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An immunocytochemical and ultrastructural study of the endocrine pancreas of Pseudemys scripta elegans (chelonia)
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
60, 95-103 (1985)
An lmmunocytochemical and Ultrastructural Study of the Endocrine Pancreas of Pseudemys scripta elegans (Chelonia) B. ACULLEIRO, Department
of Cytology
A. GARC~AAYALA, and Histology,
University
ANDM.
E. ABAD
of Murcia.
Murcia,
Spain
Accepted February 4, 1985 Immunocytochemical and ultrastructural methods have shown four cell types in the endocrine pancreas of the turtle Pseudemys scripta elegans: insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-immunoreactive cells. Each endocrine cell type was distributed differently in the duodenal or splenic regions of the turtle pancreas. Round or fusiform insulin- and glucagon-containing ceils could be seen as single scattered cells which were more numerous in the duodenal regions, and the cell groups becoming progressively smaller from splenic to duodenal region. Round or fusiform somatostatin cells with thick processes and spindly pancreatic polypeptide cells with long protrusions were less numerous the nearer they were to the splenic regions; they were isolated in the duodenal zone. Insulin cells were surrounded by somatostatin cells and an outer layer of glucagon cells around the cell groups could be seen. Insulin cells were characterized by their round secretory granules which contained a polygonal, irregular or rod-shaped dense core. They also contained numerous clustered mitochondria, large multivesicular bodies, and cilium. Glucagon cells, joined by desmosomes to adjacent ones, had numerous filamentous mitochondria with longitudinal cristae and round electron-dense secretory granules with closely applied membrane. Somatostatin cells contained two kinds of secretory granules, some of which showed an electron-dense core, while others had moderately electron-dense floccular material PP cells were characterized by round secretory granules, smaller than those of other cell types, and a large euchromatinic nucleus. Lysosomes, microtubules, bundles of microfilaments, a well-developed Golgi apparatus, and scarce rough endoplasmic reticulum were present in the cytoplasm of all these endocrine cell types. D 1985 Academic Press, IIIC.
During the last few years, endocrine pancreas of lower vertebrates have mostly been studied by immunocytochemical methods although only a few reptiles (lizards and crocodile) (Rhoten and Smith, 1978; Rhoten and Hall, 1981; El-Salhy and Grimelius, 1981a; Buchan ef al., 1982; ElSalhy el al., 1983) have been investigated. Chelonia endocrine pancreas has been described on the basis of its light microscope tinctorial properties and electron microscopic characteristics (Kano, 1961; Titlbach, 1966; Epple and Brinn, 1975; Yaglov and Eletski, 1975; Yaglov, 1976). The present study, carried out first using peroxidase-antiperoxidase (PAP) technique (Sternberger, 1979) and then by electron microscope, reports on the endocrine pancreas of the turtle Pseudemys scripta ele-
gans, previously
microscopy
only investigated (Miller, 1962).
MATERIALS
by light
AND METHODS
Ten young specimens tles (P. scripta elegans), anesthetized with ethyl ventral caparaces were
of both sexes of red-eared turabout 6-8 gin body wt, were ether or chloroform and their removed. Immunocytochemistry. The pancreas was divided into two pieces (splenic and duodenal portions), fixed by immersion for 16 hr in Bouin’s fluid and embedded in’paraplast Plus. The sections were processed by the indirect immunocytochemical method (PAP) (Sternberger, 1979). Sections were rinsed in phosphate-buffered saline (PBS) of pH 7.2 and incubated for 24 hr at 4” in a humid atmosphere with the respective antisera diluted with PBS of pH 7.2: guinea pig anti-porcine insulin (Immunonuclear Corp.) 1:50, porcine anti-pancreatic glucagon (Milab) 1: 1000 and 1:1500, rabbit antisomatostatin (Immunonuclear Corp.) 1:500. rabbit
95 0016~6480/85 $1.50 Copyright All tights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
96
AGULLEIRO,
GARCIA
anti-BPP (gift of Dr. R. E. Chance, Eli Lilly, Indianapolis, Ind.) 1:20,000, and porcine anti-GIP (Milab) 1:320. They were washed several times in PBS, incubated for 30 min to 3 hr at room temperature with goatanti-rabbit IgG (Polysciences. Inc.) diluted 1: 10 with PBS, then washed with PBS and incubated for 30 min to 3 hr at room temperature with rabbit peroxidaseantiperoxidase complex (Polysciences, Inc.) diluted 1:40 with PBS. After rinsing in PBS and subsequently in 0.05 M Tris-HCl buffer of pH 7.6 peroxidase activity was demonstrated with a solution of 0.05% 3,3’diaminobenzidine in Tris-HCI buffer containing 0.01% HzO, for 15 min. All positive results were controlled by the omission of the primary antiserum and by a parallel incubation with antisera that had been preabsorbed with an excess of the respective peptide, additional control procedures were carried out with the GIP antiserum which was saturated with glucagon peptide. Ekctron microscopy. Samples of pancreatic tissue were fixed by immersion for 5 hr at 4” in 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 to 7.4, then postfixed in 1% buffered osmium tetroxide, and embedded in a mixture of epoxy resins. Ultrathin sections were cut using a LKB III ultramicrotome, stained with uranyl acetate and lead citrate, and examined by ZEISS EM 1OC electron microscope.
RESULTS
Light Microscopy Isolated and clustered endocrine cells were found throughout the pancreas. A distinct connective capsule was not present. Four cell types were identified using antisera against mammalian insulin, pancreatic glucagon, somatostatin, and pancreatic polypeptide. Glucagon-containing cells also reacted with gastric inhibitory polypeptide
AYALA,
AND ABAD
(GIP) antiserum which showed a lighter stain than glucagon antiserum. When the GIP antiserum was saturated with glucagon no staining was observed. Each endocrine cell type was distributed differently in the duodenal and splenic regions of the turtle pancreas. Insulin immurtoreactive cells were numerous in the splenic region where large or medium solid islets occurred as strands and single scattered cells (Fig. la). In the duodenal region some medium or small-sized cell groups and isolated cells were found (Fig. lb). All cells were round or fusiform. Glucagon immunoreactive cells occurred mainly at the splenic region where round or ovoid, large and medium-sized islets with no immunoreactive core and scarce single cells were found (Fig. 2a). In the duodenal region similar cell arrangements were seen but there were smaller islets and more numerous single cells (Fig. 2b). The cells were round or fusiform. Somatostatin immunoreactive cells were numerous in the duodenal zone and decreased the nearer the splenic region (Figs. 3a, b). Cells were found either isolated or in groups of two or three in the duodenal zone while in the splenic region they were in islets with no immunoreactive core. The cells were wedge-shaped with a distinct cytoplasmic protrusion. Pancreatic polypeptide immunoreactive cells were numerous in the duodenal region and seldom seen in the splenic zone (Figs. 4a, b). Wedge-shape cells with a long cy-
FIG. 1. Insulin-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) Large cell groups of the splenic region. (b) Cell groups of the duodenal region. PAP method. x 200, x 200. FIG. 2. Glucagon-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) Cells surrounding a non-immunoreactive zone of the splenic region. (b) Strands and islet-like peripheral glucagon-immunoreactive cells of the duodenal region. PAP method. x 200. x 200. FIG. 3. Somatostatin-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) Cell groups of the splenic region. (b) Numerous somatostatin-containing cells of the duodenal region. PAP method. x90, x 30. FIG. 4. Pancreatic polypeptide-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) The only PP cells of the splenic region. (b) Numerous isolated PP cells of the duodenal region. PAP method. x 90, x 30.
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AYALA,
AND
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toplasmic process were located among the exocrine parenchyma isolated or in groups of two or three. Electron Microscopy B cells contained a large oval euchromatinic nucleus with close deep indentations and nucleolus (Fig. 5). Juxtanuclear dictyosomes displayed cisternae with dilated borders containing an osmiophilic material. Numerous small vesicles were scattered throughout the cytoplasm. Clustered filamentous mitochondria with dense matrix and longitudinal or transversal cristae, free ribosomes, scarce rough endoplasmic reticulum, multivesicular bodies, some large lysosomes, centrioles, and a cilium could be seen (Fig. 6). Striated fibers having a regular cross banding with a repeating period composed of parallel microfilaments, microtubules, and some microfilaments were present (Fig. 7). Characteristic B secretory granules were round and sometimes irregular, of uniform size (500 nm in diameter) and scattered throughout the whole cell or clustered in the basal cytoplasm near capillaries. Most of the granules had a round, irregular, or rod core of varying size and electron density and a light or medium dense matrix (Fig. 7). Some granules showed a homogeneous, moderately dense content extended to their limiting membrane . A cells showed a large, oval, scalloped, eccentric euchromatinic nucleus with a dense
99
nucleolus (Fig. 8). Several juxtanuclear dictyosomes next to the cell membrane had cisternae with dilated borders with an electron-dense content (Fig. 9). Scattered numerous mitochondria with dense matrix and longitudinal cristae, scarce long cisternae of rough endoplasmic reticulum, free ribosomes, lysosomes, microtubules, and bundles of microfilaments were found (Figs. 9 and 10). Secretory granules (500 nm in diameter), with or without a clear halo, were electron-dense, round and dispersed or, if numerous, grouped at the cell pole next to capillaries. Granules which were pyriform or in contact with the cell membrane (Fig. 10) and desmosomes were seen. D cells were found surrounding B cells, and in turn surrounded by A cells, or isolated in the pancreatic parenchyma. The large central euchromatinic nucleus, with a small nucleolus, had both slight and deep invaginations (Fig. 11). A well-developed Golgi apparatus, scarce filamentous mitochondria, numerous free ribosomes, and short cisternae of rough endoplasmic reticulum next to the cell periphery could be seen. Multivesicular bodies, microtubules, striated fibers having a regular crossbanding composed of parallel microfilaments, centriole, and lysosomes were also found (Fig. 12). Two types of secretion granules were seen. The most numerous were round or ovoid, frequently with incomplete surrounding membrane, and containing a medium electron-dense material (Fig. 12). These granules were larger (700
FIG. 5. Electron micrograph of B cells showing an indented nucleus (N), a well-developed Golgi apparatus (G), and the secretory granules with an irregular dense core. x 6300. FIG. 6. Detail of the cytoplasm of a B cell. Note the centriole (cn), developing cilium. groups of small vesicles, and the secretory granules. cl: cilium. x 20,000. FIG. 7. Mitochondria, secretion granuies, Iysosomes, and striated tibres (D of a B cell. x 20,000. FIG. 8. Electron micrograph of an A cell (A) showing euchromatinic nucleus with a dense nucleolus (nu) and electrondense secretory granules. D: cytoplasm of a D cell. x 6300. FIG. 9. Detail of the cytoplasm of an A cell showing Golgi apparatus (G), lysosomes (ly), and secretory granules. Note the dense material inside a golgian cistema (arrow). x 20,000. FIG. 10. Mitochondria (m) and secretion granules next to plasmatic membrane of an A cell of Pseudemys scripra elegans. x 20,000.
AGULLEIRO,
GARCIA AYALA,
AND ABAD
FIG. 11. Electron micrograph of a D cells of endocrine pancreas of Pseudemys scripra elegans showing euchromatinic nucleus (N) and secretion granules of different electrondensity. G: Golgi apparatus. x 7500. FIG. 12. Golgi apparatus, centriole (cn), striated fibre (f), and characteristic medium electron-dense granules of a D cell. x 20,000. FIG. 13. Detail of cytoplasm of a D cell. Note the dense granules with round core. x 20.000.
nm in diameter) and less dense than the other granular type (300 nm in diameter) (Fig. 13). A clear halo separated the membrane and core in both granular types. PP cells were joined by desmosomes to the adjacent exocrine pancreatic cells. An extremely light nucleus with dense nucleolus, showing deep invagination was present (Fig. 14). The Golgi apparatus had
some dilated cisternae containing a dense material similar to the secretion granules (Fig. 16). Scattered mitochondria with dense matrix, some long cisternae of rough endoplasmic reticulum, free ribosomes, multivesicular bodies, lysosomes, microtubules, and striated fibers having a regular cross banding composed of parallel microfilaments were found. Secretion granules
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IMMUNOCYTOCHEMISTRY
were round, pyriform, or almost irregular. They were small (350 nm in diameter) and had a very dense core (Fig. 15). DISCUSSION
Although only alpha and beta cells have been previously reported in the endocrine pancreatic tissue of P. scripta elegans (Miller, 1962), immunocytochemical meth-
ods have been able to show insulin-, glucagon-, somatostatin-, and pancreatic-polypeptide immunoreactive cells similar to those described in the lizards Anofis carolinensis (Rhoten and Smith, 1978; Rhoten and Hall, 1981), Mabuya quinquetaeniata and Uromastyx aegyptia (El-Salhy and Grimelius, 198 la), and Chalcides ocellatus (ElSalhy et al., 1983), and in the crocodile Alligator mississippiensis (Buchan et al.,
FIG. 14. PP cell surrounded by exocrine pancreatic cells. Note the extremely light indented nucleus (N) and small secretory granules. x 4000. FIG. 15. Small electron-dense secretory granules of a PP cell. x 20,000. FIG. 16. Note a vesicule with dense content near the Golgi apparatus (arrow), small electron-dense secretory granules, and lysosomes (ly) of a PP cell of endocrine pancreas of Pseudemys scripta elegnns.
X 20,000.
101
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AGULLEIRO,
GARCIA
AYALA,
AND
ABAD
1982). The glucagon-immunoreactive cells These cells, as in lizards (El-Salhy and Grialso showed GIP immunoreactivity. Glumelius, 1981a, b), were numerous and isocagon/GIP immunoreactive cells were lated in the duodenal pancreatic region found in trout (Wagner and McKeown, of P. scripta elegans. PP cells, isolated 1981) where, as in P. scripta elegans, or in groups of two or three, were seen blocking GIP antiserum by glucagon no throughout the pancreas among the exostain was observed. GIP immunoreactivity crine cells in P. scripta elegans as in other was found in specific pancreatic cells of U. reptilia (Rhoten and Smith, 1978; Rhoten aegyptia (El-Salhy and Grimelius, 1981b) and Hall, 1981; Buchan et al., 1982) and where the stain was not blocked by glu- were numerous in the duodenal region, as cagon. GIP and glucagon peptides contain in lizards which have PP cells located in the islet periphery of the splenic zone (El-Salhy several common sequences (Sjolund et al., and Grimelius, 1981a; El-Salhy et al., 1983) and therefore in A cells of the turtle 1983). pancreas a precursor peptide which crossreacts with antisera against both GIP and B, A, D, and PP cells were identified by glucagon could exist. Bovine pancreatic ultrastructural criteria in the pancreas of polypeptide antiserum showed PP cells in P. scripta elegans although only B, A, and D have been found in reptilia (Cardeza, P. scripta elegans just as in lizards while only the avian pancreatic polypeptide an- 1960; Titlbach, 1966, 1967, 1968; Rhoten, tiserum gave positive results in A. missis- 1973) and PP cells have only recently been sippiensis (Buchan et al., 1982). seen using electron immunocytochemical Islet-like structures, not surrounded by methods (Rhoten and Hall, 1981, 1982; connective tissue, were smaller and the iso- Buchan et al., 1982). B cells were characlated cells more numerous in the duodenal terized by their indented nucleus and round region of the pancreas of P. scripta elegans secretory granules containing an irregular while the endocrine tissue of lizards was or rod-shaped dense core similar to granmostly confined to the splenic zone (Elules described in some reptilia (Titlbach, Salhy and Grimelius, 1981a, b; El-Salhy et 1966, 1969). Other granules with homogeal., 1983) and in the case of A. mississip- neous matrix have also been found in B piensis no distinct frequency of the indicells of P. scripta elegans as previously vidual cells types was seen between the noted in other reptilia (Cardeza, 1960; Wapancreatic regions (Buchan et al., 1982). tari et al., 1970; Rhoten, 1971, 1973). A Isolated and scattered insulin- and glucells of P. scripta elegans had a scalloped cagon-containing cells were found to be nucleus, mitochondria with longitudinal more numerous in the duodenal region cristae as in some reptilia (Cardeza, 1960). where the cells groups were smaller than in and secretion granules, similar to other repthe splenic one. Insulin cells occurred in tilia, were round and with dense round core the islet center and the glucagon cells in the (Cardeza, 1960; Titlbach, 1966, 1968, 1969; periphery as in M. quinquetaeniata (ElRhoten, 1971). In D cells of P. scriptu eleSalhy and Grimelius, 1981a) and C. ocel- gans’two secretory granular types of dislatus (El-Salhy et al., 1983). Scarce so- tinct density could been seen although only matostatin cells were located between in- moderately dense secretory granules have sulin and glucagon cell layers in the islets been seen in some reptilia (Cardeza, 1960; of P. scripta elegans while in lizards they Rhoten, 1971, 1973). were mostly found in the islet periphery In previous ultrastructural studies of the (Rhoten and Smith, 1978; El-Salhy and Gri- endocrine pancreatic cells of reptilia, PP from A melius, 1981a, b; El-Salhy et al., 1983). cells could not be distinguished
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PANCREAS
cells but in P. scripta elegans they were easily characterized by their extremely light and indented nucleus, a long cytoplasmic protrusion, and the small round electron-dense granules similar to those found in most vertebrates (Solcia et al., 1978) although they were irregular in A. mississippiensis (Buchan et al., 1982) and were large and ellipsoidal in A. carolinensis (Rhoten and Hall, 1981). REFERENCES Buchan, A. M. J., Lance, V., and Polak, J. M. (1982). The endocrine pancreas of Alligator mississippie&s.
Cell
Tissue
Res. 224,
117- 128.
Cardeza, A. E (1960). Citologia de1 pancreas de la serpiente Xenodon merremii. Rev. Sot. Argent. Biol. 36, 108-117. El-Salhy, M., and Grimelius, L. (1981a). Histological and immunohistochemical studies of the endocrine pancreas of lizards. Histochemistry 72, 237247. El-Salhy, M., and Grimelius, L. (1981b). Immunohistochemical localization of Gastrin C-terminus, Gastric inhibitory peptide (GIP) and Endorphin in the pancreas of lizards with special reference to the hibernation period. Regul. Pept. 2, 97- 111. El-Salhy, M., Abu-Sinna, G., and Wilander, E. (1983). The endocrine pancreas of a squamate Reptile, the desert lizard (Chalcides ocellatus). A histological and immunocytochemical investigation. Histochemistry 78, 391-397. Epple, A., and Brinn, J. E., Jr. (1975). Islet histophysiology: Evolutionary correlations. Gen. Comp. Endocrinol.
27, 320-349.
Kano, K. (1961). Histologische, cytologische und elektronenmikroskopische untersuchungen tiber die langerhansschen inseln der Schildkrote (Clemmys
japonica).
Arch.
Histol.
Japon.
22,
123-180. Miller, M. R. (1962). Observations on the comparative histology of the reptilian pancreatic islet. Gen. Comp. Endocrinol. 2, 407-414. Rhoten, W. B. (1971). Light and electron microscopic studies on pancreatic cells of the lizard Lygosoma laterale. I. Normal and glucose-loaded animals. Gem Comp. Endocrinol. 17, 203-219. Rhoten, W. B. (1973). The cellular response of saurian pancreatic islets to chronic insulin administration. Gen.
Compl.
Endocrinol.
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20, 474-489.
Rhoten, W. B., and Hall, C. E. (1981). Four hormones
in the pancreas of the lizard, Anolis Anat.
Rec.
carolinensis.
199, 89-97.
Rhoten, W. B., and Hall, C. E. (1982). An immunocytochemical study of the cytogenesis of pancreatic endocrine cells in the lizard, Anolis carolinensis. Amer. J. Anat. 163, 181-193. Rhoten, W. B., and Smith, P H. (1978). Localization of four polypeptide hormones in the Saurian pancreas. Amer. J. Anat. 151, 595-602. Sjolund, K., Ekelund, M.. Hakanson, R., Moody, A. J., and Sundler, F. (1983). Gastric inhibitory peptide-like immunoreactivity in Glucagon and Glicentin cells: Properties and Origin. An immunocytochemical study using several antisera. J. Histochem. Cytochem. 31, 811-817. Solcia, E., Polak, J. M., Pearse, A. G. E., Forsmann, W. G., Larsson, L. I., Sundler, F., Lechago, J., Grimelius, L., Fujita, T., Creutzfelt, W., Gepts, W., Falkmer, S., Lefranc, G., Heitz, Ph., Hage, E., Buchan, A. M. J., Bloom, S. R., and Grossman, M. I. (1978). Lausanne 1977 classification of gastroenteropancreatic endocrine cells. In “Gut Hormones” (Sr. Bloom, ed.), pp. 40-48. Churchill Livingstone, Edinburgh. Sternberger, L. A. (1979). “Immunocytochemistry.” 2nd Ed. Wiley, New York. Titlbach, M. (1966). Licht-und elektronenmikroskopische untersuchungen der Langerhansschen inseln von Schildkroten (Testudo graeca. Emys orbicularis L.). Z. Zellforsch. 70, 21-35. Titlbach, M. (1968). Licht-und elektronenmikroskopische untersuchungen der Langerhansschen inseln von nattern (Natrix natrix L., Natrix tessellata Laurenti). Z. Zeliforsch. 90, 519-534. Titlbach, M. (1969). The identification of the types of Langerhans islets cells Part II. Electron microscopical study. Folia Morphol. 17, 17-22. Wagner, G. F., and McKeown, B. A. (1981). Immunocytochemical localization of hormone-producing cells within the pancreatic islets of the rainbown trout (Salmo gairdneri). Cell Tissue Res. 221, 181-192. Watari, N., Tsukagoshi, N., and Yoshiharu, H. (1970). The correlative light and electron microscopy of the islets of Langerhans in some lower Vertebrates. Arch. Histol. Japan. 31, 371-392. Yaglov, V. V. (1976). Morphology of the endocrine portion of the reptile pancreas. Arkh. Anat. Gistol. Embriol. 71, 89-93. Yaglov, V. V.. and Eletski, Y. K. (1975). The morphology and classification of acinar-islet cells of the pancreas. Arkh. Anat. Gistol. Embriol. 69, 20-23.
AND
COMPARATIVE
ENDOCRINOLOGY
60, 95-103 (1985)
An lmmunocytochemical and Ultrastructural Study of the Endocrine Pancreas of Pseudemys scripta elegans (Chelonia) B. ACULLEIRO, Department
of Cytology
A. GARC~AAYALA, and Histology,
University
ANDM.
E. ABAD
of Murcia.
Murcia,
Spain
Accepted February 4, 1985 Immunocytochemical and ultrastructural methods have shown four cell types in the endocrine pancreas of the turtle Pseudemys scripta elegans: insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-immunoreactive cells. Each endocrine cell type was distributed differently in the duodenal or splenic regions of the turtle pancreas. Round or fusiform insulin- and glucagon-containing ceils could be seen as single scattered cells which were more numerous in the duodenal regions, and the cell groups becoming progressively smaller from splenic to duodenal region. Round or fusiform somatostatin cells with thick processes and spindly pancreatic polypeptide cells with long protrusions were less numerous the nearer they were to the splenic regions; they were isolated in the duodenal zone. Insulin cells were surrounded by somatostatin cells and an outer layer of glucagon cells around the cell groups could be seen. Insulin cells were characterized by their round secretory granules which contained a polygonal, irregular or rod-shaped dense core. They also contained numerous clustered mitochondria, large multivesicular bodies, and cilium. Glucagon cells, joined by desmosomes to adjacent ones, had numerous filamentous mitochondria with longitudinal cristae and round electron-dense secretory granules with closely applied membrane. Somatostatin cells contained two kinds of secretory granules, some of which showed an electron-dense core, while others had moderately electron-dense floccular material PP cells were characterized by round secretory granules, smaller than those of other cell types, and a large euchromatinic nucleus. Lysosomes, microtubules, bundles of microfilaments, a well-developed Golgi apparatus, and scarce rough endoplasmic reticulum were present in the cytoplasm of all these endocrine cell types. D 1985 Academic Press, IIIC.
During the last few years, endocrine pancreas of lower vertebrates have mostly been studied by immunocytochemical methods although only a few reptiles (lizards and crocodile) (Rhoten and Smith, 1978; Rhoten and Hall, 1981; El-Salhy and Grimelius, 1981a; Buchan ef al., 1982; ElSalhy el al., 1983) have been investigated. Chelonia endocrine pancreas has been described on the basis of its light microscope tinctorial properties and electron microscopic characteristics (Kano, 1961; Titlbach, 1966; Epple and Brinn, 1975; Yaglov and Eletski, 1975; Yaglov, 1976). The present study, carried out first using peroxidase-antiperoxidase (PAP) technique (Sternberger, 1979) and then by electron microscope, reports on the endocrine pancreas of the turtle Pseudemys scripta ele-
gans, previously
microscopy
only investigated (Miller, 1962).
MATERIALS
by light
AND METHODS
Ten young specimens tles (P. scripta elegans), anesthetized with ethyl ventral caparaces were
of both sexes of red-eared turabout 6-8 gin body wt, were ether or chloroform and their removed. Immunocytochemistry. The pancreas was divided into two pieces (splenic and duodenal portions), fixed by immersion for 16 hr in Bouin’s fluid and embedded in’paraplast Plus. The sections were processed by the indirect immunocytochemical method (PAP) (Sternberger, 1979). Sections were rinsed in phosphate-buffered saline (PBS) of pH 7.2 and incubated for 24 hr at 4” in a humid atmosphere with the respective antisera diluted with PBS of pH 7.2: guinea pig anti-porcine insulin (Immunonuclear Corp.) 1:50, porcine anti-pancreatic glucagon (Milab) 1: 1000 and 1:1500, rabbit antisomatostatin (Immunonuclear Corp.) 1:500. rabbit
95 0016~6480/85 $1.50 Copyright All tights
0 1985 by Academic Press. Inc. of reproduction in any form reserved.
96
AGULLEIRO,
GARCIA
anti-BPP (gift of Dr. R. E. Chance, Eli Lilly, Indianapolis, Ind.) 1:20,000, and porcine anti-GIP (Milab) 1:320. They were washed several times in PBS, incubated for 30 min to 3 hr at room temperature with goatanti-rabbit IgG (Polysciences. Inc.) diluted 1: 10 with PBS, then washed with PBS and incubated for 30 min to 3 hr at room temperature with rabbit peroxidaseantiperoxidase complex (Polysciences, Inc.) diluted 1:40 with PBS. After rinsing in PBS and subsequently in 0.05 M Tris-HCl buffer of pH 7.6 peroxidase activity was demonstrated with a solution of 0.05% 3,3’diaminobenzidine in Tris-HCI buffer containing 0.01% HzO, for 15 min. All positive results were controlled by the omission of the primary antiserum and by a parallel incubation with antisera that had been preabsorbed with an excess of the respective peptide, additional control procedures were carried out with the GIP antiserum which was saturated with glucagon peptide. Ekctron microscopy. Samples of pancreatic tissue were fixed by immersion for 5 hr at 4” in 4% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 to 7.4, then postfixed in 1% buffered osmium tetroxide, and embedded in a mixture of epoxy resins. Ultrathin sections were cut using a LKB III ultramicrotome, stained with uranyl acetate and lead citrate, and examined by ZEISS EM 1OC electron microscope.
RESULTS
Light Microscopy Isolated and clustered endocrine cells were found throughout the pancreas. A distinct connective capsule was not present. Four cell types were identified using antisera against mammalian insulin, pancreatic glucagon, somatostatin, and pancreatic polypeptide. Glucagon-containing cells also reacted with gastric inhibitory polypeptide
AYALA,
AND ABAD
(GIP) antiserum which showed a lighter stain than glucagon antiserum. When the GIP antiserum was saturated with glucagon no staining was observed. Each endocrine cell type was distributed differently in the duodenal and splenic regions of the turtle pancreas. Insulin immurtoreactive cells were numerous in the splenic region where large or medium solid islets occurred as strands and single scattered cells (Fig. la). In the duodenal region some medium or small-sized cell groups and isolated cells were found (Fig. lb). All cells were round or fusiform. Glucagon immunoreactive cells occurred mainly at the splenic region where round or ovoid, large and medium-sized islets with no immunoreactive core and scarce single cells were found (Fig. 2a). In the duodenal region similar cell arrangements were seen but there were smaller islets and more numerous single cells (Fig. 2b). The cells were round or fusiform. Somatostatin immunoreactive cells were numerous in the duodenal zone and decreased the nearer the splenic region (Figs. 3a, b). Cells were found either isolated or in groups of two or three in the duodenal zone while in the splenic region they were in islets with no immunoreactive core. The cells were wedge-shaped with a distinct cytoplasmic protrusion. Pancreatic polypeptide immunoreactive cells were numerous in the duodenal region and seldom seen in the splenic zone (Figs. 4a, b). Wedge-shape cells with a long cy-
FIG. 1. Insulin-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) Large cell groups of the splenic region. (b) Cell groups of the duodenal region. PAP method. x 200, x 200. FIG. 2. Glucagon-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) Cells surrounding a non-immunoreactive zone of the splenic region. (b) Strands and islet-like peripheral glucagon-immunoreactive cells of the duodenal region. PAP method. x 200. x 200. FIG. 3. Somatostatin-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) Cell groups of the splenic region. (b) Numerous somatostatin-containing cells of the duodenal region. PAP method. x90, x 30. FIG. 4. Pancreatic polypeptide-immunoreactive cells of the pancreas of Pseudemys scripta elegans. (a) The only PP cells of the splenic region. (b) Numerous isolated PP cells of the duodenal region. PAP method. x 90, x 30.
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AYALA,
AND
ABAD
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IMMUNOCYTOCHEMISTRY
toplasmic process were located among the exocrine parenchyma isolated or in groups of two or three. Electron Microscopy B cells contained a large oval euchromatinic nucleus with close deep indentations and nucleolus (Fig. 5). Juxtanuclear dictyosomes displayed cisternae with dilated borders containing an osmiophilic material. Numerous small vesicles were scattered throughout the cytoplasm. Clustered filamentous mitochondria with dense matrix and longitudinal or transversal cristae, free ribosomes, scarce rough endoplasmic reticulum, multivesicular bodies, some large lysosomes, centrioles, and a cilium could be seen (Fig. 6). Striated fibers having a regular cross banding with a repeating period composed of parallel microfilaments, microtubules, and some microfilaments were present (Fig. 7). Characteristic B secretory granules were round and sometimes irregular, of uniform size (500 nm in diameter) and scattered throughout the whole cell or clustered in the basal cytoplasm near capillaries. Most of the granules had a round, irregular, or rod core of varying size and electron density and a light or medium dense matrix (Fig. 7). Some granules showed a homogeneous, moderately dense content extended to their limiting membrane . A cells showed a large, oval, scalloped, eccentric euchromatinic nucleus with a dense
99
nucleolus (Fig. 8). Several juxtanuclear dictyosomes next to the cell membrane had cisternae with dilated borders with an electron-dense content (Fig. 9). Scattered numerous mitochondria with dense matrix and longitudinal cristae, scarce long cisternae of rough endoplasmic reticulum, free ribosomes, lysosomes, microtubules, and bundles of microfilaments were found (Figs. 9 and 10). Secretory granules (500 nm in diameter), with or without a clear halo, were electron-dense, round and dispersed or, if numerous, grouped at the cell pole next to capillaries. Granules which were pyriform or in contact with the cell membrane (Fig. 10) and desmosomes were seen. D cells were found surrounding B cells, and in turn surrounded by A cells, or isolated in the pancreatic parenchyma. The large central euchromatinic nucleus, with a small nucleolus, had both slight and deep invaginations (Fig. 11). A well-developed Golgi apparatus, scarce filamentous mitochondria, numerous free ribosomes, and short cisternae of rough endoplasmic reticulum next to the cell periphery could be seen. Multivesicular bodies, microtubules, striated fibers having a regular crossbanding composed of parallel microfilaments, centriole, and lysosomes were also found (Fig. 12). Two types of secretion granules were seen. The most numerous were round or ovoid, frequently with incomplete surrounding membrane, and containing a medium electron-dense material (Fig. 12). These granules were larger (700
FIG. 5. Electron micrograph of B cells showing an indented nucleus (N), a well-developed Golgi apparatus (G), and the secretory granules with an irregular dense core. x 6300. FIG. 6. Detail of the cytoplasm of a B cell. Note the centriole (cn), developing cilium. groups of small vesicles, and the secretory granules. cl: cilium. x 20,000. FIG. 7. Mitochondria, secretion granuies, Iysosomes, and striated tibres (D of a B cell. x 20,000. FIG. 8. Electron micrograph of an A cell (A) showing euchromatinic nucleus with a dense nucleolus (nu) and electrondense secretory granules. D: cytoplasm of a D cell. x 6300. FIG. 9. Detail of the cytoplasm of an A cell showing Golgi apparatus (G), lysosomes (ly), and secretory granules. Note the dense material inside a golgian cistema (arrow). x 20,000. FIG. 10. Mitochondria (m) and secretion granules next to plasmatic membrane of an A cell of Pseudemys scripra elegans. x 20,000.
AGULLEIRO,
GARCIA AYALA,
AND ABAD
FIG. 11. Electron micrograph of a D cells of endocrine pancreas of Pseudemys scripra elegans showing euchromatinic nucleus (N) and secretion granules of different electrondensity. G: Golgi apparatus. x 7500. FIG. 12. Golgi apparatus, centriole (cn), striated fibre (f), and characteristic medium electron-dense granules of a D cell. x 20,000. FIG. 13. Detail of cytoplasm of a D cell. Note the dense granules with round core. x 20.000.
nm in diameter) and less dense than the other granular type (300 nm in diameter) (Fig. 13). A clear halo separated the membrane and core in both granular types. PP cells were joined by desmosomes to the adjacent exocrine pancreatic cells. An extremely light nucleus with dense nucleolus, showing deep invagination was present (Fig. 14). The Golgi apparatus had
some dilated cisternae containing a dense material similar to the secretion granules (Fig. 16). Scattered mitochondria with dense matrix, some long cisternae of rough endoplasmic reticulum, free ribosomes, multivesicular bodies, lysosomes, microtubules, and striated fibers having a regular cross banding composed of parallel microfilaments were found. Secretion granules
TURTLE
PANCREAS
IMMUNOCYTOCHEMISTRY
were round, pyriform, or almost irregular. They were small (350 nm in diameter) and had a very dense core (Fig. 15). DISCUSSION
Although only alpha and beta cells have been previously reported in the endocrine pancreatic tissue of P. scripta elegans (Miller, 1962), immunocytochemical meth-
ods have been able to show insulin-, glucagon-, somatostatin-, and pancreatic-polypeptide immunoreactive cells similar to those described in the lizards Anofis carolinensis (Rhoten and Smith, 1978; Rhoten and Hall, 1981), Mabuya quinquetaeniata and Uromastyx aegyptia (El-Salhy and Grimelius, 198 la), and Chalcides ocellatus (ElSalhy et al., 1983), and in the crocodile Alligator mississippiensis (Buchan et al.,
FIG. 14. PP cell surrounded by exocrine pancreatic cells. Note the extremely light indented nucleus (N) and small secretory granules. x 4000. FIG. 15. Small electron-dense secretory granules of a PP cell. x 20,000. FIG. 16. Note a vesicule with dense content near the Golgi apparatus (arrow), small electron-dense secretory granules, and lysosomes (ly) of a PP cell of endocrine pancreas of Pseudemys scripta elegnns.
X 20,000.
101
102
AGULLEIRO,
GARCIA
AYALA,
AND
ABAD
1982). The glucagon-immunoreactive cells These cells, as in lizards (El-Salhy and Grialso showed GIP immunoreactivity. Glumelius, 1981a, b), were numerous and isocagon/GIP immunoreactive cells were lated in the duodenal pancreatic region found in trout (Wagner and McKeown, of P. scripta elegans. PP cells, isolated 1981) where, as in P. scripta elegans, or in groups of two or three, were seen blocking GIP antiserum by glucagon no throughout the pancreas among the exostain was observed. GIP immunoreactivity crine cells in P. scripta elegans as in other was found in specific pancreatic cells of U. reptilia (Rhoten and Smith, 1978; Rhoten aegyptia (El-Salhy and Grimelius, 1981b) and Hall, 1981; Buchan et al., 1982) and where the stain was not blocked by glu- were numerous in the duodenal region, as cagon. GIP and glucagon peptides contain in lizards which have PP cells located in the islet periphery of the splenic zone (El-Salhy several common sequences (Sjolund et al., and Grimelius, 1981a; El-Salhy et al., 1983) and therefore in A cells of the turtle 1983). pancreas a precursor peptide which crossreacts with antisera against both GIP and B, A, D, and PP cells were identified by glucagon could exist. Bovine pancreatic ultrastructural criteria in the pancreas of polypeptide antiserum showed PP cells in P. scripta elegans although only B, A, and D have been found in reptilia (Cardeza, P. scripta elegans just as in lizards while only the avian pancreatic polypeptide an- 1960; Titlbach, 1966, 1967, 1968; Rhoten, tiserum gave positive results in A. missis- 1973) and PP cells have only recently been sippiensis (Buchan et al., 1982). seen using electron immunocytochemical Islet-like structures, not surrounded by methods (Rhoten and Hall, 1981, 1982; connective tissue, were smaller and the iso- Buchan et al., 1982). B cells were characlated cells more numerous in the duodenal terized by their indented nucleus and round region of the pancreas of P. scripta elegans secretory granules containing an irregular while the endocrine tissue of lizards was or rod-shaped dense core similar to granmostly confined to the splenic zone (Elules described in some reptilia (Titlbach, Salhy and Grimelius, 1981a, b; El-Salhy et 1966, 1969). Other granules with homogeal., 1983) and in the case of A. mississip- neous matrix have also been found in B piensis no distinct frequency of the indicells of P. scripta elegans as previously vidual cells types was seen between the noted in other reptilia (Cardeza, 1960; Wapancreatic regions (Buchan et al., 1982). tari et al., 1970; Rhoten, 1971, 1973). A Isolated and scattered insulin- and glucells of P. scripta elegans had a scalloped cagon-containing cells were found to be nucleus, mitochondria with longitudinal more numerous in the duodenal region cristae as in some reptilia (Cardeza, 1960). where the cells groups were smaller than in and secretion granules, similar to other repthe splenic one. Insulin cells occurred in tilia, were round and with dense round core the islet center and the glucagon cells in the (Cardeza, 1960; Titlbach, 1966, 1968, 1969; periphery as in M. quinquetaeniata (ElRhoten, 1971). In D cells of P. scriptu eleSalhy and Grimelius, 1981a) and C. ocel- gans’two secretory granular types of dislatus (El-Salhy et al., 1983). Scarce so- tinct density could been seen although only matostatin cells were located between in- moderately dense secretory granules have sulin and glucagon cell layers in the islets been seen in some reptilia (Cardeza, 1960; of P. scripta elegans while in lizards they Rhoten, 1971, 1973). were mostly found in the islet periphery In previous ultrastructural studies of the (Rhoten and Smith, 1978; El-Salhy and Gri- endocrine pancreatic cells of reptilia, PP from A melius, 1981a, b; El-Salhy et al., 1983). cells could not be distinguished
TURTLE
PANCREAS
cells but in P. scripta elegans they were easily characterized by their extremely light and indented nucleus, a long cytoplasmic protrusion, and the small round electron-dense granules similar to those found in most vertebrates (Solcia et al., 1978) although they were irregular in A. mississippiensis (Buchan et al., 1982) and were large and ellipsoidal in A. carolinensis (Rhoten and Hall, 1981). REFERENCES Buchan, A. M. J., Lance, V., and Polak, J. M. (1982). The endocrine pancreas of Alligator mississippie&s.
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