A cut was made on the middorsal skin of mice of various ages of strain C57BL/10J using fine iridectomy scissors. Specimens from the wounded skins were fixed at various days after wounding and were subjected to the dopa reaction and to the combined dopa-premelanin reaction. When the dorsal skins of 1·5-day-old mice were wounded, the melanocyte population positive to the dopa reaction as well as the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction increased dramatically in the epidermis adjacent to a skin wound. Pigment-producing melanocytes in mitosis were frequently found in the vicinity of a wound immediately after wounding. When the dorsal skins of 4·5-day-old mice were wounded, the increase in the melanocyte and melanoblast-melanocyte populations was smaller than that of 1·5-day-old mice. The increase in number of pigment-producing melanocytes in mitosis was reduced and delayed as compared to 1·5-day-old mice. When the dorsal skins of 8·5-, 20·5-, and 60·5-day-old mice were wounded, the increase in the melanocyte and melanoblast-melanocyte populations was much smaller than the newborn mice. Moreover, pigmentproducing melanocytes in mitosis were never found. These results indicate that the proliferative response of mouse epidermal melanocytes to skin wounding becomes delayed and diminished with development.

It has been reported that the number of functioning melanocytes in the epidermis of mammalian skin is increased by external stimuli, such as ultraviolet irradiation (Quevedo & Smith, 1963; Quevedo et al. 1965), carcinogen treatment (Szabó, 1963; Iwata et al. 1981) and skin wounding (Staricco, 1961; Snell, 1963; Giacometti & Allegra, 1967; Giacometti et al. 1972). However, whether the increase in the number of melanocytes is due to differentiation of precursor melanoblasts or to mitotic division of melanocytes was not resolved. Recent studies using adult mouse ear skin show that the epidermal melanocytes are continuously renewed by mitotic division in normal circumstances (Rosdahl & Lindstrom, 1980; Rosdahl & Bagge, 1981) and that the proliferative activity of epidermal melanocytes is stimulated by ultraviolet irradiation (Rosdahl & Szabó, 1976, 1978; Rosdahl, 1978). In the dorsal skin of newborn mice, the pigment-producing melanocytes were shown to undergo mitotic division during the healing of skin wounds (Hirobe, 1983). Mitotic melanocytes were never found in normal epidermis. These results indicate that the differentiated melanocytes in the mouse epidermis can proliferate with or without external stimuli. However, it is not known whether the proliferative activity of differentiated melanocytes changes with development. The present study was designed to solve this problem through the wounding of the skin during the postnatal development of mice. The response of epidermal melanocytes to skin wounding was analysed by means of light microscopy using techniques of histochemistry and of conventional histology.

The animals used in this study were the mouse, Mus musculus, of strain C57BL/10J (substrain C57BL/10JHir). They were given water, fed ad libitum on a commercial diet (Clea Japan) and maintained at 24 ± I °C with 40–60 % relative humidity; 12 h of fluorescent light was provided daily.

The method of skin wounding was reported previously (Hirobe. 1983). A full-thickness cut 7mm long was made anteroposteriorly on the middorsal skin of mice of 1·5, 4·5, 8·5,20·5 and 60·5 days of age using fine iridectomy scissors. The incision extended from the epidermis to the deepest layer of macrophages under the panniculus carnosus muscle. Immediately after the incision was made, the margins retracted and the wound cavity widened to about 2 mm. The wounds were not sutured or dressed. The animals were killed at various times thereafter. The entire wound area, including the bed, was then removed from the animals. Biopsy specimens from the wounded skins and from corresponding fields of skin from intact control animals were fixed with 16 % formalin in phosphate buffer (pH 7·0) for 20–24 h at 2°C. Each age group was represented by three mice and one sample was obtained from the wounded skin area of each animal. One wounded skin consisted of two regenerating epidermes, namely right and left sides of the wound bed. The experiments were repeated three times. The specimens were washed with distilled water and incubated with 0·1% i.-dopa (3,4-dihydroxy-phenylalanine, Wako) solution in phosphate buffer (pH 7·4) for 20–24 h at 37°C. This staining reveals tyrosinase-containing differentiated melanocytes (Hirobe, 1982). The specimens were oriented transversely to the wound edge and 10 μm serial sections were deparaffinized and counterstained with eosin. For combined dopa-premelanin reaction (combined dopa-ammoniacal silver nitrate staining), deparaffinized sections after the dopa treatment were incubated with 10% ammoniacal silver nitrate (Wako) solution for 10 min at 58°C (Mishima, 1960; Hirobe & Takeuchi, 1977). This preferential staining reveals undifferentiated melanoblasts that contain unmelanized stage-1 and -II melanosomes in addition to tyrosinase-containing differentiated melanocytes (Mishima, 1964; Hirobe, 1982). The specimens were also counterstained with eosin.

The number of melanocytes (cells positive to the dopa reaction) and the number of stage-1 and -11 melanosome-containing melanoblasts plus melanocytes (cells positive to the combined dopa-premelanin reaction) were estimated per 0·1 mm2 of the epidermis of each section of skin, and the calculations based on ten consecutive sections with the width of 1 mm covering the area 0·1 mm2 of the skin.

In some cases, the specimens from wounded and control animals were fixed with Bouin’s fixative and sectioned transversely to the wound edge. Serial sections, 8 μm in thickness, were stained with haematoxylin and eosin. Pigment-producing melanocytes in resting phase and mitosis were examined with the light microscope using numerous sections.

Changes in the melanocyte and melanoblast-melanocyte populations in the epidermis after wounding

When the dorsal skins of 1·5-day-old mice were wounded, the melanocyte population positive to the dopa reaction as well as the melanoblast-melanocyte population positive to the combined dopa-premelanin reaction increased dramatically in the epidermis adjacent to a skin wound (Fig. 1A,B). On day 1 after wounding, the melanocyte and melanoblast-melano-cyte populations in the epidermis within 1 mm of the wound edge significantly (P<0·05) exceeded the controls on day 0 and day 1 (Fig. 2). Both populations showed maximal number on day 3, then gradually decreased (Fig. 2). From day 3, both populations were observed in the roots of hair follicles in addition to the basal layer of epidermis. This suggests that the epidermal melanoblasts or melanocytes migrate into hair follicles. In all stages of wound healing, the number of melanocyte population did not differ significantly from that of melanoblast-melanocyte population, suggesting that all melanoblasts differentiate into melanocytes in the epidermis adjacent to a skin wound. The melanocyte and melanoblast-melanocyte populations appeared in the regenerating wound epidermis on day 3 and increased in number (Fig. 2). Both populations were observed in the roots of the advancing epidermal sheets from day 3 and lagged behind their forward edges. This suggests that epidermal melanoblasts or melanocytes increase in number adjacent to a skin wound and, thereafter, migrate into the regenerating wound epidermis. Both populations showed a maximal number on day 7 and decreased thereafter. From day 7, both populations were observed in the roots of hair follicles in addition to the basal layer of the regenerating wound epidermis, suggesting that the epidermal melanoblasts or melanocytes migrate into hair follicles. In all stages of wound healing, the melanocyte and melanoblast-melanocyte populations did not differ significantly in number, suggesting that all melanoblasts differentiate into melanocytes in the regenerating wound epidermis.

Fig. 1.

Vertical sections of the dorsal skins of C57BL/10J mice during wound healing. A cut was made on the middorsal skins of 1·5- (A,B), 4·5- (C,D), 8·5- (E,F), 20·5- (G,H) and 60·5- (I,J) day-old mice. Cells positive to the dopa reaction (A,C,E,G,I) as well as to the combined dopa-premelanin reaction (B,D,F,H,J) are shown in the dorsal skins on day 3 after wounding. Epidermal melanocytes or melanoblasts are seen in the vicinity of a wound (arrows). The number of epidermal melanocytes or melanoblasts is greater in younger mice (A–D) than in older mice (E–J) The right sides of all figures indicate the wound edge and regenerating wound epidermis. ×120.

Fig. 1.

Vertical sections of the dorsal skins of C57BL/10J mice during wound healing. A cut was made on the middorsal skins of 1·5- (A,B), 4·5- (C,D), 8·5- (E,F), 20·5- (G,H) and 60·5- (I,J) day-old mice. Cells positive to the dopa reaction (A,C,E,G,I) as well as to the combined dopa-premelanin reaction (B,D,F,H,J) are shown in the dorsal skins on day 3 after wounding. Epidermal melanocytes or melanoblasts are seen in the vicinity of a wound (arrows). The number of epidermal melanocytes or melanoblasts is greater in younger mice (A–D) than in older mice (E–J) The right sides of all figures indicate the wound edge and regenerating wound epidermis. ×120.

Fig. 2.

Changes in the number of melanocyte positive to the dopa reaction (A) and melanoblast-melanocyte positive to the combined dopa-premelanin reaction (B) in the dorsal skin of 1·5-day-old C57BL/10J mice per 0·1 mm2 of the epidermis within 1 mm of the wound edge (▪), the control epidermis (▫), and the regenerating wound epidermis (○). Bars indicate S.E.M.

Fig. 2.

Changes in the number of melanocyte positive to the dopa reaction (A) and melanoblast-melanocyte positive to the combined dopa-premelanin reaction (B) in the dorsal skin of 1·5-day-old C57BL/10J mice per 0·1 mm2 of the epidermis within 1 mm of the wound edge (▪), the control epidermis (▫), and the regenerating wound epidermis (○). Bars indicate S.E.M.

When the dorsal skins of 4·5-day-old mice were wounded, the increase in the melanocyte (Fig. 1C) and melanoblast-melanocyte populations (Fig. 1D) in the epidermis adjacent to a skin wound was smaller than that of 1·5-day-old mice (Fig. 3). In the regenerating wound epidermis, both populations appeared on day 2, increased in number until day 7, then gradually decreased (Fig. 3).

Fig. 3.

Changes in the number of melanocyte and melanoblast-melanocyte populations in the dorsal skin of 4·5-day-old C57BL/10J mice. Data presented as Fig. 2.

Fig. 3.

Changes in the number of melanocyte and melanoblast-melanocyte populations in the dorsal skin of 4·5-day-old C57BL/10J mice. Data presented as Fig. 2.

When the dorsal skins of 8·5- (Figs 1E,F, 4) or 20·5- (Figs 1G,H, 5) day-old mice were wounded, the melanocyte and melanoblast-melanocyte populations in the epidermis within 1 mm of the wound edge exceeded that of the control epidermis. However, the increase in both populations was much smaller than that of the younger mice. In the regenerating wound epidermis, the melanocyte and melanoblast-melanocyte populations appeared on day 3, increased in number until day 10, then decreased. Neither populations exceeded the initial density (Figs 4, 5).

Fig. 4.

Changes in the number of melanocyte (A) and melanoblast-melanocyte (B). Populations of the dorsal skin of 8·5-day-old C57BL/10J mice. Data presented as Fig. 2.

Fig. 4.

Changes in the number of melanocyte (A) and melanoblast-melanocyte (B). Populations of the dorsal skin of 8·5-day-old C57BL/10J mice. Data presented as Fig. 2.

Fig. 5.

Changes in the number of melanocyte (A) and melanoblast-melanocyte (B). Populations of the dorsal skin of 20·5-dav-old C57BL/10J mice. Data presented as Fig. 2.

Fig. 5.

Changes in the number of melanocyte (A) and melanoblast-melanocyte (B). Populations of the dorsal skin of 20·5-dav-old C57BL/10J mice. Data presented as Fig. 2.

When the dorsal skins of 60·5-day-old mice were wounded, no marked increase in the melanocyte and melanoblast-melanocyte populations in the epidermis adjacent to a skin wound was observed (Figs 1I,J, 6). In the regenerating wound epidermis, the melanocyte and melanoblast-melanocyte populations slightly increased in number after wounding. Both populations on day 14 significantly (P<0·05) exceeded the initial density.

Fig. 6.

Changes in the number of melanocyte (A) and melanoblast-melanocyte (B). Populations of the dorsal skin of 60·5-day-old C57BL/10J mice. Data presented as Fig. 2.

Fig. 6.

Changes in the number of melanocyte (A) and melanoblast-melanocyte (B). Populations of the dorsal skin of 60·5-day-old C57BL/10J mice. Data presented as Fig. 2.

Changes in the mitotic indices of melanocytes in the epidermis after wounding

When the dorsal skins of 1·5-day-old mice were wounded, melanocytes in mitotic division were found in the epidermis within 1 mm of the wound edge from day 1 to day 4 (Figs 7A,B, 8). Mitotic melanocytes were most frequently found on day 2 (Mitotic index =5·06%; Fig. 8). In contrast, mitotic melanocytes were never found in either the regenerating wound epidermis or control epidermis.

Fig. 7.

Vertical sections of the dorsal skins of C57BL/10J mice on day 2 (A), day 3 (B), day 5 (C) and day 7 (D) after wounding. A cut was made on the middorsal skin of 1·5- (A.B) and 4·5- (C,D) day-old mice. Melanocytes in metaphase are observed in the epidermis (arrows) adjacent to a skin wound. All specimens were fixed with Bouin’s fixative and stained with haematoxylin and eosin. No dopa or silver treatment. ×460.

Fig. 7.

Vertical sections of the dorsal skins of C57BL/10J mice on day 2 (A), day 3 (B), day 5 (C) and day 7 (D) after wounding. A cut was made on the middorsal skin of 1·5- (A.B) and 4·5- (C,D) day-old mice. Melanocytes in metaphase are observed in the epidermis (arrows) adjacent to a skin wound. All specimens were fixed with Bouin’s fixative and stained with haematoxylin and eosin. No dopa or silver treatment. ×460.

Fig. 8.

Developmental change of the mitotic indices of epidermal melanocytes of the dorsal skin of C57BL/10J mice stimulated by skin wounding. A cut was made on the middorsal skin of 1·5- (○), 4·5- (•), and 8·5- (▫) day-old mice (arrows). Specimens were fixed at various days after wounding with Bouin’s fixative. Mitotic indices of melanocytes in the epidermis within I mm of the wound edge are shown. When the dorsal skins of 20·5-, and 60·5-day-old mice were wounded, no melanocytes in mitosis were found.

Fig. 8.

Developmental change of the mitotic indices of epidermal melanocytes of the dorsal skin of C57BL/10J mice stimulated by skin wounding. A cut was made on the middorsal skin of 1·5- (○), 4·5- (•), and 8·5- (▫) day-old mice (arrows). Specimens were fixed at various days after wounding with Bouin’s fixative. Mitotic indices of melanocytes in the epidermis within I mm of the wound edge are shown. When the dorsal skins of 20·5-, and 60·5-day-old mice were wounded, no melanocytes in mitosis were found.

When the dorsal skins of 4·5-day-old mice were wounded, mitotic melanocytes were also found in the epidermis adjacent to a skin wound from day 3 to day 7 (Figs 7C,D, 8). The mitotic index was maximal on day 5 (0·59%). The increase in the mitotic indices of 4·5-day-old mice was reduced and delayed as compared to 1·5-day-old mice (Fig. 8). In contrast, mitotic melanocytes were never found in either the regenerating wound epidermis or control epidermis.

When the dorsal skins of 8·5-, 20·5- or 60·5-day-old mice were wounded, mitotic melanocytes were never found in the epidermis within 1 mm of the wound edge, the regenerating wound epidermis, and control epidermis (Fig. 8).

The present study demonstrated that the proliferative response of mouse epidermal melanocytes to skin wounding was diminished as developmental age advanced, since the increase in the melanocyte and melanoblast-melanocyte populations as well as the increase in the mitotic indices of pigment-producing melanocytes of older mice was reduced and delayed as compared to newborn mice. It has been reported that melanocytes begin to differentiate in the epidermis around the time of birth and increase in number until 4 days, then gradually decrease and disappear by 30 days of age in the dorsal skin of C57BL/10J strain mice (Hirobe & Takeuchi, 1977; Hirobe, 1984). Therefore, it is conceivable that the differentiating melanocytes or newly differentiated melanocytes which can be stimulated to undergo mitosis by skin wounding diminish in the epidermis after birth. They are thought to migrate into hair follicle in early days after birth. These potent cells, which are considered as ‘stem cell’, may undergo mitosis by external stimuli even after migrating into hair bulbs. This hypothesis is supported by the observations of Silver et al. (1969) that the melanocytes in the hair follicle began to divide when the hair growth cycle entered into new activation stage (Anagen; Dry, 1926).

In the epidermis of hairy skin, epidermal melanocytes are found only during the early weeks after birth (Quevedo et al. 1966; Takeuchi, 1968; Hirobe & Takeuchi, 1977). However, in the glabrous skin of ear, nose and tail, numerous differentiated melanocytes are found in the epidermis of adult mice. Recent studies using the mouse ear skin show that the epidermal melanocyte is normally in continuous renewal (Rosdahl et al. 1980, 1981). However, it is not known whether the mitotic activity of epidermal melanocytes in the mouse skin changes with development. Also, the epidermal melanocytes of adult human skin are reported to undergo mitotic division normally (Jimbow et al. 1975). However, it is not clear whether the mitotic activity of human melanocytes changes from newborn to adult. The solution of these problems is expected to clarify the mechanism of the proliferation of mammalian epidermal melanocytes during development.

Bucher et al. (1964) showed that in a regenerating rat liver DNA synthesis became reduced and delayed as developmental age advanced. In the postnatal development of mouse seminal vesicle, the proliferative activity of seminal vesicle cells was shown to be maximal at both 8 and 30 days of age (Okamoto et al. 1982). Shirasawa & Yoshimura (1982) reported that the mitotic rate of GH and prolactin cells increased from 5 to 70 days of age in male rat pituitary. In contrast, ACTH- and TSH-cells decreased with age. On the other hand, Takahashi et al. (1984) reported that the mitotic indices of prolactin cells decreased steadily from 20 days of age in male rat pituitary, while the mitotic rate in female rat was maximal at 60 days of age and then decreased. These studies together with the present study show that the differentiated cells in mammals can undergo mitosis normally or by external stimuli and that the proliferative activity of mammalian cells changes during development. However, it remains to be solved in a future study whatever common mechanism exists in regulating the proliferative activity of mammalian cells in development.

This work was supported in part by Grant 58740290 from the Ministry of Education, Japan.

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