of the associated apical cell membranes, might be correlated with the different ages of cells. So the electron light cells turn out to be secretory cells undergoing degeneration characterized by swollen or even reduced mitochondria as well as by porous apical cell membranes. The latter fact makes it possible to identify cells with secretory activity by means of SEM. Young (TEM electron dense) epithelial cells demonstrate a well-developed apical morphology in spite of the degenerating ones. They can be identified in SEM as clear areas caused by increased electron contamination. Thus they form a mosaic-like pattern together with dark areas of degenerating (TEM electron lucent) cells with reduced protrusions. In this way a great variability of the cell surface morphology of identical conjunctival regions in the eyelids could be traced. This is true even for the same individual demonstrating the intense cell dynamic processes of the agamid conjunctiva. Additionally this suggests - also on the base of the well-developed apical junctional complex that the lamina propria conjunctivae may be lined by a typical transport epithelium. These results include the peripheral B zone. As far as is known, the goblet cell density in all vertebrates increases in the direction of the fornix conjunctivae, but the functional significance of this regionally different occurrence is unknown. Possibly the mucous masses might obstruct the sight, if the goblet cells occurred centrally. The concentration of goblet cells in the periphery turns the agamid conjunctival epithelium into regions which can be defined also in these lizards as mucous glands (lshikuro 1903; Podhonlnyi 1966). The significance of the goblet cell activity in both higher and lower vertebrates such as reptiles is discussed extensively elsewhere (see Kessing 1968; Amend

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1991). The goblet cells of the B zone in the reptiles studied may at least serve for the cleaning and lubrication of the ocular adnexa. Finally, the lamellae in the A zone may mainly remove particles. Also most of the other types of secretory cells as demonstrated in this study can be found in higher vertebrates such as mammals. Thus Atoji et al. (1993) demonstrated in the infraorbital glands of Capricorn is crispus blebs producing secretory cells with identical morphology as shown in Agama aculeata which can be classified as apocrine cells. Both cases retain "crown-shaped structures ". The lamina propria of the A- and B zones is, in spite of the conjunctival epithelium, largely homogeneous. The course of the basal membrane is very smooth, but sometimes there are deep protrusions towards the directly underlying connective tissue, defined by Farquhar and Palade (1965) as anchoring fibrils. Similar findings are known in the lamina propria of the human cornea (Gipson et al. 1987; Khodadoust et al. 1969; Barge et al. 1991). The functional significance can be described after Burgeson (1987) and Regauer et al. (1990) as dermoepidermal junctions. This may play a special functional role in agamids because of their protruded and highly movable eyes, covered by appropriate eyelids. As in other vertebrates (Oppenheimer et al. 1958) the lamina propria of the eyelids in agamids is supplied by nervous tissue, which includes both unmyelinated and myelinated nerve axons of various calibres. Intraepithelial nerve structures like those in reptilian cutaneous mechanoreceptors (Hiller 1976, 1978), which also supply the outer epidermal generation of the eyelids, were not found . The repeatedly described "synapses en distance" (ref. Bloom and Fawcett 1986) could be shown likewise as in Chamaeleonidae and Jguanidae, too (Hiller, unpublished). The contractility of the tissue obviously leads to an optimal contact between both the conjunctiva palpebrae and the surface of the bulbus even during extensive eye movements. Furthermore, the occurrence of fenestrated capillaries in the lamina propria emphasizes the high physiological activity of the overlying epithelium. This is also true for the abundant free cells in both lamina propria and conjunctival epithelium, representing the immunological system of the ocular adnexa. The mast cell structure is similar to that of man (Iwamoto and Smelser 1965). Thus as in the human conjunctiva (Ratnakar et al. 1976), an immune activity can be assumed in the agamid lizards studied as well as in snakes (Ahne 1994) because of this cell type, as it is true for plasma cells (Walker and Isselbacher 1977). In these agamids they occur in all parts of the conjunctiva, even near the epithelial surface. This implies a direct release of the secretory product into the conjunctival sac. Lymphocytes are also to be found scattered in the lamina propria and the epithelium itself with the exception of the B zone, perhaps because of the occurrence of goblet cells. Lymphoid tissue in reptilian eyelids was described first by Leydig (1872) in Lacerta agilis and Lacerta viridis. Generally, intraepithelial lymphocyte migration is, as in this study,

frequently observed in human skin (Dustin et al. 1988). Uccini et al. (1993) described the enzymatic digestion of the epithelial basement membrane followed by the intraepithelial migration of lymphocytes. Bianchi et al. (1993) found lymphoid infiltrations in the esophageal epithelium of turtles and deduced an uptake of extracellular materials. Because of the similarly abundant intraepithelial free cells found in both the lamina propria and conjunctiva itself an immunological activity of the agamid eyelid can be strongly assumed, thus protecting the ocular adnexa from invading bacteria. However, the agamid lizards studied lack conjunctival lymphatic follicles as described in homing pigeons by Racket (1991) and in rabbits (Franklin and Remus 1984). Because only a few publications are concerned with the reptilian conjunctiva on the submicroscopic level, systematic comparative conclusions require further studies on reptiles with movable eyelids, e. g. Iguanidae, Chamaeleonidae, and Crocodylidae. Acknowledgements: I am very much indebted to PD Dr. W. Bohme, Bonn, Dr. Faust, Zoo Frankfurt/a. M., and Dr. K. Klemmer, Forschungsmuseum Senckenberg, Frankfurt/a. M., for letting me have all the samples required. Furthermore I thank Mrs. I. Schroeder for preparation of the TEM-specimens and Mrs. S. Seidel for her technical assistance in the skilful preparation of both the SEM samples and the photographic figures as well as for the PC-documentation of the literature.

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