Arch, Histol. Cytol pe 34-354 Vol. 62, No. 4 (1999) The Serous Demilune of Rat Sublingual Gland Is an Artificial Structure Produced by Conventional Fixation* Shohei YaMASHINA, Hideaki TAMAKI and Osamu Katsu MATA Department of Anatomy, Kitasato University School of Medicine, Sagamihara, Japan Received August 27, 19 ‘Summary. The ultrastructure of the seeretory end- piece of the rat sublingual gland was examined in sam- ples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphies from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved eel- lular ultrastructures, especially the membrane strue- ture, in perfect condition, and demonstrated the termi nal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells dis- tributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure, In contrast, samples fixed by the conventional immersion method showed distended mucous eells displacing the serous eells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous eells during immersion fixation. In the majority of higher animals including humans, the terminal portions of salivary glands, especially the sublingual and submandibular glands, are com: posed of both serous and mucous cells and hence are called a mixed gland, In a typical mixed salivary gland, serous cells have been believed to locate at the most distal end of the secretory end-pieces, surround: ing a group of mucous cells like a cap. This intriguing structure has been called GIANNUZz’s demilune or crescent, after its discovery in 1865 by Gianwuzzi Textbooks on histology describe the demilune as a structural landmark that characterizes the mixed glands (BARGMANN, 1977; JAMIESON, 1988; FAWCETT 1994), Janson (1988) in “The Cell and Tissue Biol ogy” explained the serous demilune as follows. “The serous cells occupy the fundus of acinar sac, and the ‘mucous cells closer to the opening to the initial duct segment (intercalated duct)”. The same concept is also illustrated in KRSTI¢’s textbook (KRsTIC, 1985) ‘The serous demilune has been commonly accepted as 1 group of serous cells forming a basophilic cap at the terminal end of the mucous acini For the structural premise of the demilune, secre: tion from the serous cells has been described as having direct access to the luminal space by a cyto plasmic process or through secretory canaliculi (LERSON, 1967). In the cow submandibular gland, ‘SCHAKLEFORD and WILBORN (1970) reported the pres: ence of cytoplasmic processes of demilune cells between mucous cells and considered them to be the passage through which the serous materials are se creted into the lumen. In the opossum submandibular gland, the significance of intercellular canaliculi has been recognized as the means to conduct secretory materials from the serous cells to the lumen (Wr. BORN and SCHAKLEFORD, 1969; LEESON et al., 1978). Controversial opinions regarding the passage of the secretory materials from demilune cells have been reviewed by PINKSTAFF (1980) ‘A previous study by one of the present authors demonstrated that the limiting membrane of secre tory granules in the acinar cells of the rat submandib- ular gland became discontinuous and appeared co: alescent by electron microscopy as the proportion of mucous to serous materials increased with the matu ration of the acinar cells during the process of post: natal development (YAMASHINA and MIZuntRA, 1976) ‘The structural modification observed might reflect “This study was partly supported by grants from the Japanese Ministry of Education, Science and Culture (No. 11470008) and the Kitasato Gakuen (AKPS No. 99-5002}SBS. Yanastuna et al Fig. 1. ‘An electron micrograph of the terminal portion of the rat sublingual gland prepared by the rapid freezing and freeze-substitution method. This fixation method enables the greatly improved preservation of the membrane structure, especially that of the mucous cells (4). All mucigen granules are round and isolated structures. In this acinus, four mucous and two serous (S) cells align to surround a common lumen (2), serous demilune can be detected. x 13,000 (All scale bars fa distension of the mucous substance in the intra: cellular granules through the incorporation of water into the mucin molecules during, conventional chemi: cal and immersion fixation for light and electron microscopy. Structural changes have been known to >be minimized by the application of rapid freezing and freoze-substitution fixation, by which fine structures are preserved without artifical intervention (Ici KAWA et al,, 1980, 1982; IcHIKAWA and Icuikawa, 1987), We therefore applied this technique for speci men preparation to the rat sublingual gland to te- examine the ultrastructure of the terminal portion. ‘The spatial relations of the serous cells with respe to the mucous cells and also the luminal space were analyzed three-dimensionally using computer recon: struction from micrographs of serial sections. ‘As a result, the demilune that has been bel be an actual histological entity for more than 130 years was concluded to be an artificial structure produced by conventional chemical fixation. ved to snd no Am) MATERIALS AND METHODS Adult male Wistar rats weighing approximately 250 were used in this study. Small pieces of the sublin gual gland were removed under ether anesthesia, ‘They were frozen rapidly by smashing against the polished surface of an ultra-pure copper block which hhad been cooled to the temperature of liquid nitrogen. ‘The device for rapid freezing was homemade with a design similar to that of HEUSER et al. (1979), The frozen samples were frecze-substituted in 2% osmi- um tetroxide in acetone at —80°C for 2 days, followed by stepwise warming to room temperature. Samples ‘were embedded in epoxy resin after dehydration in acetone. Ultra-thin sections were examined by a JEM-1200EX electron micrascope (JBOL Co., Tokyo) after double staining with uranyl acetate and lead citrate. For comparison, samples prepared by conven- tional chemical immersion fixation in 2.5% glutaral- dehyde followed by 1% osmium tetroxide were simi: larly processed and examined.Sublingual Gland Demilune is an Artificial Structure 319 Fig. 2. An electron micrograph of the terminal portion of the rat sublingual gland similar to that shown in Figure 1, but treated by conventional immersion fixation. Secretory granules of mucous cells are irregular in size {due to coalescence by the distuption of limiting membranes. Serous cells are compressed by distended mucous, cells toward the peripheral portion of the acinus, forming the demilune structure, 8,300 Reconstruction of the 3D image was performed using 2 personal computer, Dimension 400 (Dell Com- puter Co,, Round Rock, TX, USA), installed with TRI 34D reconstruction software (Ratoc System Engineer- ing Co., Ltd., Tokyo). A series of more than 100 serial sections at 2m in thickness were cut on an ultra- ‘microtome from epoxy-embedded tissue treated by rapid freezing or conventional immersion fixation, and were mounted on glass slides. Light micrographs were taken after counterstaining by toluidine blue. From a set of serial micrographs, the contour of the acini, the limiting membrane of the lumen, and the ‘outline of the mucous and serous cells were traced on transparent sheets, and the line drawings were fed into the computer with a stylus pen on a digitizing tablet. Reconstruction of one acinus usually required about 40 serial micrographs. Stereo pairs were gener- ated by tilting the images at +5° (AOVAMA et al., 1995). Enzyme histochemical demonstration of 5'-nucleo- tidase was conducted using sublingual tissues fixed in 1% glutaraldehyde for 1h. Sections, 50-ym-thick, were cut on a microslicer (Dosaka EM, Kyoto) to be incubated in a medium containing 2.0mM cerium chloride and 1.0 mM adenosine 5’-monophosphate (RoBINSON and KARNOVSKY, 1983; YAMASHINA et al., 1986) for 30min at room temperature. After the histochemical treatment, specimens were fixed in 1% osmium tetroxide for 1h followed by the routine process for electron microscopy. RESULTS ‘The ultrastructures of the exocrine cells at the termi nal portion of a rat sublingual gland appeared rather different between those sections fixed by rapid freez- ing followed by freeze substitution and those prepar- ed by conventional immersion fixation (compare Figs. Land 2). The general ultrastructures of mucous and serous acinar cells essentially confirmed those of the ‘Mongolian gerbil sublingual gland described by Ick KaWA and ICHIKAWA (1987). A characteristic finding was the extremely good preservation of cellular membranes in the mucous cell, resulting in that all secretory granules were round and clearly isolated