Light and Electron Microscopic Histochemistry of the Serous Secretory Granules in the Salivary Glandular Cells of the Mongolian Gerbil (Mongolian meridianus) and Rhesus Monkey (Macaca irus) MISAO ICHIKAWA AND ATSUSHI ICHIKAWA Department ofAnatomy, Yokohamn City University School of Medzcme, Yokohama 232, Japan
ABSTRACT Light and electron microscopic histochemistry was carried out on the serous secretory granules of the parotid acinar and sublingual demilunar cells of the Mongolian gerbil. These are bipartite in structure with a central dense core and a rim of lower density. Light microscopic techniques included staining of sections with Alcian blue and PAS reaction before and after sialidase digestion. PA-methenamine silver, dialyzed iron, colloidal thorium and ruthenium red reactions for mucosubstance were compared on control and pronase digested materials. The results obtained have been interpreted to indicate that the peripheral rim of the granules contained mainly sialomucin whereas the central dense core was rich in protein. Freeze-etched replicas and frozen thin sections of the gerbil salivary glands revealed the bipartite substructure of the granules to be a consistent structure regardless of specimen preparation procedures. The secretory granules in the serous acinar cells of the monkey submandibular gland which also have a central dense spherule and a rim of lower dense matrix were tested in the same way with the PA-methenamine silver method. A positive reaction was limited to the rim of lower density matrix, indicating that this granular matrix is rich in carbohydrates in contrast to an unstained dense central spherule which is digested with pronase. Morphological and cytochemical similarities of the granule content between species are compared. A number of studies have revealed that the parotid acinar and sublingual demilunar cells in most rodents contain homogeneous secretory granules enclosed in a closely fitting limiting membrane (Parks, '61, '62; Shackleford and Schneyer, '64; Shackleford and Wilborn, '68; Amsterdam et al., '69; Wilborn and Schneyer, '70; Enomoto and Scott, '71; Castle et al., '72; Hand, '72; Cope and Williams, '73; Rhodin, '74). We have recently found that the parotid acinar cells of the Mongolian gerbil have secretory granules with a large dense homogeneous core and the peripheral rim of lower density which appears finely granulated (Ichikawa and Ichikawa, '75). It was further noted that the bipartite substructure of these granules did not depend on specimen preparation procedures, degree of ANAT. REC., 189: 125-140.
granule maturation or age of the animal. The present study further characterizes the secretory granules of the Mongolian gerbil salivary glandular cells by light and electron microscopic histochemistry, freeze-etched replicas and frozen thin sections. In primate salivary glands, the serous secretory granules have been reported to have a bipartite substructure (Sato et al., '66; Cowley and Shackleford, '70; Kagayama, '71; Tandler and Erlandson, '72; Riva and RivaTesta, '73). In order to compare secretory granules of primates to those of the gerbil, we examined demilunar cells of the monkey submandibular gland by some of the same histochemical techniques and found similarities between granules of both species. Received Dec. 19, '75. Accepted Mar. 18, '77.
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MISAO ICHIKAWA A N D ATSUSHI ICHIKAWA MATERIALS AND METHODS
Twelve male and five female adults of the Mongolian gerbil, Meriones meridianus, ranging from 57-75 gm in body weight, raised from birth in our laboratory colony were used in this study. The animals were fed on standard rat food pellets and water ad libitum and fasted for 12 hours before fixation. While under sodium pentobarbital anesthesia, the gerbils used for electron microscopy were fixed by vascular perfusion of fixative introduced into the left ventricle and the parotid and sublingual glands were then excised. Tissue blocks were minced in the same fixative and fixed for a total of two hours at 4OC. The fixative used contained 2.0-2.5%glutaraldehyde buffered a t pH 7.2-7.4with 0.2 M cacodylate (340-385 mOS) or 0.2 M phosphate (395424 mOS). The tissues were then washed in buffer for three hours, postfixed in cacodylate or phosphate buffered 1% osmium tetroxide for one hour, dehydrated in a graded series of cold ethanol and embedded in an EponAraldite mixture. For histochemical analyses, the following methods were employed. (1) Paraffin sections, which were obtained from the materials fixed with 10% formaldehyde buffered with 2%calcium acetate or 6%HgC12 buffered with 1.3% sodium acetate, were stained with Alcian blue a t pH 1.0 and 2.5 followed by the periodic acid Schiff reaction (Spicer, '65). The sialidase digestion test (Spicer and Warren, '60) was also carried out on these sections. (2) Basic protein was examined in paraffin sections stained with 0.04% Biebrich scarlet in Laskey's glycine buffer a t various pHs (Spicer and Lillie, '61). (3) Tissues were fixed and stained with ruthenium red (Luft, '71) and then embedded for electron microscopy. (4) Thin resin sections were stained with periodic acid methenamine silver for mucosubstance (De Martino and Zamboni, '67). (5) For the dialyzed iron reaction, the tissues were taken immediately a€ter glutaraldehyde fixation and sliced into50-pm-thick sections by a tissue sectioner and treated with iron solution a t pH 2.0 (Wetzel et al., '66).(6)Some tissue blocks were embedded in methacrylate after osmification and thin sections were stained with colloidal thorium (Revel '64). (7) Some resin sections were treated with 0.5%pronase (purified Type VI of Sigma Chem. Go.) for 30 minutes to 12 hours a t 40°C before staining with uranyl acetate and lead citrate (Monneron e t Bernhard, '66).
The flat substrate method for histochemical examinations (Ichikawa and Ichikawa, '73) was applied for carrying out the histochemical methods 4, 6 and 7. For preparing freezeetched replicas, the tissue was perfused with phosphate-buffered glutaraldehyde, small pieces rinsed with buffer solution and glycerinated in 30%glycerol in Ringer solution for two hours, and frozen in Freon 22 cooled in liquid nitrogen. Frozen tissues were fractured, etched for one to two minutes and shadowed with platinum with a Hitachi HFZ1 freeze etching device. After carbon was evaporated to form a replica the tissue was removed with a solution of hypochlorite and the replica examined with JEM lOOC electron microscope operated a t 100 kv. For frozen thin sections, small tissue blocks were fixed in the same glutaraldehyde solution described earlier for ten minutes, washed in buffer, transfered into 50% sucrose in buffer for 15 to 30 minutes, frozen in Freon 22 in liquid nitrogen and sectioned on a SORVALL MT-2B ultramicrotome adapted with a cryokit t o maintain the tissue a t -90°C. The thin sections were dried and negatively stained with 0.5%uranyl acetate (Tokuyasu, '73). Thin sections of the monkey submandibular gland were obtained from two male adult animals (3.5 and 4.2 kg in body weight) by the same method as described for the gerbil parotid gland and stained with PA-methenamine silver. Some thin sections were examined in electron microscopy after pronase digestion as for the gerbil parotid gland. OBSERVATIONS
The parotid and sublingual glands of the Mongolian gerbil are generally similar in their location, development and cell architecture to those in other rodents reported by previous investigators. The acinar cells composing the terminal portion of the parotid gland are cuboidal or pyramidal in shape and have cytological characteristics of a typical serous cell (fig. 1).They contain a spherical nucleus located near the cell base, well-developed lamellar arrays of the granular endoplasmic reticulum in the basal and perinuclear regions, a prominent Golgi complex in the supranuclear area and numerous secretory granules in the apical cytoplasm. The lateral and basal borders of the cell body are provided with numerous interdigitating cell folds. The secretory granules have a bipartite substructure composed of a large dense homogeneous core
HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES
and the peripheral rim of finely granulated material (fig. 1, inset). The serous acinar cells of the sublingual gland are located a t the blind end of the tubulo-alveolar gland in the form of a demilune (fig. 2). They are low pyramidal in shape and rarely appear to border onto the duct in sections, because they have a small luminal border. They contain a spherical nucleus situated near the base, lamellar arrays of the granular endoplasmic reticulum in the lower half of the cell body and a well-developed Golgi complex in the supranuclear region. The lateral and basal cell membranes are relatively smooth in contrast to the parotid acinar cells. The membrane-enclosed secretory granules of the sublingual demilunar cells are also bipartite in substructure. They consist of a n eccentric, dense, homogeneous core of onethird to two-thirds the diameter of the granule and a peripheral component composed of a complex of pale matrix with moderately dense material of the reticulated appearance. In some planes of sections, the latter appears to be packed parallel arrays of meandering tubules (fig. 2, inset). The appearance of the secretory granules both in the parotid acinar and sublingual demilunar cells were similar in both sexes and not dependent on differences in osmoralities of the fixatives used. The serous acinar cells of the monkey submandibular gland are pyramidal in shape and also have substructural features of a typical serous cell (fig. 3). The secretory granules have an eccentrically placed dense spherule and a rim of lower dense matrix. The latter is usually composed of a homogeneously fibrillogranular material and occasionally accompanied with a randomly located crescentric structure of intermediate density.
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followed by the PAS reaction stained the peripheral component bluish purple or blue. After a long term digestion of the slide with sialidase (18 hours), the peripheral rim of the granules was stained red with the PAS reaction. The staining pattern in the thin sections of the parotid and sublingual gland acinar cells treated with PA-methenamine silver are shown in figures 4 and 5, respectively. Deposition of reduced silver was intense in the peripheral rim of the granules while the central dense core was only faintly stained. Following treatment with dialyzed iron a t pH 2.0, the acinar cells in the outermost layer of the tissue blocks took up the dye into their cytoplasm and the peripheral rim of the granules in these cells appeared denser than the central core when the sections were not stained with uranyl acetate and lead citrate (fig. 6). In control sections the whole granule is of low density and the dense core is apparent only after uranyl plus lead staining. The results obtained by the colloidal thorium technique were similar to the dialyzed iron staining though the staining was not as intense as in the latter case. Ruthenium red staining of the granules was apparent only in cells where the plasma membrane was disrupted. In such cells the peripheral rim of the granules appeared denser than that in other cells unstained in the same section. In all the EM histochemical methods for mucosubstance used in this study, deposition of staining precipitates in the sublingual demilunar cells was localized t o the material forming the reticulated network peripheral to the eccentric dense core, in contrast to a homogeneous distribution in the peripheral rim of the granules in the parotid acinar cells. In the serous acinar cells of the monkey submandibular gland stained with PA-methenamine silver, the secretory granules were Histochemical results also found to have an unreacted dense central The central dense core of the secretory spherule and a positively stained peripheral granules both in the parotid acinar and sub- matrix (fig. 7).The outer matrix which is comlingual demilunar cells of the gerbil was posed of randomly located zones of intermediweakly positive with the PAS reaction and ate and low density reacted similarly to the stained intensely red with Biebrich scarlet a t PA-methenamine silver stain but the denser pH 6.0. Electron microscopy of thin sections region stained slightly more intensely. After digested with pronase revealed that the cen- pronase digestion, the central dense spherule tral dense core lost its usual density (fig. 11). lost their proper density and appeared empty The material in the peripheral rim of the (fig. 12). granules appeared red with the PAS reaction Results of cryo-techniques and intensely blue with Alcian blue a t pH 2.5 Freeze-etched replicas of the gerbil parotid but faintly stained at pH 1.0. A combined staining reaction a t pH 2.5 with Alcian blue acinar cells showed a bipartite substructure
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MISAO ICHIKAWA AND ATSUSHI ICHIKAWA
consisting of the central dense core surrounded with relatively narrow outer zone (fig. 8). The peripheral zone appears to be more readily etched under high vacuum than the core. The reticulated appearance of the peripheral zone in the sublingual demilunar cell granules was apparent in freeze-etched replicas (fig. 9). The bipartite heterogeneity of the granules was also found in uranyl acetate stained frozen thin sections of the gerbil parotid acinar cells (fig. 10). DISCUSSION
Light microscopic histochemistry in the present study revealed that the materials in the peripheral rim of gerbil salivary gland granules were intensely positive with the PAS reaction and stained blue with Alcian blue a t pH 2.5but faintly a t pH 1.0. Prolonged digestion with sialidase destroyed this affinity for Alcian blue. These properties indicate the presence of sialomucin in the peripheral rim of the granules (Quintarelli e t al., '64; Spicer, '65). The data of the fine structural histochemistry for mucosubstance agree with the results a t light microscopic level and demonstrated with a higher resolution that staining precipitates were specifically localized in the peripheral rim of the granules whereas the central core showed little or no reaction. These results are presumed to be due to the presence of acidic groups in sialomucin of the peripheral rim of the granules. Our previous study of the gerbil parotid gland has revealed that the bipartite substructure of granule content was detectable in all the materials examined regardless of fixation and staining methods, granule maturation and animal age (Ichikawa and Ichikawa, '75). Cryo-techniques applied to this study which exclude use of dehydrating and embedding agents eliminate further treatments which may introduce an artefactual appearance of this substructure. The freezeetched replicas of the gerbil parotid acinar cells revealed secretory granules consisting of a large round central area surrounded with a rather coarse peripheral narrow zone. These two areas are considered to represent the central dense core and the peripheral rim of lower density seen in thin sections, respectively, because of similarities in their dimension and proportion. A reticulated framework of moderately dense material found in the peripheral rim of sublingual demilunar cell granules was also present in the freeze-etched replicas.
Thus, the results obtained by cryo-techniques further suggest that the bipartite substructure of granule content in both parotid acinar and sublingual demilunar cell granules is not an artefact caused during preparatory procedures after fixation but a stable structure. Light microscopic histochemistry of the salivary glandular cells in other rodents has revealed that serous secretory granules were intensely stained with the PAS reaction but remain unstained with Alcian blue and colloidal iron (Shackleford and Wilborn, '68). They have emphasized that these granules are serous in nature. Since the early ultrastructural study of Scott and Pease ('591,a number of electron microscopic observations on the salivary glandular cells have been shown that the mature secretory granules of the serous cells have a dense homogeneous interior (Shackleford and Schneyer, '64; Shackleford and Wilborn, '68; Amsterdam et al., '69; Wilborn and Schneyer, '70; Enomoto and Scott, '71; Castle e t al., '72; Hand, '72; Cope and Williams, '73; Rhodin, '74). Heterogeneity in the distribution of granule matrix density has been described by Parks ('61, '62) and Rutberg ('611, but has been attributed to fixation artefact by Robinovitch et al. ('66) and Simson et al. ('74). The general appearance of the salivary glandular cells has equated them to pancreatic exocrine cells and the granules to the zymogen granules. It is, however, known that the serous secretory granules of the salivary cells contain a fairly large carbohydrate component when compared to other serous cells, such as pancreatic acinar cells and chief cells of the gastric gland (Leblond, '50; Munger, '64). The ultrastructural appearance of salivary gland cell granules seems to vary among different species and may indicate differencies in their composition. Thus in some rodents, granules of some salivary glands have been defined as seromucous rather than serous (Munger, '64; Shackleford and Wilborn, '64). Simson et al. ('74) have reported bipartite distribution pattern of matrix density in the rat parotid acinar cell granules and suggested that it indicated the presence of a lipoidal material in the matrix. The present data revealed that the central dense core of the granules was rich in protein whereas the peripheral rim was composed mainly of sialomucin. Unlike other rodent or non-primate salivary glands, the serous cells of the gerbil parotid and sublingual glands have secretory granules with a clearly defined
HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES
heterogeneous interior consisting of chemically different compositions. This organization is rather similar to those in the monkey serous salivary cells. A similar bipartite substructure of granules has been described in the mouse Paneth cells (Spicer et al., '66) and the guinea pig pyloric glandular cells (Zeitoun et al., '72) in addition to the salivary glandular cells in primates. Spicer et al. ('66) have verified mucosubstances in the peripheral rim of granules by light and electron microscopic histochemistry. Zeitoun et al. ('72) have shown the peripheral ring of the granules was stained with Thiery's method for glycoprotein and polysaccharides (Thiery, '67) whereas the central dense core was unstained. They have also demonstrated by immunohistochemistry that pyloric pepsinogen was concentrated in the central dense core of granules. The present data are quite similar to those obtained from these glandular cells. Such a characteristic feature of chemical components of granule content is not well understood, but highly charged mucosubstances may be considered to provide a protective coat for the central zymogenic material. Mechanisms by which the granule content is separated into two phases and the intracellular pathway of two components to form a granule packed within a limiting membrane remain to be elucidated. Since the serous secretory granules of the gerbil salivary glandular cells are morphologically similar to those of the monkey and probably of man, the gerbil material may be useful as a model system for experimental manipulations correlated with a morphological study on secretory processes which may be used to understand the secretion of human salivary glands. ACKNOWLEDGMENT
The authors are indebted to Doctor Susumu Ito, Department of Anatomy, Harvard Medical School, for his invaluable advice during preparation of this manuscript. LITERATURE CITED Amsterdam, A., I. Ohad and M.Schramm 1969 Dynamic changes in the ultrastructure of t h e acinar cell of t h e r a t parotid gland during the secretory cycle. J. Cell Biol., 41:
753-773.
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and after isoprenaline-induced degranulation. J. Anat., 116: 269-284.
Cowley, L. H., and J. M. Shackleford 1970 Electron microscopy of squirrel monkey parotid glands. Alabama J. Med. Sci., 7: 273-282. De Martino, C.,and L.Zamboni 1967 Silver methenamine stain for electron microscopy. J. Ultrastr. Res., 19:
273-282. Enomoto, S., and B. L. Scott 1971 Intracellular distribution of mucosubstance in the major sublingual gland of the rat. Anat. Rec., 169: 71-96. Hand, A. H. 1972 The effects of acute starvation on the parotid acinar cells. Ultrastructural and cytochemical observation on ad-libitum-fed and starved rats. Am. J. Anat., 135: 71-92. Ichikawa, A., and M. Ichikawa 1973 An application of 'flat substrate method' for electron microscopic histochemistry with oxidative procedures. J. Electron Microscopy,
22: 361-363. Ichikawa, M., and A. Ichikawa 1975 The fine structure of the parotid gland of the Mongolian gerbil, Meriones meridianus. Arch. histol. jap., 38: 1-16. Kagayama, M. 1971 The fine structure of the monkey submandibular gland with special reference to intraacinar nerve endings. Am. J. Anat., 135: 419-433. Leblond, C. P. 1950 Distribution of periodic acid-reactive carbohydrates in t h e rat. Am. J. Anat., 86: 1-49. Luft, J.H. 1971 Ruthenium red and violet. I. Chemistry, purification methods of use for electron microscopy and mechanisms of action. Anat. Rec., 171: 347-368. Monneron, A., and W. Bernhard 1966 Action de certaines enzymes sur des tissus inclus en epon. J. Microscopie, 5:
697-714. Munger, B. L. 1964 Histochemical studies on seromucous and mucous secreting cells of human salivary gland. Am. J. Anat., 115: 411-430. Parks, H. F. 1961 On the fine structure of the parotid gland of the mouse and rat. Am. J. Anat., 108: 303-329. 1962 Morphological study of the extrusion of secretory materials by the parotid gland of the mouse and rat. J. Ultrastr. Res., 6: 449-465. Quintarelli, G., J. E. Scott and M. C. Dellovo 1964 The chemical and histochemical properties of alcian blue. 11. Dye binding of tissue polyanions. Histochemie, 4: 86-98. Revel, J. P. 1964 A stain for t h e ultrastructural localization of acid mucopolysaccharide. J. Microscopie, 3:
-
635-544. Rhodin, J. A. G. 1974 Salivary Glands and Teeth. In: Histology: A Text and Atlas. Oxford Press, New York, London, Toronto, pp. 520-526. Rinehart, J. F., and S. K. Abul-Haj 1951 An improved method for histologic demonstration of acid mucopolysaccharides in tissues. Arch. Path., 52: 189-194. Riva, A,, and F. Riva-Testa 1973 Fine structure of acinar cells of human parotid gland. Anat. Rec., 176: 149-166. Robinovitch, M. R.,L. M. Sreebny and E. A. Smuckler 1966 Preservation of the secretory granules of r a t parotid gland for electron microscopy. Exp. Cell Res., 42: 634-639. Rutberg, U. 1961 Ultrastructure and secretory mechanisms of the parotid gland. Acta odontol. scan. (19 Suppl.), 30: 1-69. Sato, M.,T. Noguchi, M. Yokoyama and M. Yotsumoto 1966 On the secretory granules of the serous cell in the human submandibular gland. J. Electron Microscopy, 15:
Castle, J. D., J. D. Jamieson and G. E. Palade 1972 Radioautographic analysis of the secretory process in the 1.14. parotid acinar cell of the rabbit. J. Cell Biol., 53: 290-311. Cope, G. H., and M. A. Williams 1973 Exocrine secretion in Shackleford, J. M., and C. A. Schneyer 1964 Structural and functional asuects of rodent salivarv glands includthe parotid gland: A stereological analysis a t the electron ing two desert species. Am. J. Anat., 115:>79-308. microscopic level of the zymogen granule content before ~
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Shackleford,J. M., and W. H. Wilborn 1968 Structural and histochemical diversity in mammalian salivary glands. Alabama J. Med. Sci., 5: 180-203. Simson, J.A. V., S. S. Spicer and B. J. Hall 1974 Morphology and cytochemistry of rat salivary gland acinar secretory granules and their alteration by isopreterenol. J. Ultrastr. Res., 48: 465-482. Spicer, S. S. 1965 Diamine methods for differentiating mucosubstances histochemically. J. Histochem. Cytochem., 13: 211-234. Spicer, S.S., and R. D. Lillie 1967 Histological identification of basic protein with Biebrich scarlet a t alkaline pH. Stain Technol., 36: 365-370. Spicer, S. S.,M. W. Stalay, M. G. Wetzel and B. K. Wetzel 1966 Acid mucosubstance and basic protein in mouse Paneth cells. J. Histochem. Cytochem., 15: 225-242. Spicer, S.S.,and L. Warren 1960 The histochemistry of sialic acid containing mucoproteins. J. Histochem. Cytochem., 8;135-137.
Tandler, B., and R. A. Erlandson 1972 Ultrastructure of the human submaxillary gland. IV. Serous granules. Am. J. Anat., 1.35: 419-434. Thiery, J. P. 1967 Mise en evidence des polysaccharides sur coures fines en microscopie electronique. J. Microscopie, 6: 987-1018. Tokuyasu, K. T. 1973 A technique for ultracryotomy of cell suspension and tissues, J. Cell Biol., 57: 551-565. Wetzel, M. G.,B. K. Wetzel and S.S. Spicer 1966 Ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains. J. Cell Biol., 30:
299-315. Wilborn, W.H., and C. A. Schneyer 1970 Ultrastructural changes of rat parotid gland induced by a diet of liquid metrecal. Z. Zellforsch., 103: 1-11. Zeitoun, P.,N. Duclert, F. Liautaud, F.Potet and L. Zylberberg 1972 Intracellular localization of pepsinogen in guinea pig pyloric mucosa by immunohistochemistry. Lab. invest.. 27: 218-225.
PLATE 1 EXPLANATION OF FIGURE
1 The serous acinar cells of the Mongolian gerbil parotid gland a t low magnification. Fixation: phosphate-buffered glutaraldehyde and osmium tetroxide. X 4,660. The inset shows the bipartite substructure of the secretory granule in higher magnification. X 14,500.
HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE I
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PLATE 2 EXPLANATION OF FIGURE
2 The serous demilunar cells in the Mongolian gerbil sublingual gland at low magnification. Fixation: phosphate-buffered glutaraldehyde and osmium tetroxide. X 4,000. The inset shows the bipartite substructure of a granule content. Note the reticulated appearance of moderately dense material in the peripheral rim of the granules. X 14,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE 2
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PLATE 3 EXPLANATION OF FIGURE
3 The serous acinar cells of the monkey submandibular gland. Fixation: cacodylatebuffered glutaraldehyde and osmium tetroxide. The secretory granules have a characteristic heterogeneity being composed of a small dense spherule and surrounding material of lower density. X 8,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
4 Secretory granules in the gerbil parotid acinar cell stained with the PA-methenamine silver. Note that staining precipitates are heavily deposited in the peripheral rim of granules in contrast to the faintly positive central core. X 24,800. 5 Secretory granules in the serous demilunar cell of the gerbil sublingual gland stained with PA-methenamine silver. Reaction products appear to be localized corresponding to a reticulated framework of moderately dense material in the peripheral rim of the granules. X 24,800. 6 Secretory granules in a serous demilunar cell of the sublingual gland treated with dialyzed iron. No uranyl acetate and lead staining. The peripheral rim of the granules are intensely stained in contrast to unstained central core. X 22,000.
7 Secretory granules in the serous demilunar cell of the monkey submandibular gland stained with PA-methenamine silver. Stained precipitates are localized specifically in the less dense material surrounding the spherule. X 15,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE
4
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PLATE 5 EXPLANATION OF FIGURES
8
Freeze-etched replica of the secretory granules in the gerbil parotid acinar cell. Smooth surfaced central areas and coarsely narrow outer zone (arrow) are clearly defined. X 14,800.
9 Freeze-etched replica of the secretory granules in the sublingual demilunar cell. Bipartite nature of granule content is demonstrated and reticulated appearance of moderately dense material in the outer zone of granule is detectable (arrows). X
14,800.
10 Bipartite substructure of the secretory granules in the gerbil parotid acinar cell, demonstrated by frozen thin section technique. Section is stained negatively with uranyl acetate. X 25,000. 11 Secretory granules of the gerbil parotid acinar cell after pronase digestion for six hours. The central dense core is selectively digested and appear empty. X 14,000.
12 Secretory granules of the monkey submandibular demilunar cell after pronase digestion for six hours. X 22,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE 5
139
ABSTRACT Light and electron microscopic histochemistry was carried out on the serous secretory granules of the parotid acinar and sublingual demilunar cells of the Mongolian gerbil. These are bipartite in structure with a central dense core and a rim of lower density. Light microscopic techniques included staining of sections with Alcian blue and PAS reaction before and after sialidase digestion. PA-methenamine silver, dialyzed iron, colloidal thorium and ruthenium red reactions for mucosubstance were compared on control and pronase digested materials. The results obtained have been interpreted to indicate that the peripheral rim of the granules contained mainly sialomucin whereas the central dense core was rich in protein. Freeze-etched replicas and frozen thin sections of the gerbil salivary glands revealed the bipartite substructure of the granules to be a consistent structure regardless of specimen preparation procedures. The secretory granules in the serous acinar cells of the monkey submandibular gland which also have a central dense spherule and a rim of lower dense matrix were tested in the same way with the PA-methenamine silver method. A positive reaction was limited to the rim of lower density matrix, indicating that this granular matrix is rich in carbohydrates in contrast to an unstained dense central spherule which is digested with pronase. Morphological and cytochemical similarities of the granule content between species are compared. A number of studies have revealed that the parotid acinar and sublingual demilunar cells in most rodents contain homogeneous secretory granules enclosed in a closely fitting limiting membrane (Parks, '61, '62; Shackleford and Schneyer, '64; Shackleford and Wilborn, '68; Amsterdam et al., '69; Wilborn and Schneyer, '70; Enomoto and Scott, '71; Castle et al., '72; Hand, '72; Cope and Williams, '73; Rhodin, '74). We have recently found that the parotid acinar cells of the Mongolian gerbil have secretory granules with a large dense homogeneous core and the peripheral rim of lower density which appears finely granulated (Ichikawa and Ichikawa, '75). It was further noted that the bipartite substructure of these granules did not depend on specimen preparation procedures, degree of ANAT. REC., 189: 125-140.
granule maturation or age of the animal. The present study further characterizes the secretory granules of the Mongolian gerbil salivary glandular cells by light and electron microscopic histochemistry, freeze-etched replicas and frozen thin sections. In primate salivary glands, the serous secretory granules have been reported to have a bipartite substructure (Sato et al., '66; Cowley and Shackleford, '70; Kagayama, '71; Tandler and Erlandson, '72; Riva and RivaTesta, '73). In order to compare secretory granules of primates to those of the gerbil, we examined demilunar cells of the monkey submandibular gland by some of the same histochemical techniques and found similarities between granules of both species. Received Dec. 19, '75. Accepted Mar. 18, '77.
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MISAO ICHIKAWA A N D ATSUSHI ICHIKAWA MATERIALS AND METHODS
Twelve male and five female adults of the Mongolian gerbil, Meriones meridianus, ranging from 57-75 gm in body weight, raised from birth in our laboratory colony were used in this study. The animals were fed on standard rat food pellets and water ad libitum and fasted for 12 hours before fixation. While under sodium pentobarbital anesthesia, the gerbils used for electron microscopy were fixed by vascular perfusion of fixative introduced into the left ventricle and the parotid and sublingual glands were then excised. Tissue blocks were minced in the same fixative and fixed for a total of two hours at 4OC. The fixative used contained 2.0-2.5%glutaraldehyde buffered a t pH 7.2-7.4with 0.2 M cacodylate (340-385 mOS) or 0.2 M phosphate (395424 mOS). The tissues were then washed in buffer for three hours, postfixed in cacodylate or phosphate buffered 1% osmium tetroxide for one hour, dehydrated in a graded series of cold ethanol and embedded in an EponAraldite mixture. For histochemical analyses, the following methods were employed. (1) Paraffin sections, which were obtained from the materials fixed with 10% formaldehyde buffered with 2%calcium acetate or 6%HgC12 buffered with 1.3% sodium acetate, were stained with Alcian blue a t pH 1.0 and 2.5 followed by the periodic acid Schiff reaction (Spicer, '65). The sialidase digestion test (Spicer and Warren, '60) was also carried out on these sections. (2) Basic protein was examined in paraffin sections stained with 0.04% Biebrich scarlet in Laskey's glycine buffer a t various pHs (Spicer and Lillie, '61). (3) Tissues were fixed and stained with ruthenium red (Luft, '71) and then embedded for electron microscopy. (4) Thin resin sections were stained with periodic acid methenamine silver for mucosubstance (De Martino and Zamboni, '67). (5) For the dialyzed iron reaction, the tissues were taken immediately a€ter glutaraldehyde fixation and sliced into50-pm-thick sections by a tissue sectioner and treated with iron solution a t pH 2.0 (Wetzel et al., '66).(6)Some tissue blocks were embedded in methacrylate after osmification and thin sections were stained with colloidal thorium (Revel '64). (7) Some resin sections were treated with 0.5%pronase (purified Type VI of Sigma Chem. Go.) for 30 minutes to 12 hours a t 40°C before staining with uranyl acetate and lead citrate (Monneron e t Bernhard, '66).
The flat substrate method for histochemical examinations (Ichikawa and Ichikawa, '73) was applied for carrying out the histochemical methods 4, 6 and 7. For preparing freezeetched replicas, the tissue was perfused with phosphate-buffered glutaraldehyde, small pieces rinsed with buffer solution and glycerinated in 30%glycerol in Ringer solution for two hours, and frozen in Freon 22 cooled in liquid nitrogen. Frozen tissues were fractured, etched for one to two minutes and shadowed with platinum with a Hitachi HFZ1 freeze etching device. After carbon was evaporated to form a replica the tissue was removed with a solution of hypochlorite and the replica examined with JEM lOOC electron microscope operated a t 100 kv. For frozen thin sections, small tissue blocks were fixed in the same glutaraldehyde solution described earlier for ten minutes, washed in buffer, transfered into 50% sucrose in buffer for 15 to 30 minutes, frozen in Freon 22 in liquid nitrogen and sectioned on a SORVALL MT-2B ultramicrotome adapted with a cryokit t o maintain the tissue a t -90°C. The thin sections were dried and negatively stained with 0.5%uranyl acetate (Tokuyasu, '73). Thin sections of the monkey submandibular gland were obtained from two male adult animals (3.5 and 4.2 kg in body weight) by the same method as described for the gerbil parotid gland and stained with PA-methenamine silver. Some thin sections were examined in electron microscopy after pronase digestion as for the gerbil parotid gland. OBSERVATIONS
The parotid and sublingual glands of the Mongolian gerbil are generally similar in their location, development and cell architecture to those in other rodents reported by previous investigators. The acinar cells composing the terminal portion of the parotid gland are cuboidal or pyramidal in shape and have cytological characteristics of a typical serous cell (fig. 1).They contain a spherical nucleus located near the cell base, well-developed lamellar arrays of the granular endoplasmic reticulum in the basal and perinuclear regions, a prominent Golgi complex in the supranuclear area and numerous secretory granules in the apical cytoplasm. The lateral and basal borders of the cell body are provided with numerous interdigitating cell folds. The secretory granules have a bipartite substructure composed of a large dense homogeneous core
HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES
and the peripheral rim of finely granulated material (fig. 1, inset). The serous acinar cells of the sublingual gland are located a t the blind end of the tubulo-alveolar gland in the form of a demilune (fig. 2). They are low pyramidal in shape and rarely appear to border onto the duct in sections, because they have a small luminal border. They contain a spherical nucleus situated near the base, lamellar arrays of the granular endoplasmic reticulum in the lower half of the cell body and a well-developed Golgi complex in the supranuclear region. The lateral and basal cell membranes are relatively smooth in contrast to the parotid acinar cells. The membrane-enclosed secretory granules of the sublingual demilunar cells are also bipartite in substructure. They consist of a n eccentric, dense, homogeneous core of onethird to two-thirds the diameter of the granule and a peripheral component composed of a complex of pale matrix with moderately dense material of the reticulated appearance. In some planes of sections, the latter appears to be packed parallel arrays of meandering tubules (fig. 2, inset). The appearance of the secretory granules both in the parotid acinar and sublingual demilunar cells were similar in both sexes and not dependent on differences in osmoralities of the fixatives used. The serous acinar cells of the monkey submandibular gland are pyramidal in shape and also have substructural features of a typical serous cell (fig. 3). The secretory granules have an eccentrically placed dense spherule and a rim of lower dense matrix. The latter is usually composed of a homogeneously fibrillogranular material and occasionally accompanied with a randomly located crescentric structure of intermediate density.
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followed by the PAS reaction stained the peripheral component bluish purple or blue. After a long term digestion of the slide with sialidase (18 hours), the peripheral rim of the granules was stained red with the PAS reaction. The staining pattern in the thin sections of the parotid and sublingual gland acinar cells treated with PA-methenamine silver are shown in figures 4 and 5, respectively. Deposition of reduced silver was intense in the peripheral rim of the granules while the central dense core was only faintly stained. Following treatment with dialyzed iron a t pH 2.0, the acinar cells in the outermost layer of the tissue blocks took up the dye into their cytoplasm and the peripheral rim of the granules in these cells appeared denser than the central core when the sections were not stained with uranyl acetate and lead citrate (fig. 6). In control sections the whole granule is of low density and the dense core is apparent only after uranyl plus lead staining. The results obtained by the colloidal thorium technique were similar to the dialyzed iron staining though the staining was not as intense as in the latter case. Ruthenium red staining of the granules was apparent only in cells where the plasma membrane was disrupted. In such cells the peripheral rim of the granules appeared denser than that in other cells unstained in the same section. In all the EM histochemical methods for mucosubstance used in this study, deposition of staining precipitates in the sublingual demilunar cells was localized t o the material forming the reticulated network peripheral to the eccentric dense core, in contrast to a homogeneous distribution in the peripheral rim of the granules in the parotid acinar cells. In the serous acinar cells of the monkey submandibular gland stained with PA-methenamine silver, the secretory granules were Histochemical results also found to have an unreacted dense central The central dense core of the secretory spherule and a positively stained peripheral granules both in the parotid acinar and sub- matrix (fig. 7).The outer matrix which is comlingual demilunar cells of the gerbil was posed of randomly located zones of intermediweakly positive with the PAS reaction and ate and low density reacted similarly to the stained intensely red with Biebrich scarlet a t PA-methenamine silver stain but the denser pH 6.0. Electron microscopy of thin sections region stained slightly more intensely. After digested with pronase revealed that the cen- pronase digestion, the central dense spherule tral dense core lost its usual density (fig. 11). lost their proper density and appeared empty The material in the peripheral rim of the (fig. 12). granules appeared red with the PAS reaction Results of cryo-techniques and intensely blue with Alcian blue a t pH 2.5 Freeze-etched replicas of the gerbil parotid but faintly stained at pH 1.0. A combined staining reaction a t pH 2.5 with Alcian blue acinar cells showed a bipartite substructure
128
MISAO ICHIKAWA AND ATSUSHI ICHIKAWA
consisting of the central dense core surrounded with relatively narrow outer zone (fig. 8). The peripheral zone appears to be more readily etched under high vacuum than the core. The reticulated appearance of the peripheral zone in the sublingual demilunar cell granules was apparent in freeze-etched replicas (fig. 9). The bipartite heterogeneity of the granules was also found in uranyl acetate stained frozen thin sections of the gerbil parotid acinar cells (fig. 10). DISCUSSION
Light microscopic histochemistry in the present study revealed that the materials in the peripheral rim of gerbil salivary gland granules were intensely positive with the PAS reaction and stained blue with Alcian blue a t pH 2.5but faintly a t pH 1.0. Prolonged digestion with sialidase destroyed this affinity for Alcian blue. These properties indicate the presence of sialomucin in the peripheral rim of the granules (Quintarelli e t al., '64; Spicer, '65). The data of the fine structural histochemistry for mucosubstance agree with the results a t light microscopic level and demonstrated with a higher resolution that staining precipitates were specifically localized in the peripheral rim of the granules whereas the central core showed little or no reaction. These results are presumed to be due to the presence of acidic groups in sialomucin of the peripheral rim of the granules. Our previous study of the gerbil parotid gland has revealed that the bipartite substructure of granule content was detectable in all the materials examined regardless of fixation and staining methods, granule maturation and animal age (Ichikawa and Ichikawa, '75). Cryo-techniques applied to this study which exclude use of dehydrating and embedding agents eliminate further treatments which may introduce an artefactual appearance of this substructure. The freezeetched replicas of the gerbil parotid acinar cells revealed secretory granules consisting of a large round central area surrounded with a rather coarse peripheral narrow zone. These two areas are considered to represent the central dense core and the peripheral rim of lower density seen in thin sections, respectively, because of similarities in their dimension and proportion. A reticulated framework of moderately dense material found in the peripheral rim of sublingual demilunar cell granules was also present in the freeze-etched replicas.
Thus, the results obtained by cryo-techniques further suggest that the bipartite substructure of granule content in both parotid acinar and sublingual demilunar cell granules is not an artefact caused during preparatory procedures after fixation but a stable structure. Light microscopic histochemistry of the salivary glandular cells in other rodents has revealed that serous secretory granules were intensely stained with the PAS reaction but remain unstained with Alcian blue and colloidal iron (Shackleford and Wilborn, '68). They have emphasized that these granules are serous in nature. Since the early ultrastructural study of Scott and Pease ('591,a number of electron microscopic observations on the salivary glandular cells have been shown that the mature secretory granules of the serous cells have a dense homogeneous interior (Shackleford and Schneyer, '64; Shackleford and Wilborn, '68; Amsterdam et al., '69; Wilborn and Schneyer, '70; Enomoto and Scott, '71; Castle e t al., '72; Hand, '72; Cope and Williams, '73; Rhodin, '74). Heterogeneity in the distribution of granule matrix density has been described by Parks ('61, '62) and Rutberg ('611, but has been attributed to fixation artefact by Robinovitch et al. ('66) and Simson et al. ('74). The general appearance of the salivary glandular cells has equated them to pancreatic exocrine cells and the granules to the zymogen granules. It is, however, known that the serous secretory granules of the salivary cells contain a fairly large carbohydrate component when compared to other serous cells, such as pancreatic acinar cells and chief cells of the gastric gland (Leblond, '50; Munger, '64). The ultrastructural appearance of salivary gland cell granules seems to vary among different species and may indicate differencies in their composition. Thus in some rodents, granules of some salivary glands have been defined as seromucous rather than serous (Munger, '64; Shackleford and Wilborn, '64). Simson et al. ('74) have reported bipartite distribution pattern of matrix density in the rat parotid acinar cell granules and suggested that it indicated the presence of a lipoidal material in the matrix. The present data revealed that the central dense core of the granules was rich in protein whereas the peripheral rim was composed mainly of sialomucin. Unlike other rodent or non-primate salivary glands, the serous cells of the gerbil parotid and sublingual glands have secretory granules with a clearly defined
HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES
heterogeneous interior consisting of chemically different compositions. This organization is rather similar to those in the monkey serous salivary cells. A similar bipartite substructure of granules has been described in the mouse Paneth cells (Spicer et al., '66) and the guinea pig pyloric glandular cells (Zeitoun et al., '72) in addition to the salivary glandular cells in primates. Spicer et al. ('66) have verified mucosubstances in the peripheral rim of granules by light and electron microscopic histochemistry. Zeitoun et al. ('72) have shown the peripheral ring of the granules was stained with Thiery's method for glycoprotein and polysaccharides (Thiery, '67) whereas the central dense core was unstained. They have also demonstrated by immunohistochemistry that pyloric pepsinogen was concentrated in the central dense core of granules. The present data are quite similar to those obtained from these glandular cells. Such a characteristic feature of chemical components of granule content is not well understood, but highly charged mucosubstances may be considered to provide a protective coat for the central zymogenic material. Mechanisms by which the granule content is separated into two phases and the intracellular pathway of two components to form a granule packed within a limiting membrane remain to be elucidated. Since the serous secretory granules of the gerbil salivary glandular cells are morphologically similar to those of the monkey and probably of man, the gerbil material may be useful as a model system for experimental manipulations correlated with a morphological study on secretory processes which may be used to understand the secretion of human salivary glands. ACKNOWLEDGMENT
The authors are indebted to Doctor Susumu Ito, Department of Anatomy, Harvard Medical School, for his invaluable advice during preparation of this manuscript. LITERATURE CITED Amsterdam, A., I. Ohad and M.Schramm 1969 Dynamic changes in the ultrastructure of t h e acinar cell of t h e r a t parotid gland during the secretory cycle. J. Cell Biol., 41:
753-773.
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and after isoprenaline-induced degranulation. J. Anat., 116: 269-284.
Cowley, L. H., and J. M. Shackleford 1970 Electron microscopy of squirrel monkey parotid glands. Alabama J. Med. Sci., 7: 273-282. De Martino, C.,and L.Zamboni 1967 Silver methenamine stain for electron microscopy. J. Ultrastr. Res., 19:
273-282. Enomoto, S., and B. L. Scott 1971 Intracellular distribution of mucosubstance in the major sublingual gland of the rat. Anat. Rec., 169: 71-96. Hand, A. H. 1972 The effects of acute starvation on the parotid acinar cells. Ultrastructural and cytochemical observation on ad-libitum-fed and starved rats. Am. J. Anat., 135: 71-92. Ichikawa, A., and M. Ichikawa 1973 An application of 'flat substrate method' for electron microscopic histochemistry with oxidative procedures. J. Electron Microscopy,
22: 361-363. Ichikawa, M., and A. Ichikawa 1975 The fine structure of the parotid gland of the Mongolian gerbil, Meriones meridianus. Arch. histol. jap., 38: 1-16. Kagayama, M. 1971 The fine structure of the monkey submandibular gland with special reference to intraacinar nerve endings. Am. J. Anat., 135: 419-433. Leblond, C. P. 1950 Distribution of periodic acid-reactive carbohydrates in t h e rat. Am. J. Anat., 86: 1-49. Luft, J.H. 1971 Ruthenium red and violet. I. Chemistry, purification methods of use for electron microscopy and mechanisms of action. Anat. Rec., 171: 347-368. Monneron, A., and W. Bernhard 1966 Action de certaines enzymes sur des tissus inclus en epon. J. Microscopie, 5:
697-714. Munger, B. L. 1964 Histochemical studies on seromucous and mucous secreting cells of human salivary gland. Am. J. Anat., 115: 411-430. Parks, H. F. 1961 On the fine structure of the parotid gland of the mouse and rat. Am. J. Anat., 108: 303-329. 1962 Morphological study of the extrusion of secretory materials by the parotid gland of the mouse and rat. J. Ultrastr. Res., 6: 449-465. Quintarelli, G., J. E. Scott and M. C. Dellovo 1964 The chemical and histochemical properties of alcian blue. 11. Dye binding of tissue polyanions. Histochemie, 4: 86-98. Revel, J. P. 1964 A stain for t h e ultrastructural localization of acid mucopolysaccharide. J. Microscopie, 3:
-
635-544. Rhodin, J. A. G. 1974 Salivary Glands and Teeth. In: Histology: A Text and Atlas. Oxford Press, New York, London, Toronto, pp. 520-526. Rinehart, J. F., and S. K. Abul-Haj 1951 An improved method for histologic demonstration of acid mucopolysaccharides in tissues. Arch. Path., 52: 189-194. Riva, A,, and F. Riva-Testa 1973 Fine structure of acinar cells of human parotid gland. Anat. Rec., 176: 149-166. Robinovitch, M. R.,L. M. Sreebny and E. A. Smuckler 1966 Preservation of the secretory granules of r a t parotid gland for electron microscopy. Exp. Cell Res., 42: 634-639. Rutberg, U. 1961 Ultrastructure and secretory mechanisms of the parotid gland. Acta odontol. scan. (19 Suppl.), 30: 1-69. Sato, M.,T. Noguchi, M. Yokoyama and M. Yotsumoto 1966 On the secretory granules of the serous cell in the human submandibular gland. J. Electron Microscopy, 15:
Castle, J. D., J. D. Jamieson and G. E. Palade 1972 Radioautographic analysis of the secretory process in the 1.14. parotid acinar cell of the rabbit. J. Cell Biol., 53: 290-311. Cope, G. H., and M. A. Williams 1973 Exocrine secretion in Shackleford, J. M., and C. A. Schneyer 1964 Structural and functional asuects of rodent salivarv glands includthe parotid gland: A stereological analysis a t the electron ing two desert species. Am. J. Anat., 115:>79-308. microscopic level of the zymogen granule content before ~
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Shackleford,J. M., and W. H. Wilborn 1968 Structural and histochemical diversity in mammalian salivary glands. Alabama J. Med. Sci., 5: 180-203. Simson, J.A. V., S. S. Spicer and B. J. Hall 1974 Morphology and cytochemistry of rat salivary gland acinar secretory granules and their alteration by isopreterenol. J. Ultrastr. Res., 48: 465-482. Spicer, S. S. 1965 Diamine methods for differentiating mucosubstances histochemically. J. Histochem. Cytochem., 13: 211-234. Spicer, S.S., and R. D. Lillie 1967 Histological identification of basic protein with Biebrich scarlet a t alkaline pH. Stain Technol., 36: 365-370. Spicer, S. S.,M. W. Stalay, M. G. Wetzel and B. K. Wetzel 1966 Acid mucosubstance and basic protein in mouse Paneth cells. J. Histochem. Cytochem., 15: 225-242. Spicer, S.S.,and L. Warren 1960 The histochemistry of sialic acid containing mucoproteins. J. Histochem. Cytochem., 8;135-137.
Tandler, B., and R. A. Erlandson 1972 Ultrastructure of the human submaxillary gland. IV. Serous granules. Am. J. Anat., 1.35: 419-434. Thiery, J. P. 1967 Mise en evidence des polysaccharides sur coures fines en microscopie electronique. J. Microscopie, 6: 987-1018. Tokuyasu, K. T. 1973 A technique for ultracryotomy of cell suspension and tissues, J. Cell Biol., 57: 551-565. Wetzel, M. G.,B. K. Wetzel and S.S. Spicer 1966 Ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains. J. Cell Biol., 30:
299-315. Wilborn, W.H., and C. A. Schneyer 1970 Ultrastructural changes of rat parotid gland induced by a diet of liquid metrecal. Z. Zellforsch., 103: 1-11. Zeitoun, P.,N. Duclert, F. Liautaud, F.Potet and L. Zylberberg 1972 Intracellular localization of pepsinogen in guinea pig pyloric mucosa by immunohistochemistry. Lab. invest.. 27: 218-225.
PLATE 1 EXPLANATION OF FIGURE
1 The serous acinar cells of the Mongolian gerbil parotid gland a t low magnification. Fixation: phosphate-buffered glutaraldehyde and osmium tetroxide. X 4,660. The inset shows the bipartite substructure of the secretory granule in higher magnification. X 14,500.
HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE I
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PLATE 2 EXPLANATION OF FIGURE
2 The serous demilunar cells in the Mongolian gerbil sublingual gland at low magnification. Fixation: phosphate-buffered glutaraldehyde and osmium tetroxide. X 4,000. The inset shows the bipartite substructure of a granule content. Note the reticulated appearance of moderately dense material in the peripheral rim of the granules. X 14,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE 2
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PLATE 3 EXPLANATION OF FIGURE
3 The serous acinar cells of the monkey submandibular gland. Fixation: cacodylatebuffered glutaraldehyde and osmium tetroxide. The secretory granules have a characteristic heterogeneity being composed of a small dense spherule and surrounding material of lower density. X 8,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
4 Secretory granules in the gerbil parotid acinar cell stained with the PA-methenamine silver. Note that staining precipitates are heavily deposited in the peripheral rim of granules in contrast to the faintly positive central core. X 24,800. 5 Secretory granules in the serous demilunar cell of the gerbil sublingual gland stained with PA-methenamine silver. Reaction products appear to be localized corresponding to a reticulated framework of moderately dense material in the peripheral rim of the granules. X 24,800. 6 Secretory granules in a serous demilunar cell of the sublingual gland treated with dialyzed iron. No uranyl acetate and lead staining. The peripheral rim of the granules are intensely stained in contrast to unstained central core. X 22,000.
7 Secretory granules in the serous demilunar cell of the monkey submandibular gland stained with PA-methenamine silver. Stained precipitates are localized specifically in the less dense material surrounding the spherule. X 15,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE
4
137
PLATE 5 EXPLANATION OF FIGURES
8
Freeze-etched replica of the secretory granules in the gerbil parotid acinar cell. Smooth surfaced central areas and coarsely narrow outer zone (arrow) are clearly defined. X 14,800.
9 Freeze-etched replica of the secretory granules in the sublingual demilunar cell. Bipartite nature of granule content is demonstrated and reticulated appearance of moderately dense material in the outer zone of granule is detectable (arrows). X
14,800.
10 Bipartite substructure of the secretory granules in the gerbil parotid acinar cell, demonstrated by frozen thin section technique. Section is stained negatively with uranyl acetate. X 25,000. 11 Secretory granules of the gerbil parotid acinar cell after pronase digestion for six hours. The central dense core is selectively digested and appear empty. X 14,000.
12 Secretory granules of the monkey submandibular demilunar cell after pronase digestion for six hours. X 22,000.
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HISTOCHEMISTRY OF SEROUS SECRETORY GRANULES Misao Ichikawa and Atsushi Ichikawa
PLATE 5
139
