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Effects of Exogenous Thyroid Hormone on the Postnatal Morphogenesis of the Rat Parotid Gland.

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THE ANATOMICAL RECORD 291:94–104 (2007)
Effects of Exogenous Thyroid Hormone
on the Postnatal Morphogenesis of the
Rat Parotid Gland
RIE IKEDA,1* SHIGEO AIYAMA,1 AND ROBERT S. REDMAN2
1
The Nippon Dental University, Tokyo, Japan
2
Department of Veterans Affairs Medical Center, Washington, DC
ABSTRACT
Administration of thyroid hormone has been shown to accelerate the
early postnatal development of the rat parotid gland, but these studies
have dwelt almost entirely on biochemical changes. The objective of this
study was to describe the effects of exogenous thyroid hormone on morphologic aspects of the developing parotid gland, in particular the transient appearance of scattered mucous cells in this otherwise serous gland.
Pups were given a daily subcutaneous injection of thyroxine (T4) of 0.1,
0.5, or 5.0 mg/g body weight, vehicle only (injection control), or no injection (normal control) beginning at 4 days, and killed for the collection of
blood and parotid glands at intervals through 15 days. The serum was
analyzed for T4 and the glands were examined by light and electron microscopy. The results indicated that both serum T4 and the pace of gland
development were proportional to the dose of T4. In particular, T4 accelerated decreases in acinar size and gland area occupied by stroma and
translocation of a subset of cells with small secretory granules, deeply
stained with periodic acid–Schiff, from acini to intercalated ducts. However, the chronology of mucous cell disappearance was indifferent to
treatment. In addition, signs of toxicity, including slower gain in body
weight and greatly increased apoptosis and vacuoles in the glands,
occurred with the higher doses of T4. Anat Rec, 291:94–104,
2007. Ó 2007 Wiley-Liss, Inc.
Key words: apoptosis; morphogenesis; parotid gland; rats; thyroid; hormone
The mature parotid gland of the rat is classified as serous by Young and van Lennep (1978), with the carbohydrates of the secretory glycoproteins being almost
entirely of the neutral variety. Accordingly, in sections of
formaldehyde-fixed, paraffin-embedded tissues, the acinar secretory granules are compact and mainly eosinophilic, stain modestly with the periodic acid–Schiff
procedure, and not at all with Alcian blue. By transmission electron microscopy (TEM), the secretory granules
are electron-dense. The secretory granules contain acidic
epididymal glycoprotein (AEG), parotid secretory protein
(PSP), and proline-rich proteins; several digestive enzymes, for example, a-amylase, deoxyribonuclease I, and
alkaline ribonuclease; and other enzymes, for example,
peroxidase (reviewed by Ball, 1993).
Several workers have described the transient appearance of occasional cells with mucous secretory granules
Ó 2007 WILEY-LISS, INC.
in the developing rat parotid gland at ages 1 through
8 days after birth (Lawson, 1970; Taga and Sesso, 1979;
Ikeda and Aiyama, 1997, 1999). These secretory granules stain intensely with both periodic acid–Schiff and
Grant sponsor (RI): The Nippon Dental University, Tokyo,
Japan. Grant sponsor (RSR): The National Institute of Dental
and Craniofacial Research, The National Institutes of Health,
Bethesda, MD; Grant number: DE 14995.
*Correspondence to: Rie Ikeda, Department of Histology, School
of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20
Fujimi, Chiyoda-Ku, Tokyo 102-8159 Japan. Fax: 81-3-3264-8399.
E-mail: ikedarie@tky.ndu.ac.jp
Received 11 April 2007; Accepted 15 September 2007
DOI 10.1002/ar.20620
Published online in Wiley InterScience (www.interscience.wiley.
com).
T4 EFFECTS ON RAT PAROTID MORPHOGENESIS
Alcian blue, indicating that they are rich in both neutral
and acidic glycoproteins. By TEM, these secretory granules have bipartite or tripartite structures of mostly
electron-lucent material, with not only a fine fibrillar
substructure consistent with mucins, but also eccentrically located electron-dense cores. It is noteworthy that
the secretory granules of the mucous anterior buccal
gland, which develops as a branch of the parotid duct in
rat, have a similar ultrastructural pattern (Nicholas and
Redman, 1985).
It is not clear what governs the appearance and disappearance of the parotid mucous cells. Apoptosis is rare
in the developing rat parotid gland (Kruse and Redman,
1999; Tsujimura et al., 2006). In the developing mouse
parotid gland, amylase has been immunohistochemically
localized to the mucous secretory granules, especially
the electron-dense cores, which gradually displace the
electron-lucent areas with age of pups (Takada et al.,
2001). These observations suggest that the mucous cells
redifferentiate into serous cells. Johnson et al. (1987)
reported that the parotid secretory granules of thyroxine-treated mature rats showed a markedly different
morphology from that of the vehicle-injected controls.
These secretory granules were electron-lucent with an
amorphous, rather dense core, superficially resembling
those of the early postnatal mucous cells. Although this
finding suggests that the additional thyroxine may have
brought about the re-appearance of mucous cells, the secretory granules were not analyzed for mucins. In a preliminary study (Ikeda and Aiyama, unpublished data),
rat pups were given daily injections of T4 (5 mg/g body
weight) beginning at age 5 days. Mucous cells were
observed in the parotid glands of two of the T4-treated
pups at 11 days, the oldest in the experiment. The purpose of this study was to determine the influence of exogenous thyroid hormone on the morphogenesis of the
rat parotid gland in the early postnatal period, with special attention given to the possibility that it would prolong mucous cell survival.
MATERIALS AND METHODS
All experimental procedures were approved by the
appropriate review boards of The Nippon Dental University and the Washington, DC, Veterans Affairs Medical
Center.
Experimental Animals
Sprague-Dawley rats (Rattus norvegicus albinus; certified viral pathogen free) were obtained from Harlan (Indianapolis, IN), arriving on day 14 of gestation. Pups
were considered to be age 0 days on the day of birth.
Each rat dam and her litter were housed in a plastic
cage with shredded corncob bedding in a quiet room
with controlled temperature and humidity, lights on at
7:00 AM and off at 7:00 PM, and a commercial pelleted
diet and water available ad libitum. Litters were
adjusted to 10 pups at 2 days to eliminate runts. All
pups were weighed daily. Two pups in each litter were
given a daily subcutaneous injection of thyroxine (T4) of
0.1, 0.5, or 5.0 mg/g body weight (Low, Medium, and
High doses groups, respectively), vehicle only (injection
control; Vehicle group), or no injection (Normal Control
group) between 8:00 AM and 9:00 AM beginning at age 4
95
days after birth. As much as possible, the treatments
were equalized by sex. Serum samples were collected 3
hr after the last injection of T4 from four litters each
(total of 8 pups per treatment or control group) at ages
4, 7, 10, and 15 days (12 days for the High dose T4).
Additional samples of parotid glands were taken from
noninjected pups at ages 1, 3, 5, and 21 days, to determine whether the mucous cells appear and disappear
in these rats on a schedule similar to that previously
reported.
Pups were deeply anesthetized with halothane and
the abdominal wall and thoracic cage opened. Blood was
collected from the right atrium and allowed to clot at
room temperature for 10 min, then centrifuged at 700 3 g
for 10 min at 48C. The serum (supernate) was stored in
cryogenic vials at 2708C for subsequent assays.
Assays
The serum concentration of total T4 was assayed in
duplicate for each sample by chemiluminescent enzyme
immunoassay (CLEIA; Access1 Total T4) by means of an
Access1 Discrete Analyzer (Beckman Coulter, Fullerton,
CA).
Tissue Preparation
The left parotid gland from each rat was cut into
small pieces and prepared for TEM by immersion in a
mixture of 1% glutaraldehyde and 4% formaldehyde
(prepared from paraformaldehyde) in 0.05 M phosphate
buffer (PB) at pH 7.4 for 6 hr. Tissues were rinsed in PB
overnight and post-fixed in sodium cacodylate-buffered
1% osmium tetroxide for 2 hr, dehydrated with ethanol
and propylene oxide, and embedded in Embed-812 (Electron Microscopy Sciences, Fort Washington, PA). The
right parotid glands were fixed whole (small glands) or
hemisected (larger glands) as above but without glutaraldehyde and osmium, dehydrated with ethanol and
xylol, and embedded in paraffin.
Light Microscopy
Paraffin sections were cut at 5 mm, stained with periodic acid–Schiff or Alcian blue 8GX at pH 2.5, and
lightly counterstained with hematoxylin (PAS-H and
AB-H, respectively) by the methods of Mowry (1963), or
with Mayer’s mucicarmine (Luna, 1968). Mucous cells
were counted in at least two paraffin sections (one
stained with AB-H; the other, mucicarmine) from each
gland by two of us (R.I., R.S.R.) independently. Discrepancies were resolved by conferring on those slides with a
two-headed microscope.
Histometrics
Sections stained with PAS-H at ages 4, 7, 10, and 15
days (12 for High dose T4) were assessed for the proportion (percent) of gland area occupied by acini, the several types of duct, and stroma, by contact with intersections in a grid superimposed by means of an eyepiece.
Areas occupied by individual profiles of the parenchymal
structures were calculated (mm2) by analysis of tracings
of projected images (SigmaScan1, Jandel Scientific,
Chicago, IL). Details of these methods have been presented previously (O’Connell et al., 1999).
96
IKEDA ET AL.
Electron Microscopy
For selection and orientation of specimens for TEM,
thick (ca. 1 mm) sections were stained with 1.0% toluidine blue O in 1.0 % sodium borate (Todd et al., 2005).
Thin (ca. 70 nm) sections were stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963)
and examined in a JEOL-2000EX-II electron microscope.
Analysis of Apoptosis by Means of TUNEL
TUNEL (terminal deoxynucleotide transfer method
UTP/nick end-labeling) reactions were performed using
an ApopTag1Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA). Per
the kit instructions, paraffin sections were treated with
3% H2O2/PBS after proteinase K treatment, then incubated with terminal deoxynucleotidyl transferase (TdT)
and deoxyuridine triphosphate–digoxigenin. After further incubation with anti-digoxigenin peroxidase, the
reaction was developed using the DAB method, and the
sections were counterstained with hematoxylin. Negative control sections were incubated with distilled water
in the absence of TdT.
Statistical Evaluation
The presence or absence of mucous cells was subjected
to w2 analysis (Ball et al., 2003). Body weight and histometric data were subjected to one-way analysis of variance using the SPSS version 14 software package
(SPSS Japan, Tokyo) and are presented as means 6 SE.
With both methods, null hypotheses were rejected and
significance was accepted when P < 0.05.
RESULTS
Serum Total T4
The serum T4 values are presented by age and treatment group in Figure 1. The serum concentration of T4
did not differ by sex or litter within treatment groups at
the same age, so these data were combined for each
group. Serum T4 also did not differ between the Normal
and Vehicle pups at each age, so these data also were
combined into one group, labeled ‘‘Vehicle/Normal.’’
There was a rise in serum T4 (mean mg/dL 6 S.D.) in
the Vehicle/Normal pups from 1.68 6 0.16 at 4 days to
8.45 6 0.84 at 15 days. The serum T4 concentrations
were proportional to the doses injected at each age.
Peak values for the Low, Medium, and High dose groups
were 67.4 6 14.5 at 10 days, 141.6 6 22.0 at 7 days, and
637.6 6 42.8 at 4 days, respectively. Curiously, the value
for the Medium dose declined after 7 days and the value
for the High dose declined after 4 days and remained
almost constant for the Low dose, indicating that not
only was there no day-to-day accumulation of T4, but
Fig. 2. Photomicrographs of sections of parotid glands of Normal or
Vehicle-injected rats. A: At 4 days, Alcian blue and hematoxylin stains.
Several mucous cells (arrows), including one with a mitotic figure, show
‘‘robin’s egg blue’’ secretory granules. B: At 6 days, mucicarmine. Mucous cells contain magenta secretory granules (arrow). C: At 4 days.
Periodic acid–Schiff and hematoxylin (PAS-H) stains in this and subsequent light micrographs. Cells with deep magenta secretory granules
Fig. 1. Serum total T4 (mg/dL) by age and treatment. The scale for
the Vehicle/Normal (vehicle-injected and noninjected) pups is on the
right multiplied 340, as the increase with age would appear barely
above baseline if set to the scale of the T4-treated pups.
that as the pups developed, they became more efficient
in eliminating the excess T4 between injections.
Growth and Development
The body weights of the pups by age and treatment
group are shown in Table 1. Growth of the pups in the
High dose group slowed between 7 and 10 days and in
the Medium dose group between 10 and 15 days. By 11
days, the health of the High dose group was deteriorating and one pup had died. Therefore, all other pups in
this group were killed at age 12 days. Consistent with
reports by others (Gamborino et al., 2001), the growth of
fur, opening of eyes, and eruption of incisors were
noticeably accelerated in the High and Medium dose
pups. The body weights and general development of
may be mucus, as most other cells have lighter staining. D: At 10 days.
Acinar secretory granules vary from deeply (arrow) to moderately PASpositive. E: At 15 days. Cells containing small, strongly PAS-positive
granules (arrow) are predominantly localized in intercalated ducts. F: At
21 days. Acinar secretory granules are weakly or moderately PAS-positive, while the smaller granules of the intercalated ducts (arrow) are
strongly stained. Magnification 5 A, 3500, B, 3450; C–F, 3410.
97
T4 EFFECTS ON RAT PAROTID MORPHOGENESIS
TABLE 1. Body weights (mean g 6 SE) by age and T4 treatmenta
Age (days)
Treatment
High T4
Medium T4
Low T4
Vehicle/Normal
a
4
10.8
10.8
10.3
10.2
6
6
6
6
7
0.2
0.2
0.2
0.2
16.1
16.6
16.3
15.8
6
6
6
6
10
0.3
0.3
0.4
0.3
19.1
21.9
22.8
22.7
6
6
6
6
12
0.7*
0.5
0.6
0.8
20.1
25.1
29.0
29.2
6
6
6
6
15
1.7*
0.9*
0.6
1.0
Values marked with an asterisk differ from those not so marked in the same age column (P < 0.05).
Figure 2.
28.0
35.6
37.9
6
6
6
6
0.9*
0.6
0.7
98
IKEDA ET AL.
Vehicle/Normal group, were not significant. Thus, there
was no consistent pattern of the mucous cells staying
longer or disappearing more rapidly with any of the
treatment groups.
Morphogenesis
Fig. 3. Electron micrograph of mucous cells. At 4 days, Normal.
Secretory granules have a fibrillar substructure and electron-dense
cores. Magnification, 327,000.
TABLE 2. Number of pups with parotid mucous cells
present or absent by age and T4 treatment
Age (days)
1 2 4 5 7 10 12 15 21
Treatment
High T4
Mucous cells
Present
Absent
Present
Medium T4
Absent
Present
Low T4
Absent
Vehicle/Normal
Present
Absent
3
0
3
0
3
0
6
0
3
0
5
1
3
0
4
3
4
1
5
1
8
0
1
5
0
6
2
5
3
5
0
6
-
0
4
1
4
0
8
0
2
pups in the Low dose group did not differ from those of
the Normal and Vehicle groups.
Mucous Cells
Examples of mucous cells in the parotid glands of
early postnatal pups in paraffin sections stained with
AB-H, mucicarmine, and PAS-H are shown in Figure
2A–C, and in an electron micrograph in Figure 3. The
secretory granules of mucous cells stained intensely
with PAS, AB, and mucicarmine, indicating that they
are rich in acidic and neutral glycoproteins. By TEM,
the mucous granules had a bipartite or tripartite substructure with dense cores. Mucous cells were observed
beginning at age 1 day (Table 2). Mitotic figures were
seen in a few mucous cells in the younger pups (Fig.
2A). The number of pups with mucous cells peaked at 4
days and declined thereafter, the decline being significant for all groups collectively (i.e., all T4 and all T4 plus
Vehicle/Normal) and individually. Only one pup, in the
Low dose T4 group, had mucous cells after 10 days. Differences among the T4 groups, and between the T4
groups, both collectively (High 1 Medium dose, and 1
High 1 Medium 1Low dose) and individually, and the
The sections stained with PAS-H were used to assess
the effects of T4 on several aspects of morphogenesis. No
differences were seen among the Normal, Vehicle and T4
groups at age 4 days, nor between the Normal and Vehicle groups at any age.
Representative photomicrographs of glands from Normal pups are shown in Figure 2C–F and from T4-treated
groups in Figure 4. In the Normal and Vehicle groups,
the secretory granules in the developing acini were moderately to strongly PAS-positive through age 7 days,
after which there developed a dichotomy of larger, weakly
PAS-positive granules and much smaller, intensely PASstained but AB-negative granules. The cells with the
smaller granules became localized to the acinar-intercalated duct junctions between 10 and 15 days, and by
21 days all of these were localized to the granular segments
of the intercalated ducts. It is not clear whether these cells
migrate away from the acini or vice versa.
In the Low dose groups, development of the dichotomy
of larger, weakly PAS-positive secretory granules in
acini, and smaller, densely PAS-positive, AB-negative
granules in intercalated ducts began at age 10 days
(Fig. 4A) and was virtually complete by 15 days (Fig.
4B). This pattern developed between 7 and 10 days
in the Medium (Fig. 4C,D) and High (Fig. 4E,F)
dose groups. The weakly PAS-positive acinar secretory
granules were greatly diminished by 10 and 15 days in
the High and Medium dose T4 groups, respectively. Scattered vacuoles appeared in the acini of the High dose
group at 10 days and were extremely numerous at 12
days (Fig. 4F). At age 15 days, the vacuoles were common in the Medium dose group (Fig. 4D) and uncommon
in the Low dose group. Ultrastructurally (Fig. 5), the
vacuoles had a sparse substructure and were membrane-bounded.
Histometrics
The proportional areas (mean percentage of total area
per gland) occupied by parenchymal structures and
stroma are presented in Table 3. There were no significant differences in the proportional areas of parenchyma
and stroma among all groups at age 4 days, nor between
the Normal and Vehicle groups at any age. The proportional acinar areas increased progressively with age
while the proportional stromal areas decreased, with the
largest changes occurring between 10 and 15 days. Both
changes were accelerated in all of the T4 groups at 7
and 10 days except for High dose T4 at 10 days. Proportional intercalated duct areas also increased in the Low
and Medium dose T4 groups at 10 days and in all T4
groups at 12 and 15 days. Otherwise, the proportional
areas of all ducts changed little with age.
The mean areas of individual parenchymal elements
are shown in Table 4. Mean acinar areas decreased progressively with age between 4 and 15 days in the Normal, Vehicle, and Low dose T4 pups, and even more in
the Medium and High dose T4 pups. The mean areas of
the ducts changed very little with age in all groups.
T4 EFFECTS ON RAT PAROTID MORPHOGENESIS
99
Fig. 4. Photomicrographs of sections of parotid glands of T4treated rats, periodic acid–Schiff and hematoxylin (PAS-H) stains. A:
At 10 days, Low dose T4. Small, strongly PAS-positive secretory granules are partly in intercalated ducts. B: At 15 days, Low dose T4. The
gland is similar to the 21-day normal, with strongly PAS-positive secretory granules localized in intercalated ducts and lightly stained
granules in acini. C: At 10 days, Medium dose T4. Many secretory
granules in the acini have a weaker positive reaction with PAS compared with Normal day 10. Small, densely PAS-positive secretory
granules are seen in intercalated ducts and adjacent acini. D: At 15
days, Medium dose T4. Vacuoles are seen in many acinar cells. Acinar
secretory granules are decreased, and densely PAS-positive secretory
granules are located in intercalated ducts. E: At 7 days, High dose T4.
Densely PAS-positive secretory granules are located partly in acini
and partly in intercalated ducts. F: At 12 days, High dose T4. Secretory granules are largely depleted in both acini and intercalated ducts,
and vacuoles are very numerous in acini and striated ducts. Magnification (all), 3410.
Apoptosis
normal and vehicle-injected groups, and at age 4 days in
all groups. TUNEL-positive cells, mostly acinar, were
noticeably more numerous (10–40 cells per 200 3 field)
at 10 days in the Medium dose group, and strikingly
increased (50–100 cells per 200 3 field) in the High dose
Apoptosis, as indicated by the TUNEL reaction (Fig.
6), of all cell types was very rare (one to four TUNELpositive parenchymal cells per section) at all ages in the
100
IKEDA ET AL.
trend for the exogenous T4 to hasten the exit of the mucous cells, this effect was not consistent.
Serum thyroid hormone (T4) concentration is low in
the fetus and newborn rat, increases to twice the adult
level by 16 days of age, and returns to the adult level by
30 days (Clos et al., 1974; Porterfield et al., 1981; Dussault et al., 1982; Porterfield, 1985; Farwell and DubordTomasetti, 1999). The rise in serum T4 in the Vehicle/
Normal group is consistent with these figures. Fetal serum corticosterone concentration is at the adult level
during the last stage of gestation, rapidly declines after
birth, and increases back to the adult level between 15
and 25 days of age (Takeuchi et al., 1977a, 1978). Maturation of the parotid acini, as assessd by a-amylase per
unit of tissue, is delayed when the postnatal rise in either thyroid or corticosteroid hormones are surgically or
chemically prevented (Takeuchi et al., 1977b) and precocious when either hormone is injected at 6 to 8 days of
age (Sasaki et al., 1976; Takeuchi et al. 1978; Takuma
et al., 1978; Kumegawa et al., 1980). It should be noted
that there are parallel effects on general development,
e.g., eye opening and tooth eruption (Bakke et al., 1975;
Froelich and Meserve, 1982; Gamborino et al., 2001)
that may influence parotid acinar maturation by means
of early change from liquid to solid diet (Redman, 1987).
Maintenance of mature rat parotid acini also depends on
adequate levels of both hormones (Johnson et al., 1987).
As cited previously (Johnson et al., 1987), the parotid
secretory granules of thyroxine-treated mature rats
were electron-lucent with an amorphous, rather dense
core, somewhat like those of the early postnatal mucous
cells. This finding raises the possibilty that the extra
thyroxine may have brought about the re-appearance of
the mucous cells. In addition, Takuma et al. (1978) demonstrated electron-lucent secretory granules with dense
cores in parotid glands of mice at age 11 days after daily
injections of hydrocortisone starting at 6 days. This sug-
group at 10 and 12 days and the Medium dose group at
15 days.
DISCUSSION
Mucous Cells
There were as many glands (3 glands) with mucous
cells among the 8 glands of the Normal and Vehicle
groups as there were among the 19 glands of the three
T4-treated groups at 10 days, but the only mucous cells
seen in any of the glands at 15 days were in one pup in
the Low T4 group (Table 2). Thus, although there was a
Fig. 5. Electron micrograph, at 12 days, acinar cell, High dose T4.
Vacuoles are membrane-bounded and have a sparse substructure.
Magnification 326,000.
TABLE 3. Proportions of gland area (mean % 6 SE) occupied by parenchymal structures and stroma by age
and T4 treatment
Age (days)
4
7
10
15
Treatment
High
Medium
Low
Normal
Vehicle
High
Medium
Low
Normal
Vehicle
High
Medium
Low
Normal
Vehicle
Highc
Medium
Low
Normal
Vehicle
Acini
12.0
11.5
7.1
13.0
11.3
32.6
28.7
28.1
19.8
19.8
36.0
62.6
54.7
26.5
35.1
66.7
59.4
65.2
54.6
62.0
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
1.8
1.3
0.8
2.1
1.8
1.5a,b
1.5a,b
1.9a,b
1.3
1.2
2.9
2.6a,b
3.0a,b
2.1
4.4
3.9
3.4
2.7
6.2
3.7
I.D.
1.0
1.6
1.2
1.1
1.5
3.6
4.2
3.4
4.2
3.5
3.9
5.1
4.1
2.0
3.0
7.1
10.6
7.7
4.1
3.9
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
I.D., intercalated duct; S.D., striated duct; E.D., excretory duct.
Significant difference from Normal group at this age.
b
Significant difference from Vehicle group at this age.
c
High T4 pups in this row were age 12 days.
a
0.2
0.2
0.2
0.2
0.1
0.1
0.4
0.5
0.5
0.4
0.6
0.5a,b
0.4a
0.3
0.3
0.3a,b
0.3a,b
0.6a,b
0.5
0.3
S.D.
1.9
1.6
1.2
1.4
1.4
3.7
3.4
3.2
3.3
3.4
2.8
3.5
3.1
2.6
2.4
4.2
5.8
3.6
3.1
3.7
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0.2
0.4
0.3
0.4
0.3
0.4
0.5
0.5
0.3
0.5
0.4
0.6
0.6
0.5
0.5
0.7
0.8
0.8
0.9
0.6
E.D.
1.7
2.4
2.0
3.5
3.4
3.1
4.0
3.4
5.5
3.5
3.5
3.4
4.1
2.3
5.4
10.3
7.9
5.8
4.6
4.9
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Stroma
0.6
0.8
0.3
1.6
0.6
0.5
0.5
1.1
0.6
0.7
0.7
0.8
0.9
0.3
1.6
2.4
1.6
0.8
1.3
1.1
83.6
83.2
88.8
81.8
82.4
57.1
59.7
62.4
67.2
70.5
54.7
25.8
34.0
66.6
54.8
14.4
16.3
17.6
33.6
26.2
6 1.9
6 1.5
6 0.9
6 1.9
6 1.7
6 1.3a,b
6 2.3a,b
6 2.0b
6 1.2
6 1.9
6 3.4
6 3.1a,b
6 3.1a,b
6 1.8
6 2.9
6 2.7
6 2.1
62.6
6 5.7
6 2.9
101
T4 EFFECTS ON RAT PAROTID MORPHOGENESIS
TABLE 4. Areas (Mean mm2 6 SE) of parenchymal structures by age and T4 treatment
Age (days)
4
7
10
15
21
Treatment
High
Medium
Low
Normal
Vehicle
High
Medium
Low
Normal
Vehicle
High
Medium
Low
Normal
Vehicle
Highd
Medium
Low
Normal
Vehicle
Normal
Acini
81.0
76.3
72.7
76.4
81.2
51.4
53.7
54.7
64.7
55.1
31.2
37.3
42.2
42.5
45.7
28.2
31.5
44.2
39.4
39.8
50.6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
9.7
10.9
4.0
6.9
3.3
6.5
3.3
1.1
1.1
7.7
0.7b,c
2.0c
1.9
2.1
2.3
1.1b,c
1.6b,c
1.4
2.0
1.6
2.3
I.D.
40.5
39.9
39.3
35.9
38.1
35.4
33.6
31.3
33.9
30.4
31.2
33.9
33.9
33.8
35.8
26.2
34.8
35.4
31.6
34.1
36.1
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
S.D.
4.4
3.3
3.0
3.3
4.2
0.8
2.3
2.2
1.2
1.3
1.9
0.6
1.1
1.3
2.4
2.7
1.3
0.4
1.0
1.4
1.5
105.9
98.4
112.9
107.8
100.4
100.2
111.6
106.7
117.7
105.6
107.7
108.8
102.5
105.7
108.0
98.1
105.6
103.3
102.1
106.1
112.3
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
E.D.
2.9
9.8
8.0
6.2
1.8
4.3
6.8
2.1
7.5
2.6
2.8
1.6
3.6
3.8
2.5
2.1
3.6
2.4
4.3
3.5
2.9
225.3
249.3
234.1
414.5
253.0
256.2
371.4
266.6
457.0
352.4
373.9
411.5
320.6
371.8
314.6
435.1
351.4
293.4
377.5
435.3
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
49.1
32.9
44.3
28.3
37.3
22.1
21.6
23.6
50.0
22.3
36.9
48.2
31.1
14.3
27.3
25.4
I.D., intercalated duct; S.D., striated duct; E.D., excretory duct.
Excretory ducts were present in only one sample of some groups at ages 4 and 7 days; hence, the values for these groups
have no SE.
b
Significant difference from Normal group at this age.
c
Significant difference from Vehicle group at this age.
d
High T4 pups in this row were age 12 days.
a
gests that the hydrocortisone maintained these cells
past their usual disappearance by 10 days (Takada
et al., 2001). Furthermore, one might speculate that the
cited (Takeuchi et al., 1977a, 1978) adult serum corticosteroid concentration before and at birth is necessary for
the differentiation of the parotid mucous cells, and the
low concentration after birth is insufficient to sustain
them past 8 days. Administering exogenous corticosteroid to attain adult serum levels before their disappearance thus might sustain the mucous cells for awhile.
Unfortunately, however, no mucous stains were used in
either study, and it is possible that the electron-lucent
areas in the secretory granules were due to other substances. Furthermore, the disappearance of mucous
granules at 8 to 10 days coincides with the normal rise
in thyroid hormone, but precedes the rise in corticosterone (Takeuchi et al., 1977a, 1978). Thus, it is possible
that the normal thyroid hormone rise triggers the mucous cell disappearance and that exogenous T4 may
accelerate, rather than retard, this process. On the other
hand, administration of thyroid hormone to rat pups
induces a precocious rise in serum corticosterone and
corticosteroid-binding globulin (D’Agostino and Henning,
1982) and, in rat pancreas, in glucocorticoid receptors
(Lu et al., 1988). Thus, the lack of a consistent effect of
the additional T4 on the chronology of mucous cell reduction observed in the present study might be due to
opposing effects of the T4 and the corticosteroid factors
which it induces.
Morphogenesis
The replacement of stroma by parenchymal elements
in the developing rat parotid gland can occur by proportional increases in either the areas or numbers of
individual parenchymal structures, or both. That no
parenchymal structures increased in individual area,
and acini actually decreased in individual area, indicate
that, in all groups, the decrease in stromal area was due
almost entirely to a proportional increase in the number
of parenchymal structures. However, there also are
changes in the composition of the stroma during this period (Cutler, 1990; Lazowski et al., 1994) that may result
in its being less hydrophilic during histologic processing,
and thus may artifactually reduce the areas in histologic
sections. Enzymes involved in these changes have been
shown to be increased by exogenous thyroxine (Imai
et al., 1982). In any event, the processes involved in the
replacement of stroma by parenchyma were accelerated
in proportion to increased T4.
Between 10 and 14 days, a subset of cells in the parotid acini become localized near the acinar/intercalated
duct junction, and by 21 days all are in the granular
segments of the intercalated ducts (Redman and Field,
1993; Sivakumar et al., 1998). The secretory granules of
these cells can be distinguished from those in the
mature acini by a lack of salivary peroxidase (Redman
and Field, 1993) and expression of CSP-1 and SMGB
(Sivakumar et al., 1998). These granules are smaller
and stain more intensely with PAS than those of the
mature acini, and we relied on PAS-H to follow the
effects of T4 on overall differentiation. The aspects of differentiation as outlined above, especially the change in
location of the cells with small, strongly PAS-positive secretory granules from acini to intercalated ducts, were
accelerated in proportion to increased T4. All doses of T4
initially accelerated the maturation of acinar cells, as
evidenced by the earlier and greater accumulation of
moderately PAS-positive secretory granules and relocalization of cells with small, intensely PAS-positive
102
IKEDA ET AL.
Fig. 6. Photomicrographs of sections of parotid glands of T4-treated rats, TUNEL (terminal deoxynucleotide transfer method UTP/nick end-labeling) procedure (DAB chromogen). Cells with brown nuclei are
in various stages of apoptosis. A: At 10 days, Low dose T4. B: At 15 days, Low dose T4. C: At 10 days,
Medium dose T4. D: At 15 days, Medium dose T4. E: At 7 days, High dose T4. F: At 10 days, High dose
T4. Magnification (all), 3370.
T4 EFFECTS ON RAT PAROTID MORPHOGENESIS
secretory granules from acini into intercalated ducts.
However, the number of secretory granules per acinus
was greatly diminished by 10 days in the Medium and
High dose T4 groups as compared to the Vehicle/Normal
and Low dose groups.
Apoptosis, Vacuoles, and Toxicity
The paucity of apoptotic cells at all ages in the Normal
and Vehicle groups is consistent with previous reports
(e.g., Kruse and Redman, 1999; Tsujimura et al., 2006).
Therefore, their much greater number in the Medium
and High dose groups clearly is abnormal.
The vacuoles that developed in the Medium and High
dose T4 pups are similar to the ‘‘watery vacuoles’’
described in rat parotid gland after strong secretory
stimulation (Garrett, 1978; Garrett et al., 1978). Exogenous thyroxine has been shown to induce hypersecretion
by rat salivary glands by means of adrenergic nerve
(Tumilasci et al., 1982; Medina et al., 1984), 5-hydroxytryptamine (Ostuni et al., 2003), substance P (Tumilasci
et al., 1986a), and vasoactive intestinal peptide (Tumilasci et al., 1986b) routes, and indirectly by increased
Na1, K1 -dependent adenosine triphosphatase activity
(Saito et al., 1982). The observed appearance of vacuoles
and reduction of acinar size thus are most likely due to
salivary hypersecretion caused by the induced hyperthyroidism. Hyperthyroidism-increased metabolic rate,
and an associated inability to obtain sufficient nourishment (as indicated by slowing of gain in body weight),
may also have contributed to reduced acinar size.
To some extent, the numerous vacuoles seen with the
two higher doses could be considered a physiologic
response to the increased thyroid hormone. On the other
hand, the slowing of the rate of growth, the greatly
increased number of apoptotic cells in the Medium and
High dose groups beginning at 10 days, and the death in
the High dose group at 11 days, indicate that the pups
administered the two higher doses developed a considerable degree of toxicity.
In this study, the serum T4 was measured 3 hr after
injection, and precise comparison with the normal, endogenous T4 would require taking multiple samples to
obtain a 24 hour average of serum and tissue T4. Considering this, the Low dose T4 induced a precocious
increase in serum T4 that came closest to approximating
the normal increase that occurs 6–10 days later. The
High and Medium doses of T4 produced, respectively,
severe and moderate hyperthyroidism, the resulting toxicity limiting their usefulness. In several other studies
on the effects of T4 on various aspects of rat growth and
development, the amounts administered have ranged
from 0.05 to 2.0 mg/g body weight (Dussault et al., 1982;
D’Agostino and Henning, 1982; Fitch et al., 1999;
Mutapcic et al., 2005). In general, doses of 1.0 or more
were detrimental to the organ or tissue being studied,
but little or no mention was made regarding general
effects on the pups. The observations presented here
indicate that, for rats, daily doses greater than 0.1 mg/g
body weight beginning at 4 days after birth will exceed
that which is needed to produce a precocious increase in
serum thyroid hormone similar to that occurring at 10–
15 days. Such doses risk effects that are both unnecessarily harmful to the animals and potentially confounding
to interpretation of the results of the study.
103
ACKNOWLEDGMENTS
We thank Tong Hui Mixon, Sumie Satoh, and Patricia
Shue for technical assistance, Dr. Phillipe Marmillot for
graphics, the Medical Media Service for assistance with
the color illustrations, and Dr. Kenneth Burman for
advice regarding thyroid hormone assays. The Baltimore
College of Dental Surgery, University of Maryland, provided visa assistance to Dr. Ikeda during a sabbatical
leave to the Department of Veterans Affairs Medical
Center, Washington, DC.
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