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Giant mitochondria in the seromucous secretory cells of the accessory submandibular gland of the long-haired fruit bat Stenonycteris lanosus.

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THE ANATOMICAL RECORD 237:157-162 (1993)
Giant Mitochondria in the Seromucous Secretory Cells of the
Accessory Submandibular Gland of the Long-Haired Fruit Bat,
Stenonycteris lanosus
BERNARD TANDLER AND CARLETON J. PHILLIPS
Department of Oral Biology, School of Dentistry, Case Western Reserve University,
Cleveland, Ohio 44106 (B.T.); Department of Biological Sciences, Illinois State University,
Normal, Illinois 61 761-6901 (C.J.P.)
ABSTRACT
Giant mitochondria, measuring up to 6.4 pm in diameter,
are present in the seromucous secretory cells of the accessory submandibular gland of the long-haired fruit bat, Stenonycteris lanosus. These mitochondria, as well as all of the smaller ones in the same cells, contain in their
matrix compartment an abundance of 33 nm threads that probably consist
of protein. Some mitochondria, regardless of size, contain 5 nm helical filaments within an expanded crista. Despite their altered morphology, the
enlarged mitochondria in the accessory submandibular gland of S. lanosus
must be able to function normally in energy metabolism, since the secretory
cells in which they are found elaborate numerous secretory granules.
0 1993 Wiley-Liss, Inc.
Key words: Helical filaments, Expanded cristae, Energy metabolism,
Secretory granules
Despite the basic histologic similarity of major salivary glands in most chiropterans, species-specific differences have been reported, especially with respect to
secretory granule substructure (Tandler et al., 1986;
Phillips and Tandler, 1987; Tandler et al., 1990; Tandler and Phillips, 1993) and striated duct surface modifications (Tandler et al., 1989). The cytologic machinery related to the production of proteins and
glycoconjugates slated to be discharged tends to be typical in appearance, but some mitochondria in secretory
cells of certain species of bats may exhibit morphological modifications.
For example, in the parotid gland of Carollia perspicillata, some of the acinar cell mitochondria have parallel, prismatic cristae, each connected to its nearest
neighbors by periodic intercristal bridges (Tandler et
al., 1988). Concentric, ring-shaped mitochondria are
present in acinar cells of the submandibular gland of
Tonatia syluicola (Tandler et al., 1990). Paracrystalline
plates are found within the cristae of secretory cell mitochondria of the accessory submandibular gland of
Truchops cirrhosis (Tandler et al., 1990).
In the course of a continuing large-scale study of the
comparative ultrastructure of bat salivary glands, we
have encountered highly modified mitochondria in the
accessory submandibular gland of the long-haired fruit
bat, Stenonycteris lanosus. These unusual organelles
are described in this report.
cated in the highlands of western Kenya. The animals
were killed by a n i.p. injection of 1ml T-61 Euthanasia
Solution. Extirpated salivary glands initially were
fixed in half-strength Karnovsky’s (1965)fixative buffered with cacodylate and containing 2.5% DMSO (Kalt
and Tandler, 1971). After two hours, the tissues were
transferred to cacodylate buffer and stored until refrigeration became available. The tissues then were placed
in cacodylate-buffered 3% glutaraldehyde and stored in
the refrigerator (Phillips, 1985). After several weeks,
the tissue blocks were extensively washed in phosphate
buffer, then postfixed for two hours in phosphate-buffered 2% osmium tetroxide (Millonig, 1961a). Rinsing in
distilled water was followed by overnight soaking in
cold, acidified 0.25% uranyl acetate (Tandler, 1990).
After another rinse in distilled water, the specimens
were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in
Epon-Maraglas (Tandler and Walter, 1977). Thin sections were stained with uranyl acetate (Tandler, 1990)
followed by either lead citrate (Venable and Coggeshall, 1964) or by lead tartrate (Millonig, 1961b) and
examined in a Siemens 101 electron microscope.
Epoxy sections 1 pm thick were mounted on glass
slides, stained with toluidine blue (Bjorkman, 1960),
and examined and photographed in a Zeiss Ultraphot.
A portion of a n accessory gland from each of the two
MATERIALS AND METHODS
Two specimens of the relatively uncommon and difficult to obtain long-haired fruit bat, Stenonycteris
(also known a s Rousettus) lanosus, one male and one
female, were live-trapped in the Kagamega Forest lo0 1993 WILEY-LISS, INC
Received November 15, 1990; accepted May 18, 1993.
Address reprint requests to Dr. Carleton J. Phillips, Department of
Biological Sciences, Felmley Hall, Room 206, Illinois State University, Normal, IL 61761-6901.
158
B. TANDLER AND C.J. PHILLIPS
double limiting membranes and occasional cristae (Fig.
3); also their development can be traced from normalsized mitochondria. The enlarged mitochondria display
a paucity of cristae; instead their matrix contains an
abundance of dense threads measuring -33 nm in diameter (Fig. 3). The threads, which are of indeterminate length, usually are randomly oriented, but in
some mitochondria they show a degree of parallelism
(Fig. 4).
Because of their distinctive morphology, the development of the giant mitochondria can readily be followed. Such organelles can be traced back to normalsized ones through a spectrum of intermediate-sized
mitochondria. Regardless of size or shape, each and
every mitochondrion in the seromucous cells contains
some matrical threads (Fig. 5).
Again, independent of size, some mitochondria have
one or more expanded cristae (Fig. 6) in which is found
a series of plectonemic helical filaments, each of which
is about 5 nm thick; the helices measure 13 nm in
diameter and have a helical angle of approximately 40”
(Fig. 7). In a very few of the expanded cristae, the
included filaments may lack a helical configuration
(Fig. 6).
Fig. 1. Photomicrograph of a semithin section of Stenonyctens accessory submandibular gland. An acinus consisting wholly of serous
cells is indicated by the long arrow; all other cells are seromucous
cells, some in demilunes, others not. Short arrows point to several of
the large, densely stained cytoplasmic inclusions in the seromucous
cells. Toluidine blue. x 815.
bats used in this study was fixed directly in phosphatebuffered 10% formalin. After conventional processing,
the specimens were embedded in paraffin, sectioned at
6 pm, and stained by the PAS technique for glycogen,
using diastase controls (Lillie and Fullmer, 1976).
RESULTS
The accessory submandibular gland, a mixed gland,
of S. lanosus consists of a few serous acini that are
capped by seromucous demilunes, as well as a preponderance of acini that are composed wholly of seromucous cells [classification of cell types is based not only
on their ultrastructural appearance but also on a series
of histochemical tests’ (C.A. Pinkstaff, personal communication)]. Regardless of whether the seromucous
cells are in a demilunar or acinar position, they often
contain one or several large (up to 6.4 pm in diameter)
cytoplasmic inclusions that are deeply stained in semithin epoxy sections by toluidine blue (Fig. 1). They
were present in equal numbers in the male and female.
In paraffin sections, these cytoplasmic bodies are completely PAS-negative, both before and after exposure to
diastase.
At the ultrastructural level, the cytoplasmic inclusions are seen to be mitochondria, albeit of a highly
modified sort. Such organelles may be as big as or bigger than the secretory cell nuclei (Fig. 2). These mitochondria can be identified as such by virtue of their
‘In a n earlier publication (Tandler and Phillips, 1993),we identified the cells in Stenonycteris lanosus that contained giant mitochondria as serous cells on purely morphological grounds. In keeping with
our dictum, as enunciated in that work, we now defer to the more
accurate histochemical identification of the cells in question as being
seromucous.
DISCUSSION
Giant mitochondria have been observed in a variety
of normal tissues, including ctenophore comb plate
(Horridge, 1964);human uterine glands (Merker et al.,
1968); flight muscle of the house fly (Sohal et al.,
1972);human myocardial cells (Kraus and Cain, 1980);
Schwann cells of bovine splenic nerve (Thureson-Klein,
1972); urinary bladder of the mole (Aumuller and
Forssmann, 1973); insect spermatozoa (Afzelius et al.,
1976); and in lining epithelium of gerbil trachea and
bronchioles (Spicer et al., 1990). They have never been
seen in any previously examined mammalian salivary
gland cell, but were described in a pleomorphic adenoma of the human submandibular gland (Tandler and
Erlandson, 1983).
Although it is true that mitochondria1 giantism can
be evoked by a variety of drugs and hormones and by
nutritional manipulation (reviewed by Tandler and
Hoppel, 1986; Hoppel and Tandler, 1993), those found
in the accessory submandibular gland of Stenonycteris
appear to be a normal component of the seromucous
cells, whether demilunar or acinar, since mitochondria
in the immediately adjacent serous acinar cells are normal in size and appearance, and mitochondria in the
other major salivary glands of this bat also are of normal size.
The giant mitochondria that we found in the longhaired fruit bat differ from virtually all megamitochondria previously described in other organs in that they
contain an abundance of dense, more or less randomly
oriented threads in their inner compartment. In this
respect, they resemble giant mitochondria with dense
“filamentous” inclusions recently described in three benign thyroid lesions (two goiters and a hyalinizing trabecular adenoma) of human origin (Matias et al.,
1991). In addition to randomly disposed filaments,
these giant thyroid mitochondria often contained one
or several electron-dense spheres. When ultrathin sections of these lesions were exposed to proteolytic enzymes, both types of inclusions (filaments and spheres)
Fig. 2. Survey electron micrograph of several seromucous cells, each of which contains one or more
cytoplasmic bodies, which may nearly match the nuclei in size. x 4,175.
Fig. 3. A cytoplasmic body a t higher magnification. The double limiting membrane and the presence
of occasional cristae (arrow) stamp it as a mitochondrion. The inner compartment contains numerous
dense threads, which appear as dots (right side of micrograph) when sectioned transversely. x 37,000.
160
B. TANDLER AND C.J. PHILLIPS
underwent a marked reduction in density, indicating
that both consisted in a major way of proteinaceous
material. By analogy, it may be surmised that the
dense threads in the bat giant mitochondria probably
also consist of protein; the PAS-negativity of these organelles in paraffin sections argues for the absence of
any significant amounts of polysaccharides. Other giant mitochondria have been described as containing
thread-like inclusions, but these usually exhibit a regular, paracrystalline packing (Tandler and Hoppel,
1972, 1986).
Helical filaments within cristae have been seen in
normal size mitochondria in several tissues, including
acinar and duct cells in rat parotid and von Ebner’s
glands, where they are present in fully one-quarter of
these organelles (Hand, 1970). Helical filaments have
been found in giant mitochondria in the aforementioned case of a human submandibular pleomorphic adenoma (Tandler and Erlandson, 1983), and in hepatic
megamitochondria evoked in rats by a low protein diet
(Svoboda and Higginson, 1964). The significance of intracristal helical filaments in any mitochondrion is unknown a t this time.
Despite their peculiar morphology, the enlarged mi-
Fig. 4. An enlarged mitochondrion in which the inner compartment
threads have aggregated to form several crystalloid-like structures.
x 40,000.
GIANT MITOCHONDRIA
Fig. 6. An enlarged mitochondrion with two expanded cristae, the upper one of which contains straight
filaments. The expanded crista a t center left contains filaments of the helical variety. This organelle
exhibits numerous, dense intramatrical threads that are randomly oriented. x 37,500.
Fig. 7. Helical filaments within a n expanded crista viewed at high magnification. x 77,000.
Fig. 5. Seromucous cells with mitochondria in a spectrum of sizes; all of these organelles, from the
smallest to the largest, contain dense threads. Note the virtual absence of normal-appearing mitochondria. x 9.000.
161
162
B. TANDLER AND C.J. PHILLIPS
tochondria in Stenonycteris accessory submandibular
gland must be able to carry out their normal role in
energy metabolism, since the cells in which they are
found obviously are capable of producing secretory
granules and show no degenerative changes. It is of
interest to note that we have never observed such bizarre mitochondria in the major or minor salivary
glands in the nearly 200 other species of chiropterans
or in any other species from other mammalian Orders
that we have examined ultrastructurally.
This work was supported in part by NIDR grant DE
07648. The authors are grateful to Thomas J . Slabe and
Douglas Johnston for technical assistance. We are indebted to Dr. Carlin A. Pinkstaff, West Virginia University, for carrying out a battery of histochemical procedures to unequivocally identify the nature of the
secretory endpieces in this bat.
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