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The occurrence of an unusual tubular organelle in surface epithelial cells of the mouse ascending colon after injection of diazo-oxo-norleucine.

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THE ANATOMICAL RECORD 196:413-420 (1980)
The Occurrence of an Unusual Tubular Organelle in
Surface Epithelial Cells of the Mouse Ascending
Colon after Injection of Diazo-0x0-Norleucine
JOHN E. MICHAELS
Department ofdnatomy, Uniuersity of Cincinnati College of Medicine,
Cincinnati. Ohio 45267
ABSTRACT
Tubular structures were observed in surface epithelial cells of
mice that had been injected with high dosages of diazo-0x0-norleucine(DON), a
glutamine antagonist. The tubules often occurred in bundles which contained a
variable number of tubules, often as many as one hundred being present. Within
the bundles, the tubules were oriented either randomly or parallel t o one another.
They measured 25 to 35 nm in diameter with angular or circular profiles and were
as long as 1to 2 pm. In the center of each tubule, a smaller tubule-like component
was evident that measured 5 to 7 nm in diameter. With the exception of endoplasmic reticulum, often with attached ribosomes, organelles were excluded from the
bundles. Since the tubules and the endoplasmic reticulum occasionally were observed to be continuous, it is suggested that the tubules may originate from this
organelle.
There have been many reports of modified
endoplasmic reticulum containing formed elements, some of which are tubular. Such modifications have occurred in a variety of plant
and animal cells derived from normal, pathological, or drug treated specimens.
A tubular organelle, which may be a modified form of endoplasmic reticulum, was observed in surface epithelial cells of murine ascending colon after injection of high dosages of
the drug diazo-0x0-norleucine (DON). The
current report will be limited to a description of
this structure.
DON is a glutamine antagonist that interferes with glycosylation of forming glycoproteins, including those that comprise the cell
coat (Greene and Pratt, '77). The compoundbinds irreversibly (Rando, '75) t o transamidases, such as those responsible for the glutamine dependent conversion of fructose to glucosamine, a precursor of two carbohydrates of
cell coat glycoproteins, N-acetylglycosamine
and N-acetylgalactosamine. Both of these carbohydrates apparently are added to peptides
within rough endoplasmic reticulum (Whur et
al., '69). Absence of the compounds would prevent further addition of sugars to forming glycoproteins. DON also inhibits purine synthesis
(Livingston et al., '70).
The effects of DON on the surface columnar
epithelial cells of the ascending colon were of
interest, since these cells have a n abundant cell
coat composed of glycoproteins on their apical
surface, and they rapidly incorporate carbohydrate precursors of glycoproteins (Bennett et
al., '74). Moreover, the mode ofmigrationof cell
coat glycoproteins from the Golgi apparatus to
the cell surface has been established for these
cells (Michaels and Leblond, '76).
MATERIALS AND METHODS
Thirteen adult male CD-1 outbred albino
mice were injected intraperitoneally with
diazo-0x0-norleucinein a saline solution as follows. One pair of mice was given a single dosage
of 10 mglkg body weight 18 hours before sacrifice. Four pairs of mice received a total dosage
of 5, 10, 20, or 100 mg/kg body weight; these
mice were given two injections of equal quantity a t a 24 hour interval and sacrificed 24
hours later. Three additional mice received a
total dosage of 150 mgkg body weight; these
mice were given three injections of equal quantity a t 24 hour intervals and sacrificed 24 hours
later. Two control animals received three injections at 24 hour intervals of a volume of saline
equivalent to that injected into the animals
dosed with 150 mgkg of DON, 0.5 ml. At all
times, mice were given feed and water ad libitum.
000-3276X/80/1964-0413$02.400 1980 ALAN R. LISS, INC.
Received April 3, 1979; accepted July 30, 1979
413
414
JOHN E. MICHAELS
In order to prepare the tissue for electron
microscopy, the ascending colon of each anesthetized mouse was incised about 1 cm from the
cecum and fixative was injected into the lumen
of the intestine 2 cm distally. A 2 cm segment of
jejunum was fixed in a similar manner. The
fixative consisted of 2.0% glutaraldehyde and
0.5% formaldehyde (prepared from paraformaldehyde) buffered with 0.1 M sodium
cacodylate, pH 7.2. After about two minutes of
continuous intraluminal injection of fixative a t
room temperature, each segment of intestine
was dissected, cut into small pieces with a razor
blade, and additionally fixed by immersion a t
room temperature for about 30 minutes and
then at 4°C for two to three hours. The tissue
was washed in 0.2 M sodium cacodylate buffer
(pH 7.2) for one hour and postfixed for one and a
half hours with 1.5%osmium tetroxide in 0.1 M
sodium cacodylate buffer (pH 7.2) a t 4°C. Dehydration was initiated with cold alcohols and
specimens were brought to room temperature
in 100% alcohol. Tissue was immersed in two
rinses of propylene oxide for a total of 20 minutes, then infiltrated in a mixture of propylene
oxide and Epon 812 (Luft, '611, and embedded in
Epon. Sections were cut with a diamond knife
on a Reichert OMU-3 Ultramicrotome mounted
on uncoated grids and stained with uranyl acetate (Watson, '58) and lead citrate (Reynolds,
'63). A Philips EM 301 electron microscope operated a t 60 or 80 kV was used to observe the
specimens.
OBSERVATIONS
In the basal half of normal surface epithelial
cells of the mouse ascending colon (Fig. 11,
mitochondria are the most predominant organelle and profiles of rough endoplasmic reticulum are frequent. At the lateral surfaces,
processes from adjacent cells interdigitate (Fig.
1).
After injection of DON, long tubular structures, composed of unit membrane, appeared in many of the surfaae epithelial cells
(Fig. 2) as well as mid-crypt cells that had characteristics of surface cells. The tubular profiles
measured 25-35 nm in diameter and as long as
2-3 pm (Figs. $7) before passing out of the
plane of section. The tubules occurred singly,
but more often in bundles containing as few as
three or four (Fig. 2, upper left corner) to as
many as one hundred or more tubules (Fig. 3 ) .
The bundles of tubules were most abundant
and largest in mice that received the highest
dosages of DON (150 mg/kg body weight) and
occurred, to a lesser extent, in animals that had
been injected with an intermediate dosage (100
mg/kg body weight); but they were absent in
mice receiving lower dosages.
The tubules, singular or in bundles, were not
found in goblet cells nor in argentaffin cells.
Many absorptive cells from the jejunum of mice
that had received the highest dosage of DON
were observed for the presence of the peculiar
tubular structures, but the modified organelle
was never apparent in these cells. Since only
stomach, jejunum, and ascending colon were
fixed and observed, it is not known whether
cells of other organs from animals injected with
DON exhibited the tubular organelle.
The tubules were not uniform in orientation;
in each bundle they were oriented either randomly (Fig. 3 ) or more or less parallel to one
another for 1-2 pm (Fig. 4). Most often, a single
bundle of tubules was observed in a cell (Fig. 2).
The bundles frequently occurred basal to or
near the nucleus, but they were not limited to
this region. Some bundles occurred above the
nucleus and others near the lateral cell membrane. In sections that included the subnuclear
region of the cells, bundles were observed in
many adjacent cells (Fig. 2). When this region
was not present in the section, the bundles were
less numerous. Therefore, the overall frequency of the structures in sections of cells varied from 1&15% t o 80-9W0, depending on the
region sectioned. It is possible that serial sectioning would have revealed the structures in
every absorptive cell. The bundles were approximately circular in cross section (Fig. 2) and
measured as large as 1-2 pm in diameter. In
cells that contained more than one bundle, the
orientation of each bundle of tubules was independent of other bundles in the cell.
Most large organelles were excluded from the
bundles. However, profiles of rough endoplasmic reticulum were located near and within the
bundles (Figs. 2-6) and lipid droplets occasionally appeared to be associated with the bundles
(Figs. 3, 5).
The distance between many tubules in each
of the bundles of parallel tubules varied between 10 to 20 nm (Fig. 4). In some instances,
connections appeared to occur between one or
more tubules and smooth or rough endoplasmic
reticulum (Figs. 4-6). Yet, the tubules themselves always remained agranular.
In cross section (Figs. 6,7),the tubulesvaried
in shape, some appearing triangular, others
polygonal or round. In the center of each tubule,
a very small circular structure ( 5 7 nm in diameter) was evident (Figs. 6,7),composed of a n
electron opaque wall and an electron lucent
core, therehy appearing tubular. The wall of
the central tubule was approximately 1.5 nm
thick. In every tubule that was sectioned transversely, the inner 7 nm tubule was evident.
Once the central tubule was recognized in
transverse sections, close observation of
TUBULAR ORGANELLE IN CELLS OF ASCENDING COLON
415
Fig. 1. In this eleetmn miciugraph of normal epithelial cells of the ascending colon, the region basal ta the nucleus is
illustrated in longitudinal section. Mitochondria (M)are numerous and profiles of rough endoplasmic reticulum (R) are
frequent. At the lateral surfaces (LS) processes from adjacent cells interdigitate. x 17,750.
The following electron micrographswere obtained from sectionsof surface epithelial cells of murine ascending
colons. The mice had received dosages of DON that totalled either 100 or 150 mgikg body weight.
Fig. 2. This low magnification micrograph shows cells sectioned transversely at a level just basal to the
nucleus. In most cells, bundles (B) of tubule-like structures are evident. Many of the tubules are sectioned
transversely; a few longitudinally sectioned tubules (LT) also appear. Profiles of rough endoplasmic reticulum (R)
lie close to or within the bundles of tubules. Other organelles are not observed within the bundles of tubules.
x 17.750.
Fig. 3. In this cell, a large bundle of irregularly arranged tubules is located at the basal end of the nucleus.
Numerous tubules are evident, some of which are sectioned transversely (T),and a few profiles of rough
endoplasrnic reticulum are present. Two lipid droplets occur in the bundle (LD). Two microtubules (arrows) are
smaller in diameter than the tubules. x 37,500.
Fig. 4. A portion of a bundle of parallel tubules, sectioned longitudinally, is illustrated. Many of the tubules
maintain a rather uniform distance from each other. A connection between a profile of rough endoplasmic
reticulum and a tubule is evident (large arrow). The central tubule may be observed in many of the parallel tubules
(small arrow). x 102.000.
418
JOHN E. MICHAELS
TUBULAR ORGANELLE IN CELLS OF ASCENDING COLON
tubules that were sectioned longitudinally revealed faint images of the central tubules also
oriented longitudinally (Fig. 4).
DISCUSSION
The tubular structures, containing a central
tubule-like component as described in this report, a r e apparently unique. A few generalizations concerning their characteristics
may be suggested. The most common location of
the bundles of tubules, basal to and near the
nucleus, implies that this region had the most
suitable environment for formation of the
structure, although neither smooth nor rough
endoplasmic reticulum was especially abundant a t this site. This region was also the most
prevalent location of another very different and
infrequent modification of endoplasmic reticulum observed in the same cell type in normal
mice (Michaels, '75). (This modification consisted of stacks of multiple parallel cisternae.
The cisternae were separated by a regular spacing in which a single layer of small vesicle was
found).
Few organelles were observed between the
tubules of a single bundle with the exception of
the many profiles of rough endoplasmic reticulum, indicating that the bundles, regardless of
the orientation of the tubules, were fairly cohesive. The profiles of endoplasmic reticulum
within the bundles had occasional sites where
connections to the tubules were evident,
suggesting that the tubules may be formed
from endoplasmic reticulum. At the subnuclear
location, the tubules were oriented either randomly or parallel to each other. In either case,
the tubules themselves maintained the same
diameter and the 7 nm central tubule was always present.
A superficial resemblance between the
tubules and microtubules was apparent, especially in the instances of parallel tubules (Fig.
4). However, the diameter of microtubules is
somewhat smaller (Fig. 3); microtubules are
not formed from unit membrane; continuity between microtubules and endoplasmic reticulum is not observed; microtubules tend not to
occur in irregularly oriented bundles (Fig. 3);
and their profiles in transverse sections are
more uniformly round t h a n those of the
419
tubules. Moreover, a central tubule is lacking
in microtubules.
Previously tubular elements (e.g., Uzman et
al., '71; Valeri et al., '71; Baringer and Swoveland, '72; Quan et al., '74; Hruban et al., '76) or
fibers (Baic et al., '73; Wang, '74) have been
observed within endoplasmic reticulum, occurring in different cell types of diverse organisms.
Several types of tubules have been described,
many of which conform to two broad categories,
but only on the basis of their morphological
appearance. In one group, multiple tubules in
endoplasmic reticulum have been described
that were highly curved and branched and had
diameters of 2530nm. Examples of this type of
intracisternal tubules were found in 1)tumor
cells or cells of animals that had been infected
with a virus (Uzman et al., '71, who also reviewed earlier work; Baringer and Swoveland,
'72; Hruban et al., '76), and 2) in human 1 . ~ phoid cells treated with 5-brom0-2-deoxpr1dine (Grimley et al., '73). In some cases, the
tubules apparently were formed as invaginations of the endoplasmic reticulum and therefore consisted of unit membrane (Baringer and
Swoveland, '72; Hruban et al., '76).
In a second category, the intracisternal
tubules were multiple, straight, and most had
diameters of 20-45 nm. Examples of these occurred in pars intermedia of toad hypophysis
(Valeri et al., '71), in a thyroid tumor of a dog
(Deutschlander, '72), and in a variety of plant
cells, each instance of which differed from the
others (Jansen, '68; Hepler and Newcomb, '69;
Schnepf and Diechgraber, '72; Quan et al., '74;
Heywood, '72; Cresti et al., '74).
In addition, other arrangements of intracisternal tubules have been described, such as
triangular prism-shaped inclusions in rough
endoplasmic reticulum of malpighian tubules
of the mosquito, Culex tarsalis (Houk, '77). Recently, tubular structures with a diameter similar to the tubules reported here and containing a central 7 nm core were observed in
Langerhans cells (Kobayashi and Hoshino,
'78). However, long profiles were not observed
and the cores were not tubular. The authors
suggested that the cored tubules were distinct
from Langerhans cell granules.
Fig. 5. This oblique section through a bundle of tubules includes profiles of rough endoplasmic reticulum. A
few tubules (arrowheads) appear to connect with the membrane that forms the endoplasmic reticulum. x 62,500.
Fig. 6. In this micrograph, a bundle of tubules has been sectioned transversely. The profiles measure about 30
nm in diameter and reveal circular, triangular, and irregular shapes. Each tubule displays the central tubule-like
element (small arrow) measuring about 7 nm in diameter. Many tubules maintain a fairly uniform distance from
one another. Toward the center of the bundle, the membrane of a profile of agranular endoplasmic reticulum is
continuous with two tubules (arrowhead). x 62,500.
Fig. 7. At higher magnification, the tubules may be observed to be composed of unit membrane (arrows).The
central tubular element is depicted more clearly in this micrograph. x 100,000,
420
JOHN E. MICHAELS
The tubules possibly derived from endoplasmic reticulum and those containing central
tubules in the mice treated with DON, are
clearly different from the endoplasmic reticulum that contains intracisternal tubules described by others. The relationship of the
tubules to DON, whether they arise as a primary response to the drug o r a secondary toxic
reaction to DON or its metabolic products, can
only be speculated upon. The tubules did not
stain positively for the presence of glycoproteins with periodic acid-chromic acid silver
methenamine technique (not illustrated), but
this was also the case for other endoplasmic
reticulum as well. Whatever the relationship to
DON, it is worth noting that another gastrointestinal cell type which contains a significant
amount of glycoproteins, the parietal cells, exhibited an abnormal structure-i.e., concentric
saccules (Michaels,’79)-after injection of high
dosages of DON.
The difference in response of the jejunum and
the ascending colon epithelial cells to DON or
its metabolites is interesting. These two cell
types have different functions, but morphological similarity, including a well developed
cell coat composed of glycoproteins. Yet, the
bundles were never observed in epithelial cells
of the jejunum. It is also of interest that malignant tumors occur much more frequently in the
colon and they may be accompanied by alterations of surface glycoproteins (Gold and
Freedman, ’65).Further analysis of the effects
of DON on these cells would seem to be warranted.
Gold, P., and S.O. Freedman (1965) Demonstration of
tumor-specific antigens in human colonic carcinomata by
immunological tolerance and absorption techniques. J.
Exp. Med., 121:43%462.
Greene, R.M., and R.M. Pratt (1977)Inhibition of diazo-oxonorleucine (DON)of rat palatal glycoprotein synthesis and
epithelial cell adhesion invitro. Exp. Cell Res., 105:27-37.
Grimley, P.M., D.W. Barry, and Z. SchafT(1973)Induction of
tubular structures in the endoplasmic reticulum of human
lymphoid cells by treatment with 5-bromo-2’-deoxyuridine. J. Natl. Canc. Instit., 51:1751-1760.
Hepler, P.K., and E.H. Newcomb (1964) Microtubules and
fibrils in the cytoplasm of coleus cells undergoing secondary wall deposition. J. Cell Biol., 20t529-533.
Heywood, P. (1972) Structure and origin of flagellar hairs in
Vucuoluria uirescens. J. Ultrastruct. Res., 39:60%623.
Houk, E.J. (1977) Endoplasmic reticulum inclusions in the
Malpighian tubules of the mosquito Culex tarsalis. J.
Ultrastruct. Res., 60:6%70.
Hruban, Z., T.-W. Wong, M. Itabashi, and E.S. Lyon (1976)
Glomiform and fibrillar cytoplasmic inclusions. Virchows
Archiv b Cell. Pathol., 20.9-102.
Jensen, W.A. (1968) Cotton embryogenesis embryogenesis:
The tube-containing endoplasmic reticulum. J. Ultrastruct. Res., 22:2=302.
Kobayashi, M., and T. Hoshino (1978) Occurrence of cored
tubule in the Birbeck granule-containing cells of mice. J.
Electron Microsc., 27,199-205,
Livingston, R.B., J.M. Venditti, D.A. Cooney, andS.K. Carter (1970) Glutamine antagonists in chemotherapy. Adv.
Pharmacol. Chemother., 8:57- 120.
Luft, J.H. (1961) Improvements in epoxy resin embedding
methods. J. Biophys. Biochem. Cytol., 9:40%414.
Michaels, J.E. (1975) An unusual modification of endoplasmic reticulum in epithelial cells of the mouse ascending
colon. Anat. Rec., 183:27-38.
Michaels, J.E. (1979) Formation of concentric saccules in
murine parietal cells after injection of diazo-oxo-norleucine. Anat. Rec., 193:775-790.
Michaels, J.E., and C.P. Leblond (1976) Transport of glycoprotein from Golgi apparatus to cell surface by means of
“carrier” vesicles, as shown by radioautography of mouse
colonic epithelium after injection of 3H-fucose.J. Microsc.
Biol. Cell., 25:243-248.
Quan, S.G., E.Y. Chi, and S.M. Caplin (1974) Tubular
ACKNOWLEDGMENTS
structures in endoplasmic reticulum cultured in broccoli.
It is a pleasure to thank Mrs. Julia T. Hung
J. Ultrastruc. Res., 48t9Z-101.
for her excellent and dedicated technical assis- Rando, R.R. (1975)On the mechanism of action of enzymes
which act a s irreversible enzyme inhibitions. Biochem.
tance.
Pharmacol., 24: 1153- 1160.
This work was supported initially by a grant Reynolds, E.S. (1963)Theuseof lead citrate a t high pH as a n
from the University of Cincinnati Research
electron opaque stain in electron microscopy.J. Cell Biol.,
13r405-421.
Council and subsequently by a grant from the
Schnepf, E., and G. Deichgraber (1972)Tubular inclusions in
National Institutes of Health, GM-24604.
the endoplasmic reticulum of the gland hairs of Onionis
repens. L. (Fabaceac)J. Microscopie, 14:361-364.
LITERATURE CITED
Uzman, B.G., H. Saito, and M. Kasae (1971) Tubular arrays
in endoplasmic reticulum in human tumor cells. Lab. InBaic, D., B.E. Frye, and B.G. Ladewski (1973) Intracisternal
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fibers in the liver of starved frogs. J. Ultrastruct. Res.,
43;478-482.
Valeri, V., R.P. Goncalves, A.R. Cruz, and E.M. Laicine
Baringer, J.R., and P. Swoveland (1972)Tubular aggregates
(1971)Tubular structure within the granular endoplasmic
in endoplasmic reticulum: Evidence against their viral
reticulum of the pars intermedia of toad hypophysis. J.
Ultrastruct. Res., 35:197-200.
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Bennett, G., C.P. Leblond, and A. Haddad (1974) Migration Wang, N.S. (1974)Fine structural alterations in mesothelial
of glycoprotein from the Golgi apparatus to the cell surface
cells associated with cardiac anomaly. Virchows Archiv b
Call. Pathol., 15:217-222.
of various cell types as shown by radioautography after
labeledfucoseinjectioninto rats. J. Cell Bio1.,60:258-284. Watson, M.L. (1958) Staining of tissue sections for electron
Cresti, M., E. Pacini, and C. Simoncioli (1974) Uncommon
microscopy with heavy metals. J. Biophys. Biochem.
paracrystalline structures formed in the endoplasmic reCytol., 4:475-478.
ticulum of the integumentary cells ofDipZotaziserucoides Whur, P., A. Herscovics, a n d C.P. Leblond (1969)
ovules. J. Ultrastrud. Res., 49:21%223.
Radioautograpbic visualization of the incorporation of
Deutschlander, N. (1972) Ungewohnliche Tubuli im endogalactose-% and mannose-3H by rat thyroids in vitro in
plasmatischen Reticulum von Schildriisentumorzellen.
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Biol., 43;289-3 11.
Virchows Arch. B Zellpath., 1l:ll-18.
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