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Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors.

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THE ANATOMICAL RECORD 217:107-123 (1987)
Immunocytochemical and Radioautographic
Evidence for Secretion and lntracellular
Degradation of Enamel Proteins by Ameloblasts
During the Maturation Stage of Amelogenesis
in Rat Incisors
ANTONIO NANCI, HAROLD C. SLAVKIN, AND CHARLES E. SMITH
Dkpartements de stomatologie et d’anatomie, Universitt de Montreal, Montrkal, Quebec,
Canada H3C 3J7 (A.N.); Laboratory for Developmental Biology, Graduate Program in
Craniofacial Biology, School of Dentistry, University of Southern California, Los Angeles, C A
90089 (H. C.S.); and Departments of Anatomy and Oral Biology, McGill University, Montreal,
Quebec, Canada H3A 2B2 (C.E.S.)
ABSTRACT
In the continuously erupting rat incisor the ameloblasts progress
through distinct stages associated with the secretion and maturation of enamel. We
have examined the possibility that the so-called “postsecretory” ameloblasts of the
maturation stage of amelogenesis remain biosynthetically active and are engaged
in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel
organ cells during the early maturation stage of amelogenesis in rat incisors by
using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late
secretion through the first complete modulation from ruffle-ended to smooth-ended
ameloblasts were examined. Specific immunolabelling was found within the rough
endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout these regions. The heaviest intracellular immunolabelling was
found within secretory granules and lysosomes (multivesicular type). Quantitative
analyses showed that the Golgi saccules and the multivesicular lysosomes of modulating ameloblasts were generally less immunoreactive compared to similar organelles in ameloblasts secreting the inner enamel layer. Radioautographic studies
confirmed that ameloblasts of the maturation stage incorporated 3H-leucineand 3Hmethionine and secreted labelled proteins into the enamel layer. Grain counts
indicated that ameloblasts from the first ruffle-ended band incorporated about twofold less 3H-methionine and secreted about tenfold less labelled proteins into the
enamel compared to ameloblasts secreting the inner enamel layer. The results of
this study confirm that ameloblasts do not terminate biosynthesis and secretion of
enamel proteins once the final layer has been deposited on the surface of the
developing enamel. They continue to form and release new proteins during the
maturation stage which intermix with older proteins laid down initially during the
secretory stage of amelogenesis. Secretory activity for enamel proteins has been
detected in ameloblasts up to at least the second ruffle-ended phase of maturation,
at which point the enamel matrix is partially soluble in EDTA.
Several authors have suggested that certain epithelia1
cells have the capacity to down regulate the production
of specific gene products using intracellular degradative
mechanisms (reviewed in Bienkowski, 1983). One model
for critically examining these possibilities can be found
in developing enamel in which ectodermally derived
ameloblasts produce an extracellular matrix of finite
thickness (reviewed in Smith, 1979).
The organic matrix of enamel is believed to contain
two main subfamilies of proteins termed amelogenins
and enamelins (reviewed in Eastoe, 1979; Termine et al.,
0 1987 ALAN R. LISS, INC.
1980; Robinson et al., 1983; Shimizu and Fukae, 1983;
Fincham and Belcourt, 1985; Robinson and Kirkham,
1985). Amelogenins are found in highest concentrations
within newly formed and partially mineralized “young”
enamel. Enamelins comprise the major protein associated with the mineral in fully “mature” enamel (references above). Current biochemical evidence suggests
that small amounts of enamelins are present along with
Received June 2, 1986; accepted August 12, 1986.
108
A. NANCI, H.C. SLAVKIN. AND C.E. SMITH
the amelogenins in newly formed enamel (Termine et
al., 1980; Robinson et al., 1983; Shimokawa et al., 1984;
Zeichner-David et al., 1985). Hence, enamel maturation
is viewed as a process which results in the breakdown
and selective loss of amelogenins with the preferential
retention of the enamelins (reviewed in Robinson and
Kirkham, 1985).
Radioautographic studies by several workers have indicated that ameloblasts incorporate the greatest
amounts of labelled amino acids during the secretory
stage of amelogenesis (Slavkin et al., 1976; Warshawsky, 1979; Blumen and Merzel, 1982; Takano and
Ozawa, 1984; see also Robinson et al., 1982). Such findings are consistent with the idea that these cells actively
synthesize and secrete large quantities of matrix proteins (amelogenins and enamelins) in order to create a n
enamel layer by appositional growth. There is additional evidence suggesting that ameloblasts also form
exportable proteins after the enamel layer has developed to its full thickness (Weinstock, 1972; Warshawsky,
1979; Warshawsky and Josephsen, 1981). The exact
chemical nature of these proteins remains obscure, but
it is believed that they may represent components of a
basal-lamina-like structure which appears on the surface of the enamel near the beginning of the maturation
stage of amelogenesis (Weinstock, 1972; Takano, 1979).
It is also possible that a portion of these proteins could
constitute newly formed enzymes such as proteases
(“amelogenases”) which some workers believe are secreted extracellularly to cleave the amelogenins into
small molecular weight fragments thereby promoting
enamel maturation (reviewed in Carter et al., 1984;
Crenshaw and Bawden, 1984). No evidence presently
exists that part, or all, of this material is newly synthesized enamel proteins secreted by the ameloblasts during the maturation stage of amelogenesis.
We originally undertook this investigation to probe
the distribution of antigenic sites to enamel proteins
with the lysosomal system of the ruffle-ended ameloblasts of the maturation stage of amelogenesis. These
cells are believed to internalize fragments of partially
degraded amelogenins and digest them via the lysosomes (Reith and Cotty, 1967; Kallenbach, 1974; Ozawa
et al., 1983; Sasaki, 1984b). We assumed that if these
cells resorb enamel proteins, then there should be a
subpopulation of lysosomes containing material with
sflicient antigenicity to be detected by high-resolution
immunocytochemistry. Over the course of this investigation it became apparent that ameloblasts associated
with maturing enamel showed not only immunoreactive
lysosomes but also a pattern in the intracellular distribution of antigenic sites to enamel proteins reminiscent
of the one reported recently for ameloblasts from secretory stage of amelogenesis (Nanci et al., 1985). Hence, it
became necessary also to reevaluate the synthetic potential of the modulating ameloblasts by conventional light
microscopic radioautography.
barbital (M.T.C. Pharmaceuticals, Hamilton, Ontario).
The left ventricle was cannulated and the vascular system was flushed for about 30 seconds with lactated Ringer’s solution (Abbott Laboratories, Montreal). Four
animals were perfused for 10 minutes with 1% glutaraldehyde in 0.1 M or 0.08 M sodium cacodylate buffer, pH
7.3, containing 0.05% CaC12.Two animals were perfused
with 4% paraformaldehyde in 0.1 M sodium cacodylate
buffer, pH 7.4. The mandibles were removed and immersed for 1 hour in fresh fixative at 4°C. They were
then washed briefly in cold 0.1 M sodium cacodylate
buffer, pH 7.4, containing 4% sucrose (CS buffer) and
decalcified in 4.13% disodium EDTA (BDH Chemicals,
Montreal) for 14 days at 4°C (Warshawsky and Moore,
1967).The mandibles were washed for 2 days in frequent
changes of cold CS buffer. Excess bone and molars were
removed, and most incisors with surrounding alveolar
bone were subdivided into four segments about 5 mm in
length. Each segment was then split in half by passing
a razor blade down the midline of the segment (Smith,
1974). Some of the hemisegments were postfixed for 1
hour a t 4°C in 1.5%potassium ferrocyanide/l% osmium tetroxide (reduced osmium; Karnovsky, 1971). The
hemisegments were dehydrated in graded acetone, infiltrated, and flat embedded with Epon 812 substitute
(MECA Laboratories, Montreal) so as to produce longitudinal sections of the enamel organ. Other unosmicated tooth specimens were dehydrated in graded
methanol, infiltrated, and embedded in Lowicryl K4M
(Bendayan, 1984)for cross sections of the enamel organ.
Thin sections of selected areas were cut with a diamond
knife on a Reichert Ultracut E microtome, mounted on
200-mesh nickel grids having a carbon-coated Formvar
film, and processed for immunocytochemistry.
Radioautographicstudies
Twelve male Wistar rats weighing 109
5 g were
anesthetized with sodium pentobarbital and injected via
the external jugular vein with 0.1 ml of a normal saline
solution containing 1mCi of either [meth~l-~HI-L-methionine (s.a. 80 Ci/mM) or [3,4,5, -3H (NI]-L-leucine (s.a.
147 Ci/mM) (Dupont NEN, Boston). The radioactive
amino acids had been concentrated in a Speed Vac (Savant Instrument Inc., Farmingdale, NY) and resuspended in normal salinejust prior to use. After 6 minutes
the rats were injected via the opposite external jugular
vein with 0.1 ml of a normal saline solution containing
a n excess of %old” L-methionine or L-leucine (10 mM)
(Sigma Chemical Co., St. Louis, MO). The rats were
killed in pairs at 20 minutes, 1hour, and 4 hours after
the initial injection of radioactive tracer by first perfusing the vascular system via the left ventricle with lactated Ringer’s for about 30 seconds and then with
fixative for 20 minutes. Rats from the 20-minute group
were perfused for 4 minutes with 4% paraformaldehyde
in 0.08 M sodium cacodylate buffer, pH 7.4., containing
0.05% CaC12 (done to avoid nonspecific binding of amino
acids to protein; see Josephsen and Warshawsky, 1982).
MATERIALS AND METHODS
This was followed by 5% glutaraldehyde in 0.05 M soTissue Preparation
dium cacodylate buffer, pH 7.3, containing 0.05% CaC12
for 16 minutes. The remaining rats were perfused dilmrnunocytochemicalstudies
rectly for 20 minutes with the glutaraldehyde fixative
Six male Wistar rats weighing 100 f 10 g body weight solution after vascular prewashing. The mandibles were
(Charles Rivers Canada, St.-Constant, Quebec) were an- removed and immersed for a total of 4 hours in fresh
esthetized by intraperitoneal injection of sodium pento- fixative a t 4°C. The mandibles were washed overnight
SECRETORY ACTIVITY IN MODULATING AMELOBLASTS
at 4°C in CS buffer on a rotator, decalcified in 4.13%
disodium EDTA for 16 days, washed, and split into
hemisegments as described above. All hemisegments
were postfixed for 2 hours at 4°C in reduced osmium,
dehydrated in graded alcohol and propylene oxide, and
flat embedded in Epon 812 substitute. A series of l-pmthick sections was cut with glass knives on a Reichert
Omu4 or Ultracut E microtome. The first sections from
each block were placed on separate glass slides and
stained with toluidine blue. The remaining sections were
mounted on alcohol-cleaned glass slides and stained with
Regaud’s hematoxylin. Three slides were prepared from
each block with 3 to 4 sections per slide. The slides were
dipped in Kodak NTB2 liquid emulsion, exposed for 4
days (two slides, for quantitation) or 14 days (one slide,
for photography), and developed (Kopriwa and Leblond,
1962).
Cytochemical studies
Eleven male Sprague-Dawley or Wistar rats weighing
100-150 g were anesthetized with chloral hydrate (0.4
mg/g body weight) and perfused for 8-10 minutes via
the left ventricle with 2% or 2.5% glutaraldehyde in 0.1
M or 0.08 M sodium cacodylate buffer, pH 7.3, containing 0.05% CaC12. In some experiments 2% dextran
(40,000 MW, Pharmacia, Montreal) was included in the
fixative solution, and the vascular system was flushed
with lactated Ringer’s prior to perfusion with fixative.
The mandibles were removed and immersed in fresh
fixative for 1 hour a t 4°C. They were washed with cold
CS buffer for about 2 hours and then decalcified in 4.13%
EGTA (Eastman Kodak Co., Rochester) for 5 or 14 days
a t 4°C with constant agitation. The mandibles were
washed in frequent changes of CS buffer for 2 days a t
4°C on a rotator. In some cases enamel organs were
dissected from the labial surfaces of incisors in three
parts corresponding to the secretory stage and the early
and the late maturation stages of amelogenesis. In other
cases whole incisors were cut grossly into three segments containing one of these same three stages. The
location of cross cuts demarcating these stages was established by measurements taken with a ruler through
the eyepieces of a dissecting microscope. All dissections
were done in a petri dish filled with ice-cold CS buffer.
The segments were embedded in 7% agar and cut at 5070 pm thickness on a Sorvall TC-2 tissue chopper, and
the sections were washed in CS buffer and processed for
cytochemistry.
lmmunocytochemical Labelling
The protein A-gold technique was used for detecting
antigenic sites in thin sections from Lowicryl- or Eponembedded material (Bendayan, 1984). Thin sections cut
from osmicated and Epon-embedded material were
treated with sodium metaperiodate for 1 hour at room
temperature, washed with distilled water, and incubated with rabbit polyclonal antibody against SDS-denatured mouse amelogenins (U10 dilution) followed by
protein A-gold as described previously (Nanci et al.,
1985). The sections then were grid stained with uranyl
acetate and lead citrate and examined with a Philips
300 or 410 electron microscope. Controls consisted of
incubating sections with 1)antibody that had been exposed to a n excess of amelogenin, 2) pre-immune serum,
or 3) protein A-gold alone (see Nanci et al., 1985). The
specificity of the antibody used in this study has been
109
documented elsewhere (Slavkin et al., 1982, 1984). It
has been found to cross-react with amelogenins and enamelins derived from mouse as well as a variety of other
mammalian species (Slavkin et al., 1984). Only results
from glutaraldehyde-fixed, osmicated, and Epon-embedded material are presented in this report because this
procedure gave the best balance between labelling intensity, background, and resolution.
Cytochemistry
Tissue chopper sections were washed and incubated
for 15, 30, 60, or 90 minutes at 37°C and pH 5.0 in
conventional acid phosphosphatase media by using pglycerophosphate (BGP) (Gomori, 1950) or cytidine 5‘monophosphate (CMP) (Novikoff, 1963) as substrates.
Some sections were also incubated for 20 or 40 minutes
a t 37°C and pH 3.9 in the medium described by Doty et
al. (19771, with inorganic trimetaphosphate (TMP) as
substrate. Controls for all experiments consisted of incubating separate sections in duplicate media prepared
with all components except substrate. Following incubations the sections were washed, osmicated, and processed for embedding in Epon as described previously
(Smith, 1981). Thin sections were cut on a Reichert
Omu4 ultramicrotome, grid stained with lead citrate,
and examined in a Philips 400 electron microscope. Some
thin sections were mounted on nickel grids, treated with
sodium metaperiodate, and incubated with antibody as
described above.
Quantitative Analyses
Distribution of gold particles (immunocytochernistry)
Nonoverlapping and randomly selected micrographs
were taken in the supranuclear compartment and along
the apical surfaces of ameloblasts situated near the junction between a n initial smooth-ended band (SEo) and the
first true ruffle-ended band of maturation (RE1) (see Fig.
2 and Results sections for details). Similar micrographs
were also taken from ameloblasts engaged in early and
mid-inner enamel secretion (IES). Prints were made and
a Zeiss MOP-3 manual image analyzer was then used to
compute profile areas within which gold particles were
located (Tables 1, 2). Data were expressed directly in
micrometers squared by using the internal correction
capability of the MOP, and data for organelles other
than lysosomes were pooled across individual prints.
Density of labelling was computed separately for each
set of individual (lysosomes) or pooled (other organelles)
area measurements and particle counts. Statistical tests
were carried out on resultant means by using H P Statistical Library routines run on a n HP series 9000 model
216 microcomputer (Hewlett-Packard Desktop Computer Division, Fort Collins, CO). It was estimated that
a total of at least 100 ameloblasts were sampled at each
site quantitated for this study (Tables 1, 2; IES, SEo,
RE1).
Distribution of silver grains (radioautography)
Silver grains were counted with the aid of a Whipple
ocular grid mounted in a self-focusing eyepiece on Wild
light microscope. The counts were done by using a n oilimmersion objective lens within rectangular areas of the
grid measuring 1 vertical x 3 horizontal squares or 8.6
pm x 25.8 pm a t the magnification used. The location
for sampling within the secretory zone of amelogenesis
110
A. NANCI, H.C. SLAVKIN, AND C.E. SMITH
TABLE 1. Immunocytochemistry: Density of gold labelling over enamel and organelles in ameloblasts following
incubation with antiamelogenin antibody
No. of
Compartment
Secretion stage
Inner Enamel Secretion (IESI3
En ame1
Ameloblast
Nucleus
Mitochondria
Endoplasmic reticulum
Golgi saccules5
Secretion granules6
Lysosomes
Totals
Maturation stage
Smooth-ended (SE01
Enamel4
Ameloblast
Nucleus
Mitochondria
Endoplasmic reticulum
Golgi saccules5
Secretion granules6
Lysosomes
Totals
Ruffle-ended (REJ
Enamel4
Ameloblast
Nucleus
Mitochondria
Endoplasmic reticulum
Golgi saccules5
Secretion granules6
Lysosomes
Totals
individual
or
summed
fields’
Total
No. of
particles
counted
(No.)
Total
enclosed
area
Density of
labellins2
(pm2)
(No./pm )
31
1,024
9.7
121.4 f 53.7
8
21
28
50
24
408
440
233
452
2,769
408
2,240
7,566
186.3
59.1
53.5
206.4
3.1
116.1
634.2
2.6 f 1.4
3.8 f 2.7
8.4 2.3
15.1 f 7.1
137.6 & 40.7
17.4 f 40.7
15
620
6.1
101.1 f 22.4
14
51
22
46
15
458
278
620
314
973
183
1,638
4,626
67.1
166.5
36.7
100.1
1.5
169.1
547.1
4.0 f 1.9
3.8 f 2.3
8.5 f 2.5
8.9 f 5.7
135.5 f 63.1
10.4 & 17.4
15
463
8.1
33
55
36
58
9
599
607
712
743
1,305
81
3,707
7,618
133.8
156.1
76.9
136.7
4.1
230.8
746.5
Background
Background
58.8
=
3.4 f 2.47
=
3.8 f 2.Z7
=
4.8 f 3.37
20.8
5.3 f 4.5
4.4 f 2.2
9.6 f 2.9
10.6 5.7
140.2 f 83.5
19.0 f 25.8
Background
‘Samples were pooled in all cases except for lysosomes where the number of fields equals the number of individual lysosomal profiles measured.
‘Mean k standard deviation.
3Data pooled from early and mid-inner enamel secretion,
4Data from small areas of matrix sampled just distal (deep)to the Tomes’ processes (IES)or the basal lamina covering the surface of the enamel
layer (SEo,RE,).
5Area measurements included the cytoplasmic spaces between the saccules.
‘Immature and/or mature secretory granules encountered near the Golgi apparatus.
7Average of nuclear and mitochondrial labelling; other values were significantly higher compared to this background as evaluated by the
Student’s t-test (P< ,051.
was selected arbitrarily a t a point where the thickness
of enamel from the base of interrod prongs to the dentino-enamel junction (DEJ) occupied seven vertical rectangles. This location corresponded roughly to the
thickness of enamel through which newly formed proteins seemed to diffuse by 4 hours after injection of 3Hmethionine. Ameloblasts at this position were slightly
beyond the midpoint in the secretory zone, and they
were forming inner enamel. For animals at 20 minutes
after injection the horizontal lines of the grid were
aligned parallel to DEJ and the edges of the prongs of
interrod material a t the apex of the ameloblasts, and
the number of silver grains over the Tomes’ processes
andor matrix in each rectangle was counted. The grid
was then moved and superimposed over the cells adjacent to the area of enamel just counted. Care was taken
to align the top of the grid at the same position corresponding to the base of the preceding counts. The num-
ber of silver grains over cytoplasm andor nuclei of cells
was scored across all rectangles extending vertically
from the apex of ameloblasts to the outer basement
membrane of the enamel organ. This process was repeated for two sections per slide, and for each of two
slides per tooth yielding a total of 16 samples per position per time interval from which means and standard
deviations were computed after corrections for background labelling (estimated at 0.75 & 1.2 grains per
rectangle based on counts done over areas of plastic
devoid of tissue; therefore, the correction factor for background = 0.75 x [the number of rectangles occupying a
compartment of interest]). For animals a t 1and 4 hours
after injection the grid lattice was advanced incisally
from the seven-rectangle-thick position a n amount equal
to the estimated average migration occurring relative to
the animals at 20 minutes after injection (baseline) (18
pm and 99 pm, respectively; estimated from data in
111
SECRETORY ACTIVITY IN MODULATING AMELOBLASTS
TABLE 2. Immunocytochemistry: Distribution of reactive and unreactive lysosomes in ameloblasts following incubations
with antiamelogenin antibody
Overall
No.
Lysosomal t y p e
No.
labelled
No.
No.
Density of
labelling3
weakly
heavily
labelled4
labelled5
observed
Frequency
density of
labelling’
137
159
112
34
39
27
8.5 f 17.7
11.1 & 18.5
37.0 & 68.3
52 (38)
75 (47)
69 (62)
22.5 f 22.8
22.6 f 21.8
59.5 f 79.2
26 (50)
39 (52)
27 (39)
l(2)
l(1)
13 (19)
206
85
167
45
19
36
4.5 & 7.1
9.8 k 21.5
18.1 20.9
+
70 (34)
37 (44)
124 (74)
12.3 f 7.3
21.6 28.7
23.9 k 21.4
48 (69)
23 (62)
56 (45)
0 (0)
l(3)
2 (2)
198
137
264
33
23
44
7.4 & 13.1
9.8 k 14.3
32.4 f 31.0
82 (41)
70 (51)
214 (81)
17.2 & 15.9
18.0 5 16.2
39.5 f 30.3
49 (60)
40 (57)
46 (21)
0 (0)
0 (0)
11 (5)
above
background’
Secretion stage
Inner enamel secretion (IES)~
Dark
Pale
Multivesicular
Maturation state
Smooth-ended (SE01
Dark
Pale
Multivesicular
Ruffle-ended (RE1)
Dark
Pale
Multivesicular
‘Mean number of gold particles per pm2 (+ standard deviation) for all lysosomes in each class.
%ee Table 1 for definition of background; values in parentheses indicate percent labelled lysosomes in each class.
3Mean number of gold particles per pm2 (* standard deviation) for labelled lysosomes in each class.
4Lysosome~showing ten + background or fewer gold particles per pm2; values in parentheses indicate the percent of labelled lysosomes that
were weakly labelled.
5Lysosome~showing 100+background or more gold particles per pm2; values in parentheses indicate the percent of labelled lysosomes that
were heavily labelled.
‘Data pooled from early and mid-inner enamel secretion.
a
BL
cv
dl
IES
inv
isg
mvb
n
Pl
S
sg
tP
Abbreviations
Apical surface of ameloblast
Basal lamina
Coated vesicle
Dense lysosome
Inner enamel secretion (secretory stage)
Apical invagination
Immature secretory granule
Multivesicular body
Nuclear compartment of ameloblast
Pale lysosome
Supranuclear compartment of ameloblast
Secretory granule
Tomes’ process
Regional classification (from most apical to most incisal)
NTP
ASH
PST
RE
SE
LPG
NPG
RE0
GM
No Tomes’ process
Ameloblasts shrink in height
Postsecretory transition (combined NTP ASH)
RuMe-ended (subscript indicates sequence beginning
with the initial or ‘0’band)
Smooth-ended (as above)
Large pigment granules
No large pigment granules
Reduced enamel organ
Gingival margin
+
Smith and Warshawsky, 1975). The sampling position
for counts within the maturation stage of amelogenesis
was located at a point 1mm incisally from where Tomes’
processes were no longer evident at the apices of the tall
columnar ameloblasts (see Figs. 1, 3). Ameloblasts at
this site in all teeth counted were engaged in the first
ruffle-ended phase of maturation (RE,; see Fig. 3).
Counts were made vertically in rectangular areas across
enamel and adjacent cells of the enamel organ as described above. The density of silver grains was estimated by dividing the total number of grains over
enamel or cells by the total number of rectangular areas
within which grains were distributed (Table 3). Al-
Fig. l . Mesial side of the left mandible from a male Wistar rat
weighing about 100 g body weight. The apical (NTP) and incisal (GM)
limits of regions defined relative to the maturation stage of amelogenesis in the incisor are indicated by the white arrows. The site marked
“NTP’ corresponds to the origin, or zero point, for the length measurements presented in Figures 2-4. X 3 .
though only the data from animals injected with 3Hmethionine are presented in this report the qualitative
findings from animals injected with dH-leucine were
identical to those for methionine.
Length measurements
Lengths of various regions within the maturation zone
of amelogenesis (Figs. 2,3) were estimated directly from
a light microscope by using 1-pm-thick toluidine-bluestained sections. The microscope, equipped with a drawing tube, was mounted over the magnetic tablet of the
MOP, and a light-emitting cursor was traced along the
apices of ameloblasts to measure the apicalhncisal
112
A. NANCI, H.C. SLAVKIN. AND C.E. SMITH
Mandibular Incisor
Mandibulor Incisor
-3
U
lOOg rots
-
)
.
u
m
+’ 1600-
m
0)
W
0
-E, soo-
-2
I 1200-
b.
.+
I
s
- 1
w
E
t
-
400-
.-
6
2
0
z
Q
0
f
-0 0
.A-
O - Enomel
o
Enomel
w
NO
Enamel
w
-
-
Timemes
I
2
3
4
5
7
6
10
9
S
ii
12
Giniivol
morgin
Distance ( rnm 1
process
Fig. 2. Bar graph summarizing data from length measurements in
various sequential regions defined by the appearance of the enamel
matrix in EDTA-decalcifiedincisors. The height of each bar represents
the mean length standard deviation (SD). The right ordinate shows
a time scale based on the cell migration studies by Smith and Warshawsky (1975). At the bottom of the figure the length of each region
(fSD) is represented to scale along a n abcissa beginning at the point
where the Tomes’ processes were no longer visible at the apices of
ameloblasts and extending incisally to the gingival margin.
O
3200-
i5
Mondibular Incisor
I
n
2800+m
C
2400-
--E,
I
2000 -
I
1600.
0
C
‘5 1200-
-
V
E
.A-
0
800-
f
w
.
NTP ASH RE,
PST
!E4 SE, R E 5 L f f i NPG R E 0
I
M
I i
w
‘
I
N o Tomes
process
... ... . .. . . .
.... ... .... ...
.. .
.
.. . .. .. .. .. .. . . .. .
SEO
........
...................
.
hY
\
2
3
4
5
6
7
8
...
...
.
.. :
hW
N
1 1
cc--(w
9
Distance ( m m I
1
0
1
1
.
.....
.
:.
I ]
w
1
1
2
Gingival
rnarqm
Fig. 3. Bar graph summarizing data from length measurements in
various sequential regions defined by the morphology of ameloblasts.
The height of each bar represents the mean length standard deviation (SD). The right ordinate shows a time scale based on the cell
migration studies by Smith and Warshawsky (1975). At the bottom of
the figure the lengths of each region are represented to scale along a n
abcissa beginning at the point where the Tomes’ processes were no
longer visible at the apices of ameloblasts and extending incisally to
the gingival margin. The lines under the long bar indicate the mean
position (k SD) for points of transition from smooth-ended ( S E p 4 )
(shaded) to ruffle-ended (REo-5) bands, and for the location of the
gingival margin. The region marked “REo/SEo” (stippled bar third
from left) was variable in its composition and contained ameloblasts
displaying either an incompletely ruffled apical surface or a true
smooth-ended apical surface or a combination of both (with smooth-end
cells usuallv abuttine RE,). The uresence of distinct accumulations of
mitochondria at the Gase df apicaf imaginations provided one criterion
by which ameloblasts of the first true ruffle-ended band (RE,) could be
distinguished from those forming a partially ruffle-ended band (REo)
when smooth-ended cells (SE,) were not present in this region.
+
I
2
3
4
5
6
7
8
9
1
0
1
1
1
2
Distance (rnrn )
Fig. 4. Probability maps showing the location of boundaries between
sequential regional compartments measured relative to the enamel
matrix (top panel) or the ameloblasts (bottom panel). The line bisecting
each map represents the average or mean location of a boundary in all
incisors (50% probability), as is illustrated for the cellular compartments by the horizontal bar running across the middle panel. In these
maps a vertical boundary line would mean that the compartment
began (or terminated) at the same location in all teeth whereas a tilted
line means the reference point was distributed over a wider range of
values between incisors. The map in the lower panel further demonstrates that certain compartments were not observed on all teeth (e.g.,
an SE4 band was present on only about 75% of all incisors used in this
study). Note these maps suggest that there is a 100% probability the
first complete modulation (RE, to SE1) will occur relative to matrix
which is insoluble in EDTA and in which the enamel rods usually are
clearly visible. There is a 70% probability the second modulation (RE2
to SE2)will occur before the matrix is soluble in EDTA.
length of various regions. Fields were enlarged at x 200
magnification and data were converted into micrometers by using the internal correction capability of the
MOP. Measurements were taken on one section per segment per animal, and the data from 21 teeth were used
to obtain the means and standard deviations shown in
Figures 2 and 3. The probability lines in Figure 4 were
computed by first mapping the position of the regions
for each incisor on graph paper and then determining
the cumulative number of teeth containing the region
in sequential 250-pm steps from the apical reference
point (no Tomes’ process) incisally to the gingival margin. In Figures 3 and 4 the transitional areas from
ruffle-endedto smooth-endedameloblasts (Josephsen and
Fejerskov, 1977) were included with the ruffle-ended
bands.
RESULTS
Location and Morphological Features of Regions Sampled
Relative to Early Maturation
The classifications Of Warshawsky and Smith (1974)9
Kalknbach (197419 Josephsen and FeJerskov (19771, Takano (1979), and Inage and Toda (1984) were used to
SECRETORY ACTIVITY IN MODULATING AMELOBLASTS
divide the maturation zone of amelogenesis into regions
arrayed sequentially from the point marking the site
where Tomes’ processes were no longer visible at the
apices of arneloblasts’ to the gingival margin (Figs. 14). Across these regions characteristic changes in the
appearance of the enamel matrix and in the morphology
of ameloblasts were evident at the level of the light
microscope in EDTA-decalcified incisors. Compartments
defined relative to the enamel matrix (Fig. 2) included
regions within which the matrix 1)was deeply stained
and the outlines of enamel rods were invisible or indistinct, 2) contained clearly visible enamel rods, 3) shrank
in thickness and the rods became pale staining, and 4)
was absent and represented by a clear space. Compartments defined relative to ameloblasts (Fig. 3) included
regions within which the cells 1)were tall in height but
lacked Tomes’ process (NTP- a list of abbreviations and
their meanings precedes Fig. 11, 2) shrank in height
(ASH), 3) displayed variable apical morphology consisting either entirely of a poorly defined ruffle-end (mostfrequent) or a short segment of incompletely ruffled cells
followed by true smooth-ended ameloblasts (REdSEo) 4)
possessed a true ruffled apex (RE1, RE2, etc.), 5) were
smooth-ended with lateral intercellular spaces (SE1, SE2,
etc.), 6) contained large pigment granules in both the
supranuclear and infranuclear compartments (LPG), 7)
were devoid of these granules (NPG), or 8)were cuboidal
in shape (REO). Measurements taken from 1-pm-thick
plastic sections showed that all regions could be identified fairly consistently within incisors from different
animals despite wide variations in the exact location of
certain transition points such as the start of EDTAsoluble enamel or the location of smooth-ended bands
(Figs. 2-4). These data also indicated that the length of
a n incisor over which detectable quantities of enamel
proteins were present in maturing enamel (regions of
indistinct rods + visible rods partially soluble enamel)
on the average was almost identical to the exact length
of the secretory zone of amelogenesis (4,688*520 pm
vs. 4,618 f 296 pm, respectively, as measured in Wistar
rats weighing 1OOg). Furthermore, ameloblasts generally completed only two modulations, or they were engaged in the third ruffle-ended phase, relative to the
change from EDTA-insoluble to EDTA-soluble enamel
(Fig. 4).Regions of interest to this study included those
representing transition and reorganization from secretion to maturation (NTP, ASH, REdSEo in Fig. 3) and
initial modulation (RE1, SE1, RE2 in Figs. 3 and 4).
These regions occupied about 2.5 mm at the apical aspect of the maturation zone of amelogensis in rat
incisors.
+
lrnrnunocytochernicalLabelling
Distribution of gold particles
Enamel organ sections from regions of early maturation incubated with antiamelogenin antibodies showed
specific labelling with gold particles. Gold particles were
found in the heaviest concentration over the enamel
layer, over granular material situated in the extracellular spaces along the lateral sides or within apical
invaginations of ruffle-ended ameloblasts and over the
protein synthetic (rough ER, Golgi saccules, secretory
granules) and degradative (lysosomes) organelles of
ameloblasts (Table 1,Figs. 5-12). Nuclei and mitochondria (Fig. 6) rarely contained labelling above a n amount
113
attributed to background (Table 1; see also Nanci et al.,
1985). Occasional degenerating ameloblasts were observed which showed accumulations of immunoreactive
material in the cytoplasm, and small focal patches of
immunoreactive granular material were sometimes observed between papillary layer cells near the bases of
ameloblasts (not shown). No significant labelling was
found over coated vesicles a t the apical surfaces of ameloblasts (Fig. 6) or over other intracellular and extracellular sites related to cells of the papillary layer including
macrophages. Gold particles were almost completely absent over enamel and over ameloblasts within control
sections incubated under conditions where antibody was
preabsorbed with excess antigen (Fig. 9).
Distribution of irnmunoreactiveameloblasts
Ameloblasts with obvious immunoreactive Golgi saccules, secretory granules, and lysosomes were observed
continuously across all regions up to the beginning of
the second ruffle-ended phase of maturation (NTP, ASH,
REdSEo, REl,SEz,REZ, Fig. 3; Figs. 10, 11).Although
no major differences in the pattern of intracellular labelling were obvious in these morphologically different
ameloblasts, a gradual decrease in the intensity of immunolabelling was evident incisally across these regions especially in relation to Golgi saccules, small
secretory granules, and the invaginations at the apical
surfaces of ruffle-ended ameloblasts (e.g., Figs. 7, 8).
Ameloblasts which contained ferritin pigment in the
cytoplasm or the lysosomes (RE2 and beyond) showed
little immunolabelling except over lysosomes (Fig. 16)
and occasional residual granular material in the apical
invaginations of ruffle-ended cells.
Distribution of irnrnunoreactive lysosornes in arneloblasts
Lysosomes were identified on the basis of cytochemical
localizations by using acid phosphatase (AcPase) and
inorganic trimetaphosphatase (TMPase) as enzyme
markers (Novikoff, 1963; Doty et al., 1977). Based on
these experiments the lysosomes in ameloblasts were
divided into three morphologically distinct groups-the
consistently TMPase-positive dark lysosomes and the
variably TMPase-positive and AcPase-positive pale lysosomes and multivesicular bodies (Figs. 13-15). All
types were variably immunoreactive, but the multivesicular bodies showed the greatest frequency and intensity in labelling with gold particles (Table 2). Immunoreactive lysosomes were found in ameloblasts
throughout all regions where these cells abutted EDTAinsoluble enamel (Fig. 4). Ameloblasts sampled from
areas of EDTA-soluble enamel (probably late RE2 or RE3)
still contained some immunoreactive lysosomes. Dual
immunocytochemical and cytochemical localizations
‘It is a n oversimplification to equate the disappearance of Tomes’
processes with the exact beginning of the maturation stage of amelogenesis. For example, the final enamel layer is rod-free and secreted
by ameloblasts which have no Tomes’ process. The definition of postsecretory transition given by Warshawsky and Smith (1974) (used in
this study) takes into account the overlap of events between cessation
in appositional growth of the enamel layer and the completion of
morphological changes associated with maturation of the enamel.
Other classifications, such as the one given by Kallenbach (1974),
define postsecretory transition exclusively in the context of the physical shortening of ameloblasts. The difference between these two
classifications constitutes 255 f 87 pm linear distance or about 9.4
hours of cell time in the rat incisor.
114
A. NANCI, H.C. SLAVKIN, AND C.E. SMITH
SECRETORY ACTIVITY IN MODULATING AMELOBLASTS
115
Fig. 9. Immunocytochemical preparations showing oblique cuts
through the apical portions of ruffle-ended ameloblasts @El) incubated
with antibody (A) and with antibody that was preabsorbed with excess
antigen (B).The reaction present over the enamel and granular material within the infoldings of apical invaginations (inv) is abolished
under these control conditions thereby demonstrating the specificity of
labelling with gold. Note in Figure 9A that some structures (arrows)
contain immunoreactive material which lack the halos normally seen
in apical invaginations (inv). ~26,000.
suggested that heavily labelled lysosomes were usually
TMPase negative (Figs. 13, 14). Analysis of lysosomal
types In early MIlooth-ended(SEO) and IWEIe-ended (RE11
ameloblasts indicated that the smooth-ended cells contained more dense type and fewer mult~Vesiculartype
compared to ruffle-ended ameloblasts (Table 2). The 1YSOsomes in ruffle-endedcells also appeared more heavily labelled than in smooth-ended ameloblasts (Table 2).
Radioautographic Studies
Sites of incorporation of tracer at 20 minutes after injection
At 20 minutes after injection of SH-methionine(or 3Hleucine) silver grains were present Over all cells of the
enamel organ throughout the entire maturation zone.
The heaviest reaction was seen over the supranuclear
compartments and apical surfaces of ameloblasts abutting EDTA-insolubleenamel esDeciallv in reeions at the
begnning of the maturation zine (NYP, ASH, SE&Ed
RE1) (Figs. 3, 4, 17). Ameloblasts from smooth-ended
bands (SEo,SE1),and those from the second and subsequent ruffle-ended bands (RE& showed less intense labelling at their apical surfaces compared to ameloblasts
Figs. 5-8. Immunocytochemial preparations illustrating the distribution of gold labelling at the apical surfaces of ameloblasts from the from the most apically positioned ruffle-endedband (RE0
initial, partially ruffle-ended (RE1, Fig. 5) and/or smooth-ended (SEo, andor RE11 (Fig. 17).
Fig. 6) band, and near the beginning (RE1 <) or the end (RE1>) of the
first true ruflle-ended band of maturation. Numerous gold particles Distribution of silver grains at 4 hours after injection
are present over the enamel layer which is separated from the cells by
By 4 hours fewer silver grains were evident over the
a basal lamina-like structure (BL). Small immunoreactive secretory
granules (sg) are found near the apical surface or among the invagi- supranuclear regions and apical surfaces of ameloblasts
nations (inv) of the plasma membrane in ruffleended cells. Granular (Table 3, Fig. 18). Silver grains were seen over previmaterial between the infoldings of the apical invaginations is heavily ously unlabelled areas of the enamel matrix both adjalabelled with gold. Granular material appears heaviest in the apical
invaginations of ameloblasts at the beginning of the first true ruffle- cent to, and at a distance from, the apical surfaces of
ended band (compare Figs. 5 and 6 ; a difference in staining intensity ameloblasts. The intensity of labelling over the matrix
along the apical surface can be seen in light micrographs such as those did not always appear uniform along the same region
illustrated in Fig. 18 [E,F,H]). Immunoreactive multivesicular bodies ('hot' and 'cold' spots as illustrated for regions ASH and
(mvb)are found near the apices of the ruffle-ended cells (e.g., Figs. 7,8).
Coated vesicles (cv) and mitochondria (m) show little, or no, immuno- RE1 in Fig. 18). The intensity of the radioautographic
reaction also seemed to decline in an incisal direction
labelling (Figs. 5,6). Figures 5-7: ~26,000.Figure 8: x 17,750.
116
A. NANCI, H.C. SLAVKIN, AND C.E. SMITH
117
SECRETORY ACTIVITY IN MODULATING AMELOBLASTS
TABLE 3. Radioautography: Density of silver grains over enamel and enamel organ cells following a single injection
of 3H-Methionine
Compartment
Secretion stage3
Enamel
Cells
Ameloblasts
Tomes' process
Distal part
Proximal part
Supranuclear
Nuclearhnfranuclear
Papillary layer
cells
Maturation stage4
Enamel
Cells
Ameloblasts
Apex
Supranuclear
Nuclearhnfranuclear
Papillarylayer
cells
No. of
silver
grains'
(No.)
20 minutes
Percent
Area
total
counted2 grains
(R units)
(%)
61 f 29
389 f 83
361 f 79
119 f 44
85 f 35
34 f 15
181 f 38
62 f 23
7
14
10.5
3
2
1
4
3.5
14
86
80
26
19
7
40
14
29 f 14
3.5
6
7 f 6
145 & 36
108 f 25
36 k 11
43 f 10
28 f 13
7
10
5
1
2
2
5
95
71
24
28
18
37 f 14
5
24
Density of
labelling'
(No./R)
No. of
silver
grains'
(No.)
8.8 f 4.2 485 f 110
28.1 f 6.0 187 f 49
36.9 k 8.4 156 f 41
39.6 f 14.6 85 f 28
40.6 k 18.2 75 f 30
33.8 f 15.1 10 f 5
44.1 f 11.6 40 f 16
17.7 f 6.7
31 10
8.7 f 4.2
31 f 14
4 hours
Percent
Area
total
counted2 grains
(R units)
(%)
Density of
labelling'
(No./R)
Change in
density
of
labelling
7.3
14
10.5
3
2
1
4
3.5
72
28
23
13
11
1
6
5
66.4 f 15.1
13.0 f 3.6
14.7 f 3.8
28.2 f 9.4
37.6 f 15.2
9.5 f 5.4
10.0 f 4.1
8.7 f 2.9
7.5
.5
.4
.7
.9
.3
.2
.5
3.5
5
8.9 k 3.9
1.0
0.8 f 0.7
45 f 16
14.5 f 3.1 120 f 27
21.1 k 4.9
75 f 20
35.6 f 11.2 24 f 9
26 f 6
21.7 5.0
13.3 f 5.4
24 f 11
7
10
5
1
2
2
27
73
45
14
16
15
6.5 f 2.3
11.9 f 2.3
15.2 f 3.0
23.5 f 9.1
13.2 f 3.2
12.7 f 3.9
8.1
45 f 11
5
27
8.8 f 2.7
1.1
7.8 f 2.5
.8
.7
.7
.6
1.0
'Mean f standard deviation; all values were corrected for a n average background labelling of 7.5 f 3.0 grains per 10R.
'One R unit = 221.9 pm' (see Materials and Methods).
3Samples were taken from mid inner enamel secretion (see Materials and Methods).
*Samples were taken from the apical part of first ruffle-ended band (RE1) (see Materials and Methods).
across the various regions related to EDTA-insoluble
enamel (compare panels B-H in Fig. 18). Silver grains
were evident in the matrix above ameloblasts from both
smooth-ended and ruffle-ended bands (Fig. 18 D-H).
DISCUSSION
Interpretations of results from biochemical andor
morphological studies of the maturation stage of amelogenesis have been based to a large extent on the assumption that the ameloblasts do not secrete additional
enamel proteins into the matrix once the final layer of
enamel has been deposited. Hence, a phenomenon such
as the maturation-related shift from predominantly intermediate components to higher proportions of low and
high molecular weight components in enamel has been
Figs. 10-12. Immunocytochemical preparations of the supranuclear
compartment of ruffle-ended (Figs. 10, 12) and smooth-ended (Fig. 11)
ameloblasts. The protein synthetic organelles (endoplasmic reticulum,
Golgi saccules, and secretory granules [isg, sg]) and degradative organelles (dark lysosomes [dl], pale lysosomes [pl], and multivesicular
bodies [mvb]) of ameloblasts are immunoreactive throughout the initial ruffle-/smooth-ended band (REdSEo) and first true ruffle-ended
band (RE1). Gold particles associated with the endoplasmic reticulum
are sometimes seen within the cisternal lumen, but more often along
the cisternal membrane or near ribosomes (inset). The lysosomes show
variable labelling. Multivesicular bodies are frequently and intensely
labelled with gold. The arrows in Figure 11 indicate occasional dense
structures, presumably elements of the lysosomal system, that were
difficult to classify by the method used in Table 2 (not included in
quantitative analyses). Figure 10: ~ 3 5 , 0 0 (inset
0
X36,750). Figures 11
and 12: ~ 2 6 , 0 0 0 .
attributed exclusively to the breakdown of preexisting
proteins in the matrix (amelogenins)(reviewed in Robinson and Kirkham, 1985).Similarly, the presence of granular material at the surfaces or within invaginations
and granules at the apices of ruffle-ended ameloblasts
has been postulated to be indicative of resorptive activity by these cells CReith, 1970; Kallenbach, 1974; Takano, 1979). The data presented in this study suggest
that the maturation process is more complicated, and a
portion of the changes believed to result solely from
degradation may derive from continued secretory activity by the ameloblasts (Kallenbach, 1974).
The strongest evidence for continued biosynthesis of
enamel proteins by modulating ameloblasts from the
maturation stage is based on our observations that these
cells contain significant immunoreactivity (above background) in the endoplasmic reticulum, Golgi saccules,
and small secretory granules. Evidence that this material is secreted is provided by the radioautographic studies which document similiarities in the pattern of uptake
of labelled amino acid precursors and subsequent passage and diffusion of labelled proteins within the matrix
when comparing modulating ameloblasts to those from
the secretory stage (Table 3). We assume that the lower
immunoreactivity within modulating ameloblasts is due
to less overall biosynthetic activity as opposed t o alterations in the antigenic properties of enamel proteins being
formed by these cells. This conclusion is suggested by
data from the radioautographic studies which show that
these ameloblasts incorporate about two times less radioactive tracer and release about ten times less labelled
118
A. NANCI, H.C. SLAVKIN, AND C.E. SMITH
SECRETORY ACTIVITY IN MODULATING AMELOBLASTS
Fig. 16. Immunocytochemical preparation of the supranuclear compartment of a n ameloblast from the second smooth-ended band. (SE2;
see Fig. 3). The intensity of the immunocytochemical reaction over
Golgi saccules declines to background levels as ameloblasts progress
farther into the maturation stage of amelogenesis. These cells (SE2)
show ferritin both throughout the cytoplasm and accumulated within
numerous lysosomes that appear unlabelled with gold (arrows). However, some multivesicular bodies (mvb) remain intensely immunoreactive. ~24,875.
Figs. 13, 14. Dual cytochemical and immunocytochemical preparations illustrating the distribution of TMPase-positive and immunoreactive lysosomes in the supranuclear compartment of ruffle-ended
ameloblasts (RE1). Dark lysosomes (dl) often show TMPase activity (+)
but little immunolabelling with gold. This is in contrast to the pale
lysosomes (pl) and multivesicular bodies (mvb) which show spotty, or
no (-), TMPase activity and moderate or heavy labelling with gold.
Focal areas of smooth endoplasmic reticulum, or SER (Ozawa et al.,
1983)show TMPase activity but no immunoreactivity. ~49,000.
Fig. 15. Cytochemical preparation of the supranuclear compartment
of ameloblasts illustrating the distribution of AcPase-positive lysosomes using P-glycerophosphate as substrate. Multivesicular bodies
react unpredictably for AcPase activity but many are positive thereby
confirming their lysosomal nature. Within the multivesicular bodies
the vesicles (arrowhead) sometimes show heavy deposits of reaction
product. ~38,000.
119
material into the matrix over 4 hours compared to ameloblasts forming the inner enamel layer (based on density of labelling; Table 3).
Presently we are unsure where modulating ameloblasts terminate biosynthesis and secretion of the
enamel proteins. Data in this study have indicated only
that the immunoreactivity and intensity of radioautographic reactions over ameloblasts decline gradually in
a n incisal direction, and both are evident at least up to
the second ruffle-ended phase, at which point the cells
are associated with partially EDTA-soluble enamel.
Similarly, the molecular characteristics and the longterm fate of the enamel proteins released by modulating
ameloblasts into maturing enamel are unclear at this
time. A portion, or all, of these newly formed proteins
presumably would undergo degradation as occurs for
most proteins added to the matrix during its original
appositional growth phase (secretory stage).
While our classification system for lysosomes was
crude, it was capable of revealing similarities and differences for various components of the lysosomal system in
ameloblasts derived from the secretory and maturation
stages of amelogenesis. It is evident from Table 2 that
the lysosomes with dark contents are the leaset immunoreactive in all cases. These are the same population
of lysosomes which generally show strong and consistent TMPase activity in cytochemical localizations
(Smith, 1979; Sasaki et al., 1984; Figs. 13, 14). This
suggests that dark lysosomes likely correspond to typical secondary lysosomes where degradation of proteins
has been ongoing for some time (Bainton, 1981). We
believe that the more immunoreactive pale lysosomes
and intensely immunoreactive multivesicular bodies
represent components of the lysosomal system positioned functionally closer to the endosomal compartment than the dense lysosomes (deDuve, 1983).The high
content of antigenic sites and high frequency of labelling
observed in these structures are consistent with a prelysosomal compartment in which there is limited degradation. The cytochemical findings that a portion of
these structures shows spotty AcPase activity (Smith,
1979; Takano and Ozawa, 1980; Ozawa et al., 1983; Fig.
15) and TMPase activity (Smith, 1979; Figs. 13, 14) are
also consistent with their functional transition toward
secondary lysosomes (reviewed in Bainton, 1981; deDuve, 1983; Bergeron et al., 1985). Studies by several
workers have shown that the lysosomes in ruffle-ended
ameloblasts will accumulate exogeneous protein tracers
from 10 minutes to 1 hour or more after injection (Kallenbach, 1980; Takano and Ozawa, 1980; Sasaki, 1984b;
Sasaki et al., 1984). Based on this rationale, the data in
Tables 1and 2 add more direct support to the hypothesis
that modulating ameloblasts endocytose enamel proteins during the maturation stage (Reith and Cotty,
1967).These data further support the contention of Takano and Ozawa (1980) that smooth-ended ameloblasts
may be lessendocytotically active thanruffle-ended ameloblasts, albeit only marginally according to our data
(Table 2). An observation in Table 2 that cannot be explained easily at this time concerns why the multivesicular bodies, and to a lesser extent the pale and dark
lysosomes, show more intense immunoreactivity on a
per lysosome basis in ameloblasts during the secretory
stage compared to the maturation stage. This clearly
implies that there is likely substantial endocytotic (re-
120
A. NANCI, H.C. SLAVKIN, AND C.E. SMITH
Fig. 17. Light microscope radioautographs showing the distribution
of silver grains across contiguous regions of the maturation stage (BH) up to the second ruffle-ended band (H) at 20 minutes after an
intravenous injection of meth~l-~H-rnethionine.
Panel A shows ameloblasts from the secretory stage of amelogenesis (IES) for comparison.
An intense radioaugograpbic reaction is evident in the supranuclear
compartment@ and along the apical portions (a) of ameloblasts
undergoing postsecretory transition (B, C) and modulation (D-H).
Fewer silver grains appear to be present over these compartments in
ameloblasts positioned more incisally (compare panels D and H). Reactions along the apical surface appear less intense in smooth-ended
ameloblasts versus ruffle-ended ameloblasts (compare panels F-H). In
panel D the apical morphology of ameloblasts which are illustrated
was incompletely r m e d (REo). The enlarged space laterally between
ameloblasts are artifacts due to initial perfusion fixation with paraformaldehyde. All radioautographs were dipped in the same emulsion
and exposed equally (14days). ~ 7 0 0 .
SECRETORY ACTIVITY IN MODULATING AMELOBLASTS
Fig. 18. Light microscope radioautographs showing the distribution
of silver grains across contiguous regions of the maturation stage (BH) up to the second rume-ended band (H)at 4 hours after a n intravenous injection of methyl-3H-methionine. Panel A shows ameloblasts
from the secretory stage of amelogenesis (IES) for comparsion. Radioactive proteins have been secreted by the ameloblasts and these have
diffused deep into the maturing enamel layer. Less intense, and more
patchy, radioautographic reactions are evident over the matrix where
enamel rods are clearly visible (panels G and H) and where amelob-
121
lasts are changing in height prior to initial modulation (panel C).
Silver grains are present over the matrix near both smooth-ended and
ruffle-ended ameloblasts (panels G and H). In panel D the apical morphology of ameloblasts which are illustrated was incompletely rumed
(RE,$ The data in Table 3 were obtained from cells positioned similarly to those illustrated in panel E (same for Fig. 17). All radioautographs were dipped in the same emulsion and exposed equally (14
days). ~ 7 0 0 .
122
A. NANCI, H.C. SLAVKIN, AND C.E. SMITH
sorptive) activity during appositional growth of the
enamel layer (reviewed in Smith, 1979; Blumen and
Merzel, 1982; Ozawa et al., 1983; Sasaki, 1984a; Robinson and Kirkham, 1985). The major correlation evident
from data in Tables 1 and 2 suggests that when the
biosynthetic organelles show high immunoreactivity
then the lysosomes also show substantial immunoreactivity.
Although the focus of this study was not on banding
(modulation) per se, there are three points about this
phenomenon that pertain to the data presented herein.
First, over the course of this investigation easily the
most recurring theme from incisor to incisor was that of
variability. This included 1)variability in length of ruffle bands (Josephsen and Fejerskov, 1977) and in the
amount and density of immonoreactive material present
in the apical invaginations of ruffle-ended ameloblasts
(most dramatic across RE,); 2) variability in the number
and exact location of smooth-ended bands in the maturation zone (at least three to as many as seven including
SEo; the best match in number and position was usually
between the incisors from the same animal; see Josephsen and Fejerskov, 1977); 3) variability in the existence of true smooth-ended ameloblasts in the initial
region of modulation (about 20% of incisors showed a
SEo region, and these were not always present on both
mandibular incisors from the same animal; Josephsen
and Fejerskov, 1977; Crenshaw and Takano, 1982); and
4) variability in immunoreactivity and intensity of radiautographic reactions (declining gradient incisally).
Second immunocytochemical, cytochemical , or radioautographic comparisons of rufne-ended ameloblasts to
smooth-ended ameloblasts were inconclusive. Cells with
either apical morphology showed immunoreactive Golgi
saccules, secretory granules, and lysosomes as well as
the ability to incorporate radioactive amino acids and
secrete labelled proteins into the enamel martrix. With
the exception of data given in Table 2 and the radioautographic finding of less intense apical reactions over
smooth-ended ameloblasts at early times after injection
of radioactive amino acids, any differences that were
noted seemed more closely related to incisal gradients
than to profound functional differences between these
cells. Lastly, on the average the ameloblasts undergo
only two of the several characteristic modulations over
the inverval of time when the majority of the organic
material disappears from the maturing matrix as visualized in EDTA-decalcified incisors. A smooth-ended
band is often found near the point where the matrix
changes appearance from indistinct to distinct enamel
rods (SE11 and where the matrix changes from EDTAinsoluble to EDTA-soluble (SE?).
The reason(s1for continued biosynthesis and secretion
of enamel proteins by modulating ameloblasts is unclear
at this time. On the one hand, it could be argued that
this activity is unimportant and merely reflects the inability of the cells to completely shut down the genes
coding for enamel proteins (residual secretion). On the
other hand, this activity could be a n expression of something more subtle such as a change in the type or molecular characteristics of the newly formed proteins (to
high or low molecular weights or both). Irrespective of
the reasons, the identification of such secretory activity
in ameloblasts during the early phases of the maturation stage introduces additional factors that must be
taken into account when defining global events related
to the process of amelogenesis.
ACKNOWLEDGMENTS
Several people made important contributions to this
investigation. We thank Annie BBlanger and Micheline
Fortin for their technical assistance in animal preparation and sectioning, Dr. Beatrix Kopriwa and Fernando
Evaristo for their assistance in dipping and processing
of radioautographs, Denis Kay for instruction on the use
of the Speed Vac, Margo Oeltzschner for making the line
drawings, and Tony Graham and Gaston Lambert for
their photomicrography and photocopy work. We also
thank Dr. MoYse Bendayan for his advise concerning
immunocytochemical procedures. Portions of this investigation were supported by grants from the Medical
Research Council of Canada (A.N. and C.E.S.), by grant
DE-02848 from the National Institutes of Health
(H.C.S.), and by the E.D. Broughton Research Fund
(Faculty of Dentistry, McGill).
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degradation, enamel, rat, immunocytochemical, stage, evidence, protein, radioautography, intracellular, maturation, secretion, amelogenesis, incisors, ameloblasts
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