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The effects of vinblastine on the secretory ameloblastsAn ultrastructural cytochemical and immunocytochemical study in the rat incisor.

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THE ANATOMICAL RECORD 219:113-126 (1987)
The Effects of Vinblastine on the Secretory
Ameloblasts: An Ultrastructural, Cytochemical, and
lmmunocytochemical Study in the Rat Incisor
Dkpartements de Stomatologie et d‘Anatomie, UniuersitC de MontrCat, Montrbal, QuCbec H3C
357 (A.N.) and Department ofdnatomy, McGill University, Montreal (l!U., H. W), Quebec,
H3A 2B2 Canada
Secretory ameloblasts synthesize the organic matrix of enamel and
secrete it at two distinct “putative secretory sites” characterized by membrane
infoldings (Nanci and Warshawsky, 1984a).The antimicrotubular agent vinblastine
sulphate interferes with secretion. We have examined the effect of this drug on the
ameloblast secretory sites and reevaluated the effect on the intracellular organization of the cell by using conditions that optimize fixation, cytochemistry (ZIO), and
immunocytochemistry.Associated with the disappearance of secretory granules and
Golgi-related structures from Tomes’ process was the loss of membrane infoldings at
secretory sites. The Golgi apparatus appeared fragmented and numerous granule
clusters were found throughout the cell body. These clusters were often seen in
relation to extracellular patches of material in which no crystallites were seen.
Immunocytochemistry revealed the presence of enamel proteins in the protein synthetic organelles, including various granule types, in lysosomes and in the extracellular patches. These data suggest that ameloblasts under the effect of vinblastine
carry on secretory activities, but the product is not routed to the usual sites. It was
confirmed that membrane infoldings characterize the sites where enamel proteins
are normally secreted.
The ameloblasts of inner enamel secretion in the rat
incisor were postulated to possess two distinct putative
secretory sites (Nanci and Warshawsky, 1984a). The interrod secretory site is located on the proximal portion
of Tomes’ process and forms a cooperative front for the
organization of interrod enamel (Warshawsky et al.,
1981; Nanci and Warshawsky, 1984a).The rod secretory
site is present on one surface of the interdigitating portion of Tomes’ process and organizes the individual rods
that fill the interrod cavities (Warshawsky et al., 1981;
Nanci and Warshawsky, 1984a). These putative secretory sites are characterized by three structural features:
membrane infoldings at the growth front of enamel,
accumulation of secretory granules in the proximity of
the infoldings, and the association of granules with tubular and vesicular structures (Weinstock and Leblond,
1971;Smith, 1979;Warshawsky et al., 1981;Simmelink,
1982;Nanci and Warshawsky, 1984a).
Vinblastine sulphate has been shown to interfere with
the secretory process in various cell types (Ekholm et
al., 1974; Ericson, 1980; Williams, 1981; Miake et al.,
1982; Bennett et al., 1984) and specifically the secretory
ameloblast (Moe and Mikkelsen, 1977a,b; Moe, 1979;
Takuma et al., 1982; Takuma et al., 1984). The latter
studies, as well as those using colcemid, an agent producing similar effects (Karim and Warshawsky, 19791,
suggest that directional movement of secretory granules
originating from the Golgi apparatus is affected. This
results in accumulation of secretory granules close to
0 1987 ALAN R. LISS, INC.
the Golgi saccules, which are now found throughout the
cell body but not in Tomes’ process, and the possible
release of these granules at abnormal sites along the
ameloblast surface.
In view of the usefulness of vinblastine in affecting
the secretory process and the possibility of altering the
distribution of secretory sites, inner and outer enamel
secretory ameloblasts (Warshawsky and Smith, 1974)
were examined after short-term administration of vinblastine, under conditions previously described to optimize fixation (Nanci and Warshawsky, 198413). The zinc
iodide-osmium tetroxide method (ZIO) (Maillet, 1963;
Kallenbach et al., 1963; Ozawa et al., 1983)was used to
assess the redistribution of membranous profiles associated with the Golgi apparatus. Immunocytochemistry
(Nanci et al., 1985) was used to verify whether enamel
proteins persist in the cell at the various sites along the
synthetic and secretory pathway, and whether the ectopically released material represents enamel proteins.
Received September 19,1986; accepted March 2, 1987.
Dr. Uchida’s present address is Department of Anatomy, Yamanashi Medical School, Tamaho, Yamanashi, 409-38, Japan.
Address reprint requests to Dr. Antonio Nanci, Departement de
Stomatologie, Facult6 de Medecine Dentaire. Universit6 de Montreal,
C.P. 6128, Succ. A., Montreal, QuBbec, Canada H3C 357.
chondria (m). Numerous cisternae of rough endoplasmic reticulum
(rER) and various types of granules (g) appear de nouo in the cytoplasm.
The proximal cell web (pew) of the ameloblast and the desmosomes (d)
associated with the cells of the stratum intermedium (SI) have been
retained. Some nuclei (N) seem to have been displaced distally. fg,
Fig. 1. The infranuclear compartment of inner enamel secretory granule with flocculent content, 17,500,
ameloblasts appears to contain fewer than normal numbers of mito-
Figs. 1-7. Electron micrographs of inner and outer enamel secretory
ameloblasts two hours after vinblastine injection and fixed with a
mixture of acrolein, formaldehyde, and glutaraldehyde. The sections
were stained with uranyl acetate and lead citrate.
Male Sherman or Wistar rats, 100 g weight, were
injected through the jugular vein with 5 mg/100 g body
weight of vinblastine sulphate (Sigma Chemical Company) in physiological saline Woe and Mikkelsen,
1977a). One or 2 hours after the injection, the rats were
anesthetized with an intraperitoneal injection of sodium
pentobarbital and perfused through the left ventricle
with lactated Ringer’s solution (Abbott)for about 30-45
seconds followed by the fixative for 10 minutes.
Tissue Preparation for Ultrastructural Analysis
Three rats were used to study the ultrastructural effects of vinblastine sulphate 2 hours after injection. The
fixative used was a mixture of 2% acrolein, 2.5% glutaraldehyde, and 3%formaldehyde in 0.06 M sodium cacodylate buffer containing 0.05% CaC12, pH 7.3 (Nanci and
Warshawsky, 198413). After perfusion, the mandibles
were dissected and immersed in the same fixative for 3
hours at 4°C. The incisors were then dissected from the
alveolar bone and washed in 0.1M sodium cacodylate buffer containing 5% sucrose, pH 7.3. All teeth were
postfixed in osmium tetroxide reduced with potassium
ferrocyanide (Karnovsky, 1971) for 2 hours at 4°C. Tissues were then left overnight at 4°C in 0.1 M sodium
cacodylate washing buffer, dehydrated in acetone and
embedded in Epon. Thin sections in the cross and tangential plane of the incisor were cut with a diamond
knife, stained with uranyl acetate and lead citrate,
and examined with a Philips 400 electron microscope
at 80 kV.
Zinc Iodide-Osmium Tetroxide Method (ZIO)
Four vinblastine sulphate and 2 normal saline injected rats were used for ZIO staining. One hour after
the injection, the animals were perfused with a mixture
of 2% acrolein, 2.5% glutaraldehyde, and 3% formaldehyde in 0.05 M sodium phosphate buffer, pH 7.3. The
mandibles were dissected and immersed in the same
fixative for 2 hours at room temperature. The incisors
were then dissected from the alveolar bone and washed
in 0.1 M sodium phosphate buffer. The ZIO staining was
performed according to Reinecke and Walther (1978).
dark granule
granule with flocculent material
Golgi apparatus
stacks of Golgi saccules
infranuclear compartment
interrod growth site
interdigitating portion of Tomes’ process
proximal cell web
pale granule
particulate material
proximal portion of Tomes’ process
rod growth site
rough endoplasmic reticulum
Tomes’ process
stratum intermedium
Briefly, the incisors were washed in a tris-HC1 buffer
solution, pH 3.8 at 4°C. They were then incubated in
the ZIO reagent for 18 hours at 4°C in the dark, washed
in tris-HC1buffer, dehydrated in acetone, and embedded
in Epon. Ultrathin sections were cut with a diamond
knife, stained with uranyl acetate and lead citrate,
and examined with a Philips 400 electron microscope
at 80 kV.
lmmunocytochemicalMethod Using the Protein A-Gold
Two vinblastine injected rats were fixed with 2% glutaraldehyde in 0.08 M cacodylate buffer containing
0.05% CaC12, pH 7.3. The mandibles were dissected and
further fixed by immersion in the same fixative for 2
hours at 4°C. They were then decalcified in 4.13%EDTA
for 14 days at 4°C (Warshawsky and Moore, 1967). Longitudinal segments of the incisor were cut, washed in
0.1 M cacodylate buffer containing 5% sucrose, and postfixed in potassium ferrocyanide reduced osmium tetroxide (Karnovsky, 1971). They were dehydrated in graded
acetone and flat embedded in Epon. Thin sections were
cut with a diamond knife and mounted on 200-mesh
nickel grids having a carbon-coated formvar film. The
sections were processed for immunocytochemistry using
the modified protein A-gold immunocytochemical technique for the detection of antigenic sites on osmium
postfixed tissues (Bendayan and Zollinger, 1983; Bendayan, 1984; Nanci et al., 1985) Briefly, the sections
were pretreated with sodium metaperiodate for 1 hour
and then incubated with a rabbit polyclonal antibody
against SDS-denatured mouse amelogenins (Slavkin et
al., 1982)at a 1/30 dilution in 0.01 M phosphate-buffered
saline containing 1%ovalbumin for 3 hours at room
temperature followed by protein A-gold complex for 30
minutes. These antibodies have been found t o crossreact with several mammalian species; however, they
do not distinguish between amelogenins and enamelins
(Slavkin et al., 1982). As a control, the antibody was
absorbed with excess antigen. Control experiments with
protein A-gold alone and preimmune serum were reported previously (Nanci et al., 1985). After the immunocytochemical procedure, the sections were stained
with uranyl acetate and lead citrate, and examined with
a Philips 410 electron microscope at 80kV.
Morphological Alterations Induced by Vinblastine
Although the external conformation of ameloblasts
exposed to high doses of vinblastine sulphate was not
significantly affected after 1 or 2 hours, their internal
organization was disrupted. Ameloblasts from both regions of inner (Figs. 1,2,3,4,6,7)and outer (Figs. 5,111
enamel secretion seemed to have been affected in a
similar manner. Microtubules were rarely observed, but
the filaments associated with junctional complexes (particularly the proximal one) in many cases still appeared
to persist (Figs. 1,14). Occasional accumulations of filaments were observed throughout the cytoplasm. The
infranuclear mitochondria1 compartment contained
fewer mitochondria than normal, but abundant profiles
of rough endoplasmic reticulum and numerous granules
of varying size and density were now present (Fig. 1).
Nuclei appeared to have been displaced distally (Figs. 1,
141, resulting in an elongated infranuclear comDartment. The normal tubula; configuration of the Golgi
Fig. 2. Cross-sectioned inner enamel secretory ameloblasts. In some cells the Golgi apparatus (G)is no
longer a cylindrical tube but is fragmented into separate stacks of saccules that have been displaced to
the periphery of the cell. Clusters of granules (arrows) are also seen near the cell membrane and are often
associated with extracellular patches of granular material (*). ly, lysosome; N, nucleus. x 14,850.
Fig. 3. Distal portion of inner enamel secretory ameloblasts. The rough endoplasmic reticulum (rER)
extends into the proximal portion of Tomes’ process ( p a , but the interdigitating portion (iT) appears
devoid of organelles. A fine particulate material (pm) accumulates in some processes. The membrane
associated with both the interrod (ir) and rod (r) growth sites is not infolded. g, granule; ly, lysosome.
x 13,750.
apparatus was broken into stacks of saccules distributed out the entire cell and extended into Tomes’ process
throughout the supranuclear compartment (Figs. 2,11, (Figs. 33). Clusters of secretory-like, dense-content
16). Occasionally, membrane profiles resembling Golgi granules were found throughout the cell pigs. 2,3,7,
saccules appeared in the infranuclear compartment. 11). These granules were either dark or pale-staining
The rough endoplasmic reticulum was dispersed through- (Figs. 2,7) similar to those found in normal ameloblasts
Fig. 4. Tomes’ processes (Tomes) from inner enamel secretory amelo- points (arrows). Some granules with flocculent material (fg) and phablasts appear devoid of organelles and the membrane associated with gosomes (ph) are also seen in the proximal portion. The membrane
interrod (ir) and rod (r) growth sites lacks significant infolding. x 14,000. associated with the growing interrod (ir) and rod (r) enamel is not
significantly infolded. x 13,750.
Fig. 5. Distal portion of outer enamel secretory ameloblasts. Rough
Fig. 6.Cross section of the interdigitating portions of Tomes’ proendoplasmic reticulum (rER) extends into the proximal portion of
Tomes’ process (pT) and occasionally some cisternae reach the interdi- cesses in inner enamel secretion showing “fissures” (arrows). The rod
gitating portion (iT). This latter portion appears fissured at several (R) associated with these processes seem to be subdivided. x 13,750.
Fig. 7. Electron micrograph showing the various types of granules
Fig. 8 . Thin section of inner enamel secretory ameloblasts fixed with
observed in ameloblasts treated with vinblastine. The cluster of dense- glutaraldehyde only and incubated with antiamelogenins antibodies
content granules in cell 1contains both pale (pg) and dark (dg) gran- revealed by the protein A-gold complex. The section was stained with
ules. In cell 2, two clusters of granules with flocculent material (fg), uranyl acetate and lead citrate. Both pale (pg) and dark (dg) granules
are seen. Between the cells there is a patch of granular material (*I m, in cell 1 and granules with a core of flocculent material (fg) in cell 2
mitochondria; N, nucleus. X29,250.
are labeled by gold particles. Only the core portion of these granules is
labeled. The rough endoplasmic reticulum (rER), the patch of extracellular material (*) and a larger lysosome-like granule (ly) also show
labeling by gold particles. x37,200.
Fig. 9. Immunocytochemical preparation as Figure 8. Profiles of
Tomes’ process of inner enamel secretion, cut in different planes of
section, are devoid of organelles and show no significant labeling. An
accumulation of filaments (f) is seen in one process. The cell surfaces
associated with rod growth sites are smooth (r). The enamel shows an
intense labeling by gold particles. x 16,350.
Fig. 10. Immunocytochemical preparation as Figure 8. This electron
micrograph shows three patches of granular material (*I found laterally between cells of inner enamel secretion and labeled by gold
particles. Clusters of dense-content granules (g), labeled by gold particles, are associated with these patches. X42,250.
Fig. 11. Supranuclear compartment of outer enamel secretory ameloblasts from vinblastine injected animals fixed with the aldehyde
mixture. Several phagosomes (ph) and lysosomes (ly) are seen in proximity to the Golgi apparatus (GI. Some of these phagosomes appear to
have engulfed intact granules (arrows). ~22,500.
Fig. 12.Immunocytochemical preparation as Figure 8. The granules
(arrows) present in phagosomes (ph) are labeled by gold particles. The
Golgi apparatus (G) shows some labeling. In some instances the saccules (curved arrows) contain accumulations of labeled material.
Figs. 13-18.Inner enamel secretory ameloblasts from normal untreated (Figs. 13,15,17) and vinblastine injected (Figs. 14,16,18) rats
stained for Golgi-related structures with the zinc iodide-osmium tetroxide method (210).Sections were stained with uranyl acetate and lead
Fig. 13. Few stained structures (arrows) are seen in the infranuclear
(in) compartment of ameloblasts from untreated normal rats. N, nucleus. x 11,600.
Fig. 14. The infranuclear compartment (in) of vinblastine-treated
ameloblasts contains many stained tubular and vesicular structures
(arrows). These structures are clustered similar to the granules with
flocculent content seen in figure 1. N, nucleus. ~ 1 1 , 6 0 0 .
Fig. 15. The Golgi apparatus of normal untreated ameloblasts is a
large, centrally located, cylindrical structure consisting of heavily
stained interconnected stacks (GS)of saccules. X24,300.
Fig. 16.After vinblastine treatment, the Golgi apparatus appears
fragmented into separate stacks of saccules fGS) clearly visualized by
the ZIO staining. x 11,600.
(Nanci and Warshawsky, 1984a). Another granule-type
was also observed; these were irregular in shape and
had a core of flocculent material (Figs. 1,7). Such granules were often seen in close association with Golgi
saccules and were occasionally interconnected by empty
membranous channels. The dense-content granule clusters were often in proximity to extracellular patches of
granular material found laterally between cells (Figs.
2,7). No crystal-like structures were seen in this material. Large lysosome-like granules (Figs. 2,3,11)and phagosomes (Figs. 5,11) containing cellular debris or
granules were also seen, particularly in outer enamel
secretory ameloblasts.
The most dramatic alterations were observed in Tomes’
process. The area corresponding to the proximal portion
of Tomes’ process (Figs. 3,5) and occasionally the interdigitating portion (Fig. 5) contained rough endoplasmic
reticulum and occasional granules. However, the interdigitating portion of Tomes’ process was completely devoid of other organelles (Figs. 3,4,5,). Rod and interrod
crystallites abutted on the membrane of Tomes’ process;
however, no characteristic membrane infoldings (Nanci
and Warshawsky, 198413)were associated with either the
rod or interrod growth sites (Figs. 3,4,5). In many instances, Tomes’ process and the associated enamel rod
appeared fissured (Fig. 6). Occasionally, fine particulate
material (Fig. 3) or filaments (Fig. 9) accumulated in
Tomes’ process.
ZIO Staining of Golgi-RelatedStructures
In untreated animals, ZIO stained the Golgi apparatus, which appeared as a large interconnected and centrally located cylindrical structure (Fig. 15). In Tomes’
process tubular and vesicular elements in the central
core of organelles were also reactive. Some secretorylike granules stained; others did not (Fig. 17). Few
stained structures were present in the infranuclear compartment (Fig. 13.)
In vinblastine injected rats the Golgi apparatus was
fragmented into several ZIO stained bodies dispersed
throughout the supranuclear cytoplasm (Fig. 16).
Whereas no reactive elements were seen in Tomes’ process (Fig. 18),stained vesicular elements were now present in the infranuclear compartment (Fig. 14).
lmmunocytochemical Labeling
Sections of tissue from vinblastine treated animals
incubated with the antiamelogenins antibody were specifically labeled (Figs. 8,9,10,12).The enamel (Fig. 9) and
extracellular patches of granular material (Figs. 8,101
were labeled with numerous gold particles. Intracellularly, the rough endoplasmic reticulum (Fig. 8) and the
Golgi apparatus showed some labeling (Fig. 12). Gold
particles were also present over the various granule
types (Figs. 8,101 and over larger lysosome-likegranules
(Fig. 8). Occasionally, lysosomes containing small secretory-like granules were encountered. The small granules were labeled, but the matrix of the lysosome was
not (Fig. 12). Only few, randomly dispersed gold particles were observed over the tissue section when the
antibody was absorbed with excess antigen.
Morphological Alterations Induced by Vinblastine
The present study has shown that vinblastine sulphate, which disrupts microtubules, produces severe ef-
fects on secretory ameloblasts as early as 1 to 2 hours
after injection. Both inner and outer enamel secretory
ameloblasts appeared similarly affected, emphasizing
the functional similarity between these regions of amelogenesis. In accordance with previous reports (Moe and
Mikkelsen, 1977a; Takuma et al., 1982), and similar to
the effects of colcemid (Karim and Warshawsky, 19791,
the major ultrastructural alterations produced by this
drug are the disorganization of organelles within ameloblasts and the loss of directional movement resulting
in the accumulation of secretory granules at Golgi sites
throughout the cell. The build-up of these granules eventually results in their ectopic release. ZIO, although
nonspecific, stains Golgi-related structures (Ozawa et
al., 1983). It is thus particularly suited to visualize the
alterations of the Golgi network induced by vinblastine.
The present study suggests that in normal ameloblasts
there are fine, membrane-lined tubular channels that
are associated with the Golgi apparatus and extend into
Tomes’ process, possibly related to one type of secretory
granule. After vinblastine administration, the Golgi apparatus fragments into separate stacks of saccules,
which disperse throughout the supranuclear region of
the cell. The tubular channels related to the Golgi apparatus, which normally extend into Tomes’ process, are
lost. This implies that the network of channels turns
over at the same rate as the secretory granules in Tomes’
The appearance of ZIO structures in the infranuclear
compartment reflects the appearance of Golgi related
structures, presumably de nouo, since intracellular migration is arrested. We postulate that this is an attempt
by the cell to revert to its embryonic orientation where
the Golgi apparatus occupied the infranuclear cytoplasm.
Radioautographic studies using 3H-fucose(Bennett et
al., 1984)have shown that glycoprotein synthesis continues after vinblastine administration, but there is a temporary inhibition of migration and directional exocytosis
of secretory granules. Colcemid, a drug that produces
similar ultrastructural changes in secretory ameloblasts, was also shown not to abolish protein synthesis
but to delay protein migration and secretion (Karim and
Warshawsky, 1979).It has been suggested that secretory
ameloblasts possess two secretory sites and that these
are characterized by membrane infoldings (Nanci and
Warshawsky, 1984a). We have found that vinblastine
produces a dramatic loss of these infoldings. Membrane
infoldings may reflect a general attribute of all secretory
sites, or they may be specifically involved with events
leading to the organization of enamel. In cases of rapid
secretory activity, excess membrane could result and
become infolded as a mechanism for its removal. The
loss of infolded membrane at the ameloblast secretory
sites after vinblastine could be the result of secretory
granules no longer reaching and fusing with the membrane at these sites. The fact that infoldings were not
found at ectopic sites may reflect insaicient exocytosis
or it may argue against this being a general feature of
secretory sites. On the other hand, the loss of membrane
infoldings concomitant with the loss of secretory granules and other organelles from Tomes’ process suggests
that infoldings reflect cellular activity related to secretion and extracellular organization of enamel. Regardless of the origin of these membrane infoldings, they are
always associated with the growing end of rod and inter-
Fig. 17. In normal animals, Tomes’ process (Tomes) contains a subFig. 18. Vinblastine treatment abolishes most stained structures
stantial network of stained tubular and vesicular elements. Whereas from Tomes’ process Vomes). x 11,600.
some secretory-like granules stain, others do not. X 15,900.
rod enamel (Weinstock and Leblond, 1971; Warshawsky
et al., 1981; Nanci and Warshawsky, 1984a) and they
disappear when secretion is arrested. It is thus confirmed that membrane infoldings are a characteristic
feature of the natural sites where enamel proteins are
released, and we favor the view that membrane infoldings may represent intense cellular motility related in
some way to the structuring of enamel, either by orienting the matrix or the crystallites.
lmmunocytochernical Localization of fnarnel Proteins
The immunocytochemical labeling obtained indicates
that the small granules forming clusters, irrespective of
their shape and density, contain enamel proteins. The
labeling of large lysosome-like granules raises the possibility that secretory ameloblasts degrade a portion of
their secretory product (posttranslational degradation,
crinophagy) or have a resorptive activity (Nanci et al.,
1985). The presence of secretory granules in lysosomes
(Figs. 11,121represents classical crinophagy (Smith and
Farquhar, 1966). Such a presence has never been reported in normal ameloblasts and seems to be induced
or accentuated by vinblastine treatment. Likewise, the
extracellular patches of material found laterally between cells consist of enamel proteins. This material is
generally not found, at least with such a frequency,
between secretory ameloblasts. The patches may thus
represent ectopic secretion (Moe and Mikkelsen, 1977a;
Takuma et al., 1982, 1984; Karim and Warshawsky,
1979).As reported by Takuma et al. (19841, we also have
seen no crystal-like structures within these patches. Assuming that both classes of enamel protein (amelogenins and enamelins) are secreted in tandem, the absence
of mineralization within the patches suggests that the
enamel layer, as opposed to the patches, must represent
a microenvironment that favors crystal initiation and
growth and that de nouo crystal formation requires more
than the simple presence of enamel proteins.
The authors acknowledge the technical assistance of
Ms. Annie Belanger. We are grateful to Dr. H.C. Slavkin
of the University of Southern California for having generously supplied the antibodies. This work was supported by grants from the Medical Research Council of
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ultrastructure, effect, secretory, stud, vinblastine, rat, incisors, cytochemical, immunocytochemical, ameloblasts
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