Histochem Cell Biol (2010) 133:577–584
DOI 10.1007/s00418-010-0698-6

ORIGINAL PAPER

Possible involvement of melatonin in tooth development:
expression of melatonin 1a receptor in human and mouse toothgerms
Shuku Kumasaka · Masashi Shimozuma · Tadafumi Kawamoto ·Kenji Mishima · Reiko Tokuyama · Yoko Kamiya ·Purevsuren Davaadorj · Ichiro Saito · Kazuhito Satomura

Accepted: 25 March 2010 / Published online: 7 April 2010
© Springer-Verlag 2010

Abstract Melatonin is known to regulate a variety ofphysiological processes including control of circadianrhythms, regulation of seasonal reproductive function, regulation of body temperature, free radical scavenging, and soforth. Accumulating evidence from in vitro and in vivo
experiments has also suggested that melatonin may have an
inXuence on skeletal growth and bone formation. However,
little is known about the eVects of melatonin on toothdevelopment and growth, which thus remain to be elucidated. This study was performed to examine the possibility
that melatonin might exert its inXuence on tooth development as well as skeletal growth. Immunohistochemical
analysis revealed that melatonin 1a receptor (Mel1aR) was
expressed in secretory ameloblasts, the cells of the stratum
S. Kumasaka · M. Shimozuma · R. Tokuyama · Y. Kamiya ·K. Satomura (&)
Second Department of Oral and Maxillofacial Surgery,
Department of Oral Medicine, School of Dental Medicine,
Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku,
Yokohama, Kanagawa 230-8501, Japane-mail: satomura-k@tsurumi-u.ac.jp
T. Kawamoto
RI Research Center, School of Dental Medicine,
Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku,
Yokohama, Kanagawa 230-8501, Japan
K. Mishima · I. Saito
Department of Pathology, School of Dental Medicine,
Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku,
Yokohama, Kanagawa 230-8501, Japan
P. Davaadorj
Department of Oral and Maxillofacial Surgery,
Institute of Health Biosciences,
The University of Tokushima Graduate School,
Kuramoto-cho 3-18-15, Tokushima,
Tokushima 770-8504, Japan

intermedium and stellate reticulum, external dental epithelial cells, odontoblasts, and dental sac cells. Reverse transcription-polymerase chain reaction and Western blot
analysis showed that HAT-7, a rat dental epithelial cell line,
expressed Mel1aR and its expression levels increased after
the cells reached conXuence. These results strongly suggestthat melatonin may play a physiological role in tooth development/growth by regulating the cellular function of odontogenic cells in tooth germs.
Keywords Melatonin · Melatonin 1a receptor ·Tooth development · Immunohistochemistry · Gene
expression

Introduction
Melatonin was discovered by Lerner et al. (1958), and issecreted by the pineal gland primarily during the night. Its
secretion is controlled by the light:dark cycle in such amanner that it is released in large amounts during the night,
but only minimally during the day (Lynch et al. 1987;
Reiter 1991; Reiter et al. 1996). Melatonin has a variety ofphysiological actions such as control of circadian rhythms(Redman et al. 1983; McArthur et al. 1997), regulation ofbody temperature (Dollins et al. 1994), hormone secretion
inXuencing sexual development (EsquiWno et al. 1987;
Batmanabane and Ramesh 1996) and the reproductive
cycle in seasonally breeding animals (Kennaway and Rowe
1995), activation of the immune system (Garcia-Maurino
et al. 2000; Raghavendra et al. 2001; Guerrero and Reiter
2002), and anti-tumor activity (Hill and Blask 1988;
Martínez-Campa et al. 2006; Sánchez-Barceló et al. 2005).
In addition, melatonin and its metabolites potentially exertanti-aging eVects due to its ability to function as a free

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radical scavenger and antioxidant (León et al. 2005; Tanet al. 2007). Moreover, there are some reports that melatonin controls the secretion of human growth hormone(Forsling et al. 1999) and adrenocortical hormones(Kostoglou-Athanassiou et al. 1998; Cagnacci et al. 1995).
Furthermore, it has been reported that melatonin enhances
the diVerentiation of osteoblasts in vitro and promotes boneformation in vivo (Roth et al. 1999; Koyama et al. 2002;
Turgut et al. 2005; Machida et al. 2006; Nakade et al. 1999;
Satomura et al. 2007). These data strongly suggest thatmelatonin is involved in the development and/or growth ofother hard tissues as well as bone tissue. However, there are
no reports on the involvement of melatonin in tooth development or growth.
Two genes encoding membrane melatonin receptors havebeen identiWed in mammals including humans: one is the melatonin 1a receptor (Mel1aR) (Reppert et al. 1994) and theother is the 1b receptor (Mel1bR) (Reppert et al. 1995). These
receptors are characterized by seven transmembrane-spanningdomains and coupling to a trimeric G protein. Both receptors
inhibit adenylate cyclase activity via a Gi protein (Reppert
et al. 1994, 1995). Of the two melatonin receptors, Mel1aR
was originally identiWed in the suprachiasmatic nucleus andpituitary, but recently its expression was conWrmed in otherorgans and cells including the cerebellum (Al-Ghoul et al.
1998), kidney (Drew et al. 1998), leptomeninges of the brain
and spinal cord (Drew et al. 1997), monocytes (Raghavendra
et al. 2001), and lymphocytes derived from the thymus or
spleen (Pozo et al. 1997). However, there are no reports on
the expression of melatonin receptors in teeth.
During mammalian tooth development, a complex series
of interactions occur between the oral epithelium and neuralcrest-derived ectomesenchyme (Gilbert 2000; Jernvall and
ThesleV 2000; ThesleV and Mikkola 2002). In the initialstage of tooth development, the process begins with odontogenic diVerentiation of ectomesenchyme, which is inducedby signals from the oral epithelium. Thereafter, tooth morphogenesis is regulated by sequential and reciprocal interactions between the epithelial and mesenchymal tissues(Ten Cate 1994). Such interactive processes between oral
epithelium and ectomesenchyme are mediated by a variety ofmacromolecules of the extracellular matrix together with
diVusible signaling molecules, and continue until a tooth isformed with organ-speciWc mesenchymal cells and organspeciWc epithelial cells (Jernvall and ThesleV 2000; ThesleVand Mikkola 2002; ThesleV et al. 1991). Accumulating evidence from in vitro and in vivo experiments has demonstrated that paracrine signaling molecules, including bonemorphogenetic proteins (BMPs), Wbroblast growth factors(FGFs), sonic hedgehog (Shh), Wnts, tumor necrosis factors(TNFs) and activin, play important roles in the sophisticatedepithelial–mesenchymal interaction of the tooth development(Jernvall and ThesleV 2000; ThesleV and Mikkola 2002).

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Histochem Cell Biol (2010) 133:577–584

Development of the tooth germ comprises extensive
tissue growth and expansion in the developing jaw bones. It
is well known that skeletal growth is regulated not only by
local factors such as BMPs (Yamaguchi et al. 1991), IGFs
(Shinar et al. 1993) and FGFs (Nakamura et al. 1995) but
also by systemic hormones including growth hormone(Kassem et al. 1994), parathyroid hormone (Isogai et al.
1996) and estrogen (Jilka et al. 1998). Recently, melatonin
was proven to enhance the diVerentiation of human osteoblasts in vitro, promoting mouse bone formation in vivo
(Satomura et al. 2007). Cells of the tooth germ have to form
the tooth in harmony with the growth of the surrounding
jaw bone. This raises the possibility that systemic
hormones such as melatonin could regulate tooth development as well as skeletal development.
From this point of view, it is conceivable that melatoninwould involve in tooth development as well as in boneformation. In this study, to conWrm this possibility, we
examined immunohistochemically whether the odontogenic
cells in human and mouse tooth germs express Mel1aR,
and also investigated the expression of Mel1aR in a rat
dental epithelial cell line, HAT-7.

Materials and methods
Human tissue samples
The tooth germs of the mandibular third molar in the latebell stage, extracted from Wve Japanese boys and girls(9 or 10 years of age) during orthodontic treatment, wereused in this study. The experimental protocol wasapproved by the Ethical Review Committee of theTokushima University Hospital, and informed consent
was obtained for the use of the tooth germ. Extirpated
tooth germs were Wxed with 10 N Mildform® (Wako Pure
Chemical Industries, Ltd, Osaka Japan), decalciWed in
10% EDTA, and embedded in paraYn. Sections were cut
5 mm thick, deparaYnized, and stained with hematoxylin
and eosin (Fig. 1). The data presented herein are representative of reproducible results from Wve diVerent
donors.
Mouse tissue samples
The housing care and experimental protocol wereapproved by the Animal Care and Use Committee of the
University of Tokushima School of Dentistry. The mandibles of neonatal mice (ICR, male) were extirpated, Wxedwith 10 N Mildform® (Wako), decalciWed brieXy in 10%
EDTA, and embedded in paraYn. Sections were cut 5 mm
thick, deparaYnized, and stained with hematoxylin andeosin.

Histochem Cell Biol (2010) 133:577–584

579(DMEM/F12HAM: Sigma Chemical Co, St. Louis, MO,
USA) containing 10% FBS (Filtron, Brooklyn, Australia),
100 mg/ml ascorbic acid (Wako Pure Chemical Industries,
Osaka, Japan), 1£ Glutamax® (Invitrogen, Carlsbad, CA,USA), 100 U/ml penicillin (Invitrogen) and 100 mg/ml
streptomycin (Invitrogen). The culture was maintained at
37°C in a humidiWed atmosphere of 5% CO2 in air andmedium was changed twice a week.
Analysis of expression of melatonin Mel1aR receptor

Fig. 1 (a) Panoramic radiograph showing a representative case of a
10-year-old girl with tooth germs of the mandibular third molars. The
tooth germs (arrowheads) were extirpated under local anesthesia andused for histological analysis. (b) Transverse sections of a human tooth
germ at the late bell stage (hematoxylin and eosin staining). Higher
magniWcation views of several portions indicated by alphabetizedsquares are shown in Fig. 2. (FW = 10 mm)

Immunohistochemistry
Some sections of human tooth germs and mouse mandibles
were transferred onto poly-L-lysine-coated glass slides.
After deparaYnization with xylene and rehydration withdescending concentrations of ethanol, endogenous peroxidase was blocked by treatment with 3% H2O2 in methanolfor 1 h at room temperature (RT). After treatment with 10%
normal goat serum at RT for 1 h, sections were incubatedwith rabbit anti-melatonin receptor 1a (MT1) polyclonal
antibody (CIDtech Research Inc., Cambridge, Canada) at aconcentration of 1 mg/ml in phosphate-buVered saline(PBS, pH 7.4) containing 1% bovine serum albumin at RT
for 1 h. Sections were washed with PBS, and localization of
Mel1aR was visualized using a HistoWne SAB-PO(R) Kit(Nichirei Corporation, Tokyo, Japan) and a 3,3⬘diaminobenzidine (DAB) Substrate Kit (Nichirei Corporation). Sections were counterstained with hematoxylin andmounted. The speciWcity of the immunoreaction wasconWrmed by (a) incubation with normal rabbit IgG insteadof the primary antibody and (b) preabsorption of primary
antibody with synthetic peptides which was used as an antigen for anti-Mel1aR antibody.
Cell culture
HAT-7, which is a dental epithelial cell line originatingfrom a rat incisor, was a generous gift from Dr. Hidemitu
Harada, Iwate Medical University, Iwate, Japan (Kawano
et al. 2002). HAT-7 cells were cultured in Dulbecco’s ModiWed Eagle’s Medium Nutrient Mixture F-12 HAM

Cells were seeded at a density of 1 £ 105 cells/dish in
60 mm Petri dishes and cultured until they reached conXuence. The expression of Mel1aR was conWrmed by reverse
transcription-polymerase chain reaction (RT-PCR) andWestern blot analysis. Total RNA was extracted using TRIzol
reagent (Invitrogen, Carlsbad, CA). cDNA synthesis from
1 mg total RNA was performed using the SUPERSCRIPT
Pre-ampliWcation System (Invitrogen) after DNase I treatment. The ampliWcation of primer sequences used to detect the
rat Mel1aR gene (NM_053676) were F: 5⬘-(515)TCAAGTT
AGTGCCTTCCTAATGG(537)-3⬘ and R: 5⬘-(1058)GACG
AGGAAGTGGAAAACCACCAC(1081)-3⬘. PCR products
were analyzed by ethidium bromide staining on a 2.0% agarose gel. For Western blot analysis, the cells were lysed in
RIPA buVer (10 mM Tris–HCl, 1% NP-40, 0.1% SDS,
150 mM NaCl and 1 mM EDTA) containing 1 mM phenylmethylsulfonyl Xuoride, 5 mM leupeptin and 10 mM aprotinin. The samples (100 mg protein) were separated by the
NuPAGE System (Invitrogen) using a 4–12% Bis–Tris Gel,and electroblotted onto a nitrocellulose membrane (HybondECL, Amersham Pharmacia Biotech, Buckinghamshire,UK). After blocking with 5% skimmed milk for 1 h at 4°C,
membranes were incubated with 1 mg/ml of rabbit antiMel1aR monoclonal antibody (CIDtech Research Inc.) overnight at 4°C. Membranes were then washed several times
and incubated with horseradish peroxidase-conjugated antirabbit IgG antibody (Dako, Glostrup, Denmark) for 1 h atroom temperature. After thorough washes, the immunocomplex was visualized using ECL Western Blotting Detection
Reagents (Amersham Pharmacia Biotech) and an ECL miniCamera (Amersham Pharmacia Biotech). The brain tissuesof neonatal rats (Sprague–Dawley, male) were used as apositive control for Mel1aR expression. The housing careand experimental protocol were approved by the Animal
Care and Use Committee of the University of Tokushima
School of Dentistry. Extirpated brain tissues were rinsed
with ice-cold PBS several times and then homogenized at on
ice in RIPA buVer containing protease inhibitor cocktail(Roche Diagnostics, Mannheim Germany) for Western blot.
The tissue homogenate in RIPA buVer was centrifuged for
10 min at 4,000£g and supernatants were used for Western
blot as mentioned above.

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Results
Localization of Mel1aR in human tooth germs
at the late bell stage
In the tooth germs of human mandibular third molars at thelate bell stage, some initial dentin and enamel were present
in the area where the cusp would later be formed (Fig. 1).
General histology revealed that enamel organs in the extirpated tooth germs contained mature ameloblasts secreting

Fig. 2 Higher magniWcation
views of characteristic areas of
the tooth germ (hematoxylin andeosin staining). a Mature ameloblasts (asterisk), stratum intermedium (arrows) and stellate
reticulum (arrowhead).
b Mature odontoblasts (arrows)
and dentin (d). c External dental
epithelium (asterisk) and stellate
reticulum cells (arrowhead).
Immunohistochemical localization of Mel1aR in human tooth
germ at the late bell stage (DAB
staining). d, h Negative controls.
Primary antibody was preabsorbed with antigenic peptide.
e Mature and secretory ameloblasts and stratum intermedium
cells were positive for Mel1aR.
The cells of the stratum intermedium and stellate reticulum werealso noted to be positive forMel1aR. f Odontoblasts werenoted to be positive for Mel1aR.
g External dental epithelial cells
were positive for Mel1aR (a, d, e
FW = 210 m; b, c, f, h
FW = 270 m; g FW = 300 m)

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Histochem Cell Biol (2010) 133:577–584

enamel matrix, epithelial cells in the stratum intermedium,
epithelial cells in the stellate reticulum and external dental
epithelium as the epithelial components of tooth germs(Fig. 2a, c). Moreover, dentin-forming odontoblasts, dental
papilla cells and dental sac cells were noted as mesenchymal components of the tooth germs (Fig. 2b). Immunohistochemistry for Mel1aR showed that the secretory
ameloblasts, the cells of the stratum intemedium and theexternal dental epithelium had pronounced immunoreactivity for Mel1aR (Fig. 2e–g). In contrast, the cells of theHistochem Cell Biol (2010) 133:577–584

stellate reticulum showed only slight immunoreactivity forMel1aR (Fig. 2e). Interestingly, no evident expression of
Mel1aR was noted in the odontogenic epithelial cells of thecervical loop region (data not shown). Of the mesenchymal
cells in the human tooth germs, the secretory odontoblasts
and their processes were positive for Mel1aR (Fig. 2f).
Weak positive staining for Mel1aR was found in the cellsof the dental papillae. The cells of the dental sac were also
moderately positive for Mel1aR (data not shown).

581

Localization of Mel1aR in mouse tooth germs
Similar to the human tooth germs, a positive immunoreaction for Mel1aR was observed in secretory ameloblasts,
cells of the stratum intermedium, secretory odontoblasts
and cells of the dental papillae (Fig. 3a, b, d, e). Moreover,
osteoblasts were also noted to be positive for Mel1R(Fig. 3c, f). This staining pattern was substantially similar
to that in human tooth germs.

Fig. 3 Immunohistochemical
localization of Mel1aR in mouseincisors. a, b Mature and secretory ameloblasts (asterisk), stratum intermedium (arrow) andmature odontoblasts (arrowheads) can be seen (hematoxylin
and eosin staining). a, c Mandibular bone around the incisor containing osteoblasts (arrows) andosteocytes (arrowheads) was
noted (hematoxylin and eosin
staining). d, e Ameloblasts, cellsof the stratum intermedium andodontoblasts were positive forMel1aR. Osteoblasts of the mandibular bone were noted to bepositive for Mel1aR. a–c Hematoxylin and eosin staining. d–g
DAB staining, g negative control. Primary antibody was preabsorbed with antigenic peptide(a, d: FW = 600 m; b, c, e, f, g:
FW = 300 m)

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Fig. 4 a The expression of mRNA for Mel1aR in HAT-7 cells.
SigniWcant expression of Mel1aR was not detected before conXuence,
whereas Mel1aR expression increased after the cells reached conXuence. ¡3d 3 days of culture before conXuence, and +1d, +3d, +1w and+2w 1 day, 3 days, 1 week and 2 weeks, respectively, of culture
after reaching conXuence. MM molecular marker (X174/HaeIII).
b Expression of Mel1aR protein in HAT-7 cells. HAT-7 cells were cultured for 2 weeks after reaching conXuence. Thereafter, the expressionof Mel1aR protein was analyzed by Western blot. A major band forMel1aR was observed at »37 kDa. The amount of protein loaded for
HAT-7 and rat brain was 80 and 100 g, respectively

Expression of Mel1aR in rat dental epithelial cells
Using semi-quantitative RT-PCR, we studied the mRNA
expression of Mel1aR in HAT-7 cells, a rat dental epithelial
cell line. The expression levels of Mel1aR mRNA
increased signiWcantly after the cells reached conXuence(Fig. 4a). Western blot analysis also conWrmed the expression of Mel1aR protein in HAT-7 cells (Fig. 4b).
Discussion
There are many previous studies showing that melatoninhas a variety of physiological actions such as control of circadian rhythms (Redman et al. 1983; McArthur et al.
1997), regulation of body temperature (Dollins et al. 1994),
activation of the immune system (Garcia-Maurino et al.
2000; Raghavendra et al. 2001; Guerrero and Reiter 2002),
anti-tumor activity (Hill and Blask 1988; Martínez-Campaet al. 2006; Sánchez-Barceló et al. 2005), function as a free
radical scavenger and antioxidant (León et al. 2005; Tanet al. 2007) and anabolic eVects on bone formation (Rothet al. 1999; Koyama et al. 2002; Turgut et al. 2005;
Machida et al. 2006; Nakade et al. 1999; Satomura et al.
2007). However, no data are available about its eVect on
tooth development. In this study, to investigate the possibility
that melatonin plays a physiological role in tooth development

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Histochem Cell Biol (2010) 133:577–584

as well as in skeletal development, we examined immunohistochemically whether Mel1aR, which is the most potent
transmembrane receptor for melatonin, was expressed inhuman and mouse tooth germs. The present study demonstrates for the Wrst time that Mel1aR is expressed in a variety of human and mouse dental structures during toothdevelopment. Mel1aR is also localized in osteoblasts which
are forming bone of the jaws around the tooth germ. The
expression pattern of Mel1aR in the developing mouse
tooth germs is consistent with the pattern observed inhuman tooth development. We also conWrmed immunohistochemically that Mel1aR was expressed in tooth germs ofanother strain of mouse (C57Bl/6J) and F344 rat (data notshown). In addition, HAT-7, a rat dental epithelial cell line,
was also conWrmed to express Mel1aR at the protein levelby Western blot analysis. Interestingly, the expression levels of mRNA for Mel1aR in HAT-7 cells increased signiWcantly after the cells reached conXuence. This phenomenon
may suggest that melatonin exerts its inXuence not on the
proliferation but on the diVerentiation and/or function ofodontogenic cells and be consistent with the Wndings that
Mel1aR was not expressed in the cervical loop.
The physiological or developmental signiWcance of thepresence of Mel1aR in the tooth germs remains unknown.
The issue of whether Mel1aR expressed in tooth germs
has normal function should be addressed in the future.
The physiological role of melatonin itself in toothdevelopment also remains unknown in the present study.
Nevertheless, our observations strongly suggest that melatonin might exert its inXuence on tooth development atleast partly through Mel1aR, and that tooth development
and growth may be under regulation by systemic hormones in the same way as skeletal growth. It is possiblethat melatonin may regulate the proliferation, diVerentiation and/or function of odontogenic cells in the tooth
germs in order to maintain the expanding tooth morphology. In fact, it is known that ameloblasts slowly form
prismatic enamel with cross striation (4–8 m) under the
regulation of circadian clock (Smith 2006). The eVects ofmelatonin could be conceivably related to this secretory
behavior. Another possibility is that melatonin could
harmonize the growth of the tooth with the growth of thesurrounding jaw bone through physiological regulationof odontogenic cells, judging from the fact that skeletal
growth is also controlled by melatonin. To elucidate
these issues, in vitro studies using odontogenic cells
would be very helpful, and should be performed in thenear future.
Acknowledgments This work was supported by a Grant-in-Aid for
ScientiWc Research (16390591, 17791467 and 21592575) from the
Ministry of Education, Culture, Sports, Science, and Technology ofJapan.

Histochem Cell Biol (2010) 133:577–584

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