close

Вход

Забыли?

вход по аккаунту

?

Strain-Specific and Endocrine Control of Granular Convoluted Tubule Cells and Epidermal Growth Factor Expression in the Mouse Submandibular Gland.

код для вставкиСкачать
THE ANATOMICAL RECORD 291:105–113 (2007)
Strain-Specific and Endocrine Control
of Granular Convoluted Tubule Cells
and Epidermal Growth Factor
Expression in the Mouse
Submandibular Gland
YUICHI MIYAJI, SHIGEO AIYAMA, AND SHINGO KURABUCHI*
Department of Histology, The Nippon Dental University, School of Life Dentistry at Tokyo,
Tokyo, Japan
ABSTRACT
Submandibular glands (SMGs) of 11-week-old mice from four strains,
ICR, C57BL/6J, BALB/c, and C3H/HeN were examined by immunohistochemistry for epidermal growth factor (EGF). In addition to sex-related
differences in granular convoluted tubules (GCTs), the GCT cells were
significantly larger in ICR mice than in other three strains. In males
from each of the strains, almost all the GCT cells were strongly positive
for EGF. The EGF-positive cells in the females, however, were markedly
fewer in number, and were stained weaker in C57BL/6J, BALB/c, and
C3H/HeN mice than in ICR mice. The GCT cells and their EGF expression in the F1 progeny from ICR and C3H/HeN strains were approximately intermediate between those of the parent strains of the same sex.
T3 and/or dihydrotestosterone (DHT) enhanced the GCT phenotype in the
C3H/HeN mice, and remarkably increased the EGF-positive cells in
females. Electron microscopy revealed that gold-labeling of EGF was confined to the secretory granules, and that the GCT cells in females, given
T3 1 DHT, had a well-developed Golgi apparatus and net-like RER but
few basal infoldings, whereas the equivalent cells in the untreated
females had poor RER and prominent basal infoldings. These results
suggest that the EGF concentration in SMGs is genetically high in ICR
mice and low in other strain mice and that, considering the same
response of GCT cells to T3 and/or DHT between the high and low EGF
strains, the low EGF concentrations might be partly caused by a lesser
sensitivity of the GCT cells to thyroid hormones. Anat Rec, 291:105–
113, 2007. Ó 2007 Wiley-Liss, Inc.
Key words: epidermal growth factor (EGF); submandibular
gland (SMG); granular convoluted tubule (GCT);
thyroid hormone; mouse strain
The granular convoluted tubule (GCT) cells in the submandibular glands (SMGs) of mice have been studied
extensively in terms of morphological sexual dimorphism
and their stored bioactive secretory products, which
include growth factors, renin, and members of the kallikrein gene family (reviewed by Chrétien, 1977; Barka,
1980; Gresik, 1994; Kurabuchi and Hosoi, 2001). Several
studies, many of which used ICR (CD-1) mice, have
shown that GCT cells are under the multihormonal control of androgens, thyroid hormones, and adrenocortical
Ó 2007 WILEY-LISS, INC.
*Correspondence to: Shingo Kurabuchi, Department of Histology, The Nippon Dental University, School of Life Dentistry at
Tokyo, Fujimi 1-9-20, Chiyoda-ku, Tokyo 102-8159, Japan.
Fax: 81-3-3264-8399. E-mail: kurab-fm@tokyo.ndu.ac.jp
Received 13 July 2007; Accepted 29 September 2007
DOI 10.1002/ar.20617
Published online in Wiley InterScience (www.interscience.wiley.
com).
106
MIYAJI ET AL.
hormones. Generally, these hormones act as strong promoters of the GCT phenotype and act as strong inducers
of GCT-specific secretory products. Consequently, each
GCT cell in males is large and contains abundant large
secretory granules, whereas each GCT cell in females is
small and contains fewer and smaller secretory granules
than those observed in males, thus presenting a sexually
dimorphic pattern. In accordance with the morphological
differences of GCT cells according to sex, the concentration
of many GCT-specific secretory products, including growth
factors, renin and kallikrein gene family members (with
the exception of a true tissue kallikrein, mK1) are also
higher in the glands of males than in those of females. In
addition, growth hormone has been recently suggested to
up-regulate epidermal growth factor (EGF) expression and
the granular cell phenotype in rodent salivary glands (Hiramatsu et al., 1994; Young et al., 2004).
Variations in mouse strains are an important topic of
study in this field. SMG renin, but not kidney renin, is
well known to be under genetic as well as hormonal control (Bing and Poulsen, 1971; Wilson et al., 1977; Bing
et al., 1980), and inbred strains of mice have been divided into two groups according to whether they exhibited a low or high basal activity of SMG renin (Wilson
et al., 1981). The SMG of C57BL/6J mice, which generally exhibit a low renin activity (Wilson et al., 1981),
showed no specific staining for renin (Tanaka et al.,
1980). The concentration of mK1 (recently renamed proteinase F) was higher in the SMGs of males than in
those of females among the strains of mice with a low
renin level, including BALB/CA, C3H/HeN, C57BL/10N,
and DBA/2N (Murai, 1987). In contrast, the mK1 concentration in an outbred ICR strain, which was classified into the high renin group, was higher in the SMGs
of females than in males (Hosoi et al., 1983). Furthermore, the response of mK1 expression to androgen was
suggested to be opposite between ICR and C3H/HeN
females, and such a difference may be caused by the
lesser sensitivity of C3H/HeN females to thyroid hormone (Kurihara et al., 1999). To our knowledge, the renin regulator (Rnr) gene and the synthesis of GCT-specific secretory products are unrelated, other than the
involvement of renin. However, if the low renin levels are
due to a low sensitivity to hormones in the low renin
strains, the concentration of many secretory products,
such as EGF studied herein, should differ between the low
and high renin strains. On the basis of these findings, we
examined the localization and distribution of EGF and the
GCT phenotype in the SMGs of four mice strains, C57BL/
6J, BALB/c, C3H/HeN, and ICR, using high-resolution
immunocytochemical techniques for light and electron microscopy. Next, to confirm whether EGF expression is
under genetic control, the SMGs of F1 mice (the offsprings
of ICR males 3 C3H/HeN females and of C3H/HeN males
3 ICR females) were also examined. Finally, the effect of
thyroid hormone and androgen on the GCT phenotype and
EGF expression in GCT cells was also examined in C3H/
HeN mice, and the results were compared with well-documented observations made in ICR mice.
MATERIALS AND METHODS
Animals and Experimental Procedures
All animal experiments were conducted in compliance
with the Nippon Dental University and the National
Institutes of Health (NIH) Guidelines for the Care and
Use of Laboratory Animals. Animals were housed under
controlled environmental conditions (238C, 12-hr lightdark cycle) and were provided access to food and water
ad libitum.
Male and female mice of four strains, C57BL/6J,
BALB/c, C3H/HeN, and ICR (CD-1), were purchased
from Japan Clea (Tokyo, Japan). To examine whether
the GCT phenotype and EGF expression are genetically
regulated, two types of F1 progeny were prepared by
mating male ICR mice 3 female C3H/HeN mice, and
male C3H/HeN mice 3 female ICR mice. Furthermore,
to examine hormonal control, male and female C3H/HeN
mice were injected subcutaneously with 3,30 , 5 triiodo-Lthyronine (T3; ICN Biochemicals Inc., Aurora, OH) and/
or 5a-dihydrotestostrone (DHT; Wako Ltd., Osaka,
Japan) as described below. At least five animals of each
sex, strain or F1, and injected hormone combination
were prepared.
T3 was dissolved in a small amount of 0.1 N NaOH
and then diluted with Ringer’s solution, while 5a-dihydrotestostrone (DHT: Wako Ltd.) was dissolved in sesame oil; these hormones were then injected subcutaneously at the following doses per kilogram of body weight:
T3, 1 mg; DHT, 20 mg. The injections were performed
every other day for 2 weeks (7 injections) before killing.
At 11 weeks of age, the male and female mice of the
four strains, the two types of F1 mice, and the hormoneinjected mice were subjected to an overnight fast before
killing. Under Nembutal anesthesia (1.7 mg/kg body
weight, intraperitoneally), the animals were killed in
the morning by exsanguination, and then, a cold fixative
solution consisting of a 2% glutaraldehyde and 2% paraformaldehyde mixture in a 0.05 M cacodylate buffer, pH
7. 4, was perfused through the vasculature. The SMGs
were removed, minced, and immersed in the same fixative solution for 5 hr at 48C. The SMG fragments were
then dehydrated with a graded ethanol series and embedded in resin: either Technovit 8100 (Heraeus Kulzer
GmbH, Wehrheim, Germany) for light microscopy, or
LR-white (London Resin Co. Ltd, Berkshire, England)
for transmission electron microscopy.
Light Microscopic Immunohistochemistry
Sections of 2-mm-thick Technovit-8100-embedded tissue were mounted on silane-coated slides (Matsunami
Co., Osaka, Japan). The sections were incubated in a solution of 0.3% H2O2 in methanol for 30 min to inhibit
endogenous peroxidase activity, and washed with distilled water (DW) and phosphate-buffered saline (PBS:
0.01 M sodium phosphate buffer and 0.14 M NaCl, pH
7.5). They were then sequentially immunostained using
the avidin–biotin–peroxidase complex method in a moist
chamber at room temperature with the following
reagents according to the kit manufacturer’s protocols
(Vectastain Elite ABC Kit; Vector Lab., Burlingame,
CA): normal goat serum (NGS, Vectastain Kit) for 1 hr;
rabbit anti-mouse EGF antiserum (diluted 1:3,000;
Upstate Biotechnology, Lake Placid, NY) for overnight;
biotinylated goat anti-rabbit IgG (Vectastain Kit) for
1 hr; and finally peroxidase-conjugated streptavidin (Vectastain Kit) for 2 hr. Peroxidase activity was detected
using 20 mg of 3,30 -diaminobenzidine tetrahydrochloride
(DAB, Dojin Lab., Kumamoto, Japan) and 0.01% H2O2
TUBULE CELLS OF MOUSE SUBMANDIBULAR GLAND
107
Fig. 2. A,B: Bar graphs showing the size (cellular height) of granular convoluted tubule (GCT) cells (A) and the percentage of epidermal
growth factor (EGF) -positive GCT cells occupying the GCT segments
(B) in intact male (dark bars) and female (white bars) mice of four
strains: C57BL/6J, BALB/c, C3H/HeN, and ICR. All values are the
mean 6 SD of five animals. *P < 0.001, significantly different from the
ICR strain of the same sex.
each sex, mouse strain, F1 progeny, and hormoneinjected mice (n 5 5 for each group). The significance of
the observed differences (P < 0.01 P < 0.001) was
determined using Student’s t-test.
Immunogold Staining for Electron Microscopy
Fig. 1. a–h: Nomarski image of 2-mm-thick sections of submandibular glands (SMGs) of intact male and female mice of C57BL/6J (a,b),
BALB/c (c,d), C3H/HeN (e,f), and ICR (g,h) mice; immunostaining with
anti–epidermal growth factor (EGF). Almost all the granular convoluted
tubule (GCT) cells are positively stained for EGF in the glands of males
(a,c,e,g), whereas some of the GCT cells are EGF-positive, but others
are negative, producing a cellular mosaic expression pattern of EGF in
the glands of females (b,d,f,h). The arrows show EGF-negative GCT
cells, and the arrowheads show transition granular cells. DAB color
was augmented using 0.5% osmium tetroxide in b, d, and f because
the original immunoreactions were faint. Scale bar 5 50 mm.
in 0.05 M Tris-HCl buffer, pH 7.6. As the peroxidase
reaction was very weak in the sections from female
C57BL/6J, BALB/c, and C3H/HeN mice, these sections
were incubated with 0.5% osmium tetroxide in PBS for
3 min to emphasize the DAB color, according to the protocol described by Johansson and Backman (1983). Finally,
the sections were lightly counterstained with Mayer’s
hematoxylin, dehydrated with ethanol, mounted in Canada Balsam (Wako, Tokyo, Japan) and examined under
a BX-50 microscope equipped with Nomarski differential
interference-contrast optics (Olympus, Tokyo, Japan).
In the immunostained sections, the distance (cell
height) between the apical and basal edge of more than
1,000 GCT cells per animal was measured. In addition,
the total number of GCT cells and the number of EGFimmunopositive GCT cells was counted, and the percentage of EGF-immunopositive cells in GCT segments were
calculated per gland in each animal. The mean 6 standard deviation (SD) of these values were calculated for
Ultrathin sections of LR-white-embedded tissue were
cut using an ultramicrotome (Ultrotome V: LKB, Sweden) and mounted on gold grids. The sections on the
grids were etched in 3% H2O2 in DW for 30 sec. After
rinsing with DW, the sections were incubated with NGS
(Vectastain Kit) for 1 hr, and transferred into a droplet
of rabbit anti-mouse EGF antiserum (diluted 1:2,000)
and incubated overnight at room temperature. After
rinsing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vectastain Kit) for 1 hr.
Finally, the sections were incubated with streptavidin
conjugated with 20 nm gold particles (diluted 1:600; ICN
Biochemicals) for 2 hr and then rinsed with PBS and
DW. The immunolabeled sections were stained with uranyl acetate; after washing the sections with DW and drying, they were examined in a 2000 EX-II transmission
electron microscope (JEM-2000EXII, JEOL, Tokyo,
Japan) at an accelerating voltage of 80 kv.
RESULTS
Intact Male and Female Mice From the
Four Strains
Under light microscopy, the GCT cells in the SMGs of
males appeared larger than those of females in each
strain (Fig. 1). The difference in cell size was due to the
number of secretory granules, which were more abundant in large cells than in small cells. In addition to
such sexual differences, the GCT cells in the ICR strain
were larger than those in the C57BL/6J, BALB/c, and
C3H/HeN strains, especially in females (Fig. 1b,d,f,h).
As summarized in Figure 2A, the cellular height (which
corresponds to the cellular size in this study) of the GCT
cells was approximately one and a half times larger in
both sexes of ICR mice than in the other three strains,
and no differences were observed between the latter
108
MIYAJI ET AL.
Fig. 3. a–d: Nomarski image of 2-mm-thick sections of submandibular glands (SMGs) from two types of F1 progeny bred by crossing
C3H/HeN males 3 ICR females (a,b), and ICR males 3 C3H/HeN
females (c,d); immunostaining with anti-EGF. The GCT cells are larger,
the immunoreactions are stronger, and the EGF-positive GCT cells are
more abundant, especially in the female F1 (b,d), compared with the
female C3H/HeN mice shown in Figure 1f. The arrows show the EGFnegative GCT cells, and the arrowheads show the transition granular
cells. Scale bar 5 50 mm.
three strains. Specific immunostaining for EGF was
detected exclusively in the secretory granules of the
GCT cells in male and female mice from each strain.
Nomarsky differential-interference contrast microscopy
revealed that almost all of the GCT cells in the males of
each strain were immunostained for EGF, and EGF-negative GCT cells were rarely seen (Fig. 1a,c,e,g). Only a
few of the negatively stained GCT cells were typical
GCT cells, and many of them were slender in shape and
contained only a few small secretory granules. We previously named this cell type ‘‘slender cell’’ (Kurabuchi
et al., 1999), and such cells have been characterized as a
transition type of granular duct cell (Hazen-Martin and
Simson, 1987). EGF-positive GCT cells accounted for
more than 98% of the population of GCT cells in males
from each strain (Fig. 2B). In females, on the other
hand, EGF-negative GCT cells were conspicuously seen
in each strain, and a cellular mosaic expression pattern
of EGF was prominent (Fig. 1b,d,f,h). Additionally, the
EGF-staining reaction was very weak in C57BL/6J,
BALB/c, and C3H/HeN mice (Fig. 1b,d,f). In ICR
females, approximately half of the total population of
GCT cells was negatively stained for EGF and the other
half was EGF-positive (Fig. 2B). Compared with ICR
females, this percentage was lower in the females of the
other three strains: the EGF-positive cells accounted for
approximately 20% of the total GCT cell population in
C3H/HeN mice, and for less than 10% in C57BL/6J and
BALB/c mice (Fig. 2B).
Congenic Mice From ICR and C3H/HeN Strains
The size of the GCT cells and the localization and distribution of EGF in the SMGs was almost the same
between ICR male 3 C3H/HeN female offspring and
C3H/HeN male 3 ICR female offspring, when compared
Fig. 4. A,B: Bar graphs showing the size (cellular height) of granular convoluted tubule (GCT) cells (A) and the percentage of epidermal
growth factor (EGF) -positive GCT cells occupying the GCT segments
(B) in two types of F1 progeny bred by crossing C3H/HeN males 3
ICR females, and ICR males 3 C3H/HeN females. The dark bars represent F1 males, and the white bars represent F1 females. All values
are the mean 6 SD of five animals. *P < 0.001, significantly different
from the ICR strain of the same sex shown in Figure 2A, B. **P <
0.01, significantly different from the female C3H/HeN mice shown in
Fig. 2A.
according to sex (Figs. 3, 4). The GCT cells in males of
both types of F1 progeny (Fig. 3a,c) were similar in
appearance to those in ICR males (Fig. 1g) and C3H/
HeN males (Fig. 1e). Many GCT cells were immunostained for EGF in the males of both types of F1 progeny, and a few EGF-negative cells were sometimes seen,
some of which were transition-type cells (Fig. 3a,c). As
shown in Figure 4A, the GCT cells in both types of F1
males were larger than those of C3H/HeN males but
were smaller than those of ICR males. More than 98% of
the population of GCT cells was immunostained for EGF
in both types of F1 progeny (Fig. 4B), similar to the
results seen in mice of the same sex but from the parent
strains (Fig. 2B). In females, the GCT cells in both types
of F1 females (Fig. 3b,d) were a little larger than those
in C3H/HeN females (Fig. 1f) but were smaller than
those in ICR females (Fig. 1h). Nomarsky differential-interference contrast microscopy revealed that the cellular
mosaic distribution of EGF was prominent and that
EGF immunostaining was more intensive in the F1
females (Fig. 3b,d) compared with the observations in
the C3H/HeN females (Fig. 1f). Approximately 30% of
the total GCT cells were EGF-positive cells in both types
of F1 females (Fig. 4B); this percentage was lower than
that seen in ICR females but higher than that seen in
CH3/HeN females (approximately 50% and 20%, respectively; Fig. 2B).
Repeated Injections of T3 and DHT in
C3H/HeN Mice
After seven repeated injections of T3 alone, DHT
alone, or a combination of these two hormones in C3H/
HeN mice on alternating days for 2 weeks, the body
TUBULE CELLS OF MOUSE SUBMANDIBULAR GLAND
109
Fig. 6. A,B: Bar graphs showing the size (cellular height) of granular convoluted tubule (GCT) cells (A) and the percentage of epidermal
growth factor (EGF) -positive GCT cells occupying the GCT segments
(B) in T3-injected, 5a-dihydrotestostrone (DHT) -injected, and T3 1
DHT injected C3H/HeN mice. The dark bars represent males, and the
white bars represent females. All values are the mean 6 SD of five
animals. *P < 0.01, significantly different from control C3H/HeN mice
(Fig. 2A,B) of the same sex.
Fig. 5. a–f: Nomarski image of 2-mm-thick sections of submandibular glands (SMGs) of T3-injected (a,b), 5a-dihydrotestostrone (DHT) injected (c,d), and T31DHT-injected (e,f) C3H/HeN mice; immunostaining with anti–epidermal growth factor (EGF) granular convoluted tubule
(GCT) antiserum. GCT cells and their secretory granules were larger in
each of the hormone-injected groups, compared with those in control,
untreated C3H/HeN mice (Fig. 1e,f) in both sexes. EGF-positive GCT
cells were more abundant and the immunoreactions were stronger in
each of the hormone-injected group of female mice (b,d,f), compared
with those in the control female mice (Fig. 1f). Arrows show EGF-negative GCT cells. Scale bar 5 50 mm.
weight of the mice showed no significant differences
among T3 alone, DHT alone, T3 1 DHT, and control
groups: among male mice, the body weights were 22.6 6
1.6 g, 23.0 6 1.0 g, 23.2 6 1.2 g, and 22.2 6 1.4 g for respective groups; among the female mice, the body
weights were 18.6 6 0.8 g, 17.4 6 1.4 g, 18.8 6 1.2 g,
and 17.5 6 1.8 g for the respective groups. On the other
hand, the GCT cells in each of the hormone-injected
groups were significantly larger and contained numerous large secretory granules (Fig. 5), compared with control male and female mice of the same strain (Fig. 1e,f).
In males, the GCT cells were approximately one and a
half times larger in mice given T3 alone and mice given
DHT alone (Fig. 6A), compared with untreated C3H/
HeN males (Fig. 2A). The concomitant injection of both
hormones increased the size of the GCT cells to nearly
twice that of the control GCT cells (Figs. 2A, 6A).
Nomarsky differential-interference contrast microscopy
revealed that almost all of the GCT cells in the males of
the hormone injected groups were immunostained for
EGF, and EGF-negative GCT cells were rarely seen, similarly to the results seen in control males (Figs. 1e,
5a,c,e). EGF-positive GCT cells accounted for more than
98% of the population of GCT cells in males from each
hormone-injected group (Fig. 6B) and control untreated
males (Fig. 2B). In females, the GCT cells of the hormone-injected groups (Fig. 5b,d,f) were larger than those
of control C3H/HeN females (Fig. 1f). The GCT cells
were 1.5–2 times larger in the DHT alone group, and T3
1 DHT groups than in the control untreated females
(Fig. 6A). The average of the cell size in the T3 alone
group (Fig. 6A) was a little higher than that of the control (Fig. 2A), but the significant difference was not
obtained. The EGF immunoreactivity was stronger in
each of the hormone-injected groups, and EGF-positive
GCT cells were markedly more abundant (Fig. 5b,d,f),
compared with observations in control untreated females
(Fig. 1f). More than 80% of the total GCT cells were
EGF-positive in each of the hormone-injected groups
(approximately 80%, 84%, and 87% for the T3 alone
group, the DHT alone group, and the T3 1 DHT group,
respectively); these percentages were significantly
higher than that observed for control untreated females
(Figs. 2B, 6B).
Electron Microscopic Immunocytochemistry
Based on the light microscopy findings, an ultrastructural analysis using immunogold staining was used to
examine comparatively the SMGs of untreated C3H/
HeN females, and those of C3H/HeN females, concomitantly injected with T3 1 DHT. The GCT cells of the
intact female mice contained electron-dense secretory
granules, which were sparsely distributed in the apical
cytoplasm (Figs. 7, 8). Golgi apparatuses with flattened
cisternae and several fragments of rough endoplasmic
reticulum (RER) were poor, and numerous deep membrane infoldings associated with elongated mitochondria
were usually present at the base (Fig. 8). Gold particles,
indicating the presence of EGF, were restricted to the
secretory granules in EGF-positive cells (Fig. 7). The
secretory granules were generally larger in the EGFpositive cells, but they were sometimes the same in the
EGF-positive and -negative cells (Fig. 7). On the other
hand, the GCT cells in the glands of the DHT 1 T3
group were closely packed with abundant large electrondense secretory granules in the apical two-thirds of the
110
MIYAJI ET AL.
Fig. 8. Electron micrograph of a cross section of a granular convoluted tubule (GCT) segment in the submandibular gland (SMG) of a
control intact C3H/HeN female; immunostaining with anti–epidermal
growth factor (EGF) antiserum. Two EGF-negative GCT cells are seen.
Membrane infoldings are present in the base (arrowheads) of the cells
and are associated with elongated mitochondria (M). BV, blood vessel;
N, nucleus. Scale bar 5 2 mm.
Fig. 7. Electron micrograph of a cross-section of a granular convoluted tubule (GCT) segment in the submandibular gland (SMG) of a
control C3H/HeN female; immunostaining with anti–epidermal growth
factor (EGF) antiserum. EGF-positive GCT cells (arrows) and EGF-negative GCT cells (an arrowhead) are visible. Almost all the secretory
granules in the EGF-positive cells are labeled with numerous gold particles, indicating the presence of EGF. Few gold particles are seen on
the secretory granules of the EGF-negative cells. L, lumen; N, nucleus.
Scale bar 5 2 mm.
cell and a round nucleus visible in the basal one-third
(Fig. 9). There were, however, variations in the size of
these secretory granules. The gold particles, indicating
the presence of EGF, were restricted to these secretory
granules and were labeled in many of the large secretory
granules and in fewer small ones, producing an intergranular variation. Large Golgi apparatuses with swollen
cisternae, and mesh-like cisternae on the RER were visible in the perinuclear region. A few short membrane
infoldings were often observed in the basal membrane.
DISCUSSION
The results of this study suggest that GCT cells in the
SMGs of ICR, C57BL/6J, BALB/c, and C3H/HeN mice
exhibit a cellular mosaic expression pattern of EGF that
is prominent in the females of each strain. Furthermore,
some GCT cells were positively stained for EGF, but
others were negatively stained, and both of these cells
were ultrastructurally similar. The principal site of
GCT-specific secretory products is the secretory granules
of typical GCT cells (Bing et al., 1980; Watson et al.,
1985), with the exception of a few dark cells, pillar cells,
and transition type granular cells (reviewed by Mori
et al., 1992; Gresik, 1994); the former two cell types are
nongranulated, and the latter, which contain a few small
secretory granules, may be in the process of differentiation into typical GCT cells (Hazen-Martin and Simson,
1987). The transition type cells are positively stained
only for mK1 (Kurabuchi et al., 1999, 2001, 2002, 2004;
Kurabuchi and Hosoi, 2001) and were confirmed to be
EGF-negative in the present study. Variations in EGF
staining have been previously observed in SMG GCT
cells from ICR and BKW strains (van Noorden et al.,
1977; Gresik and Barka, 1977). Renin staining in the
SMGs of SWR/J mice also showed considerable intercellular variation in the intensity of immunostaining, and
some GCT cells apparently were negative (Tanaka et al.,
1980, 1981). mK1 also showed an unusual sexually
dimorphic mosaic pattern in GCT segments of SMGs in
ICR mice (Kurabuchi et al., 1999). Together, this immunocytochemical evidence suggests that GCT cells, which
are morphologically similar in appearance, are not homogeneous with respect to these secretory substances.
We proposed in our previous report that variations in
receptors for androgen, thyroid hormone, adrenocortical
hormone, and growth hormone might divide the GCT
cells into various cell types, resulting in the mosaic cellular distribution of mK1 (Kurabuchi et al., 2004). It is
conceivable that the GCT cells might/might not have
developed into full-fledged granular cells and lost/not
lost their ability for EGF synthesis depending upon the
expression levels of these hormone receptors. The
expression level of the androgen receptor has been
shown to vary among GCT cells (Morrell et al., 1987;
Sawada and Noumura, 1995). To the best of our knowl-
TUBULE CELLS OF MOUSE SUBMANDIBULAR GLAND
Fig. 9. Electron micrograph of a granular convoluted tubule (GCT)
cells in the submandibular gland (SMG) of a female C3H/HeN mouse,
after repeated injection with T315a-dihydrotestostrone (DHT); immunostaining with anti–epidermal growth factor (EGF) antiserum. A welldeveloped mesh-like rough endoplasmic reticulum (RER) and a large
Golgi apparatus (G) with swelled cistern are visible close to the nucleus (N). A large apical portion of the cells is closely packed with
numerous secretory granules, which are labeled with gold particles,
indicating the presence of EGF. Short membrane infoldings are sometimes found in the base (arrowheads) of the cell. L, lumen; N, nucleus.
Scale bar 5 2 mm.
edge, however, no studies have investigated the expression of other hormone receptors in rodent salivary
glands.
Male mice are known to have larger GCT cells, containing abundant secretory granules, in their SMGs,
and stereologic analyses have shown that the GCTs in
the glands are much larger in males than in females
(reviewed by Gresik, 1994). This finding agrees with the
much higher concentration of secretory products in
males, compared with in females (Barka, 1980). These
sex-related differences were also confirmed in each
strain, C57BL/6J, BALB/c, C3H/HeN, and ICR, used in
this study. In addition, the GCT cells were much larger
in both sexes of ICR mice than in both sexes of C57BL/
6J, BALB/c, and C3H/HeN mice. Furthermore, most of
the GCT cells were EGF-positive in the males of each
strain, whereas EGF-negative GCT cells were prominent
in the females. Considering the size of the GCT cells, the
percentage of EGF-positive cells, and the EGF-immunoreactivity, the EGF concentration was remarkably lower
in both sexes of C57BL/6J, BALB/c, and C3H/HeN mice
than in both sexes of ICR strain. This finding suggests
111
that the basal concentration of EGF in SMGs is strainspecific as well as sex-specific, and that C57BL/6J,
BALB/c, and C3H/HeN mice belong to a low EGF group,
whereas ICR mice belong to a high EGF group. Incidentally, inbred strains of mice have been classified into two
distinct groups, one with a much higher SMG renin activity than the other (Wilson et al., 1978, 1981). C57BL/
6J, BALB/c, and C3H/HeN mice belong to the low renin
group, whereas the ICR, an outbred strain, belongs to
the high renin group (Wilson et al., 1981). SMG renin is
controlled by the renin regulator (Rnr) gene, and the allele carried by strains in the high SMG renin group is
symbolized as Rnrs, while the allele carried by strains in
the low SMG renin group is termed Rnrb (Wilson et al.,
1977). To our knowledge, however, no relationship exists
between the renin regulator gene and the production of
GCT-specific secretory products, other than the involvement of renin.
The results of mating between parents derived from
ICR, a high EGF strain, and C3H/HeN, a low EGF
strain, suggest that no histological or immunohistological differences were present between the two types of F1
progeny, one type obtained from ICR males 3 C3H/HeN
females and the other type from C3H/HeN males 3 ICR
females, in either sex. However, the size of the GCT cells
and the ratio of EGF-positive cells in the F1 progeny
suggest that the EGF concentration in the glands of the
F1 progeny might be intermediate between the parent
strains, when compared within the same sex. Similar
results were obtained in a congenic strain derived from
high and low SMG renin strains. Concretely, when the
allele Rnrs from a donor AKR/J, a high SMG renin
strain, was included on the genetic background of C57L/
J, a low SMG renin strain, SMG renin activity in the F1
progeny was intermediate between that of the parent
strains, suggesting that SMG renin is controlled by
genes located in the same region of chromosome 1
(Wilson et al., 1981). Furthermore, using the same mating pairs as those used in the present study, either type
of F1 progeny inherited all of the kallikrein isozymes
from either of the parent strains (Kurihara et al., 1999).
These two previous mating experiments suggest that
EGF is directly/indirectly under genetic regulation similar to that seen for SMG renin and that the F1 progeny
seem to inherit genetic characteristics from both parent
strains.
Repeated injection with T3 and/or DHT in male and
female C3H/HeN mice, a low EGF strain, enlarged the
GCT cells and increased the number of secretory granules in both sexes, compared with the untreated controls
of the same sex; the number of EGF-positive cells also
increased remarkably, especially in females. Furthermore, the present immunoelectron microscopy examination revealed that the RER and Golgi apparatuses in the
GCT cells of the hormone-injected mice were well developed and that basal infoldings were less abundant, compared with the GCT cells in the untreated controls.
These findings show that GCT cells in both sexes of
untreated C3H/HeN mice were not as well developed as
those observed in ICR mice and that repeated hormone
injections resulted in a marked enhancement of the GCT
phenotype, promoting EGF synthesis in many GCT cells
especially in females. An even stronger effect was
obtained in the group that received both of the hormones: the GCT phenotype and EGF expression in the
112
MIYAJI ET AL.
males nearly reached the levels seen in ICR males,
while the levels in females were much higher than the
levels seen in ICR females. It has been established in
ICR mice that the additive and/or synergistic actions of
androgen and thyroid hormone enhance the GCT phenotype and produce more abundant GCT-specific secretory
products, including EGF (Hosoi et al., 1992; Gresik,
1994; Kurabuchi and Hosoi, 2001). These facts suggest
that the response of SMG GCT cells in both sexes of
C3H/HeN mice to thyroid hormone and androgen was
similar to that seen in ICR mice, ruling out the possibility that the low and high concentration of EGF are
caused by the opposite actions of these hormones in ICR
and C3H/HeN mice.
It is suspected that a low EGF concentration in SMGs
may be caused by a lower level of hormones in blood.
However, according to previous studies, serum T3 levels
do not vary significantly between low and high EGF
strains in mature adults: approximately 0.45 ng/ml and
0.52 ng/ml in C3H/HeN males and females, respectively,
and 0.53 ng/ml and 0.45 ng/ml in ICR males and female,
respectively (Kurihara et al., 1999), 0.66 ng/ml in
C57BL/6J males and 0.61 ng/ml in C3H/HeN males
(Maia et al., 1995), and 0.72 ng/ml in ICR males (Burgi
et al., 1986). These facts suggest that serum T3 levels
are not responsible for the strain-specific low or high
concentration of EGF in SMG. On the other hand, serum testosterone levels are known to be strain-specific,
and age-specific (Eleftheriou and Lucas, 1974; Nelson
et al., 1975; Jean-Faucher et al., 1982). The serum levels
in mature adults were measured approximately 1.3 ng/
ml in C57BL/10J and 4.6 ng/ml in DBA/2J (Bartke,
1974) and were also reported to be approximately
2.2 ng/ml and 0.22 ng/ml in male and female C3H/HeN,
respectively (Angele et al., 1998), and 3.0 ng/ml in ICR
males (Shapiro et al., 1989). These data proved that serum level of androgen is remarkably lower in C57BL
and C3H/HeN males, compared with males of the other
strains. A low serum level of androgen may be one
source of the low EGF expression in SMGs and for a
less developed GCT phenotype. However, the present
study showed that sexual differences were evident in
GCT cells even in low EGF strains, despite the lower serum androgen levels, and a significant strain-specific difference in EGF expression was observed especially in
females. Therefore, androgen is unlikely to be a main
cause of this difference.
Walker et al. (1982) demonstrated that excessive exogenous T3 resulted in a marked increase in the SMG
EGF concentration, and concluded that T3 receptor
expression might be significantly modulated by T3.
Androgen receptors have also been reported to be upregulated by thyroid hormones in the SMGs of developing and adult mice (Minetti et al., 1986, 1987) and a
marked increase in the nuclear binding of androgen in
the SMGs of mice of both sexes has been shown using
repeated testosterone injection (Katsukawa et al., 1983).
Based on the findings of all of these previous studies,
the low concentration of EGF in the SMGs of the low
EGF group may be caused by fewer receptors for several
hormones, including thyroidal and androgenic hormones,
with fewer receptors leading to a lower hormonal sensitivity in the low EGF group. We believe that external
androgen and/or thyroid hormone may also increase
additively/synergistically the expression levels of each
other’s receptors in each GCT cell, resulting in the
enhancement of the GCT phenotype in many GCT cells
and the production of more EGF in the low EGF group.
As described above, SMG renin is known to be under
genetic and hormonal control (Bing and Poulsen, 1971;
Wilson et al., 1977). On average, the renin activity in
8-week-old females is known to be 200 times higher in
high renin strains than in low renin strains, and the
renin activity in females from low renin strains is not
induced by androgen to the intact level observed in
females from high renin strains (Wilson et al., 1981). In
the contrast, EGF expression in SMGs in the three low
renin strains used in this study was relatively low but
was restored to a high level by the injection of thyroid
hormone and/or androgen. Therefore, EGF regulation
differs from that of SMG renin in inbred mice strains. If
the low EGF level in such low renin strains was caused
by a low sensitivity to hormones, the expression of many
GCT-specific secretory products, including nerve growth
factor (NGF), the gene families of kallikrein, mK9 (EGFbinding protein), 13 (prorenin converting enzyme), 22 (bNGF endopeptidase), as well as EGF will be low in the
low renin strains. Furthermore, whether the hypothetical low sensitivity to hormones proposed in this study
may be applied only to the low renin group of inbred
mice or to all inbred mice, including the high renin
group, remains to be elucidated.
LITERATURE CITED
Angele MK, Ayala A, Cioffi WG, Blank KI, Chaudry IH. 1998. Testosterone: the culprit for producing splenocyte immune depression
after trauma hemorrhage. Am J Physiol 274:C1530–C1536.
Barka T. 1980. Biologically active polypeptides in submandibular
glands. J Histochem Cytochem 28:836–859.
Bartke A. 1974. Increased sensitivity of seminal vesicles to testosterone in a mouse strain with low plasma testosterone levels. J
Endocr 60:145–148.
Bing J, Poulsen K. 1971. The renin system in mice. Acta Pathol
Microbiol Scand (A) 79:134–138.
Bing J, Poulsen K, Hackenthal E, Rix E, Taugner R. 1980. Renin in
the submaxillary gland: a review. J Histochem Cytochem 28:874–
880.
Burgi U, Feller C, Gerber AU. 1986. Effects of an acute bacterial
infection on serum thyroid hormones and nuclear triiodthyronine
receptors in mice. Endocrinology 119:515–521.
Chrétien M. 1977. Action of testosterone on the differentiation and
secretory activity of a target organ: submaxillary gland of the
mouse. Int Rev Cytol 50:333–396.
Eleftheriou BE, Lucas LA. 1974. Age-related changes in testes, seminal vesicles and plasma testosterone levels in male mice. Gerontologia 20:231–238.
Gresik EW. 1994. The granular convoluted tubule (GCT) cell of
rodent submandibular glands. Microsc Res Tech 27:1–24.
Gresik EW, Barka T. 1977. Immunocytochemical localization of epidermal growth factor in mouse submandibular gland. J Histochem Cytochem 25:1027–1035.
Hazen-Martin DJ, Simson JAV. 1987. Immunocytochemical localization of nerve growth factor in mouse salivary glands. Histochem J
19:210–216.
Hiramatsu M, Kashimata M, Takayama F, Minami M. 1994. Developmental changes in and hormonal modulation of epidermal
growth factor concentration in the rat submandibular gland.
J Endocrinol 140:357–363.
Hosoi K, Tanaka I, Ishii Y, Ueha T. 1983. A new esteroproteinase
(proteinase F) from the submandibular glands of female mice.
Biochim Biophys Acta 756:163–170.
Hosoi K, Maruyama S, Ueha T, Sato S, Gresik EW. 1992. Additive
and/or synergistic effects of 5-a-dihydrotestosterone, dexametha-
TUBULE CELLS OF MOUSE SUBMANDIBULAR GLAND
sone, and triiod-L-thyronine on induction of proteinases and epidermal growth factor in the submandibular glands of hypophysectomized mice. Endocrinology 130:1044–1055.
Jean-Faucher C, Berger M, de Turckheim M, Veyssiere G, Jean C.
1982. Plasma and testicular testosterone and dihydrotestosterone
in mice: effect of age and HCG stimulation. IRCS Med Sci 11:26–27.
Johansson O, Backman J. 1983. Enhancement of immunoperoxidase
staining using osmium tetroxide. J Neurosci Methods 7:1217–1223.
Katsukawa H, Tanabe Y, Funakoshi M. 1983. Effects of androgens
on the activity of stroid-metabolizing enzymes in the murine submaxillary gland. J Dent Res 62:725–727.
Kurabuchi S, Hosoi K. 2001. Sexual dimorphism and hormonal regulation of granular convoluted tubule (GCT) cells of mouse submandibular gland. Immunocytochemical analysis. (In Japanese).
J Masticat Health 10:61–70.
Kurabuchi S, Tada J, Gresik EW, Hosoi K. 1999. An unusual sexually dimorphic mosaic distribution of a subset of kallikreins in the
granular convoluted tubule of the mouse submandibular gland
detected by an antibody with restricted immunoreactivity. Histochem J 31:19–28.
Kurabuchi S, Hosoi K, Gresik EW. 2001. Androgen regulation of the
cellular distribution of the true tissue kallikrein mK1 in the submandibular gland of the mouse. J Histochem Cytochem 49:801–
802.
Kurabuchi S, Hosoi K, Gresik EW. 2002. Developmental and androgenic regulation of the immunocytochemical distribution of mK1,
a true tissue kallikrein, in the granular convoluted tubule of the
mouse submandibular gland. J Histochem Cytochem 50:135–145.
Kurabuchi S, Gresik EW, Hosoi K. 2004. Additive and/or synergistic
action (downregulation) of androgens and thyroid hormones on
the cellular distribution and localization of a true tissue kallikrein, mK1, in the mouse submandibular gland. J Histochem Cytochem 52:1437–1446.
Kurihara K, Maruyama S, Nakanishi N, Sakagami H, Ueha T.
1999. Thyroid hormone (3,5,30 -triiodo-L-thyronine) masking/inversion of stimulatory effect of androgen on expression of mK1, a
true tissue kallikrein in the mouse submandibular gland. Endocrinology 140:3003–3011.
Maia AL, Kieffer JD, Harney JW, Larsen PR. 1995. Effect of 3,5,30 triiodothyronone (T3) administration on dio 1 gene expression
and T3 metabolism in normal and type 1 deiodinase-deficient
mice. Endocrinology 136:4842–4849.
Minetti CA, Valle LB, Fava-de-Moraes F, Romaldini J, OliveiraFilho R. 1986. Ontogenesis of androgen receptors in the mouse
submandibular gland: correlation with the development profiles
of circulating thyroid and testicular hormones. Acta Endocrinol
112:290–295.
Minetti CA, Valle LB, Romaldini J, Fava-de-Moraes F, OliveiraFilho R. 1987. Thyroidal modulation of androgenic expression in
mice submandibular gland. Horm Metab Res 19:146–151.
113
Mori M, Takai Y, Kunikawa M. 1992. Review: biologically active
peptide in the submandibular gland—role of the granular convoluted tubule. Acta Histochem Cytochem 25:325–341.
Morrell JI, Gresik EW, Barka T. 1987. Autoradiographic localization
of dihydrotestosterone binding in the major salivary glands and
other androgen-responsive organs of the mouse. J Histochem
Cytochem 35:1053–1058.
Murai T. 1987. Studies on proteinase F in major salivary glands of
inbred mice—sex difference of proteinase F. (In Japanese). Jpn J
Oral Biol 29:416–427.
Nelson JF, Latham KR, Finch CE. 1975. Plasma testosterone levels
in C57BL/6J male mice: effects of age and disease. Acta Endocrinol 80:744–752.
Sawada K, Noumura T. 1995. Developmental pattern of androgen
receptor immunoreactivity in the mouse submandibular gland.
Zool Sci 12:243–248.
Shapiro BH, Niedermeyer TM, Babalola GO. 1989. Serum androgen
levels in senescent Crl: CD-1R(ICR) BR mice: effects of castration
and testosterone treatment. J Gerontol 44:B15–B19.
Tanaka T, Gresik EW, Michelakis AM, Barka T. 1980. Immunocytochemical localization of renin in kidneys and submandibular
glands of SWR/J and C57BL/6J mice. J Histochem Cytochem
28:1113–1118.
Tanaka T, Gresik EW, Barka T. 1981. Epidermal growth factor and
renin in mouse submandibular glands. J Histochem Cytochem
29:1229–1231.
van Noorden S, Heitz P, Kasper M, Pearse AGE. 1977. Mouse epidermal growth factor: light and microscopical localization by
immunocytochemical staining. Histochemistry 52:329–340.
Walker P, Coulbome P, Dussault JH. 1982. Time- and dose-dependent effect of triiodthyronine on submaxillary epidermal growth
factor concentration in adult female mice. Endocrinology
111:1133–1139.
Watson AY, Anderson JK, Siminoski K, Mole JE, Murphy RA. 1985.
Cellular and subcellular colocalization of nerve growth factor and
epidermal growth factor in mouse submandibular glands. Anat
Res 213:365–376.
Wilson CM, Erdos EG, Dunn JF, Wilson JD. 1977. Genetic control
of renin activity in the submaxillary gland of the mouse. Proc
Natl Acad Sci USA 74:1185–1189.
Wilson CM, Erdos EG, Wilson JD, Taylor. 1978. Location on chromosome 1 of Rnr, a gene that regulates renin in the submaxillary
gland of the mouse. Proc Natl Acad Sci USA 75:5623–5626.
Wilson CM, Cherry M, Taylor BA, Wilson JD. 1981. Genetic and endocrine control of renin activity in the submaxillary gland of the
mouse. Biochem Genet 19:509–523.
Young WG, Ramirez-Young GO, Daley TJ, Smid JR, Coshigano KT,
Kopchick JJ, Waters MJ. 2004. Growth hormone and epidermal
growth factor in salivary glands of giant and dwarf transgenic
mice. J Histochem Cytochem 52:1191–1197.
Документ
Категория
Без категории
Просмотров
3
Размер файла
504 Кб
Теги
expressions, epidermal, endocrine, convoluted, strait, growth, mouse, granular, tubules, control, cells, factors, submandibular, specific, gland
1/--страниц
Пожаловаться на содержимое документа