Importance of Electron Microscopy to reveal species-specific characteristics of gland secretion

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1 mportance of Electron Microscopy to reveal species-specific characteristics of gland secretion Gabriella Chieffi Baccari 1, Alessandra Santillo 1, and Sergio Minucci 2 1 Department of Life Sciences, Second University of aples, via Vivaldi 43, Caserta; 2 Department of Experimental Medicine, Second University of aples, via Costantinopoli 16, apoli taly Tel: , Fax: ; gabriella.chieffi@unina2.it Secretory granules of Squamata Harderian glands (HG) show a different ultrastructure as compared to those described for Chelonian HG. The special secretory granules described for the HG of the lizard Podarcis s. sicula show a composite structure. A similar structure is present in Coluber viridiflavus, a species of the same order. For the gecko Tarentola mauritanica, the osmiophilic secretory granules show a crescent-like cup of higher density. The secretory granules of Testudo graeca HG are formed from a moderately dense homogeneous material. Two types of secretory granules are found in the Pseudemys scripta HG, i.e., one that is electron-dense with an electron-lucent core and another that is moderately osmiophilic with an electron-dense core. The variety of secretory granules observed at the ultrastructural level leads to the hypothesis that they serve other possible functions besides the obvious lubrication of the eyeball. Furthermore, the presence of plasma cells among the glandular cells in the HG of T. graeca strongly supports the possible immunoresponses related role of the gland, as suggested in mammals and birds. The electron microscopy also indicated that the HG of Chelonians differs from that of Squamata in terms of the presence of salt cells among the glandular cells. These characteristic ion-transporting cells are more numerous in the terrapin. The presence of salt cells in Chelonian HGs leads us to hypothesize that this gland may play a role in osmoregulation, which would be unique among vertebrates. Keywords transmission electron microscopy; harderian gland; reptiles; secretory granules 1. ntroduction The Harderian gland (HG) is an orbital gland found in a majority of land vertebrates. t is the only orbital gland found in the anuran amphibians since the lacrimal gland develops later during phylogenesis in some reptilian species [1-2]. Among Squamata reptiles, both snakes and geckos lack nictitating membranes and lacrimal glands [3]. Reptilian HGs predominantly secrete seromucous substances that are poured into the conjunctival sac. The main function of the HG in reptiles is to lubricate the eye and the nictitating membrane. n a comparative study of the HG in different reptilian species, electron microscopy (EM) proved to be a useful, essential tool for revealing species-specific characteristics that indicate other possible functions for the HG. This mini review is intended to draw attention to the importance of ultrastructural studies to highlight unsuspected species-specific gland functions. 1. Morphology of squamata Harderian gland 1.1 Podarcis s. sicula The HG is prominent in the lizard Podarcis s. sicula [4]. The gland is surrounded by a capsule of thin connective tissue, and strands of collagenous and reticular fibres penetrate between the acini. Between the acini, variable numbers of fibroblasts and mast cells are found; additionally, myoepithelial cells are closely associated with the basal surface of the glandular epithelium. Scattered melanocytes can be found either in the connective tissue capsule or between the acini [4]. An annual cycle for the mast cell number (MC) in Podarcis s. sicula HG, i.e. MC increasing in the spring and winter, has been demonstrated [5]. Podarcis s. sicula HG consists of three distinct zones (lateral, intermediate, and medial zones), each of which showing different histochemical and ultrastructural characteristics [4]. The lateral part of the gland shows higher affinity to tests for proteins due to the serous component of the acinar cells; in fact, the histochemical tests for mucosubstances are weakly positive. At the EM level, the tubulo-acinar lateral part of the gland is formed by cells that contain only special secretory granules, owing to their strikingly unusual structure (Fig.1a). These granules consist of three components that are closely associated like atoms in a molecular model. Additionally, the granules are membrane-bound. The three different components are sharply separated and differ either in density or in structure. One component is more osmiophilic than the others. The central component is less dense and heterogeneous. The last component shows still lower density and is formed by granules arranged in concentric rings suspended in a homogeneous matrix (Fig.1a). At the EM level, the glandular epithelium of the medial part is characterized by large secretory granules that occupy most of the cytoplasm (not shown). Most of these granules contain a dense core surrounded by a zone of lower density; 221

2 A. Méndez-Vilas and J. Díaz (Eds.) sometimes, the dense core shows semiluna zones of high density. According to histochemical tests, this zone secretes predominantly acid sulphated mucosubstances. Both mucous and serous granules are secreted by the glandular cells of the intermediate part of the Podarcis s. sicula HG (Fig.1b). The EM images indicate that one type of granule is similar to what is found in the glandular cells of the medial part; the second type of granule is the same as special secretory granules described in the lateral part (Fig. 1b). The two types of secretory granules are seldom intermingled; they usually occupy different cell compartments. n all three zones, the Golgi complex is prominent; additionally, the mitochondria with lamellar cristae are numerous and confined to the basal cytoplasm among islands of rough endoplasmic reticulum (RER). Secretion is mainly merocrine or apocrine in all three zones of the gland. L a b Fig.1 a) Electron micrograph of glandular cells of the lateral zone of the Podarcis s. sicula HG. The cells are filled with special secretory granules composed of three components sharply separated. The three components exhibit various degrees of electron density. The component of lowest density shows granules arranged in concentric rings suspended in a homogeneous matrix (arrows). L, lumen. X2800. b) Two types of secretory granules are present in the intermediate zone of the Podarcis s. sicula HG: one type shows moderate homogeneous electron-density (asterisks); the second is represented by special secretory granules present in the lateral zone (arrow). X Tarentola mauritanica The HG of gecko, Tarentola mauritanica, is a nonlobate, compound, acinar gland surrounded by a very thin capsule made of connective tissue from which thinner strands containing blood vessels and nerve fibres infiltrate the glandular tissue. Several fibroblasts and mast cells of connective tissue type can be found in the connective tissue around the acini [6]. The acini of the gland are lined with myoepithelial cells and contain only one type of pyramidal or columnarshaped secretory glandular cells. At the EM level, the glandular cells shows cytoplasmic secretory granules particularly concentrated in the apex (Fig. 2). Two types of secretory granules are present in the majority of glandular cells, compound, electron-dense, nonhomogeneous granules and homogeneous, electron-lucent granules (Fig. 2). The osmiophilic secretory granules always show a crescent like cup of lower density. The composite substructure of these secretory granules may indicate a mixed type of biosynthesis, e.g., serous and mucous. This hypothesis has been supported by histochemical analyses showing that the HG of Tarentola mauritanica predominantly possess protein-secreting cells and the secretion products also contain neutral mucosubstances according to the PAS method. Specifically, the mercury-bromophenol blue reaction for proteins is strongly positive in the apical portion of the glandular cells, and the absence of sialic acid in concert with the presence of a few neutral and acidic mucosubstances indicate that the HG of Tarentola mauritanica is composed of a seromucous cell type. nterestingly, a slender interstice and intracellular secretory canaliculi is present among the secretory cells. Particularly, the presence of secretory canaliculi, which represent the machinery of massive fluid secretion and, is considered a significant tool for classifying mammalian salivary secretory cells into serous type [78]. n addition to seromucous granules, the HG contains many membrane-bound, electron-lucent granules that appear to be lipid droplets (Fig. 2). These lipid droplets are intermingled with more numerous serous granules throughout the 222

3 entire cytoplasm (Fig. 2). otably, lipid droplets appear to be more numerous in the HG of males than in those of females, which is indicative of a sex dimorphism. Lipid droplets have been identified also in the HG secretory cells of some Australian geckos [9]. Lipid secretion has been found in the HGs of mammals and the female toad (Bufo viridis), while birds show a basal aggregation of non-secretory lipid-like droplets in the HG [1-2]. The ultrastructural features of the HG in geckos Tarentola mauritanica (Gekkoninae) resemble those of Australian geckos Strophurus intermedius and ephrurus milii (Diplodactylinae), in which the secretory granules lack lamellar paracrystalline structures in their electron-lucent part, which characterizes the granules of the geckos Christinus marmoratus and Heteronotia binoei (Gekkoninae) (not shown) [9]. This finding could be due to the fact that the Tarentola mauritanica is one of the more Gekkoninae primitive genera. The Australian member of Gekkoninae are of the same subfamily, but they are more evolved. Fig.2 Electron micrograph of typical pyramidal secretory cells of Tarentola mauritanica HG, containing both seromucous secretory granules (arrows) and lipid droplets (asterisks)., nucleus. X Coluber viridiflavus n the snake Coluber viridiflavus, the HG is the only gland present in the orbit [1-2]. The secretion from the HG passes through the lacrimal canal in the conjunctival sac via narrow canaliculum, thereby contributing to the lubrication of the eyeball. The gland secretion, also passes down into the mouth to mingle with the saliva, thereby possibly contributing to digestion and functioning as a vehicle for sweet-smelling substances sensed by Jacobson s organ. The HG of Coluber viridiflavus is a compound acinar gland that is organized in lobules surrounded by a loose, connective stroma that is rich in reticular fibres, through which blood vessels and nerves pass [10]. The connective tissue surrounding the acini contains several fibroblasts and myoepithelial cells. The acini consist of columnar glandular cells (type ) that are characterized by the presence of an extraordinarily well-developed rough endoplasmic reticulum. The apical cytoplasm is filled with dense secretory granules of variable sizes and non-homogeneous appearances (Fig. 3). The granules, are bound by a unit membrane and composed of three sharply separated zones. Since the three components of the granule appear to condense at the same time, they likely correspond to different secretions rather than to different stages of condensation. One is more osmiophilic than the others, whereas the second component is homogeneous and less dense. Moreover, the third shows lower density yet and is formed of randomly arranged granules suspended in a homogeneous matrix (Fig. 3). The denser component is crystalline with a highly ordered structure that differs in pattern depending on the plane of section (Fig. 3). nterestingly, the ultrastructure of these granules is very similar to the special secretory granules found in the glandular cells in the HG of lizard Podarcis s. sicula. At the histochemical level the secretory granules show two distinct parts: a thin peripheral zone strongly positive to the Alcian/PAS reaction and to Alcian at ph 1 and 2.5, and a core that, is negative to both tests. The mercury-bromophenol blue reaction for proteins is strongly positive in both parts of the granules. At the most anterior and posterior parts of the gland, some acini are composed of Alcian/PAS-positive glandular cells (type ), which stain metachromatically with toluidine blue. These acini predominantly secrete acidic sulphated 223

4 A. Méndez-Vilas and J. Díaz (Eds.) mucosubstances and can be considered as mucous type. At the EM level, the secretory granules appear homogeneous, and of low density, typical of a mucous secretion (Fig.3). These cells form acini that often are grouped or are mixed with seromucous cells. The secretion of the gland is mainly merocrine, although holocrine secretion cannot be excluded, in fact, entire cell bodies can be seen in the secretory product of the glandular lumina. Fig.3 Electron micrograph of secretory granules from both type and type cells of Coluber viridiflavus HG. Secretory granules of type cell (arrows) shows a composite structure which consists of three closely associated components (similar to the special secretory granule of the Podarcis s. sicula HG). A crystalline structure is present in the peripheral more dense component of the granule. Secretory granules of type cells (asterisks) are homogeneous and moderately electron-dense. X Morphology of chelonian Harderian gland 2.1 Pseudemys scripta The multifunctional character of the HG of Pseudemys scripta is reflected in the complexity of the epithelium of the acini, which contains four different cell types [11]. Type cells are characterised by secretory granules with a PASpositive central core surrounded by an AB/PAS positive ring. On EM these granules have moderate electron density with an electron lucent core (Fig. 4a). Type cells contain only AB-positive granules and are more numerous. On EM, they appear smaller and more osmiophilic than the previous cell type and have an electron-dense core surrounded by a zone of lower density (Fig. 4a). Rare cells, which contain both of the above mentioned secretory granules but in different cell compartments, represent the type cell (Fig.4b). Type V cells, positive only for the PAS reaction, are less common than the type and cells. On EM, the most common form of type V cells display a dense population of mitochondria with abundant elongated cristae, clumps of glycogen, and a well developed Golgi complex (Fig.4b). The apical cytoplasm is occupied by smooth-walled tubules that are short and without anastomoses. This membranous system seems to be involved in ionic transport since it is markedly developed when the salinity of the external medium increases [12]. The apical surfaces of these cells bear short microvilli covered by a thick glycocalyx, while the lateral surfaces show numerous interdigitations (Fig.4b). A seawater adaptation actives salt cells that increase in number and transform from a unicellular form to multicellular complexes [12]. Hormonal control seems very complex since not only prolactin and cortisol inhibit and stimulate salt excretion [13] but many osmoregulatory peptides (i.e. Ang and AP) may also be implicated in this regulation [14]. Chelonian salt cells are very similar to the salt cells or chloride cells described in the salt-secreting glands of various marine vertebrates, such as the gill epithelium of teleosts fish, the rectal gland of elasmobranches, and the nasal gland of marine birds. 224

5 L V a b Fig 4 a) Electron micrograph of the Pseudemys scripta HG, showing both type and type cells. Secretory granules of type cells have moderate electron density with an electron-lucent core; secretory granules of type cells show a core as the negative of that of previous granule. Microvilli are present on the cell surface. L, lumen. X2800. b) Electron micrograph of the Pseudemys scripta HG, showing both type and type V cells. Type contains both of the above secretory granules. Type V shows numerous tightly packed mitochondria, clumps of glycogen (arrows). umerous interdigitations are present among the cells., nucleus X Testudo graeca The HG of Testudo graeca contains only one type of glandular cell (Type ), which mainly secretes glycoproteins and strongly acidic mucosubstances [11]. At EM, the glandular cells are filled with a large number of secretory granules containing a moderately dense homogeneous material (Fig.5). The membrane-bound secretory granules sometimes show a dense core surrounded by a wide zone of lower density. Glycogen in the form of coarse rosettes appears throughout the cytoplasm. Elongated mitochondria show very well developed lamellar cristae (Fig.5). Between the secretory cells, several cells containing only numerous mitochondria and abundant glycogen are seen (Fig. 5). They correspond to histochemically (strongly PAS positive) identified type cell, and are very similar to the type V cell described in the HG of Pseudemys scripta (Fig.4b). umerous plasma cells with a nucleus that contains cartwheel chromatin and vacuolated endoplasmic reticulum were found among the glandular cells (not shown). 225

6 A. Méndez-Vilas and J. Díaz (Eds.) Fig. 5 Electron micrograph of Testudo graeca HG. The glandular cells (type ) are filled with secretory granules containing a moderately dense homogeneous material. Type cell shows a nucleus with evident nucleolus and numerous mitochondria. Arrows, interdigitations., nucleus. X2800. Concluding remarks This review is intended to emphasize the importance of an ultrastructural investigation to highlight species-specific characteristics of reptilian HG. Among both Squamata and Chelonia HGs, the great variability in the substructure of secretory granules leads to the hypothesise of other possible functions besides the obvious lubrication of the eyeball. For example, the HG could be a source of pheromones. Squamata HG secretion has been suggested to play a role in vomerolfaction (gekkos) and digestion (snake). Furthermore, the presence of plasma cells among the glandular cells of the chelonian HG strongly supports the possibility of HG playing an immunoresponse role which has been previously suggested in mammals and birds. The EM data also indicated that the HG of Chelonia (Pseudemys scripta, Testudo graeca) is unique among reptiles to participate as a kidney-like-organ in terms of salt elimination, due to the presence of salt cells among the glandular cells. The salt cells are characteristic ion-transporting cells and are more numerous in the terrapin. The presence of osmoregulatory salt cells in the glandular acini of Chelonia represents the best example of how the HG adapts to a specific function in a particular group of tetrapods. n conclusion, ultrastructural approaches are appropriate for acquiring necessary knowledge about species-specific gland functions. References [1] Payne AP. The Harderian gland: A trecentennial review. J. Anat. 1994; 185:1-49. [2] Chieffi G, Chieffi Baccari G, Di Matteo L, d stria M, Minucci S, Varriale B. Cell biology of harderian gland. nt. Rev. Cytol. 1996; 168:1-80. [3] Saint-Girons H. Histologie comparée des glandes orbitaries des Lèpidosauriens. Ann. Sci. at. Zool. Biol. Anim. 1982; 4: [4] Chieffi Baccari G, Minucci S, Di Matteo L, Chieffi G. Harderian gland and the lacrimal gland of the lizard Podarcis s. sicula: histology, histochemistry, and ultrastructure. Anat. Rec.1990; 226: [5] Vitiello zzo, Chieffi Baccari G, Di Matteo L, Rusciani A, Chieffi P, Minucci S. umber of mast cells in the Harderian gland of the lizard Podarcis sicula sicula (Raf): the annual cycle and its relation to environmental factors and estradiol administration. Gen. Comp. Endocrinol. 1997; 107: [6] Chieffi Baccari G, Chieffi G, Di Matteo L, Dafnis D, De Rienzo G, Minucci S. Morphology of the Harderian gland of the Gecko, Tarentola mauritanica. J. Morphol. 2000; 244: [7] Sakai T, van Lennep FW. The Harderian gland in Australian marsupials. J. Mammal. 1984; 65: [8] Sakai T. Major ocular gland (Harderian gland and lacrimal gland) of the Musk Shrew (Suncus marinus) with a review on the comparative anatomy and histology of the mammalian lacrimal glands. J. Morphol. 1989; 201: [9] Rehorek SJ. Squamate Harderian gland: an overview. Anat. Rec. 1997; 248:

7 [10] Minucci S, Chieffi Baccari G, Di Matteo L. Histology, histochemistry, and ultrastructure of the Harderian gland of the snake, Coluber viridiflavus. J. Morphol. 1992; 211: [11] Chieffi Baccari G, Di Matteo L, Minucci S. The orbital glands of the chelonians Pseudemys scripta and Testudo greca: comparative histological, histochemical and ultrastructural investigations J. Anat. 1992; 180:1-13. [12] Chieffi Baccari G, Minucci S, Di Matteo L. The orbital glands of the terrapin Pseudemys scripta in response to osmotic stress: a light and electron microscope study. J. Anat. 1993; 180: [13] Chieffi Baccari G, Di Matteo L, Minucci S. Effects of Prolactin and Cortisol on the Harderian gland of the terrapin, Pseudemys scripta, adapted to different salinities. Anat. Rec. 1996; 244: [14] Masini MA, Chieffi Baccari G, Di Matteo L, Minucci S, Chieffi G, Uva B. Atrial natriuretic peptide, bradykinin, and angiotensin -like immunoreactivity in the harderian gland of the terrapin Pseudemys scripta: response to osmotic stress. J. Exp. Zool. 1996; 276: