/. Embryol. exp. Morph. Vol. 37, pp. 79-90, 1977
Printed in Great Britain
79
Induction of melanogenesis in the
epidermal melanoblasts of newborn mouse skin
byMSH
ByTOMOHISA HIROBE AND TAKUJI TAKEUCHI1
From the Biological Institute, Tohoku University, Japan
SUMMARY
The number of melanocytes positive to the dopa reaction in the epidermis was shown to
increase after newborn mice were injected with a-MSH or DBc-AMP. The agents seemed to
induce the initiation of melanogenesis in the pre-existing melanoblasts. Electron-microscopic
observation also demonstrated that a-MSH induced not only maturation of melanosomes but
also the formation of melanosomes.
INTRODUCTION
Fully differentiated melanocytes, characterized by their tyrosinase activity,
mature melanosomes and dendrites can be seen mainly in the hair bulbs of the
skin in adult mice, where they secrete melanosomes into the surrounding
keratinocytes, giving rise to melanized hairs. These melanocytes are considered
to be derived from melanoblasts, undifferentiated precursors of melanocytes,
located in epidermis. It has been shown that the melanoblasts originate from the
neural crest and migrate into the epidermis of all body regions in early embryonic life (Rawles, 1940, 1947, 1948). The melanoblasts, however, do not
differentiate into active melanocytes within the trunk epidermis and remain
undifferentiated throughout the animal's life except for a short period after
birth (Reynolds, 1954; Rovee & Reams, 1964; Takeuchi, 1968). Numerous cells
positive to the histochemical dopa reaction are found in the basal layer of the
epidermis only during the early weeks after birth (Takeuchi, 1968).
The object of this study is to find some factors involved in the initiation of
melanogenesis, the final step in the differentiation of melanocytes in the neonatal
epidermis. The results suggest that MSH, melanocyte-stimulating hormone, is a
possible inducing agent.
1
Author's address: Biological Institute, Tohoku University, Aoba-yama, Sendai, Japan
980.
6-2
80
T. HIROBE AND T. TAKEUCHI
MATERIALS AND METHODS
The materials used in this study were newborn infants of the house mouse,
Mus musculus, of strain C57BL/1OJ. They were injected subcutaneously at the
dorsal side with a-MSH (a gift from Ciba-Geigy) or DBc-AMP (N6,O2'dibutyryl-adenosine 3',5'-cyclic monophosphate, Sigma).
After the treatment, pieces of skin were excised from the dorsal side of the
animals and fixed with formalin in phosphate buffer (pH 7-0) for 24 h at 4 °C.
They were then washed with distilled water and incubated with 0 1 % dopa
solution in phosphate buffer (pH 7-4) for 24 h at 37 °C. Combined dopaammoniated silver nitrate staining was also performed (Mishima, 1960).
According to Mishima (Mishima, Loud & Schaub, 1962; Mishima & Loud,
1963; Mishima, 1964), this staining preferentially reveals undifferentiated
melanoblasts which contain premelanosomes in addition to differentiated
melanocytes. The specimens were sectioned and counterstained with eosin. The
number of melanocytes (i.e. the cells positive to the dopa reaction) and the
number of premelanosome-containing melanoblasts plus melanocytes (i.e. the
cells positive to the combined dopa-premelanin reaction) were counted per
0-1 mm2 of the interfollicular epidermis. All experiments were performed in
triplicate and each experiment involved three specimens.
The dorsal skins for electron microscopy were fixed with 4 % glutaraldehyde
in 0-1 M phosphate buffer (pH 7-4) for 2 h and postfixed with 1 % osmium
tetroxide in 0-1 M phosphate buffer (pH 7-4) for 2 h at 2-4 °C. After the fixation,
they were dehydrated through a series of graded ethanols and embedded in
Epon 812. The ultrathin sections were cut in an LKB Ultrotome 4802 A,
stained with uranyl acetate and lead citrate, and examined with a Hitachi
HS-9 electron microscope.
RESULTS
Changes in the number of melanocytes and melanobJasts in the epidermis of
newborn mice
The dorsal skins of C57BL/10J strain mice were fixed at various days after
birth and were subjected to the dopa reaction and combined dopa-ammoniated
silver nitrate staining. Figure 1 shows the change in the number of melanocytes
(the cells positive to the dopa reaction) and the number of melanoblasts plus
melanocytes (the cells positive to the combined dopa-premelanin reaction).
Melanocytes increased in number until day 4, then gradually decreased in
number and disappeared by day 30. On the other hand, the number of cells
positive to the combined dopa-premelanin reaction remained constant until
day 4 and then decreased. Thus the proportion of melanocytes in the melanoblast-melanocyte population in the epidermis increases from 20 % to 70 % in
the first 4 days after birth, while the number of active melanocytes declined as
the size of the melanocyte-melanoblast population decreased. From day 3,
81
Hormone-induced melanogenesis
140 -
1
2
8
10
Days after birth
19
30
Fig. 1. Change in the number of epidermal melanocytes in newborn C57BL/.10J
mice. O, number of cells positive to the dopa reaction. D, number of the cells
positive to the combined dopa-premelanin reaction per 01 mm2 in the interfoUicular
epidermis. Bars indicate S.E.M. (standard error of the mean).
Fig. 2. Vertical section of the dorsal skin of three-day-old C57BL/10J mouse.
Dendritic cells positive to the dopa reaction are observed in the basal layer of epidermis and in the hair follicle.
Fig. 3. Vertical section of the dorsal skin of a-MSH (1 /ig/g BW)-treated mouse.
Numerous dopa-positive cells were seen 2 days after the a-MSH injection.
82
T. HIROBE AND T. TAKEUCHI
140
140
120
120
100
100
•s 80
80
a
a, 60
o
•o
60
40
40
20
20
Days after birth
Fig. 4
Days after birth
Fig. 5
Fig. 4. Effect of a-MSH on epidermal melanocyte. Mice were injected with a-MSH
(A) at the dose of 1 /tg/g BW, or with Hanks BSS ( • ) . O, one-day-old null control.
a-MSH and Hanks BSS were injected on day 1 and day 2.
Fig. 5. Effect of DBc-AMP on epidermal melanocytes. Mice were injected with
DBc-AMP (A) at the dose of 30 /*g/g BW, or with Hanks BSS (#). O, one-day-old
null control. DBc-AMP and Hanks BSS were injected on day 1 and day 2.
dendritic melanocytes (positive to the dopa reaction) were observed in the
roots of hair follicles in addition to the basal layer of epidermis (Fig. 2). This
suggests that the differentiated epidermal melanocytes migrate into hair follicles
at this stage.
Effects of a-MSH and DBc-AMP
Newborn mice were injected with a-MSH of 1 /tg/g BW or DBc-AMP of
30 /tg/g BW on days 1 and 2, and examined on days 2 and 3. The number of
dopa-positive cells increased after treatment with a-MSH (Fig. 4), exceeding the
controls on days 2 and 3 (P < 0-01). The melanocytes possessed well-developed
dendrites in addition to dopa-oxidase activity (Fig. 3). Treatment with DBcAMP also resulted in a remarkable increase in the number of dopa-positive cells
in the epidermis (Fig. 5). On the other hand, no change was observed in the
number of cells positive to the combined dopa-premelanin reaction after treatment with either a-MSH or DBc-AMP (Table 1).
The inducing effects of a-MSH on epidermal melanoblasts were observed even
Hormone-induced melanogenesis
83
Table 1. Effect ofa-MSH and DBc-AMP on the melanoblastmelanocyte population in the mouse epidermis
Mice were injected with a-MSH (1 /tg/g BW), DBc-AMP (30 /tg/g BW) or
Hanks' BSS on day 1 and day 2, and were fixed on day 3.
Group
No. of cells/01 mm2
(mean ± S.E.)
Control (Hanks' BSS)
a-MSH (1 /tg/g BW)
Control (Hanks' BSS)
DBc-AMP (30 /tg/g BW)
130-2 ±5-3
130-8 + 5-7
131-4 ± 3-5
1321 ±3-2
Table 2. Effect ofa-MSH on the epidermal melanocytes in the
19-day-old mice
Mice were injected with a-MSH (1 /*g/g BW) or Hanks' BSS on day 19 and
day 20, and were fixed on day 21.
Group
Day 19 control
Day 21 control
a-MSH (1 /ig/g BW)
No. of dopa positive
cells/0-1 mm2
(mean ± S.E.)
3-37 ±0-35
3-33 ±0-40
6-66 ±1-39*
* Significantly different from the day 19 control and the day 21 control (002 < P < 005);
not significantly different from the number of cells positive to the combined dopa-premelanin
reaction of the day 19 control (6-54 ±0-25) and the day 21 control (6-62 ±0-46).
after the number of dopa-positive cells declined. In epidermis from day-19
mice injected with a-MSH, a significant (P < 005) increase in the number of
dopa-positive cells was recognized. The number, however, did not exceed that
of the cells positive to the combined dopa-premelanin reaction in the controls
(Table 2).
Electron microscopic observation
In the epidermis of newborn mice, loose cells which possessed no desmosomes
and clear cytoplasm distinct from other epidermal cells were observed. They
contained well-developed Golgi apparatus and some premelanosomes (Figs. 6,
7). Therefore, they seemed to be melanoblasts, stained with the combined dopapremelanin reaction. The melanoblasts can be distinguished from Langerhans
cells which contain Langerhans granules. In the epidermis of day-3 mice, some
melanocytes with melanized melanosomes were found (Fig. 8). On the other
hand, the epidermis of a-MSH-treated mice contained melanocytes with
numerous fully melanized melanosomes (Fig. 9).
84
BM
T. HIROBE AND T. TAKEUCHI
Hormone-induced melanogenesis
85
In order to obtain a quantitative measurement, the numbers of melanosomes
of different stages were recorded for 200 microphotograms of nucleate cells in
the a-MSH-treated skin and the control skin, respectively. The stage of the
melanosome maturation was categorized according to Fitzpatrick, Hori,
Toda & Seiji (1969). Stages I and II include immature premelanosomes, while
melanized melanosomes are classified as stages III and IV. The a-MSH-treated
cells contained many more stage III-IV melanosomes than the control cells,
which included a considerable number of cells with no stage III-IV melanosomes
(Fig. 10, Table 3). In the a-MSH-treated cells, an increase in the number of
melanosomes per cell was also detected (Table 3). This result seems to indicate
that a-MSH stimulates not only the maturation of melanosomes but also de novo
formation of melanosomes.
DISCUSSION
The present report demonstrated that a large quantity of dopa-positive cells
were present in the epidermis of C57BL/10J mouse during the 3 weeks after
birth. Coleman (1962) estimated the tyrosinase activity of skin slices from the
C57BL/6J strain mice from neonatal to 30 days of age. The tyrosinase activity
was maximal at 4-5 days, and then gradually decreased and disappeared totally
by day 30. The change in the number of dopa-positive cells observed in our
study agrees well with the change in tyrosinase activity reported by Coleman.
It is well established that MSH causes reversible dispersion of melanosomes
in the melanocytes of amphibians (Shizume, Lerner & Fitzpatrick, 1954;
Wright & Lerner, 1960) and fishes (Chavin, 1956). In mammals, enhancement of
melanogenesis has been reported for guinea-pig (Snell, 1962; Clive & Snell,
1967) and human melanocytes (Lerner, Shizume & Bunding, 1954; Lerner &
McGuire, 1961) in response to MSH. Reversible dispersion of melanosomes
was also found in dispersed human epidermal melanocytes treated with a-MSH
or DBc-AMP (Kitano, 1973, 1974). However, there has been little indication
that the initiation of melanin synthesis in melanoblasts is induced by MSH.
The increase in the number of dopa-positive cells in the epidermis after treatment with a-MSH in our study seems to be the result of the induction of tyrosinase synthesis in the melanoblasts previously located in the epidermis. The
possibility that the treatment with a-MSH or DBc-AMP leads to the proliferation of active melanocytes present in the epidermis at birth can be excluded by
FIGURES 6 AND 7
Fig. 6. Electron micrograph of the epidermal melanoblast. BM, basement membrane; M, melanoblast; K, basal layer keratinocyte. x 29500.
Fig. 7. Golgi area of the epidermal melanoblast (Fig. 6). Small arrow: stage I
melanosome; large arrow: stageII melanosome; G, Golgi body; M, mitochondrion;
L, lysosome; RER, rough endoplasmic reticulum. x 69000.
86
T. HIROBE AND T. TAKEUCHI
87
Hormone-induced melanogenesis
(B) 2-day a-MSH (1 /ig/gBW)
(A) 2-day control (Hanks)
X = 82-52
X = 54-82
,
,
80 -
60-
40 -
20 -
80 100
20 40
Hl+lV/I+n + III+lV (%)
60
80
100
Fig. 10. Maturation of melanosomes by a-MSH. The percentages of stage III, IV
melanosomes against total melanosomes are shown. (A) control; (B) a-MSH
treated. The number of melanosomes (stage I-IV) were counted for 200 figures of
nucleate cells in the control skin and the a-MSH-treated skin.
Table 3. Effect of a-MSH on melanosome maturation
Classification
of melanosomes
2-day control
(Hanks)
2-day a-MSH
(1 fig/g BW)
/-test
I+11
III + IV
Total
6-41 ±0-41
9-88 ±0-68
16-29±114
3•22 ±0-23
17 •38 ±0-83
20 •60 ±1-06
P < 0001
P < 0001
0005 < P < 001
Each value is the number of melanosomes per cell (mean ± S.E. of 200 cells).
the observation that the number of dopa-positive cells after treatment with
either a-MSH or DBc-AMP was comparable to that of the melanoblastmelanocyte population (about 140 cells/0-1 mm2) in the epidermis. Migration of
melanocytes from the dermis in response to a-MSH is also unlikely since no
migrating figure through the basement membrane was observed at this stage.
This was confirmed by electron microscopic observation. In the control group,
FIGURES 8 AND 9
Fig. 8. Electron micrograph of the epidermal melanocyte (control). Small arrow:
stage I melanosome; large arrow: stage II melanosome; m3, stage III melanosome;
m4; stage IV melanosome; G, Golgi body; M, mitochondrion; RER, rough endoplasmic reticulum; C, centriole; T, tonofilament; K, basal layer keratinocyte; BM,
basement membrane, x 44600.
Fig. 9. Electron micrograph of the epidermal melanocyte (a-MSH treated). Small
arrow: stage III melanosome; large arrow: stage II melanosome; m4, stage IV
melanosome; G, Golgi body; M, mitochondrion; CV, coated vesicle; RER, rough
endoplasmic reticulum; D, desmosome; K, basal layer keratinocyte. x 25000.
88
T. HIROBE AND T. TAKEUCHI
28 % of melanocytes (melanoblasts) contained stage I and II melanosomes
without melanin deposition. On the other hand, in the a-MSH-injected group,
most of the melanocytes contained numerous stage III and IV melanosomes.
The stimulation of the initiation of melanin synthesis of melanoblasts in the
epidermis indicates that these melanoblasts are competent for induction by
MSH and that the cells respond to MSH under either normal or experimental
circumstances. Although there is no direct evidence to show the elevation of
MSH level in neonatal mouse skin, it is probable that MSH is released shortly
after birth so that the epidermal melanoblasts are stimulated to initiate melanin
synthesis. It has been reported that ACTH production in the rat pituitary begins
about the time of birth (Setalo & Nakane, 1972; Nakane, 1975) and a-MSH
production in the foetal rat pituitary begins from day 19 of gestation (Dupouy
& Dubois, 1975).
Geschwind, Huseby & Nishioka (1972) showed in yellow mice (C57BL/6JAy-) a 2-5- to 5-fold increase in tyrosinase activity in plucked adult skin
within 24 h of injection of MSH, and increases of two to three times in the
normally elevated levels of the unplucked 5-day-old mice. If the increases in the
tyrosinase activity in both cases mentioned above are caused by an increase in
the number of functional cells, it can be supposed that MSH induces the
initiation of melanin synthesis in the melanoblasts of yellow mice as well as in
those of black mice.
It is generally accepted that the response of melanocytes to MSH is mediated
through c-AMP: MSH activates membrane-bound adenyl cyclase which converts ATP to c-AMP. This concept came from the fact that the stimulating
effects of MSH on the cells are also achieved by c-AMP. The dispersion of
melanosomes was observed in melanocytes of fishes (Novales & Fujii, 1970;
Abramowitz & Chavin, 1974) and of amphibians (Bitensky & Burstein, 1965;
Novales & Davis, 1967; Abe et al 1969 a, b) when treated with c-AMP. It was
also reported in mouse melanoma cells that MSH and c-AMP elevated tyrosinase activity and melanin content (Johnson & Pastan, 1972; Kreider, Rosenthal
& Lengle, 1973; Wong & Pawelek, 1973; Pawelek et al. 1974; Wong, Pawelek,
Sansone & Morowitz, 1974; Wong & Pawelek, 1975) and that MSH activated
adenyl cyclase (Bitensky & Demopoulos, 1970; Bitensky, Demopoulos &
Russell, 1972). In our study, the effect of c-AMP mimicked that of a-MSH in
inducing melanin synthesis. It is therefore conceivable that MSH-induced
melanin synthesis in epidermal melanoblasts is mediated through c-AMP. This
assumption is supported by the results with theophylline, which induced in
organ culture an increase in the number of epidermal melanocytes, similar to
that induced by a-MSH and DBc-AMP (Hirobe & Takeuchi, 1977). We also
demonstrated that the number of the dopa-positive cells in organ-cultured skin
did not increase when treated with a-MSH or DBc-AMP in the presence of
actinomycin D or cycloheximide (Hirobe & Takeuchi, 1977).
Hormone-induced melanogenesis
89
The authors express their thanks to Dr R. H. Kahn of the University of Michigan for
reading the manuscript. We are also grateful to Mr F. Sato for his technical assistance. This
work was in part supported by Grant 944008 from the Ministry of Education.
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(Received 18 June 1976; revised 29 September 1976)