Ultrastructure of the exocrine pancreas in the snake Waglerophis merremii (Wagler)

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Ultrastructure of the exocrine pancreas in the snake Wag/erophis me"emii (Wag/er) Dagoberto Sottovia-Filho and Rumio Taga Departamento de Morfologia, Faculdade de Odontologia de Bauru, Universidade de Sao Paulo, Bauru, CEP 17043, Caixa Postal 73, Sao Paulo, Brasil

Summary. Three types of serozymogenic cells were found in the secretory compartment of the snake exocrine pancreas. Type I cell was the most common and presented a welldeveloped granular endoplasmic reticulum arranged in cisternal and vesicular forms. The cisternal form was located predominantly in the basal regions of the cell and the vesicular form was found in the supranuclear regions of the cell next to a prominent Golgi complex. Mature secretory granules were seen at the cell apex. The cytoplasmic matrix of the Type II cells was electron dense but had only poorly-developed organelles. Secretory granules were rare. The cytoplasmic matrix of the Type III cells was electron lucent and the granular endoplasmic reticulum in the cisternal form was located predominantly in the supranuclear region, whereas the vesicular form was randoly distributed throughout the cytoplasm. The nucleus appeared pale due to the fine dispersion of the chromatin; the nucleolus was prominent. Centroacinar and intermediate cells were also examined.

Key words: Snake Pancreas - Ultrastructure - Pancreatic acinar cells

Introduction Pancreatic acinar cells have frequently been a model for the study of protein synthesis, intracellular transport, exocytosis and membrane turnover. The literature on these subjects relies almost exclusively on studies of the mammalian pancreas. That of other species, especially the lower vertebrates, has been much less frequently examined. As a class the reptiles have only rarely been examined as

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Ann. Anat. (1992) 174: 345-351 Gustav Fischer Verlag Jena

concerns these cytological aspects and Thomas' (1942) descriptive study at the light microscope level may be considered as a pioneer work. It presents details on the organization of the acinus and the excretory duct system of the snake pancreas. There are only few ultrastructural studies on the snake exocrine pancreas. Miller and Lagios (1970) presented a general description of the reptile exocrine pancreas, but their report does not include much specific information about the snake pancreas. Cells that show ultrastructural features intermediate between pancreatic endocrine and exocrine cells have been found in the exocrine pancreas of some animal species (Melmed et al. 1972; Stipp et al. 1980; Sottovia-Filho and Stipp 1985).

Material and methods Six adult males of the snake species Waglerophis merrelii (Wagler) weighing between 380 and 480 g were obtained from the Instituto Butantan, Sao Paulo, Brazil. The pancreas were collected during the summer, seven days after the last feeding. The animals were decapitated and samples of the caudal region of the pancreas placed in a cold (4°C) solution of 2 % glutaraldehyde in 0.09 M phosphate buffer at pH 7.3 for 1 hour. The tissue was post-fixed for 2 hours in cold 0.05 M phosphate-buffered 1 % OS04 plus 106 mg sucrose per ml at pH 7.3. After a quick rinse in 0.9 % NaCI containing 540 mg! 100 ml sucrose, the material was contrasted in 0.5 % uranyl acetate plus 106 mg sucrose per ml for 18 hours. After dehydration in a graded series of ethanol and propylene oxide, the specimens were embedded in araldite (Luft, 1961). Semithin sections were stained according to Richardson et al. (1960). Thin sections were contrasted with 2 % uranyl acetate (Watson 1958) for 30 min and lead citrate (Reynolds, 1963) for 10 min. Micrographs were taken at a Philips EM 301.

Results The serozymogenic cells showed morphological features according to which three cell categories can be distinguished. We classified them as cell Types I, II, and III. Type I acinar cells: The Type I acinar cells were present in large numbers. In the basal regions of these cells there were areas of well-developed granular endoplasmic reticulum (ER) arranged in highly-ordered, flattened saccular structures known as the cisternal form of the ER. This ER was frequently so densely packed that it produced a "Nebenkern"-like structure consisting of concentric lamellae. The lumina of the cisterns were dilated at the periphery of the whirls and resembled the vesicular form of the ER. The vesicular form of the ER prevailed in the supranuclear regions of the cells (Figs. 1 and 2). In these regions there

Fig. 3. Type I acinar cell. Saccular portion of Golgi complex (S). At the trans face coated vesicles lie close to the condensing vacuoles (arrows). In vacuoles CV j and CV2 the intravacuolar material is in different states of condensation. Nucleus (N). X 28560 Fig. 4. Mature secretory granule (G) in Type I acinar cell. . Mitochondria (M). x 21840 Fig. 5. Formation of coated vesicle from the trans face of Golgi complex (arrow). x 38640

Fig. 1. Basal region of Type I acinar cell. "Nebenkern" figures of the GER. Vesicular forms of the GER in the periphery (arrows). X 10080 Fig. 2. Supranuclear region of Type I acinar cell. The vesicular form of the GER predominates. Formation of transitional vesicles can be seen (arrows). Saccular portion of Golgi complex (S), condensing vacuole (CV). x 30960

were also a few cisterns of the ER and structures intermediate between cisterns and vesicles. Numerous membrane ptofiles showed signs of budding-off processes at the ribosome-free surfaces of the cisterns. Also present in the supranuclear regions were large Golgi complexes and voluminous vacuoles containing electron-lucent to electrondense flocculent material. These vacuoles appeared to be larger if the material was more lucent and smaller if the material was denser. The saccules of the trans face of the Golgi complex were frequently expanded to form vesicles that had a coating of thin radially-oriented bristles. (The

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Fig. 6. Nucleus of Type I cell. Nuclear pores (arrows) and the granular portion of the nucleolus associated with heterochromatin (arrowheads). x 6030 Fig. 7. Autophagic vacuole (L) in Type I cell . x 30820 Fig. 8. Type I (AI), II (A 2) and III (A 3) cells . In A2 there are cisterns with narrow lumens oriented parallel to the long axis of the cell. In A3 a pale nucleus (N) and an electron-lucent cytoplasmic matrix. The cisternal form of the GER (GERC) encloses vesicular forms of the GER (GERV). Mitochondria (M), basal lamina (BL).

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terminology referring to the Golgi complex is that of Ehrenreich et al. 1973 .) These were coated vesicles and were frequently closely apposed to the vacuoles (Figs. 3 and 5). Mature secretory granules were located at the cell apex (Figs. 4 and 12). The cytoplasm of a Type I cell also contained randomly distributed mitochondria of very different shapes and sizes; an occasional autophagic vacuole was seen (Fig. 7). The nucleus was located in the basal third of the cell. It was more or less spherical and had a prominent nuclear envelope. The outer membrane of the envelope carried ribosomes , the inner one was associated with clumps of heterochromatin. These clumps were interrupted at the membrane pores. The nucleolus was prominent and consisted of a granular portion and a more filamentous portion (Fig. 6) .

Fig. 9. Basal region of Type II cell. The cisternal form (GERC) and the vesicular form (GERV) of the GER. Secretory granules (G), mitochondria (M) . x 13 440 Fig . 10. Type III cell. Electron-lucent cytoplasmic matri/{. In the pale nucleus a prominent nucleolus (Nu). In the supranuclear region, the GER cisterns limit a region, with vesicular forms of the GER (GERV) . Mitochondria (M), basal lamina (BL). x 7560

Type II acinar cells: The Type II cells were less common and more slender than the Type I cells. On the whole their cytoplasm was more electron dense than that of the Type I cells. The cisterns of the granular ER were more narrow and were oriented approximately parallel to the long axis of the cells (Figs. 8 and 9). The vesicular form of the granular ER as well as large numbers of free ribosomes were randomly distributed throughout the cytoplasm. The Golgi complex was poorly developed and only a small number of secretory granules could be seen. The nucleus was so densely filled with heterochromatin that the nucleolus was obscured.

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granules were not present. The mitochondria in the Type III cells were spherical and dispersed in the cytoplasm but they had a tendency to be more frequent in the basal regions (Figs. 8 and 10). Centroacinar cells: Centroacinar cells, which were infrequent and small, were also examined (Fig. 11). They did not contain any secretory granules. Some profiles of tbe granular ER were seen mingled with mitochondria (Fig. 12). These cells were joined to neighboring cells by junctional complexes at the paraluminal border and by desmosomes in other regions of the cell membrane. Intermediate cells: Intermediate cells were also rare and found interspersed between the serozymogenic cells. Their secretory granules were generally found in small groups in the basal regions of the cells (Fig. 11). Two types of granules could be distinguished: typical zymogen granules and others resembling the granules seen in the endocrine parts of the organ. Thus there were alpha granules with a central dense core and a peripheral mantle surrounded by a membrane as well as beta granules which contained polygonal crystalloid structures in a pale homogeneous matrix surrounded by a membrane (Fig. 13).

Discussion

Fig. 11. 0.5 ftm thick section of pancreas stained according to Richardson et al. (1960). Centroacinar cell (arrowhead). Intermediate cells (arrows) with cytoplasmic granules in the basal region. X 840 Fig. 12. Section through a centroacinar cell (CA) with mitochondria (M), nucleus (N), desmosome (white arrow). The acinar lumen (L) lies at the right. Zymogen granules (Z) in a Type I cell. X 10080 Fig. 13. Detail of intermediate cell with zymogen granules (Z) intermingled with alpha (A) and beta (B) granules. Autophagic vacuole (L), nucleus (N). X 10920

Type III acinar cells: The Type III acinar cells were rare. They had an electron-lucent cytoplasmic matrix with few free ribosomes and an extremely pale nucleus with a prominent nucleolus. The granular ER was arranged as series of parallel cisterns in the supranuclear region. These cisterns enclosed a multitude of granular ER vesicles (Figs. 8 and 10). The Golgi complex was poorly developed and secretory

The ultrastructure of the pancreatic acinar cells of the snake W. merremii does not differ basically from that seen in other cells with high levels of protein synthesis. The distribution of the granular ER in the Type I cells appears to be common to pancreatic acinar cells in other species such as frogs (Slot et al. 1974; Slot and Geuze 1979; Sottovia-Filho and Stipp 1985) and fishes (Stipp et al. 1980). The presence of a "Nebenkern"-like arrangement of the granular ER is similar to that described by Haguenau (1958). This peculiar arrangement has been seen in the exocrine pancreas of other animals (mouse: Weiss 1953; bat: Fawcett 1966; Watari 1968; frog: Geuze 1970; lizard: Godet et al. 1983) and in the columnar secretory cells of the venom gland in the rattlesnake (Warshawsky et aI. 1973). The cisterns with wide lumens at the periphery of the "Nebenkern" figures seem to produce the vesicles by a pinching off process. The formation of vesicles from cisternal forms of the granular ER has been described by Weiss (1953) in the mouse pancreas. This author studied the ultrastructural modification of pancreatic acinar cells in fasted and fed mice and reported that the "Nebenkern" figures represent a center of membrane formation for the granular ER in the acinar cells of fed animals. Vesicular forms of the granular ER are predominant in the supranuclear regions of the Type I acinar cells and they probably arise from the cisterns in the basal regions of the cells. The results of several studies have shown that the presence of granular ER cisterns is related to intense synthetic activity, whereas the vesicular form is associated with intracellular transport and storage (Weiss 1953; Siekevitz and Palade 1958; Herman et al. 1964; Watari 1968; Oron and Bdolah 1973; Stipp et aI. 1980). The granular ER pattern in the

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Type I acinar cells could be related to what has been described for its arrangement and function in other cells . The ultrastructure of the Type I cells suggests that transitional vesicles form at the ER cisterns and then pass to the CIS face of the Golgi complex where they coalesce. Since the earliest description of such vesicles by Ziegel and Dalton in 1962, they have been suggested to be the transport form of proteins synthesized in the granular ER and carried to the Golgi complex . Some of the pertinent studies pointed to this relationship in the guinea pig pancreas (Jamieson and Palade 1967a, 1967b, 1968, 1971), in the rat pancreas (Van Heyningen 1964), in the frog pancreas (Slot and Geuze 1979; Slot et al. 1974, 1976, 1979; Sottovia-Filho and Stipp 1985), in the rat ameloblast (Warshawsky 1968; Weinstock and Loblond 1971), and in the rattlesnake venom gland (Warshawsky et al. 1973). The ultrastructural features of the Golgi complex in the Type I acinar cells are similar to those described in the literature on the human pancreatic acinar cell (Ekholm and Edlund 1959; Ekholm et al. 1962a; Kern and Ferner 1971), in cells of various other mammalian organs (Beams and Kessel, 1968), in rat hepatocytes (Ehrenreich et al. 1973), in the fish pancreas (Stipp et al. 1980), and in the frog pancreas (Slot et al. 1974). The vacuoles are initially large and limited by an irregular membrane, probably due to the constant fusion of additional coated vesicles . The presence of vacuoles of different sizes and content suggests that the larger ones gradually become reduced in size with a concomitant increase in the electron density of their contents. This process leads to the formation of the secretory granules. Similar observations were made in reports on the synthesis, condensation and storage of secretory proteins in other cells (Beams and Kessel 1968; Jamieson and Palade 1967 a, 1967b, 1968; Jamieson 1971; Kern and Ferner 1971; Palade 1975; Novikoff et al. 1977, 1978). The intracellular transport sequence during protein synthesis in the pancreatic acinar cells may be as follows: granular ER - transitional vesicles - saccular portion of the Golgi condensing vacuoles - secretory granules. This sequence has also been suggested to take place in acinar cells of other species (Van Heyningen 1964 ; Jamieson and Palade 1971; Novikoff et al. 1977; Slot et al. 1974, 1976, 1979). With regard to the other cytoplasmic organelles, it is important to consider the autophagic vacuoles that were present in smaller quantities. This observation confirms reports in the literature about the scarcity of this organelle in pancreatic acinar cells of animals that are not fasting or in hibernation (Miller and Lagios 1970; Geuze 1970; Stipp et al. 1980). Type II acinar cells show basically the same ultrastructural features that were found in the Type I cells, although the organelles are less prominent. The Golgi complex is poorly developed and there are only few secretory granules. In a general way, the Type I and Type II cells reveal morphological characteristics similar to the so-called light and dark cells described in the frog pancreas (Komar6my et al. 1967 ; Sottovia-Filho and Stipp 1985) in the fish pancreas (Stipp et al. 1980), in the snake venom gland (Kochva and Gans

1967), in the mouse gall bladder epithelium (Yamada 1968), and in the enamel epithelium of the rat (Warshawsky 1968). Comparing the ultrastructural characteristics of the Type I and II cells, we observed that the latter present poorlydeveloped organelles for protein synthesis and we therefore suggest that the two types may possibly express different functional states: Type I might be at a high level of synthetic activity and Type II might represent a resting phase. Cells with the structural profiles like those of the Type III acinar cells have never been described in the exocrine pancreas of other adult animals. Considering the electronlucent cytoplasmic matrix, the unusual condition of the granular ER, the discrete Golgi complex, the large vesiculous and euchromatic nucleus with a prominent nucleolus, we think that these cells represent precursor elements in the serozymogenic clone. With regard to the centroacinar cells, the electron-lucent cytoplasmic matrix, the scarcity of cytoplasmic organelles, the presence of microfilaments and the lack of secretory granules are the general features described in other species (Ekholm and Edlund 1959; Ekholm et al. 1962 b; Stipp et al. 1980; Williams and Kendall 1982). The cells are very small and may therefore not have been observed by Thomas (1942) in the snake pancreas. As far as we know, intermediate cells have not been described in earlier reports on the snake pancreas. They exhibit zymogenic, alpha and beta granules intermingled within the same cell. This is an unique characteristic for these cells and has been seen in the pancreas of many animal species by Melmed et al. (1972). According to the classification proposed by these authors, we classified the intermediate cells of the snake pancreas as acinar alpha-beta intermediate cells since the predominant ultrastructure is similar to the serozymogenic cells and both alpha and beta granules coexist. The general morphological profile of the intermediate cells in the snake pancreas is in good agreement with what has been described in the pancreas of other animals (Pietro-Diaz et al. 1967; Geuze 1970; Brow and Still 1970; Melmed et al. 1972; Stipp et al. 1980; Sottovia-Filho and Stipp 1985). The ultrastructure of the alpha and beta granules is analogous to that described for the Langerhans endocrine cells in the frog (Barrington 1951; Hellman and Hellerstrom 1962), in the snake (Cardeza 1962; Hellerstrom and Asplund 1968; Trandaburu and Calugareanu 1969; Miller and Lagios 1970; Wateri et al. 1970) and some mammals (Munger et al. 1965).

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Accepted ofter revision November 15, 1991

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