BRIEF COMMUNICATION Intracristal Helices in Salivary Gland Mitochondria ARTHUR R. HAND Laboratory o f Biological Structure, National Institute of Dental Reseurch, National Institutes of Health, Bethesda, Maryland 20014 ABSTRACT Helical filaments have been found within dilated intracristal spaces of normal rat salivary gland mitochondria. Filament diameter measured 35-45 A, and the helix diameter and pitch were 140-155 A. They were found in as many as 25% of the mitochondria of acinar and intercalated duct cells. Helices were not found in mitochondria of the striated duct cells. Mitochondria1 inclusions have been reported by a number of investigators in recent years in both normal and pathological tissues (see review by Suzuki and Mostofi, '67). The majority of these inclusions have been located within the mitochondrial matrix, and have been of a filamentous nature. Inclusions have also been found within the intracristal space or in the space between the inner and outer mitochondrial membranes. Most of these inclusions were helical filaments of a similar morphology; they were found in rat liver cells in experimental pathological states (Svoboda and Higginson, '64; Porta et al., '65; Iseri et al., '66) and in liver cells (Behnke, '65), astrocytes (Mugnaini, '64) and ameloblasts (Jessen, '68 of normal rats. The purpose of this communication is to report the finding of helices within intracristal spaces of mitochondria of normal rat salivary glands. The helices were similar in appearance to those previously reported, and were of widespread occurrence in both the parotid gland and von Ebner's gland, a minor salivary gland located in the tongue. > otomy was performed, and fixation was achieved by vascular perfusion through a cannula placed in the ascending aorta. The fixative used was a mixture of 2% formaldehyde (made from paraformaldehyde powder) and 2.5% glutaraldehyde in 0.1 M Na cacodylate buffer, pH 7.4 (Berkowitz et al., '68). The perfusion lasted for 15 to 20 minutes, the tissues were excised, and fixation continued in the cold (0-4OC) for an additional two to four hours. The tissues were then washed overnight in cold 7.5% sucrose in 0.1 M Na cacodylate, pH 7.4, and post-fixed in 1% OsOa in 0.1 M Na cacodylate with 7.5% sucrose, pH 7.4. Prior to dehydration, the tissues were treated with 0.5% uranyl acetate (Karnovsky, '67) for one to two hours. The tissues were dehydrated in ethanol and embedded in Araldite (Luft, '61). Thin sections were cut on a PorterBlum microtome, stained with uranyl acetate and/or lead citrate (Venable and Coggeshall, '65), and examined in a Siemens Elmiskop I at 80 kv. RESULTS The mitochondria of the acinar and duct cells of von Ebner's gland and the MATERIAL AND METHODS acinar and intercalated duct cells of the Parotid glands and von Ebner's glands parotid gland were typical of mitochondria were obtained from adult Sprague-Dawley found in many tissues. The matrix of the rats. The animals were maintained under mitochondria was usually denser than standard laboratory conditions on labora- that of the surrounding cytoplasm, and tory chow and water, ad libitum, but were often contained one or more dense grandeprived of food for 18-24 hours prior to ules. The cristae were numerous, occasacrifice. The animals were anesthetized sionally extending completely across the with sodium pentobarbital, 50 mg/kg, or mitochondrion, but usually ending somechloral hydrate, 400 mg/kg, i.p., a tracheReceived April 2, '70. Accepted June 17, '70. ANAT. REC., 168: 565-568. 565 566 ARTHUR R. HAND where in the middle. The intracristal space varied in width from about 100-200A, and often contained a small amount of amorphous appearing material, slightly less dense than the mitochondrial matrix. Numerous mitochondria of von Ebner's gland and the parotid gland had one or more dilated cristae, the width of which varied from slightly larger than normal to approximately 1600 A. These cristae were generally oriented lengthwise within the mitochondrion and usually appeared empty or contained an amorphous material. Occasionally, a dilated cristal space contained one to five or six helically coiled filaments, lying longitudinally within the space. (figs. 1-3). Measurements of the diameter of the filaments varied between 33 and 45 A, while the diameter of the helix and the spacing of the turns each varied between 140 and 155 A. In some cases, a dense granule similar to a matrical granule was found in the cristal space with the helices (figs. 2, 3). The percentage of mitochondria containing dilated intracristal spaces and helical inclusions could not be determined readily from survey micrographs due to the small size of the spaces and helices. However, in a few low power micrographs, as many as 25% of the mitochondria had identifiable dilated intracristal spaces or helical inclusions. The maximum length observed for an inclusion was 0.8 E"..In a mitochondrion 4-5 long, as few as 20% of non-longitudinal sections cut from it would contain part of an inclusion of that length. So it is possible that even more than 2 5 % of the mitochondria actually contain inclusions. Numerous mitochondria of the striated ducts of the parotid gland were examined, but none were found which contained dilated spaces or inclusions. However, a relatively undifferentiated cell type occurring in the basal portion of the ducts did contain mitochondria with the helical inclusions. The cytoplasm of this cell type was relatively free of organelles, containing mostly free ribosomes. The function or fate of this cell type is as yet unknown. DISCUSSION The helical intracristal inclusions found in normal rat salivary glands are very similar to those previously reported in other normal and pathological tissues. The measured filament diameter and helix diameter and pitch are close to those cited by Mugnaini ('64), for helical inclusions in mitochondria of astrocytes of the rat corpus striatum; Jessen ('68), for helical inclusions in normal rat ameloblast mitochondria; and Blecher ('67), for helical inclusions in mitochondria of rat liver cells. It is likely that all of the helical inclusions are of the same nature, because of their similar morphology, size and location. Their precise nature, however, is still under debate. Their appearance has been attributed to changes in phospholipids or proteins of the mitochondrial membranes (Svoboda and Higginson, '64; Iseri et al., '66), substances entering the mitochondrion from the cytoplasm (Svoboda and Higginson, '64), and synthesis of a macromolecule of protein within the mitochondrion (Mugnaini, '64). It has also been suggested that the helices are composed of DNA or DNA-protein complexes. On the basis of cytochemical evidence, Nass and Nass ('63) and Leduc et al. ('66) have shown the existence of DNA fibers within mitochondria. Schuster ('65), also using cytochemical methods, came to the conclusion that helices of a slightly different morphology in slime mold mitochondria were nucleoprotein. Blecher ('67) felt that the helices of rat liver mitochondria were DNA or DNA-protein, based on evidence obtained from various methods of fixation, DNAase and pepsin digestions, staining with lead and uranium salts, and enhancement of density with hydrogen peroxide treatment. Fig. 1 Mitochondrion of parotid acinar cell. Five helical filaments are lying longitudinally within a dilated intracristal space. Note areas where cristal membranes are probably continuous with the inner mitochondrial membrane (arrows). X 102,900. Inset: Cross-section of two dilated cristae containing helices (arrows). Von Ebner's gland. x 146,700. Fig. 2 Acinar cell of von Ebner's gland. A dense granule is lying in the space which contains the helical filaments (arrow). x 67,900. Fig. 3 Parotid acinar cell. A dense granule lies i n the intracristal space with the helical filaments (arrow). x 76,800. MITOCHONDRIAL HELICES Figures 1-3 567 568 ARTHUR R. HAND If the helices are DNA, their presence or absence may reflect on the ability of the mitochondrion to divide, or to synthesize its own structural and enzymatic components. Or, their presence or absence may be related to the main function of the particular cell. Until further evidence is accumulated, little else can be said about the function or significance of the helices. One last comment about the helical inclusions should be made: all of the published micrographs of helices of the type described in this report have been from rat tissues. Undoubtedly, the rat is the most widely used research animal, but unless these inclusions are actually found only in rats, reports of the occurrence of these types of inclusions in mitochondria of other animals should be appearing. ACKNOWLEDGMENT The skillful technical assistance of Mrs. Betty Ho is gratefully acknowledged. LITERATURE CITED Behnke, 0. 1965 Helical filaments i n rat liver mitochondria. Exptl. Cell Res., 37: 687-689. 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