ultrastructural study - Europe PMC

465

J. Anat. (1992) 180. pp. 465-480, with 31 figures Printed in Great Britain

The harderian gland of desert rodents: ultrastructural study

a

histological and

YASMINA DJERIDANE Institut de Biologie, Universite des Sciences et de la Technologie Houari Boumedienne, 'U.S. T.H.B.', BP 39, Algiers, Algeria

(Accepted 26 February 1992)

ABSTRACT

This study describes the structure of the harderian gland in desert rodents: 3 Gerbillidae species (Gerbillus gerbillus, Meriones crassus, Psammomys obesus) and 1 Ctenodactylidae species (Ctenodactylus vali). In all these species the gland consists of tubules lined by a single layer of epithelial cells and possesses myoepithelial cells within their basal laminae. The gland contains porphyrin which is stored as solid intraluminal deposits. The glandular epithelium presents a single cell type (type I) in Psammomys obesus, 2 cell types (I and II) in Ctenodactylus vali and 3 (I, II and III) in Gerbillus gerbillus and Meriones crassus. The type I and II cells are columnar. They are characterised by many lipid vacuoles and a well developed vesicle-like structure of smooth endoplasmic reticulum. In Gerbillus gerbillus and Meriones crassus the type I cells can be distinguished from type II cells by cytoplasmic clefts approximately 1 gm in length. In Ctenodactylus vali type I cells are characterised by cytoplasmic rod-shaped crystalloid structures approximately 0.5 gim in length which are frequently observed in the mitochondrial matrix. These structures are also present in the sole cell type of Psammomys obesus. Most of the secretory lipid vacuoles of the type I cell contain an electron-dense material, possibly porphyrin, which presents different appearances according to species: it is lamellar in Gerbillus gerbil/us, trilamellar in Meriones crassus, and amorphous in Psammomys obesus and Ctenodactylus vali. Secretory lipid vacuoles are released primarily by exocytosis, but holocrine and apocrine secretion is also observed. The type III cells are pyramidal. This cell type is characterised by the presence of an extraordinarily well developed granular endoplasmic reticulum, organised in concentric lamellae in Gerbillus gerbillus, and very numerous mitochondria. Epithelial cells are frequently binucleate. The single excretory duct contains both mucous and serous cells. Mast cells, plasma cells, macrophages, fenestrated capillaries and unmyelinated nerve endings with clear or dense-cored vesicles are present in the connective tissue. Melanocytes are very numerous in the interstices of the Gerbillidae harderian gland. The gland is surrounded by a collagenous capsule and an outer layer of endothelial cells derived from the orbital venous sinus. INTRODUCTION

The harderian gland is a large orbital organ found in vertebrates that possess a nictitating membrane (Sakai, 1981). The function of this gland is generally considered to be for the protection and lubrication of the cornea. Recent investigations have demonstrated that the gland may also be part of a retinal-pineal axis (Wetterberg et al. 1970; Reiter & Klein, 1971) involved in immune responses (Burns, 1979), a source of pheromones (Thiessen et al. 1976; Payne, 1979) and a source of thermoregulatory lipids (Thiessen & Kittrell, 1980). In rodents, the harderian gland is used

as a model for porphyrin biosynthesis since the

histochemical studies of Cohn (1955) have shown that the gland contains large amounts of porphyrin pigment bound to lipids. The cytological structure of the harderian glands has been fully studied in rodents. However, to our knowledge, only 4 ultrastructural studies have been undertaken in desert rodents. These have been for the golden hamster (Bucana & Nadakavukaren, 1972 a), the mongolian gerbils Meriones meridianus (Sakai & Yohro, 1981) and Meriones unguiculatus (Johnston et al. 1983), and the Plains mouse Pseudomys australis (Johnston et al. 1985). The present investigation is the first detailed

Correspondence to Dr Y. Djeridane, Lotissement Tournier et Baldot No. 8, Kouba, Algiers, Algeria.

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Yasmina Djeridane

Fig. 1. Photograph of the eye and the harderian gland of Psammomys obesus. The gland (H) is situated at the posterior part of the eyeball (E). Fig. 2. Photograph of the harderian gland of Psammomys obesus. The gland in this species is black-brown in colour due to the presence of numerous melanocytes in the interstices of the gland. Fig. 3. Light micrograph from a 5 gm section of the harderian gland of Meriones crassus stained with haemalum-eosin. The end sections of the gland consist of tubules with wide lumina, lined by a single layer of epithelial cells. The nuclei are situated basally. x 300. Fig. 4. Meriones crassus harderian gland. Mast cells (arrows) are frequently present in the connective tissue capsule (C). The gland is covered by the endothelium of the orbital venous sinus (arrowheads). Haemalum-picro-indigocarmine, x 500. Fig. 5. Gerbillus gerbillus harderian gland. Septa from the thin connective tissue capsule penetrate the gland, dividing it into unequal-sized lobules. Cresyl violet, x 35.

survey for Saharan rodents. It was initiated in order to determine the ultrastructural characteristics of the harderian gland from 3 species of Gerbillidae (Gerbillus gerbillus, Meriones crassus and Psammomys

obesus) and 1 species of Ctenodactylidae (Ctenodactylus vali). During this study, we noticed a number of interesting characteristics of the gland that had not previously been reported in other species.

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Animals The tissues used in this study were collected from 10 Gerbillus gerbillus, 8 Meriones crassus, 8 Psammomys obesus and 4 Ctenodactylus vali, all of which were captured in the Algerian Sahara. The animals were sexually mature. They were divided into 2 groups: (1) for ultrastructural studies: 6 Gerbillus gerbillus (3 males, 3 females), 5 Meriones crassus (3 males, 2 31

females), 5 Psammomys obesus (2 males, 3 females) and 2 Ctenodactylus vali (1 male, 1 female); (2) for histological studies: 4 Gerbillus gerbillus (2 males, 2 females), 3 Meriones crassus (1 male, 2 females), 3 Psammomys obesus (2 males, 1 female) and 2 Ctenodactylus vali (1 male, 1 female). Electron microscopy

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Fig. 8. Electron micrograph of the apical region of epithelial cells from the Psammomys obesus harderian gland. The cytoplasm contains a large number of secretory lipid vacuoles (Lv). Electron-dense material (arrows) is frequently seen coating the inside of the membrane of the vacuoles. The lumen (Lm) contains varying amounts of amorphous material (large arrow) presumably derived from the cytoplasmic vacuoles and may consist of porphyrin compounds. Coated vesicles in the process of formation (arrowheads) are seen at the base of the microvilli (Mv). Large areas of cytoplasm are filled with vesicle-like structures of smooth endoplasmic reticulum (Ser). Crystalloid structures (cn) may be seen near the lower lipid vacuole (Lv). The secretory vacuole fusing with the apical cell membrane is indicative of merocrine secretion. Jc, junctional complex, x 16000. Fig. 9. A high power electron micrograph of the nuclear region of a glandular cell from the harderian gland of Psammomys obesus. The glandular endoplasmic reticulum (Ger) surrounds the nucleus (N). Mitochondria (M) and rosettes of ribosomes (R) can be seen. Lateral membranes exhibit interdigitations (Inc). x 25000. Fig. 10. Harderian gland of Psammomys obesus. Light micrograph of a 5 iim eosin-stained section. The lumina of the gland tubules contain solid porphyrin accretions (arrows). Melanocytes are present in the interstices (arrowheads). x 300.

Harderian gland of desert rodents injection of pentobarbital sodium and perfused via the left ventricle with physiological saline followed by fixative. The fixative used was a mixture of 2.5% glutaraldehyde and 2 % paraformaldehyde buffered at pH 7.3 with 0.1 M phosphate for 30 min. The harderian glands were removed and diced into approximately 1 mm cubes and placed in the same fixative at a temperature of 4 °C for a further 90 min. The tissue blocks were rinsed in 0.1 M phosphate buffer (pH 7.3) and postfixed in 4 % osmium tetroxide in 0.1 M phosphate buffer at room temperature for 2 h. After postfixation, the tissues were dehydrated in a graded ethanol series, cleared in propylene oxide and embedded in Epon 812. Semithin sections (1 gm) for light microscopy, as well as ultrathin sections (thin sections) for electron microscopy were sectioned on a Reichert Ultracut. The semithin sections, mounted on glass slides, were stained with 1 % toluidine blue in 1% borax for 1-2 min at 60 'C. Grids with thin sections were stained with uranyl acetate followed by lead citrate (Reynolds, 1963) and examined in a Philips EM-420 transmission electron microscope.

Light microscopy Animals were killed by pentobarbital sodium. Harderian glands were immersed and fixed in toto in Bouin's fixative for 24 h. The tissues were rinsed in water, dehydrated in ethanol and processed for routine paraffin sectioning; 5 jm serial sections were cut through the entire gland. Tissues were stained with haemalum-eosin, cresyl violet and haemalum-picroindigocarmine for general morphological studies, and periodic acid-Schiff for carbohydrates. RESULTS

Within each species, there were no structural differences between the male and the female harderian glands.

Light microscopy The harderian gland (Figs 1, 2) which is located at the posterior pole of the globe of the eye, is enclosed by a connective tissue capsule (Fig. 4). Septa from this capsule penetrate into the gland dividing it into several lobules (Fig. 5). The end sections of the gland consist of branching tubules with wide lumina, lined

469

by a single layer of epithelial cells (Fig. 3). Semithin sections stained with toluidine blue demonstrate that the glandular epithelium presents 1 cell type in Psammomys obesus, 2 in Ctenodactylus vali and 3 in Gerbillus gerbillus and Meriones crassus. The 3 cell types will be designated as I, II and III cells. The type I and II cells are columnar in shape. Their cytoplasm is acidophilic and filled with lipid vacuoles (Fig. 17). The difference between both cell types is revealed only after close scrutiny of semithin plastic sections. The type I cells can be distinguished from the type II cells by the presence in some vacuoles of a strongly blue-green stained 'stringy' material. The type III cells are pyramidal in shape with basophilic cytoplasm (Fig. 17). They are situated basally to the type I and II cells and are inserted between them. These cells never reach the lumina. The glandular epithelium is comprised of type I cells in Psammomys obesus, type I and II cells in Ctenodactylus vali, and type I, II and III cells in Gerbillus gerbillus and Meriones crassus. All cell types are frequently binucleate (Fig. 17). Myoepithelial cells or their cytoplasmic extensions are sometimes observed at the base of the secretory epithelium. The nuclei of the myoepithelial cells lie parallel to the basal lamina. The lumina of the end sections contain varying amounts of homogeneous material. In Psammomys obesus and Meriones crassus, the lumina of the tubules frequently contain an accretion of reddish-brown porphyrin pigment (Fig. 10). In Gerbillus gerbillus and Ctenodactylus vali, pigment is rarely observed. If present, it consists of pale gold-yellow granules. Intact harderian glands of Gerbillus gerbillus and Ctenodactylus vali display a pale yellow-green colour when placed under ultraviolet light as compared with the typical reddish-purple colour emitted by glands of Psammomys obesus and Meriones crassus. The yellow-green fluorescence may indicate few porphyrins in Gerbillus gerbillus and Ctenodactylus vali harderian glands. In all our species, no ducts are observed within the gland. There is a single specialised extraglandular duct. Tubules are directly connected with each other. They are led into a long intraglandular and central tubule, which in turn empties into the extraglandular duct. The central tubule is lined by a single layer of typical columnar tubule cells (Fig. 6). In all our species, the beginning of the duct is lined by a

Fig. 11. Electron micrograph of the type I cell of Ctenodactylus vali harderian gland. The nucleus is large, spherical, basally located and contains a prominent nucleolus and considerable amounts of euchromatin. A nerve ending with clear vesicles (arrows) is seen within the basal lamina (Bi) between secretory and myoepithelial (Me) cells. x 8000. Fig. 12. Apical region of a tubule cell of Ctenodactylus vali harderian gland. Lipid vacuoles (VI, V2) are released into the lumen (Lm) by exocytosis. Mv, microvilli x 15000. 31-2

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Fig. 13. (a) Electron micrograph of the apical portion of the type I cell of Meriones crassus harderian gland. Straight trilamellar structures (arrows) 0.3 pm in length and 0.02 pm in width are present in 2 lipid vacuoles (Lv). x 33 500. (b) High power electron micrograph showing the trilamellar structure of the inclusions, with a lighter central core and electron-opaque outer rims (arrows), in lipid vacuoles (Lv). x 6800. Fig. 14. High power electron micrograph of electron-dense material (arrow) in a lipid vacuole (Lv) from a type I cell of Gerbillus gerbillus harderian gland. This material which lines the vacuole membrane (Vm) appears lamellar. x 100000. Fig. 15. Portion of a tubule cell from Psammomys obesus harderian gland showing rosettes of ribosomes (R) and crystalloid structures (CO. The periodicity of the crystalline material is about 17.5 nm. x 47000. Fig. 16. Apical portion of type I cell of Ctenodactylus vali harderian gland illustrating cytoplasmic and intramitochondrial (M) rod-shaped crystalloid structures (Cl) and lipid vacuoles (Lv) containing amorphous material (arrows). Mv, microvilli; Lm, lumen. x 42000.

stratified epithelium with serous cells. Near the medial border of the gland, the extraglandular duct is lined by a stratified squamous epithelium and mucussecreting cells (Fig. 7). It empties onto the concave surface of the nictitating membrane. The lumina of

the central tubule and the extraglandular duct are filled with lipid vacuoles and cellular debris (Fig. 6). The interstices of the gland in all our species contain blood vessels, fibroblasts, nerve fibres, macrophages, plasma cells and mast cells. The latter occur frequently

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Fig. 17. Harderian gland of Gerbillus gerbillus showing tubules with vacuolated acidophilic columnar cells (types I and II) and basophilic pyramidal cells (type III). Some cells are binucleate (arrowheads). Lm, lumen. Toluidine blue, x 500. Fig. 18. Electron micrograph of portion of a type III cell from Gerbillus gerbillus harderian gland showing numerous mitochondria with dense matrices (M) and abundant profiles of granular endoplasmic reticulum (Ger) which is organised into concentric lamellae. x 9800. Fig. 19. Low power electron micrograph of type III cell of Gerbillus gerbillus harderian gland. This cell type is characterised by very numerous mitochondria and well developed granular endoplasmic reticulum. Epithelial cells, here types II and III, are bound together and to the myoepithelial cell (Me) by interdigitations (arrows). x 9000.

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Fig. 20. Electron micrograph of the harderian gland of Meriones crassus showing the type I and II cells. The type I cell is characterised by cytoplasmic clefts which frequently contain a needle-like structure (arrows). The perinuclear region of both cell types is occupied by granular endoplasmic reticulum (Ger). The cytoplasm of the cells is filled with the smooth endoplasmic reticulum (Ser). Two nuclei (N) are seen in the type II cell. Both types lie on a prominent basal lamina (BO). x 9000. Fig. 21. Electron micrograph of type III cell of Meriones crassus harderian gland. Type III cells are characterised by a highly developed granular endoplasmic reticulum and considerable numbers of mitochondria. I, type I cell. x 4400. Fig. 22. Electron micrograph of type III cell of Meriones crassus harderian gland showing a paracrystalline formation related to the granular endoplasmic reticulum (Ger) which is surrounded by numerous mitochondria (M). Lg, lipofuscin granule. x 23000.

473

Harderian gland of desert rodents in the loose connective tissue, either around blood vessels or under the surface of the gland (Fig. 4), and in the thick layers of connective tissue surrounding the extraglandular duct. In addition, numerous melanocytes are present in the interstices of the Gerbillidae harderian gland, especially in Psammomys obesus (Fig. 10). They impart a dark brown/grey colour to the gland (Fig. 2). The melanocytes are densely populated around the extraglandular duct. The capsule is surrounded by an endothelium, which macroscopically produces the smooth surface of the gland (Fig. 1). It is stated by Sakai & Yohro (1981) to represent the endothelium of the orbital venous sinus.

Electron microscopy

The secretory cell types are best distinguished by electron microscopy. The type I cell is the most common. It occupies approximately 40 % of the epithelial cells in Gerbillus gerbillus and 60 % in Meriones crassus and Ctenodactylus vali, whereas it is the sole cell type in Psammomys obesus. The type I and II cells have most features in common. The apical cell border of both is covered with short microvilli (Figs 8, 12, 16) and the apical ends bulge into the lumina. The cell membranes of neighbouring cells are bound together at their apices by junctional complexes (Fig. 8). The lateral and basal membranes are generally smooth, but in some cases pronounced interdigitations (Figs 9, 19) and microvilli are seen. In Meriones crassus prominent gap junctions are identifiable between the type I and II cells. The nucleus of both cell types is large, spherical, basally located and contains a prominent nucleolus (Fig. 11). In Ctenodactylus vali, euchromatin occupies the major part of the volume of the nucleus (Fig. 11). The perinuclear region of the 2 cell types contains a relatively well-developed granular endoplasmic reticulum (Figs 9, 20). Abundant rosettes of ribosomes (Figs 9, 15) occur randomly elsewhere throughout the cytoplasm of both cell types, and large areas of this cytoplasm are filled with an extensive network of small vesicle-like structures of smooth endoplasmic reticulum (Figs 8, 20). No typical Golgi complexes are observed. Pleomorphic mitochondria with a dense matrix and irregularly spaced cristae (Fig. 9) are randomly dispersed in the 2 cell types. Coated vesicles are often seen in the process of formation at the base of the microvilli (Fig. 8) or complete in the apical and basal cell cytoplasm. The entire apical half of the cells of both types is filled with lipid vacuoles (Figs 8, 12, 13, 16) whose

maximum diameter reaches approximately 1.7 gm in Gerbillus gerbillus, 1 lim in Meriones crassus, 2.7 gm in Psammomys obesus and 2.5 gim in Ctenodactylus vali. A few lipid vacuoles are also noted in basal areas of the cells. However, the vacuoles of the type I cell are clearly different from those of the type II cell. Some vacuoles of the type I cell frequently contain an electron-dense material, either coating the inside of the vacuole membrane or lying within the vacuole. This material is superimposable on the stringy material observed by light microscopy. It presents different appearances according to the species: it is amorphous in Psammomys obesus (Fig. 8) and Ctenodactylus vali (Fig. 16), lamellar in Gerbillus gerbillus (Fig. 14) and trilamellar in Meriones crassus (Figs 13a,b). Furthermore, in both types of cell, secretory vacuoles are released in the lumina, essentially by exocytosis (Figs 8, 12), but holocrine and apocrine secretion also occurs, and nuclear and cytoplasmic debris is often seen. In Meriones crassus and Psammomys obesus, the lumina of the end sections contain varying amounts of electron-dense material, presumably derived from the cytoplasmic vacuoles, which may consist of porphyrin compounds (Fig. 8). The most characteristic feature of the type I cell of Gerbillus gerbillus and Meriones crassus is the presence of cytoplasmic clefts (Fig. 20), approximately 1 gm in length. They are not membrane-bound and frequently contain needle-like dense material approximately 0.9 gm in length, usually lying to one side. In Ctenodactylus vali the type I cells are characterised by cytoplasmic dense rod-shaped crystalloid structures (Fig. 16) approximately 0.5 gm in length. They are often seen in the mitochondrial matrix. These cytoplasmic and intramitochondrial rod-shaped crystalloid structures are also present in the sole cell type of Psammomys obesus (Figs 8, 15). The periodicity of the crystal is more evident in Psammomys obesus: it is about 17.5 nm. The type III cells are peculiar to Gerbillus gerbillus (Fig. 19) and Meriones crassus (Fig. 21). They represent, as a percentage of the total epithelial cells, approximately 15 % in Meriones crassus and 30 % in Gerbillus gerbillus. The nucleus is located in the medial part of the cell. It is spherical in shape and contains a prominent nucleolus (Fig. 21) and abundant heterochromatin (Fig. 19). The most prominent feature of this cell type is the presence of very numerous mitochondria with moderately dense matrices and an extraordinarily well developed granular endoplasmic reticulum which is organised into concentric lamellae in Gerbil/us gerbil/us (Fig. 18). some

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Fig. 23. Electron micrograph of harderian gland of Psammomys obesus showing an interstitial macrophage and an axon containing clear vesicles (arrow). x 10000. Fig. 24. Electron micrograph of a mast cell in the interstices of Psammomys obesus harderian gland. It contains granules of varying size and shape. x 10000. Fig. 25. Electron micrograph showing interstitial cells, a plasma cell (P1) and a melanocyte (MO) in the harderian gland of Psammomys obesus. They are lying close to epithelial cells (Ec). x 5000. Fig. 26. Electron micrograph of the surface of the harderian gland of Psammomys obesus. The gland is covered with the endothelium of the orbital venous sinus (End). Its surface bears small irregular projections (arrows). Unmyelinated axons are seen in the connective tissue, adjacent to a myoepithelial cell (Me). NS, nucleus of Schwann cell. x 10000.

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Fig. 27. Electron micrograph of a fenestrated capillary (Cap) in the Gerbillus gerbillus harderian gland. Fenestrations are numerous (arrowheads). The capillary and epithelial cells (Ec) are separated by the basal laminae (Bo) of the 2 cells and loose connective tissue containing collagen fibrils (Cg). The basal pole of the epithelial cells demonstrates microvilli (arrows). x 10000. Fig. 28. Electron micrograph of a myoepithelial cell (Me) in a tubule from the harderian gland of Psammomys obesus. This cell possesses an elongated nucleus and numerous myofilaments, and lies between an epithelial cell (Ec) and the basal lamina (Bo). x 10000. Fig. 29. Electron micrograph of the endothelium of the orbital venous sinus (End) surrounding a tubule in the Gerbillus gerbillus harderian gland. A dense acellular layer of collagen fibres is seen in the capsule (C). Me, myoepithelial cell; Ec, epithelial cell. x 8000. Fig. 30. Plasma cell with dilated profiles of granular endoplasmic reticulum in Ctenodactylus vali harderian gland. Russell bodies (Rb) are present in this cell. x 12400.

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Harderian gland of desert rodents structures related to the granular endoplasmic reticulum (Fig. 22). Some lipofuscin granules (Fig. 22) are also present in this cell type in both species. Several cell folds and interdigitations (Fig. 19) are present on the basal and lateral faces of the type III cell. The comparative structure of the harderian epithelial cells of all our species has been represented schematically in Figure 31 a-c. Long fusiform myoepithelial cells lie between the basal lamina and the epithelial cells (Fig. 28). They are characterised by an elongated nucleus, numerous myofilaments running in the long axis and numerous micropinocytotic vesicles on the surface of the cytoplasmic processes. These cells are attached to adjacent epithelial cells by desmosomes and interdigitations (Fig. 19). Cells which are apparently in contraction show a corrugated profile. In all our species, only unmyelinated nerve fibres are encountered (Figs 23, 26). Nerve endings with clear or dense-cored vesicles are observed in the connective tissue interstices close to the epithelial cells, myoepithelial cells and capillaries. In Ctenodactylus vali nerve endings are also observed within the basal lamina of tubules, between epithelial and myoepithelial cells (Fig. 11). They contain clear vesicles. Synapses with either epithelial cells or myoepithelial cells are not seen. The harderian gland of all our species has a very high vascular supply with numerous fenestrated capillaries which are in close relationship to the tubules (Fig. 27). The basal pole of most secretory cells adjacent to these capillaries demonstrates a greater concentration of microvilli. It is not surrounded externally by cytoplasmic expansions of the myoepithelial cells (Fig. 27). Mast cells (Fig. 24), macrophages (Fig. 23), and plasma cells containing Russell bodies (Mott cells) (Figs 25, 30) occur frequently in the interstitial areas of the harderian gland. Melanocytes are peculiarly numerous in the Gerbillidae harderian gland. Their attenuated cytoplasmic extensions (Fig. 25) occur throughout the gland. The connective tissue capsule is composed of a denser acellular layer of collagen fibres (Fig. 29). The capsule is surrounded by a delicate, endothelium derived from the orbital venous sinus (Figs 26, 29).

DISCUSSION

The present description of the Gerbillidae (Gerbillus gerbil/us, Meriones crassus, Psammomys obesus) and the Ctenodactylidae (Ctenodactylus vali) harderian

477 glands supports the descriptions in other rodent species in that their gland tubules are lined by a single layer of epithelial cells within their basal laminae and that the gland contains porphyrin which is stored as solid intraluminal deposits. These general features may be compared with the gland structure in the rat (Orban & Kelenyi, 1962; Brownsheidle & Niewenhuis, 1978), the mouse (Woodhouse & Rhodin, 1963; Watanabe, 1980; Strum & Shear, 1982 a), the golden hamster (Bucana & Nadakavukaren, 1972 a), the Plains mouse Pseudomys australis (Johnston et al. 1985), the Gerbillidae Meriones meridianus (Sakai & Yohro, 1981) and Meriones unguiculatus (Johnston et al. 1983), and the rabbit (Bj6rkman et al. 1960; Kiihnel, 1971). However, as in Pseudomys. australis, the gland in Gerbillus gerbillus and Meriones crassus is unusual in that 3 distinct cell types can be distinguished, as compared with the 2 cell types reported for the mouse, rat and male golden hamster, and documented in the present study for Ctenodactylus vali, and 1 cell type reported for the female golden hamster, the Gerbillidae Meriones meridianus, Meriones unguiculatus and described in the present study for Psammomys obesus. With its numerous lipid vacuoles, the type II cell noted in Ctenodactylus vali, Gerbillus gerbillus and Meriones crassus resembles those already reported in other species. The type I cell is characterised in Gerbillus gerbillus and Meriones crassus by cytoplasmic clefts, and in Psammomys obesus and Ctenodactylus vali by rod-shaped crystalloid structures. It is unlikely that the cytoplasmic clefts are fixation artefacts. All other ultrastructural features appear normal. The cytoplasmic clefts have been reported in another Gerbillidae species, namely Meriones unguiculatus by Johnston et al. (1983), and in the Plains mouse Pseudomys australis by Johnston et al. (1985), whereas the rod-shaped crystalloid structures described in Psammomys obesus and Ctenodactylus vali have never been reported in other rodent harderian glands. Nevertheless, Maxwell et al. (1986) described rod-shaped crystalline structures in the turkey harderian gland, but they appear to be quite different from those of the present study. The type III cell noted in Gerbillus gerbillus and Meriones crassus is characterised by numerous mitochondria and an extraordinarily well developed granular endoplasmic reticulum. These cytological features reflect the high activity of this cell type and indicate that it is oriented towards protein secretion. No cell corresponding to this has yet been reported in other rodent harderian glands. The sexual dimorphism noted in the hamster (Bucana

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not occur in Gerbillus gerbillus, Meriones crassus, Psammomys obesus or Ctenodactylus vali, as this study indicates. In all our species, the lipid vacuoles in the type I cell contain an electron-dense material which presents different features according to species. It is amorphous in Psammomys obesus and Ctenodactylus vali, as in Meriones meridianus (Sakai & Yohro, 1981), Meriones unguiculatus (Johnston et al. 1983) and Pseudomys australis (Johnston et al. 1985), lamellar in Gerbillus gerbillus and trilamellar in Meriones crassus. Electron microscopic observations in the Meriones crassus and Psammomys obesus harderian glands reveal intraluminal osmiophilic masses, corresponding to the sites of brown pigment accumulation seen by light microscopy. The bulk of the pigment masses found in the lumina of the harderian gland of these species, seems to be identical to the dense component observed in the lipid vacuoles of their type I cell, suggesting that porphyrins may be selectively secreted by this cell type. The trilamellar structures observed in Meriones crassus have been described in the rat harderian gland by Carriere (1985). This author has suggested that such material represents deposits with a high content of protoporphyrin IX. We agree with this suggestion, since the investigations of Arvy & De Lerma (1961) have demonstrated by chromatography that in the harderian gland of Meriones crassus, M. shawi, M. vinogradovi and Gerbillus pyramidum protoporphyrin IX is the major free porphyrin made by the gland in these species. Similar findings have been reported in Meriones unguiculatus by Johnston et al. (1983). The content of the vacuole is released primarily by exocytosis, but apocrine and holocrine secretion is also observed. Similar mechanisms have been noted in Pseudomys australis (Johnston et al. 1985), whereas merocrine and holocrine secretion and only merocrine secretion have been observed respectively in Meriones unguiculatus (Johnston et al. 1983) and Meriones meridianus (Sakai & Yohro, 1981). Carriere (1985) believed that the holocrine process is the principal mode of porphyrin release. He reported in the rat that harderian gland free porphyrins are released through cell death. He suggested that this degeneration may be due to the fact that free porphyrins can be toxic to cells. In addition to its exocrine function (lipids and porphyrins) the harderian gland may also have an endocrine function. The best structural evidence in support of this is the blood supply, with numerous fenestrated capillaries noted in the interstices of the glands in all our species. Fenestrated capillaries have also been observed in the mouse (Strum & Shear,

1982a) and armadillo (Weaker, 1981). In our species, fenestrated capillaries have been observed in close relationship to the tubules. The surfaces of the cells adjacent to these capillaries demonstrate a greater concentration of microvilli. This relationship indicates an active exchange between the secretory cells and the blood vascular system and can also permit release of substances such as melatonin, since this hormone has been found in the harderian gland of reptiles and mammals (Bubenik et al. 1976; Vivien-Roels et al. 1981; Reiter et al. 1983). In all our species, only unmyelinated axons were observed in the interstices of the gland, close to the epithelial cells, myoepithelial cells and capillaries, whereas in the other species of Gerbillidae, Meriones meridianus (Sakai & Yohro, 1981) and Meriones Unguiculatus (Johnston et al. 1983), both myelinated and unmyelinated axons have been observed. In Ctenodactylus vali nerve endings have also been found within the basal laminae of the tubules. This localisation has been noted in the hamster (Bucana & Nadakavukaren, 1972 b) and the rabbit (Kiihnel, 1971). Two types of nerve endings have been observed in association with the above structures, clear cholinergic vesicles and dense-cored adrenergic vesicles. Huhtala et al. (1977) and Bucana & Nadakavukaren (1972b) have already reported, in the rat the hamster respectively, that the harderian gland receives both a cholinergic and an adrenergic nerve supply. The clear vesicles contained in the nerve endings located just outside the basal laminae of the tubules may release transmitter substances which diffuse through the basal laminae. These substances could stimulate either the myoepithelial or the epithelial cells, or both, inducing the release of the glandular secretory product. Mast cells are notably present in the interstices of the gland, either around blood vessels or under the surface of the gland, and around the extraglandular duct. Mast cells have also been noted in the harderian gland of Meriones meridianus (Sakai & Yohro, 1981), Meriones unguiculatus (Johnston et al. 1983), Pseudomys australis (Johnston et al. 1985) and hamster (Payne et al. 1982). Payne et al. (1982) proposed that anticoagulants are useful where porphyrins are laid down as large intraluminal accretions which might temporarily occlude blood vessels at the site of formation of the accretion or during its passage to the main secretory duct. There is little evidence for this view.

The harderian gland of Gerbillus gerbillus, Meriones and Psammomys obesus is characterised by a

crassus

dense interstitial population of melanocytes. This excess

of melanocytes has been described

previously

Harderian gland of desert rodents in other Gerbillidae species such as Meriones meridianus (Sakai & Yohro, 1981) and Meriones unguiculatus (Johnston et al. 1983), in the desert Muridae species Lemniscomys striatus (Abou-Harb & Abou-Harb, 1964) and in the Dipodidae species Jaculus jaculus or Kangaroo rat (unpublished observation). Strum & Shear (1982b) have found that the damage and squamous metaplasia in the harderian gland in albino mice exposed to high intensity light were a direct result of a radiant energy-dependent mechanism. These observations and correlations suggest that the presence of melanocytes in the harderian gland of these different rodent species, in spite of their being derived from different families, may be a physiological adaptation to life in a desert habitat. The pigment contained in melanocytes could play a role in protecting the harderian gland from solar radiation. The single extraglandular duct terminates at the nictitating membrane. In all our species, the outer opening of the duct is lined by serous and mucous cells. Similar findings have been reported in the Gerbillidae species Meriones meridianus (Sakai & Yohro, 1981) and Meriones unguiculatus (Johnston et al. 1983) and the desert Muridae species Lemniscomys striatus (Abou-Harb & Abou-Harb, 1964) and Pseudomys australis (Johnston et al. 1985). In all these desert species, mucus-secreting cells may be morphological adaptations (i.e. protection and lubrication of the eye) to life in a desert habitat. In conclusion, this study has demonstrated that the harderian glands show ultrastructural features which are peculiar to individual species. It is interesting to suggest that these features may bring to taxonomists valuable arguments for the distinction (sometimes difficult) between diverse rodent species, especially species of the large Gerbillidae family.

ACKNOWLEDGEMENTS

The author wishes to thank Dr Paul Pevet for reviewing this manuscript, Jean Claude Artault for photographic assistance and Haciba Djeridane for secretarial assistance.

REFERENCES

ABou-HARs M, ABou-HARB N (1964) La glande de Harder chez certains rongeurs sauvages desertiques et tropicaux et l'elaboration des porphyrines. Carnets de Recherches de la SociWte de Biologie 158, 2268-2270. ARWy L, DE LERMA B (1961) Sur la presence d'un pigment fluorescent de type porphyrinique dans la glande de Harder de Meriones crassus Sundevall, de M. shawi Duvernoy, de

479 M. vinogradovi Heptner et de Gerbillus pyramidum Geoffroy. (Rongeurs, Gerbillidae). Carnets de Recherches de rAcaddmie des Sciences de Paris 253, 1012-1014. BJORKMAN N, NICANDER L, SCHANTZ B (1960) On the histology and ultrastructure of the Harderian gland in rabbits. Zeitschrift fur Zellforschung und mikrokopische Anatomie 52, 93-104. BROWNSCHEIDLE CM, NIEWENHUIS RJ (1978) Ultrastructure of the Harderian gland in male albino rats. Anatomical Record 190, 735-754. BUBENIK GA, BROWN GM, GROTA LJ (1976) Immunohistochemical localization of melatonin in the rat Harderian gland. Journal of Histochemistry and Cytochemistry 24, 1173-1177. BUCANA CD, NADAKAVUKAREN MJ (1972a) Fine structure of the hamster Harderian gland. Zeitschrift fur Zellforschung und mikroskopische Anatomie 129, 178-187. BUCANA CD, NADAKAVUKAREN MJ (1972b) Innervation of the hamster Harderian gland. Science 175, 205-206. BURNS RB (1979) Histological and immunological studies on the

fowl lacrimal gland following surgical excision of Harder's gland. Research in Veterinary Science 27, 69-75. CARRIERE R (1985) Ultrastructural vizualisation of intracellular porphyrin in the rat Harderian gland. Anatomical Record 213, 496-504. CoHN S (1955) Histochemical observations on the Harderian gland of the albino mouse. Journal of Histochemistry and Cytochemistry 3, 342-353. HUHTALA A, HUIKuiu KT, PALKAMA A, TERvO T (1977) Innervation of the rat Harderian gland by adrenergic and cholinergic nerve fibres. Anatomical Record 188, 263-272. JOHNSTON HS, MCGADEY J, THOMPSON GG, MooRE MR, PAYNE AP (1983) The Harderian gland, its secretory duct and porphyrin content in the mongolian gerbil (Meriones unguiculatus). Journal of Anatomy 137, 615-630. JOHNSTON HS, McGADEY J, THOMPSON GG, MooRE MR, BREED WG, PAYNE AP (1985) The Harderian gland, its secretory duct and porphyrin content in the Plains mouse (Pseudomys australis). Journal of Anatomy 140, 337-350. KUHNEL W (1971) Struktur und Cytochemie der Harderschen Druse von Kaninchen. Zeitschrift fur Zellforschung und mikroskopische Anatomie 119, 384-404. MAXWELL MH, ROTHWELL B, MATTHEWS SA (1986) A fine structural study of the turkey Harderian gland. Journal of Anatomy 148, 147-157. ORBAN S, KELENYI G (1962) Electron microscopic observations on the Harderian gland of the rat. Acta Biologica Hungarica 13 (suppl. 2), 57-58. PAYNE AP (1979) Attractiveness of Harderian gland smears to sexually naive and experienced male golden hamsters. Animal Behaviour 27, 897-904. PAYNE AP, McGADNEY J, JOHNSTON HS, MOORE MR, THOMPSON GG (1982) Mast cells in the hamster Harderian gland: sex differences, hormonal control and relationship to porphyrin. Journal of Anatomy 135, 451-461. REITER R, KLEIN DC (1971) Observations on the pineal gland, the Harderian glands, the retina and the reproductive organs of adult female rats exposed to continuous light. Journal of Endocrinology 51, 117-125. REITER RJ, RICHARDSON BA, MATTHEWS SA (1983) Rhythms in immunoreactive melatonin in the retina and Harderian gland of rats: persistence after pinealectomy. Life Sciences 32, 1229-1236. RIEYNOLD ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17, 208-212. SAKAI T (1981) The mammalian Harderian gland: morphology, biochemistry, function and phylogeny. Archiva Histologica Japonica 44, 299-333. SAKAI T, YOHRO T (1981) A histological study of the Harderian

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Yasmina Djeridane

gland of Mongolian gerbils Meriones meridianus. Anatomical Record 200, 259-270. STRUM JM, SHEAR CR (1982a) Harderian glands in mice: fluorescence, peroxidase activity and fine structure. Tissue and Cell 14, 135-148. STRUM JM, SHEAR CR (1982b) Constant light exposure induces damage and squamous metaplasia in Harderian glands of albino mice. Tissue and Cell 14, 149-161. THIESSEN DD, CLANCY A, GOODWIN M (1976) Harderian gland pheromones in the mongolian gerbil Meriones unguiculatus. Journal of Chemical Ecology 2, 231-238. THIESSEN DD, KITTRELL EMW (1980) The Harderian gland and thermoregulation. Physiology and Behaviour 24, 417-424. VIVIEN-ROELs B, PEVET P, DuBois MP, ARENDT J, BROWN GM (1981) Immunohistochemical evidence for the presence of

melatonin in the pineal gland, the retina and the Harderian gland. Cell and Tissue Research 217, 105-115. WATANABE M (1980) An autoradiographic, biochemical, and morphological study of the Harderian gland of the mouse. Journal of Morphology 163, 349-365. WEAKER FJ (1981) Light microscopic and ultrastructural features of the Harderian gland of the nine-banded armadillo. Journal of Anatomy 133, 49-65. WETTERBERG L, GELLER E, YUWILER A (1970) Harderian gland: an extraretinal photoreceptor influencing the pineal gland in neonatal rats. Science 167, 884-885. WOODHOUSE MA, RHODIN JAG (1963) The ultrastructure of the Harderian gland of the mouse with particular reference to the formation of its secretory product. Journal of Ultrastructure Research 9, 76-98.