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Intracristal helices in salivary gland mitochondria.

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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.
Berkowitz, L. R., 0. Fiorello, L. Kruger and D. S.
Maxwell 1968 Selective staining of nervous
tissue for light microscopy following preparation for electron microscopy. J. Histochem. Cytochem., 16: 808-814.
Blecher, S. R. 1967 Mitochondria1 chromosomes. Curr. Mod. Biol., I : 249-255.
Iseri, 0. A., C. S. Lieber and L. S. Gottlieb 1966
The ultrastructure of fatty liver induced by pro-
longed ethanol ingestion. Am. J. Pathol., 48:
535-555.
Jessen, H. 1968 The morphology and distribution of mitochondria in ameloblasts with special reference to a helix containing type. J.
Ultrastruct. Res., 22: 120-135.
Karnovsky, M. J. 1967 The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J. Cell Biol., 35: 213-236.
Leduc, E. H., W. Bernhard and P. Tournier 1966
Cyclic appearance of a typical mitochondria
containing DNA fibers i n cultures of a n adenovirus 12- induced hamster tumor. Exptl. Cell
Res., 42: 597-616.
Luft, J. H. 1961 Improvements in epoxy resin
embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414.
Mugnaini, E. 1964 Helical filaments in astrocytic mitochondria of the corpus striatum in
the rat. J, Cell Biol., 23: 173-182.
Nass, S., and M. M. K. Nass 1963 Intramitochondrial fibers with DNA characteristics 11.
Enzymatic and other hydrolytic treatments. J.
Cell Biol., 19: 613-629.
Porta, E. A,, W. S. Hartroft and F. A. de la
Iglesia 1965 Hepatic changes associated with
chronic alcoholism i n rats. Lab. Invest., 14:
1437-1455.
Schuster, F. L. 1965 A deoxyribose nucleic acid
component i n mitochondria of Didymium
nigripes, a slime mold. Exptl. Cell Res., 39:
329-345.
Suzuki, T., and F. K. Mostofi 1967 Intramitochondrial filamentous bodies i n the thick limb
of Henle of the rat kidney. J. Cell Biol., 33:
605-623.
Svoboda, D., and J. Higginson 1964 Ultrastructural changes produced by protein and related
deficiencies i n the rat liver. Am. J. Pathol., 45:
353-3 79.
Venable, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use in electron
microscopy. J. Cell Biol., 25: 407-408.
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