Original Paper Acta Anal 1992:145:93-100
M. Tahir'1 A. A. Elayat“ S. Jalalah1' M.M. El-Naggar“
Isolated Pancreatic Islets of the Rat: An Ultrastructural Study
Department of Anatomy. Faculty of Medicine and Allied Sciences. King Abdulaziz University, and King Falid Medical Research Center. King Abdulaziz University. Jeddah. Saudi Arabia
Abstract Pancreatic islets from adult Wistar rats were isolated by an improved collagenase digestion technique. Examination of the preparations showed that they contained B cells possessing secretory granules, each having an eccentric elec tron-dense core surrounded by an electron-lucent halo; the Golgi bodies with their characteristic features were located in a juxtanuclear position. Roughly surfaced endoplasmic reticulum and mitochondria were present and were mostly normal in appearance. The cell possessed all the ultrastructural attributes indicating that they were fully functional and structurally intact.
Introduction Earlier examination of the islet tissue with the transmis sion electron microscope was not helpful in differentiating cell types [Lacy. 1957a. b; 1961, 1964; Lacy ct al., 1959). The difficulty was reported to be due to unsuitable fixation procedures. Improvement in fixation techniques, however, obviated this problem to some extent. Subsequently, it had been possible to identify only two types of islet cells by the earlier investigators; namely the A and B cells [Zagury et al.. 1961; Lazarus and Volk. 1962; Zagury. 1962; Herman et al., 1964; Lacy, 1964; Schultrich. 1966], Whereas many authors agree on the ultrastructural features of B cells, different descriptions have been given regarding the ultrastructure of the A cells and the secretory granules of both B and A cells. A large number of reports described B cell granules as having a central electron-dense core with a round or slightly angular profile and surrounded by a clear electron-
Received: January 22. 1992 Accepted: March 15. 1992
lucent halo in: adult rat [Caramia. 1963; Logothetopulos. 1966; Fuller. 1969; Howell et al., 1969a. b; Aerts and Van Assche, 1975; Larsson ct al.. 1976; Sacchi et al., 1982; Halban et al.. 1982: Kruse-Jarre, 1982: Orci et al.. 1984], fe tuses [Perrier-Barta, 1983]. neonates [Dutrillaux et al.. 1982] . isolated islets [Howell et al., 1969b; Meda et al., 1983] and in vitro culture of pancreatic tissue |Orci et al.. 1973; Montesano et al., 1983]. Other investigators described the size of the B cell granules as variable [Arison et al.. 1967: Fukuma, 1974| and highly species specific [Lacy. 1957b; Lacy and Hartroft, 1959; Lacy. 1961; Naka mura et al., 1980]. The core may be quite polymorphous and could take the shape of a sphere or ball, a needle or a fragmented crystalloid or the granules may be elliptical in shape containing less electron-dense core, or may have a faint granular structure and a more closely fitting smooth sac. Different descriptions are also given about A cells both when studied with the light or electron microscope. Volk ct
Mohammad Tahir. MD. PhD Department of Anatomy Faculty of Medicine and Allied Sciences King Abdulaziz University PO Box 9029, Jeddah 21413 (Saudi Arabia)
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Key Words Isolated pancreatic islets Ultrastructure
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the integrity and viability of the islet cells. Therefore, the present study was undertaken to examine the ultra structure of the isolated islets and to evaluate their struc tural integrity and viability. At the same time, an ultrastructural comparison was made of the isolated and intact islets.
Materials and Methods Adult male Wistar rats were used to isolate the pancreatic islets. The collagenase digestion technique [Lacy and Kostianovsky, 1967] was used, employing the ductal perfusion method described by Sutton et al. in 1986. The rats were anesthetized with ether inhalation, and the common bile duct was cannulatcd with a fine polythene tube PE-50 immedi ately after incision of the abdomen. The duet was then ligated near the duodenum, and the rat was bled to death. The pancreas was infused with 7 ml of cold, freshly prepared, collagenase Sigma type V (2 mg/ ml) in medium M199. The pancreas was quickly excised and incubated at 37 °C for 22 min: after this cold medium M 199 was added to stop the process of collagenase digestion. The digested pancreatic tissue was disrupted by aspiration through a 14-gauge cannula. The aspirate was filtered through a Nytcx filter having a 1.000-um mesh and collected in the centrifuge tube which was topped up with the cold medium and centrifuged at 2.000 rprn for l() min at 4 °C. The supernatant was decanted, and the pellet of the islet tissue was suspended in the bottom layer of Ficoll having a density of 1.095; to this Fieoll. gradient layers having densities of 1.085. 1.075 and 1.045. respectively, were carefully added on top of the bottom layer and centrifuged at 2.8(H) rpm for 20 min at 4 °C. The islets usually collect at the first and second interface (between layers having densities 1.045. 1.075 and 1.085. respectively). The islets were aspirated and placed into another centrifuge tube and washed twice by using cold medium: each time these were centrifuged at 2.IHI0 rpm for 10 min at 4 °C . The pellet of the isolated pancreas was suspended in the medium and transferred to a Petri dish. The islets were then further purified by hand picking of the nonislet tissue with the help of a binocular dissecting microscope. Illumination of the preparation was obtained by two horizontal beams of fiberoptic light. The whole process was carried out under a laminar flow hood with the hope of avoiding contamination of the isolated islets. The isolated islets were immediately rinsed with Trump's fixative at 4°C and were processed for electron microscopy in accordance with the method used for the intact islets. However, the isolated islets were centrifuged at each step at 2.0(H) rpm for 10 min to make a pellet to facilitate plastic embedding. For each specimen of the isolated islets, pancreas from 2 rats were used. Small pieces (about I mm ) of pancreas were obtained for studying the ultrastructure of islet cells and for making a comparison with the isolated islet preparations. Three adult male Wistar rats were used for this purpose. The pancreas was exposed, immediately fixed in Trump's fixative at 4 °C. cut into small pieces with a sharp knife and transferred to the fresh fixative and processed as usual. The plastic blocks were trimmed to fit into the microtome chuck. Thick sections were stained with 1% aqueous solution of toluidine blue to locate the islet tissue in the pancreas and to confirm the pres ence of the islet cells in the isolated islet preparations. Thin sections were then obtained using a diamond knife and LKB ultramicrotome.
Tahir/Elayat/Jalalah/EI-Naggar
Ultrastructure of Isolated Pancreatic Islets
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at. [1955] reported that the Devenport Silver method blackened only part of A cells; this was later confirmed by Heilman and Hellerstrom [1961], and Thiery and Bader [1966], who consequently classified A cells as A t (argyrophilic) and A, (nonargyrophilic). Bjorkman and Peterson [ 1966] believed that A, and D cells of Bloom 11931] were different varieties whereas Fajita [ 1964. 1966. 1968], Epple [1966] and Solcia ct al. 119811held that the A, and D cells were the same. Caramia [1963], Fuller [1969] and Orci et al. [1973] reported, however, the presence of D cells containing mod erate electron-opaque secretory granules adhering to the limiting membrane. The existence of these cells was later confirmed by using immunocytochcmical techniques for the detection of somatostatin with light microscopy [Pelle tier et al.. 1975; Erlandsen et al.. 1976; Ito et al., 1978; Baetens et al.. 1979; Unger and Orci. 1981). Application of these methods for electron microscopy also allowed the identification of somatostatin-containing cells as those characterized with pale-looking secretory granules similar to those in D cells [Goldsmith et al., 1975; Larsson et al., 1976]. Pancreatic polypeptide-producing cells, with striking ultrastructural resemblance to gastrin-secreting cells of gas tric and duodenal mucosa as described by Orci et al. [ 1968], Forssman ct al. [1969]. Lechago and Benscome [1970] and Pearse et al. [1970]. were reported in the islet cell of the human pancreas [Dcconinck et al.. 1971], Their secretory granules vary from few and very large with variable elec tron density; high density in small ones to nearly lucid in large ones. Further, the existence of these cells in the nor mal human islets was reported by Lomsky ct al. [1969] by using immunofluorescent techniques. Deconinck et al. [1971] also reported a fifth type of cell in the human islets which resembled the general morphology of serotonin-pro ducing cells as described by Orci et al. [1968] in the gas trointestinal tract of rats and by Pearse et al. [1970] in the gastrointestinal tract of man. These cells showed typically small electron-dense and generally rounded granules, some of which were elongated. Ultrastructural studies on the isolated islets arc evi dently few; using a radioautographic technique in the ultra structure study of the isolated islets. Howell et al. 11969b] could recognize B cells as having the same characteristics of the secretory granule as found in the adult rat pancreas. A similar finding was reported by Orci et al. [ 1973] in an ultrastructural study of the monolayer cell culture of B cells of the neonatal rat pancreas. Further, the techniques which arc used to isolate the pancreatic islets in a pure state may inevitably injure the tissue in the process, thereby affecting
Fig. 1. An electron micrograph of the islet cells from the normal pancreas of the rat. The cells have a highly vacuolated appear ance on account of the large number of secre tory' granules which are variable in their size and distribution: most of these are character ized by an eccentric electron-dense core surrounded by a clear electron-lucent halo. The granules vary from 300 to 500 nm in diameter. The Golgi apparatus is well devel oped and located in a juxtanuclear position. Nearby arc some vacuoles occupied by imma ture granules, filled completely with moder ately electron-dense material. Mitochondria appear mostly as elongated or rounded dense structures. Narrow stacks or network of rough-surfaced endoplasmic reticulum can be seen between the secretory granules. The nuclei of the cells arc oval in shape. Their contours may be regular or irregular; the matrix is clear, and the chromatin clumps are small and mostly adherent to the nuclear membrane. Lamellated bodies are also seen at places, x 5,900.
Observations The preparations of the islets obtained from the normal pancreas of the rat showed cells having a highly vacuolated appearance on account of the presence of a large number of secretory granules in their cytoplasm (fig. I, 2). In the majority of the cells, these were characterized by an elec tron-dense core situated eccentrically and surrounded with an electron-lucent halo. The granules ranged from 250 to 550 nm in diameter in our preparations. Most of these cells showed all the structural attributes which characterize B
cells, and conform to the structure of highly active proteinsynthesizing cells containing a well-formed Golgi appara tus, situated in a juxtanuclear position (fig. I. 2), narrow stacks and network of the endoplasmic reticulum; mito chondria were plentiful between the secretory' granules (fig. 1,2). At some places, mitochondria appeared swollen and in some of these, the cristae appeared to be frag mented. Some cells appeared to contain secretory granules in their cytoplasm, which were either completely filled with an electron-dense material or contained an electron-dense center with a narrow electron-lucent halo (fig.2). These granules appeared to be generally smaller in size (200-350 nm in diameter) than those observed in the B cells and were regarded to belong to the A cells. Only the occasional A cell was observed as compared to numerous B cells. The cytoplasm of A cells showed rough-surfaced endoplasmic reticulum in the form of narrow stacks and network
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These were spread with chloroform vapor and ribbons with a silver or gold appearance were picked onto 200 mesh grids. The grids were dried before staining with uranyl acetate and Raynold lead citrate. The sections were then examined with a Philip's transmission electron microscope.
Fig. 2. An electron micrograph of the pancreatic islet cells front the normal pancre atic preparations of the rat. Part of endocrine cells, an exocrine cell and a capillary are seen. The cytoplasm of the endocrine cell on the right side of the micrograph shows a wellformed Golgi apparatus located in a juxtanuclear position and few secretory granules including those around the nucleus. Most of the granules are completely filled with elec tron-dense material. The cell just below the capillary also contains similar granules but some of these show a very' narrow' electronlucent halo around the central dense core; these granules vary in their distribution and size, w'hich ranges from 2(X) to 350 nm in diameter. These arc characteristically A cell granules. Some immature granules are also noticeable. On the left side of the micro graph. the cell shows large-sized granules, each with an electron-dense core mostly ec centrically located within an electron-lucent halo. Most of the granules vary from 250 to 550 nm in diameter; these granules character ize the B cell of the islet. In the endocrine cells, the granules are distributed throughout the cytoplasm; some of these are close to the nuclei. Narrow stacks and network of endo plasmic reticulum, covered with ribosomes, can be seen in between the granules. Mito chondria in these cells are seen as elongated rod-shaped or rounded bodies with cristae. The lower right part of the micrograph shows part of an exocrine cell having a well-formed and prominent rough endoplasmic reticulum: its cisternae seem to be distended at places. Mitochondria are much larger and show the usual structure. The nucleus on the right edge of the micrograph bleongs to this cell and shows a euchromatic structure; nuclear pores arc also evident. x8.5(X).
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cells, i.e. possessing an electron-dense core, mostly located in an eccentric position with a well-marked electron-lucent halo around it. Their size ranged from 200 to 450 nm in diameter (fig.3. 4). and they were generally smaller than those from intact islets. The other type of secretory gran ules contained less electron-dense material completely filling the limiting membrane (fig.3). Some of these secre tory granules possessed a narrow electron-lucent halo (fig.4). The Golgi body with its characteristic features (fig.4) was located in the juxtanuclear position as in the normal preparations. Rough-surfaced endoplasmic reticu lum appeared mostly as a network between the secretory granules (fig.3, 4). Some cells seemed to contain fewer
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between the granules (fig.2); the Golgi apparatus was located in the juxtanuclear position as in B cells and showed many immature granules (fig.2): the same features were seen in the B cell Golgi zones (fig. 1). Mitochondria were abundant, and were small rod shaped or rounded in form, showing the usual structure. The nuclei appeared as elon gated oval bodies, their matrix was clear and the chromatin clumps were mostly attached to the nuclear membrane. Our observation on the preparations from the isolated islets showed that the cells were full of granules and had a highly vacuolated appearance. The granules in the cells were of two different types: in one, the granules resembled in size and structure those described to characterize the B
Fig. 3. An electron micrograph showing parts of endocrine cells obtained from the iso lated pancreatic islets of the rat. The cyto plasm of some of these cells is darker than others. The darker cells show stacks of rough surfaced endoplasmic reticulum with fewer ribosomes and less secretory granules. The granules show different characteristics: in one type the granules are occupied by an electrondense core, mostly located eccentrically and surrounded by an electron-lucent halo: most of these range from 2(X) to 450 nm in diam eter. The other type of granules are filled completely with less electron-dense material: sonic of these also show a narrow electronlucent halo. The Golgi apparatus is located in a juxtanuclear position. Mitochondria arc rounded or rod shaped. Narrow stacks and network of endoplasmic reticulum are pres ent between the granules. The dark cell shows an irregular shaped nucleus having rel atively larger clumps of heterochromatin material. The cells present all the character istics of living and viable cells. The cell on the left side of the micrograph possesses a large number of empty-looking vacuoles. X 6.3(H).
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Fig. 4. An electron micrograph showing a cell from the isolated islet of the rat pan creas at a higher magnification. The cyto plasm looks clear on account of the large number of secretory granules which were variable in their distribution and size. Two types of granule arc observed: one type with an electron-dense core, mostly located eccen trically and surrounded by a large electronlucent halo; most of these granules have a size ranging from 2(H) to 4(H) nm in diameter. The other type shows a less electron-dense center surrounded by a clear halo. Between the granules, narrow stacks and network of endo plasmic reticulum can be seen. There is one large-sized empty-looking vacuole of an uncertain nature. Many mitochondria arc dis tended. some of these have indistinct and fragmented cristae. Well-developed Golgi apparatus occupies the juxtanuclear position, x 11,800.
Discussion Our observations on the isolated islet tissue and that obtained from the normal pancreatic preparations of the rat showed most of the cells had secretory granules. These granules had electron-dense cores surrounded by electronlucent halos of variable sizes; the majority of these granules belonged to the B cell variety. This corroborates the earlier reports on the morphological features of the B cell secre tory granules [Lacy. 1957a, b; Sacchi and Barii, 1985]. Fur ther, it is not possible to rule out the likelihood of some of these cells belonging to other cell varieties, since some reports have described A cells as having a much wider elec tron-lucent halo around the electron-dense core [Sacchi et a!.. 1982; Pcrricr-Barta, 1983; Sacchi and Bani. 1985], Larsson et al. [1976] reported D cell granules, each having a dense central core separated from the limiting membrane by a well-defined but narrow electron-lucent space similar to that of B cells. In our preparations from the normal pancreas, occa sional cells were encountered which exhibited secretory granules, each having a dense core in a tightly fitting mem brane. These granules were slightly smaller than in the other cells and generally ranged from 2(X) to 350 nm in size. These cells were regarded as A cells on the premise that similar characteristics of the A cell granules had been described by Caramia [1963], Fuller [1969] and Howell et al. [1969a], These investigations described A cell granules as dense round bodies with adhering limiting membranes. Some authors found A granules with a moderately dense halo between the dense core and limiting membrane [Orci et al.. 1973]. Other investigators described A granules simi lar to those of B cells having a dense core surrounded by an electron-lucent halo and limiting membrane. According to some, this halo is narrow [Logothetopulos. 1966; Orci et al., 1968; Howell et al., 1969a; Larsson et al., 1976; KruscJarrc, 1982]. while others have reported it as considerably wider [Fuller, 1969; Sacchi et al., 1982; Perrier-Barta, 1983]. Pleomorphism in the granules of A cells was evident in some of our preparations, where some granules con tained an electron-lucent space of a small size surrounding a dense core.
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Different explanations have been offered to explain the heterogenous character of A granules. Fixation procedures have often been blamed, suggesting that the material from the halo was extracted during the process, thereby account ing for the different density gradients of granules. It is, however, hard to accept that the procedures for processing the tissue can produce or. alternatively, eliminate the gran ule halo, since this is not so for other endocrine granules. Our findings on the islet tissue from the normal pan creas of the rat agree with the general description of the cell, given by the earlier investigators: the islet cells show all the ultrastructural characteristics present in protein-synthe sizing cells, i.e. rough-surfaced endoplasmic reticulum. Golgi apparatus, mitochondria and secretory granules. At some places, the micrograph from the islet cells of the nor mal pancreas showed mitochondria with fragmenting cristac. which is not considered to be unusual if the specimen is not osmicated very early. This is, presumably, due to the activity of lipases of the pancreas, which are not inactivated except after osmic acid treatment. Our specimen had to stay overnight in Trump's fixative in some instances. Examination of the isolated islets with the electron microscope showed that our preparations had a fair degree of purified islets, the contaminants were sparse. At a few places only acinar cells could be seen. Further, the ultra structure of the isolated islet cells showed most cells full of secretory granules of variable sizes. The nuclei and other structural characteristics were well preserved; the cells con tained endoplasmic reticulum in the form of stacks or net work between the granules and showed ribosomes at places, and Golgi bodies in juxtanuclear position. The ultrastructure of these cells was fully supportive of their being intact and viable. Our expectation was that different types of islet cells would readily lend themselves to examination in the iso lated islet preparations. But. on the contrary, we observed that cells appeared to present a fairly uniform morphology of their secretory granules, of which two types were seen. In one. these contained an electron-dense core surrounded by an electron lucent halo of a variable size; the granules ranged from 200 to 450 nm in diameter, conforming to the description given for B cells. In the second type, the gran ules were completely filled with less electron-dense mate rial; some of these granules also possessed a narrow elec tron-lucent halo. Both types of granule were encountered in the same cell. Cells with granules which characterize A cells were not seen in our preparation. Light-microscopic examination of the isolated tissue in our laboratory revealed that their peripheral part was traumatized and had lost most of its constituent cells during the process of isola
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secretory granules than others (fig.3). The former con tained rough-surfaced endoplasmic reticulum with less ribosomes. In addition, these cells appeared to have heterochromatic nuclei. Mitochondria were seen in the form of dark rods or rounded bodies with the usual structure.
tion. Bearing in mind the findings of an earlier worker [Caramia. 1963] that B cells form a central core within a mantle of non-B cells at the periphery, it is not surprising to encounter mostly B cells in these preparations. This is fur ther supported by the findings of Moskalcwsky [1965], Vance et al. ( 1968], Buchanan and Mawhinney [1973] and Slavin ct al. [1977] who reported that the peripherally located non-B cells of the isolated pancratic islet are injured during the process. Ultrastructural examination of our preparations from the isolated islets showed that the cells which had stacks of rough-surfaced endoplasmic reticulum with fewer ribo somes also contained scant secretory granules and heterochromatic nuclei. These findings point to either the reduced activity of these cells or their exhaustion after a burst of activity. It is, therefore, reasonable to conclude that our prepara tion of isolated islets showed that the cells were mostly intact, viable and fairly free of contaminants. Further, it was also observed that the morphology of the secretory
granules cannot be regarded as a dependable criterion for the identification of different types of islet cells: they may vary in form and shape under varying functional phases of the cells or under different conditions of fixations. Further, there is ample evidence to suggest that the secretory gran ules of different cell types may have a similar morphology or that the same cell may show pleomorphism in the ultrastructure of their granules and present a different appear ance in varying sectional profiles.
Acknowledgments The authors take this opportunity to express their gratitude to Mrs. Manat Jamjoom. Mr. Helmy Sliihab. Mr. Abdul Kader Kungkodcn and Mr. Mohammad Abdul Kazak Saleh for their technical assistance throughout the inverstigations. The authors would also like to express their thanks to Mrs. Helen Khoja and Mrs. Kim Ishgi for their unre lenting and efficient secretarial assistance. This work was supported by a grant 17/410 from King Abdullaziz University. Jeddah.
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Moskalewsky. S.: isolation and culture of the islets of Langerhans of the guinea pig. Gen. compar. Endocr. 5: 342-353 (1965). Nakamura. M.: Shimada. T.: Fujimori. O.: Ultrastructural studies on the pancreatic polypeptide cells of the rat with special reference to pancre atic regional difference and changes induced by alloxan diabetes. Acta anal. 108: 193-201 (1980). Orci. L.: Like. A .A .: Amhcrdt. M.; Blondel. B.; Kanazawa. Y.: Marliss. E.B.: Lambert. A.E.: Wollhcim. C.B.: Renold. A .E.: Monolayer cell culture of neonatal rat pancras. An ultrastructural and biochemical study of functioning endocrine cells. J. Ultrastrucl. Res. 43: 270-297 (1973). Orci. L.: Lunod. A .: Pictet. R.: Renold. A .E.: Rouillcr. C.: Granulolysis in A cells of endo crine pancreas in spontaneous and experimen tal diabetes in animals. J. Cell Biol. 38: 462-466 (1968). Orci. L.: Ravazolla. M.M.: Amhcrdt. M.: Yanai•hara. C.: Yanaihara. N.: Alban. P.: Renold. A .E.; Pcrrelet. A.: Insulin, not C peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell. .1. Cell Biol. 98: 222-228 (1984). Pearsc. A .G .E.: C'oulling. h: Weavers. B.: Frei sen. S.: The endocrine polypeptide cells of the human stomach, duodenum and jejunum. Gut II: 649-658 (1970). Pelletier. G.: Leclcrc. R.; Arimura. A.; Shally. A.V.: immunuhistochemical localization of somatostatin in the rat pancreas. J. I listochem. Cytochem. 23: 699-701 (1975). Perrier-Barta. H.: A morphometric study of the secretory process in the endocrine pancreas of the fetal rat. Cell Tissue Res. 229; 651-671 (1983). Sacchi. T.B.: Bani. D.: New views of the identifica tion of the various cell types in the pancreatic islets of the rat (an ultrastructural and morphometrical study). Acta anal. 122: 1-17 (1985). Sacchi. T.B.: Cortesini. C.: Lombardo. D.L.: The endocrine pancreas of the rat following porta caval shunt. J. submicrosc. Cytol. 14: 655-671 (1982). Schultrich. S.: Elcktroncnmikroskopische Beiträge zum Bau des menschlichen Insclapparatcs. Endokrinologie 49; 105-125 (1966).
Slavin. B.G.: Bcigelman. P.M.; Bessman. S.P.. Cytophysiological studies on isolated pancre atic islets in vitro. Anal. Rcc. 188: 445-452 (1977). Solda. E.: Capella. C.; Buffa. R.: Vsellini. L.: Fiocca. R.: Sessa, F.: Endocrine cells at the digestive system; in Johnson's physiology of the gastrointestinal tract, pp. 39-58 (Raven Press. New York 1981). Sutton. R.; Peter. M.: McShange. P.: Gray. D.W.R.; Morris. P.J.: An improved method for the isolation of islets of Langerhans from the adult rat pancreas. Transplant Proc. 18: 1819-1820 (1986). Thiery. J.P.; Bader. J.P.: Ultraslruclure des îlots de Langerhans du pancréas humain normal et pathologique. Annls Endocr. 27; 625-647 (1965). Unger. R,H.: Orci. L.: Glucagon and the A cell. Physiology and pathophysiology. New Engl. J. Med. 304: 1518-1524 (1981). Vance. J.E.: Buchanan. K.D.: Challoner. D.R.: Williams. R.H.: Effect of glucose concentra tion on insulin and glucagon release from iso lated islets of Langerhans of the rat. Diabetes 17: 187-193 (1968). Volk. B.W.: Goldner, M.G.: Growlev. H.F.: The effect of prolonged growth hormone adminis tration on the pancreatic alpha cells in normal and hypophyscctomized rats. Metabolism 4: 491-502 (1955). Zagury. D.: Etude des cellules sécrétoires du pan créas chez le rat et l'homme. Bull. Ass. Anal.. Paris 114: 857-862 (1962). Zagury. D.: Brux. J.; Anda. M.: Lager. L.: Etudes des îlots de Langerhans du pancréas humain au microscope électronique. Presse méd. 69; 887-890 (1961).
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Lacy. P.E.: Electron microscopic identification of different cell types in the islets of Langerhans of the guinea-pig. rat. rabhit and dog. Anal. Rec. 128: 255-268 (1957). Lacy. P.E.: Electron microscopy of the normal islets of Langerhans: studies in the dog. rabbit, guinea-pig and rat. Diabetes 6: 498-507 (1957). Lacy. P.E.: Electron microscopy of beta cells of pancreas. Am. J. Med. 31: 851-859 (1961). Lacy. P.E.: Pancreatic beta cell: in Cameron. O'Connor. Aetiology of diabetes mellitus and its complications. Ciba Foundation Colloquia on Endocrinology (Churchill. London 1964). Lacy. P.E.; Cardeza. A.F.; Wilson. W.D.: Elec tron microscopy of rat pancreas. Diabetes 8: 36-44 (1959). Lacy. P.E.: llartroft. W.S.: Electron microscopy of the islets of Langerhans. Ann. N.Y. Acad. Sci. 82: 287 (1959). Lacy. P.E.: Kostianovsky. M.: Method for the iso lation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35-39 (1967). Larsson. L.I.: Sundler. F.: Hakanson. P.: Pancre atic polypeptide. A postulate new hormone: identification of its cellular storage site by light and electron microscopic immunocyloehcmistry. Diabetologia 12: 211-226 (1976). Lazarus. S.S.: Volk. B.W.: Ultranticroscopic and histochemical studies on pancreatic beta cells stimulated by tolbutamide. Diabetes II: 2-10 (1962). Lechago. J.: Bcnscomc. S.A.: Endocrine-like cells of the dog gastric fundus, pylorus and duode num (Abstract). Lab. Invest. 22: 504 (1970). Logothetopulos. J.: Electron microscopy of the pancreatic islets of the rat. Effects of prolonged insulin injections. Diabetes 15:823-829 ( 1966). Lomsky. R.: Langr. F.: Vortel. V.: Immunohisto chemical demonstration of gastrin in mamma lian islets of Langerhans. Nature 223: 618-619 ( 1969). Meda. P.: Michaels. R.L.: Halban. P.A .:O rci. L.: Sheridan. J.D .: In vivo modulation of gap junc tions and dye coupling between B cells of the intact pancreatic islet. Diabetes 32: 858-868 (1983). Montesano. R.; Mouron. P.: Amhcrdt. M.: Orci. L.: Collagen matrix promotes reorganization of pancreatic endocrine cell monolayers into islet-like organoids. J. Cell Biol. 97: 935-939 ( 1983).
M. Tahir'1 A. A. Elayat“ S. Jalalah1' M.M. El-Naggar“
Isolated Pancreatic Islets of the Rat: An Ultrastructural Study
Department of Anatomy. Faculty of Medicine and Allied Sciences. King Abdulaziz University, and King Falid Medical Research Center. King Abdulaziz University. Jeddah. Saudi Arabia
Abstract Pancreatic islets from adult Wistar rats were isolated by an improved collagenase digestion technique. Examination of the preparations showed that they contained B cells possessing secretory granules, each having an eccentric elec tron-dense core surrounded by an electron-lucent halo; the Golgi bodies with their characteristic features were located in a juxtanuclear position. Roughly surfaced endoplasmic reticulum and mitochondria were present and were mostly normal in appearance. The cell possessed all the ultrastructural attributes indicating that they were fully functional and structurally intact.
Introduction Earlier examination of the islet tissue with the transmis sion electron microscope was not helpful in differentiating cell types [Lacy. 1957a. b; 1961, 1964; Lacy ct al., 1959). The difficulty was reported to be due to unsuitable fixation procedures. Improvement in fixation techniques, however, obviated this problem to some extent. Subsequently, it had been possible to identify only two types of islet cells by the earlier investigators; namely the A and B cells [Zagury et al.. 1961; Lazarus and Volk. 1962; Zagury. 1962; Herman et al., 1964; Lacy, 1964; Schultrich. 1966], Whereas many authors agree on the ultrastructural features of B cells, different descriptions have been given regarding the ultrastructure of the A cells and the secretory granules of both B and A cells. A large number of reports described B cell granules as having a central electron-dense core with a round or slightly angular profile and surrounded by a clear electron-
Received: January 22. 1992 Accepted: March 15. 1992
lucent halo in: adult rat [Caramia. 1963; Logothetopulos. 1966; Fuller. 1969; Howell et al., 1969a. b; Aerts and Van Assche, 1975; Larsson ct al.. 1976; Sacchi et al., 1982; Halban et al.. 1982: Kruse-Jarre, 1982: Orci et al.. 1984], fe tuses [Perrier-Barta, 1983]. neonates [Dutrillaux et al.. 1982] . isolated islets [Howell et al., 1969b; Meda et al., 1983] and in vitro culture of pancreatic tissue |Orci et al.. 1973; Montesano et al., 1983]. Other investigators described the size of the B cell granules as variable [Arison et al.. 1967: Fukuma, 1974| and highly species specific [Lacy. 1957b; Lacy and Hartroft, 1959; Lacy. 1961; Naka mura et al., 1980]. The core may be quite polymorphous and could take the shape of a sphere or ball, a needle or a fragmented crystalloid or the granules may be elliptical in shape containing less electron-dense core, or may have a faint granular structure and a more closely fitting smooth sac. Different descriptions are also given about A cells both when studied with the light or electron microscope. Volk ct
Mohammad Tahir. MD. PhD Department of Anatomy Faculty of Medicine and Allied Sciences King Abdulaziz University PO Box 9029, Jeddah 21413 (Saudi Arabia)
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Key Words Isolated pancreatic islets Ultrastructure
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the integrity and viability of the islet cells. Therefore, the present study was undertaken to examine the ultra structure of the isolated islets and to evaluate their struc tural integrity and viability. At the same time, an ultrastructural comparison was made of the isolated and intact islets.
Materials and Methods Adult male Wistar rats were used to isolate the pancreatic islets. The collagenase digestion technique [Lacy and Kostianovsky, 1967] was used, employing the ductal perfusion method described by Sutton et al. in 1986. The rats were anesthetized with ether inhalation, and the common bile duct was cannulatcd with a fine polythene tube PE-50 immedi ately after incision of the abdomen. The duet was then ligated near the duodenum, and the rat was bled to death. The pancreas was infused with 7 ml of cold, freshly prepared, collagenase Sigma type V (2 mg/ ml) in medium M199. The pancreas was quickly excised and incubated at 37 °C for 22 min: after this cold medium M 199 was added to stop the process of collagenase digestion. The digested pancreatic tissue was disrupted by aspiration through a 14-gauge cannula. The aspirate was filtered through a Nytcx filter having a 1.000-um mesh and collected in the centrifuge tube which was topped up with the cold medium and centrifuged at 2.000 rprn for l() min at 4 °C. The supernatant was decanted, and the pellet of the islet tissue was suspended in the bottom layer of Ficoll having a density of 1.095; to this Fieoll. gradient layers having densities of 1.085. 1.075 and 1.045. respectively, were carefully added on top of the bottom layer and centrifuged at 2.8(H) rpm for 20 min at 4 °C. The islets usually collect at the first and second interface (between layers having densities 1.045. 1.075 and 1.085. respectively). The islets were aspirated and placed into another centrifuge tube and washed twice by using cold medium: each time these were centrifuged at 2.IHI0 rpm for 10 min at 4 °C . The pellet of the isolated pancreas was suspended in the medium and transferred to a Petri dish. The islets were then further purified by hand picking of the nonislet tissue with the help of a binocular dissecting microscope. Illumination of the preparation was obtained by two horizontal beams of fiberoptic light. The whole process was carried out under a laminar flow hood with the hope of avoiding contamination of the isolated islets. The isolated islets were immediately rinsed with Trump's fixative at 4°C and were processed for electron microscopy in accordance with the method used for the intact islets. However, the isolated islets were centrifuged at each step at 2.0(H) rpm for 10 min to make a pellet to facilitate plastic embedding. For each specimen of the isolated islets, pancreas from 2 rats were used. Small pieces (about I mm ) of pancreas were obtained for studying the ultrastructure of islet cells and for making a comparison with the isolated islet preparations. Three adult male Wistar rats were used for this purpose. The pancreas was exposed, immediately fixed in Trump's fixative at 4 °C. cut into small pieces with a sharp knife and transferred to the fresh fixative and processed as usual. The plastic blocks were trimmed to fit into the microtome chuck. Thick sections were stained with 1% aqueous solution of toluidine blue to locate the islet tissue in the pancreas and to confirm the pres ence of the islet cells in the isolated islet preparations. Thin sections were then obtained using a diamond knife and LKB ultramicrotome.
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at. [1955] reported that the Devenport Silver method blackened only part of A cells; this was later confirmed by Heilman and Hellerstrom [1961], and Thiery and Bader [1966], who consequently classified A cells as A t (argyrophilic) and A, (nonargyrophilic). Bjorkman and Peterson [ 1966] believed that A, and D cells of Bloom 11931] were different varieties whereas Fajita [ 1964. 1966. 1968], Epple [1966] and Solcia ct al. 119811held that the A, and D cells were the same. Caramia [1963], Fuller [1969] and Orci et al. [1973] reported, however, the presence of D cells containing mod erate electron-opaque secretory granules adhering to the limiting membrane. The existence of these cells was later confirmed by using immunocytochcmical techniques for the detection of somatostatin with light microscopy [Pelle tier et al.. 1975; Erlandsen et al.. 1976; Ito et al., 1978; Baetens et al.. 1979; Unger and Orci. 1981). Application of these methods for electron microscopy also allowed the identification of somatostatin-containing cells as those characterized with pale-looking secretory granules similar to those in D cells [Goldsmith et al., 1975; Larsson et al., 1976]. Pancreatic polypeptide-producing cells, with striking ultrastructural resemblance to gastrin-secreting cells of gas tric and duodenal mucosa as described by Orci et al. [ 1968], Forssman ct al. [1969]. Lechago and Benscome [1970] and Pearse et al. [1970]. were reported in the islet cell of the human pancreas [Dcconinck et al.. 1971], Their secretory granules vary from few and very large with variable elec tron density; high density in small ones to nearly lucid in large ones. Further, the existence of these cells in the nor mal human islets was reported by Lomsky ct al. [1969] by using immunofluorescent techniques. Deconinck et al. [1971] also reported a fifth type of cell in the human islets which resembled the general morphology of serotonin-pro ducing cells as described by Orci et al. [1968] in the gas trointestinal tract of rats and by Pearse et al. [1970] in the gastrointestinal tract of man. These cells showed typically small electron-dense and generally rounded granules, some of which were elongated. Ultrastructural studies on the isolated islets arc evi dently few; using a radioautographic technique in the ultra structure study of the isolated islets. Howell et al. 11969b] could recognize B cells as having the same characteristics of the secretory granule as found in the adult rat pancreas. A similar finding was reported by Orci et al. [ 1973] in an ultrastructural study of the monolayer cell culture of B cells of the neonatal rat pancreas. Further, the techniques which arc used to isolate the pancreatic islets in a pure state may inevitably injure the tissue in the process, thereby affecting
Fig. 1. An electron micrograph of the islet cells from the normal pancreas of the rat. The cells have a highly vacuolated appear ance on account of the large number of secre tory' granules which are variable in their size and distribution: most of these are character ized by an eccentric electron-dense core surrounded by a clear electron-lucent halo. The granules vary from 300 to 500 nm in diameter. The Golgi apparatus is well devel oped and located in a juxtanuclear position. Nearby arc some vacuoles occupied by imma ture granules, filled completely with moder ately electron-dense material. Mitochondria appear mostly as elongated or rounded dense structures. Narrow stacks or network of rough-surfaced endoplasmic reticulum can be seen between the secretory granules. The nuclei of the cells arc oval in shape. Their contours may be regular or irregular; the matrix is clear, and the chromatin clumps are small and mostly adherent to the nuclear membrane. Lamellated bodies are also seen at places, x 5,900.
Observations The preparations of the islets obtained from the normal pancreas of the rat showed cells having a highly vacuolated appearance on account of the presence of a large number of secretory granules in their cytoplasm (fig. I, 2). In the majority of the cells, these were characterized by an elec tron-dense core situated eccentrically and surrounded with an electron-lucent halo. The granules ranged from 250 to 550 nm in diameter in our preparations. Most of these cells showed all the structural attributes which characterize B
cells, and conform to the structure of highly active proteinsynthesizing cells containing a well-formed Golgi appara tus, situated in a juxtanuclear position (fig. I. 2), narrow stacks and network of the endoplasmic reticulum; mito chondria were plentiful between the secretory' granules (fig. 1,2). At some places, mitochondria appeared swollen and in some of these, the cristae appeared to be frag mented. Some cells appeared to contain secretory granules in their cytoplasm, which were either completely filled with an electron-dense material or contained an electron-dense center with a narrow electron-lucent halo (fig.2). These granules appeared to be generally smaller in size (200-350 nm in diameter) than those observed in the B cells and were regarded to belong to the A cells. Only the occasional A cell was observed as compared to numerous B cells. The cytoplasm of A cells showed rough-surfaced endoplasmic reticulum in the form of narrow stacks and network
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These were spread with chloroform vapor and ribbons with a silver or gold appearance were picked onto 200 mesh grids. The grids were dried before staining with uranyl acetate and Raynold lead citrate. The sections were then examined with a Philip's transmission electron microscope.
Fig. 2. An electron micrograph of the pancreatic islet cells front the normal pancre atic preparations of the rat. Part of endocrine cells, an exocrine cell and a capillary are seen. The cytoplasm of the endocrine cell on the right side of the micrograph shows a wellformed Golgi apparatus located in a juxtanuclear position and few secretory granules including those around the nucleus. Most of the granules are completely filled with elec tron-dense material. The cell just below the capillary also contains similar granules but some of these show a very' narrow' electronlucent halo around the central dense core; these granules vary in their distribution and size, w'hich ranges from 2(X) to 350 nm in diameter. These arc characteristically A cell granules. Some immature granules are also noticeable. On the left side of the micro graph. the cell shows large-sized granules, each with an electron-dense core mostly ec centrically located within an electron-lucent halo. Most of the granules vary from 250 to 550 nm in diameter; these granules character ize the B cell of the islet. In the endocrine cells, the granules are distributed throughout the cytoplasm; some of these are close to the nuclei. Narrow stacks and network of endo plasmic reticulum, covered with ribosomes, can be seen in between the granules. Mito chondria in these cells are seen as elongated rod-shaped or rounded bodies with cristae. The lower right part of the micrograph shows part of an exocrine cell having a well-formed and prominent rough endoplasmic reticulum: its cisternae seem to be distended at places. Mitochondria are much larger and show the usual structure. The nucleus on the right edge of the micrograph bleongs to this cell and shows a euchromatic structure; nuclear pores arc also evident. x8.5(X).
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cells, i.e. possessing an electron-dense core, mostly located in an eccentric position with a well-marked electron-lucent halo around it. Their size ranged from 200 to 450 nm in diameter (fig.3. 4). and they were generally smaller than those from intact islets. The other type of secretory gran ules contained less electron-dense material completely filling the limiting membrane (fig.3). Some of these secre tory granules possessed a narrow electron-lucent halo (fig.4). The Golgi body with its characteristic features (fig.4) was located in the juxtanuclear position as in the normal preparations. Rough-surfaced endoplasmic reticu lum appeared mostly as a network between the secretory granules (fig.3, 4). Some cells seemed to contain fewer
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between the granules (fig.2); the Golgi apparatus was located in the juxtanuclear position as in B cells and showed many immature granules (fig.2): the same features were seen in the B cell Golgi zones (fig. 1). Mitochondria were abundant, and were small rod shaped or rounded in form, showing the usual structure. The nuclei appeared as elon gated oval bodies, their matrix was clear and the chromatin clumps were mostly attached to the nuclear membrane. Our observation on the preparations from the isolated islets showed that the cells were full of granules and had a highly vacuolated appearance. The granules in the cells were of two different types: in one, the granules resembled in size and structure those described to characterize the B
Fig. 3. An electron micrograph showing parts of endocrine cells obtained from the iso lated pancreatic islets of the rat. The cyto plasm of some of these cells is darker than others. The darker cells show stacks of rough surfaced endoplasmic reticulum with fewer ribosomes and less secretory granules. The granules show different characteristics: in one type the granules are occupied by an electrondense core, mostly located eccentrically and surrounded by an electron-lucent halo: most of these range from 2(X) to 450 nm in diam eter. The other type of granules are filled completely with less electron-dense material: sonic of these also show a narrow electronlucent halo. The Golgi apparatus is located in a juxtanuclear position. Mitochondria arc rounded or rod shaped. Narrow stacks and network of endoplasmic reticulum are pres ent between the granules. The dark cell shows an irregular shaped nucleus having rel atively larger clumps of heterochromatin material. The cells present all the character istics of living and viable cells. The cell on the left side of the micrograph possesses a large number of empty-looking vacuoles. X 6.3(H).
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Fig. 4. An electron micrograph showing a cell from the isolated islet of the rat pan creas at a higher magnification. The cyto plasm looks clear on account of the large number of secretory granules which were variable in their distribution and size. Two types of granule arc observed: one type with an electron-dense core, mostly located eccen trically and surrounded by a large electronlucent halo; most of these granules have a size ranging from 2(H) to 4(H) nm in diameter. The other type shows a less electron-dense center surrounded by a clear halo. Between the granules, narrow stacks and network of endo plasmic reticulum can be seen. There is one large-sized empty-looking vacuole of an uncertain nature. Many mitochondria arc dis tended. some of these have indistinct and fragmented cristae. Well-developed Golgi apparatus occupies the juxtanuclear position, x 11,800.
Discussion Our observations on the isolated islet tissue and that obtained from the normal pancreatic preparations of the rat showed most of the cells had secretory granules. These granules had electron-dense cores surrounded by electronlucent halos of variable sizes; the majority of these granules belonged to the B cell variety. This corroborates the earlier reports on the morphological features of the B cell secre tory granules [Lacy. 1957a, b; Sacchi and Barii, 1985]. Fur ther, it is not possible to rule out the likelihood of some of these cells belonging to other cell varieties, since some reports have described A cells as having a much wider elec tron-lucent halo around the electron-dense core [Sacchi et a!.. 1982; Pcrricr-Barta, 1983; Sacchi and Bani. 1985], Larsson et al. [1976] reported D cell granules, each having a dense central core separated from the limiting membrane by a well-defined but narrow electron-lucent space similar to that of B cells. In our preparations from the normal pancreas, occa sional cells were encountered which exhibited secretory granules, each having a dense core in a tightly fitting mem brane. These granules were slightly smaller than in the other cells and generally ranged from 2(X) to 350 nm in size. These cells were regarded as A cells on the premise that similar characteristics of the A cell granules had been described by Caramia [1963], Fuller [1969] and Howell et al. [1969a], These investigations described A cell granules as dense round bodies with adhering limiting membranes. Some authors found A granules with a moderately dense halo between the dense core and limiting membrane [Orci et al.. 1973]. Other investigators described A granules simi lar to those of B cells having a dense core surrounded by an electron-lucent halo and limiting membrane. According to some, this halo is narrow [Logothetopulos. 1966; Orci et al., 1968; Howell et al., 1969a; Larsson et al., 1976; KruscJarrc, 1982]. while others have reported it as considerably wider [Fuller, 1969; Sacchi et al., 1982; Perrier-Barta, 1983]. Pleomorphism in the granules of A cells was evident in some of our preparations, where some granules con tained an electron-lucent space of a small size surrounding a dense core.
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Different explanations have been offered to explain the heterogenous character of A granules. Fixation procedures have often been blamed, suggesting that the material from the halo was extracted during the process, thereby account ing for the different density gradients of granules. It is, however, hard to accept that the procedures for processing the tissue can produce or. alternatively, eliminate the gran ule halo, since this is not so for other endocrine granules. Our findings on the islet tissue from the normal pan creas of the rat agree with the general description of the cell, given by the earlier investigators: the islet cells show all the ultrastructural characteristics present in protein-synthe sizing cells, i.e. rough-surfaced endoplasmic reticulum. Golgi apparatus, mitochondria and secretory granules. At some places, the micrograph from the islet cells of the nor mal pancreas showed mitochondria with fragmenting cristac. which is not considered to be unusual if the specimen is not osmicated very early. This is, presumably, due to the activity of lipases of the pancreas, which are not inactivated except after osmic acid treatment. Our specimen had to stay overnight in Trump's fixative in some instances. Examination of the isolated islets with the electron microscope showed that our preparations had a fair degree of purified islets, the contaminants were sparse. At a few places only acinar cells could be seen. Further, the ultra structure of the isolated islet cells showed most cells full of secretory granules of variable sizes. The nuclei and other structural characteristics were well preserved; the cells con tained endoplasmic reticulum in the form of stacks or net work between the granules and showed ribosomes at places, and Golgi bodies in juxtanuclear position. The ultrastructure of these cells was fully supportive of their being intact and viable. Our expectation was that different types of islet cells would readily lend themselves to examination in the iso lated islet preparations. But. on the contrary, we observed that cells appeared to present a fairly uniform morphology of their secretory granules, of which two types were seen. In one. these contained an electron-dense core surrounded by an electron lucent halo of a variable size; the granules ranged from 200 to 450 nm in diameter, conforming to the description given for B cells. In the second type, the gran ules were completely filled with less electron-dense mate rial; some of these granules also possessed a narrow elec tron-lucent halo. Both types of granule were encountered in the same cell. Cells with granules which characterize A cells were not seen in our preparation. Light-microscopic examination of the isolated tissue in our laboratory revealed that their peripheral part was traumatized and had lost most of its constituent cells during the process of isola
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secretory granules than others (fig.3). The former con tained rough-surfaced endoplasmic reticulum with less ribosomes. In addition, these cells appeared to have heterochromatic nuclei. Mitochondria were seen in the form of dark rods or rounded bodies with the usual structure.
tion. Bearing in mind the findings of an earlier worker [Caramia. 1963] that B cells form a central core within a mantle of non-B cells at the periphery, it is not surprising to encounter mostly B cells in these preparations. This is fur ther supported by the findings of Moskalcwsky [1965], Vance et al. ( 1968], Buchanan and Mawhinney [1973] and Slavin ct al. [1977] who reported that the peripherally located non-B cells of the isolated pancratic islet are injured during the process. Ultrastructural examination of our preparations from the isolated islets showed that the cells which had stacks of rough-surfaced endoplasmic reticulum with fewer ribo somes also contained scant secretory granules and heterochromatic nuclei. These findings point to either the reduced activity of these cells or their exhaustion after a burst of activity. It is, therefore, reasonable to conclude that our prepara tion of isolated islets showed that the cells were mostly intact, viable and fairly free of contaminants. Further, it was also observed that the morphology of the secretory
granules cannot be regarded as a dependable criterion for the identification of different types of islet cells: they may vary in form and shape under varying functional phases of the cells or under different conditions of fixations. Further, there is ample evidence to suggest that the secretory gran ules of different cell types may have a similar morphology or that the same cell may show pleomorphism in the ultrastructure of their granules and present a different appear ance in varying sectional profiles.
Acknowledgments The authors take this opportunity to express their gratitude to Mrs. Manat Jamjoom. Mr. Helmy Sliihab. Mr. Abdul Kader Kungkodcn and Mr. Mohammad Abdul Kazak Saleh for their technical assistance throughout the inverstigations. The authors would also like to express their thanks to Mrs. Helen Khoja and Mrs. Kim Ishgi for their unre lenting and efficient secretarial assistance. This work was supported by a grant 17/410 from King Abdullaziz University. Jeddah.
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