Induction of melanogenesis in the epidermal melanoblasts of newborn mouse skin bymsh

Transcription

1 /. Embryol. exp. Morph. Vol. 37, pp , Printed in Great Britain Induction of melanogenesis in the epidermal melanoblasts of newborn mouse skin bymsh ByTOMOHISA HIROBE AND TAKUJI TAKEUCHI 1 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

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 (N 6,O 2 '- 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 01 % 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 mm 2 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 2h 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,

3 Hormone-induced melanogenesis Days after birth 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 mm 2 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.

4 82 T. HIROBE AND T. TAKEUCHI s 80 a, o o 60 a 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 DBc- AMP 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

5 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 mm 2 (mean ± S.E.) Control (Hanks' BSS) ±5-3 a-msh (1 /tg/g BW) Control (Hanks' BSS) ± 3-5 DBc-AMP (30 /tg/g BW) 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 No. of dopa positive cells/0-1 mm 2 (mean ± S.E.) Day 19 control 3-37 ±0-35 Day 21 control 3-33 ±0-40 a-msh (1 /ig/g BW) 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).

6 84 BM T. HIROBE AND T. TAKEUCHI

7 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 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

8 86 T. HIROBE AND T. TAKEUCHI

9 Hormone-induced melanogenesis (A) 2-day control (Hanks) X = (B) 2-day a-msh (1 /ig/gbw) X = 82-52,, Hl+lV/I+n + III+lV (%) 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 ± ± ± ± ± ±1-06 P < 0001 P < < 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 mm 2 ) 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 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

10 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/6J- A y -) 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).

11 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 from the Ministry of Education. REFERENCES ABE, K., BUTCHER, R. W., NICHOLSON, W. E., BAIRD, C. E., LIDDLE, R. A. & LIDDLE, G. W. (1969a). Adenosine 3',5'-monophosphate (cyclic AMP) as the mediator of the actions of melanocyte stimulating hormone (MSH) and norepinephrine on the frog skin. Endocrinology 84, ABE, K., ROBISON, G. A., LIDDLE, G. W., BUTCHER, R. W., NICHOLSON, W. E. & BAIRD, C. E. (19696). Role of cyclic AMP in mediating the effects of MSH, norepinephrine, and melatonin on frog skin color. Endocrinology 85, ABRAMOWITZ, J. & CHAVIN, W. (1974). In vitro response of goldfish (Carassius auratus L.) dermal melanophores to cyclic 3', 5'-nucleotides, nucleoside 5'-phosphates and methylxanthines. J. cell. Physiol. 84, BITENSKY, M. W. & BURSTEIN, S. R. (1965). Effects of cyclic adenocine monophosphate and melanocyte-stimulating hormone on frog skin in vitro. Nature, Lond. 208, BITENSKY, M. W. & DEMOPOULOS, H. B. (1970). Activation of melanoma adenyl cyclase by MSH. /. invest. Derm. 54, 83. BITENSKY, M. W., DEMOPOULOS, H. B. & RUSSELL, V. (1972). MSH-responsive adenyl cyclase in the Cloudman S-91 melanoma. Pigmentation: Its Genesis and Biologic Control (ed. V. Riley), pp New York: Appleton-Century-Crofts. CHAVIN, W. (1956). Pituitary-adrenal control of melanization in xanthic goldfish, Carassius auratus L. /. explzool. 133, CLIVE, D. & SNELL, R. (1967). Effect of alpha melanocyte stimulating hormone on mammalian hair color. /. invest. Derm. 49, COLEMAN, D. L. (1962). Effect of genie substitution on the incorporation of tyrosine into the melanin of mouse skin. Archs Biochem. Biophys. 96, DUPOUY, J. P. & DUBOIS, M. P. (1975). Ontogenesis of the a-msh, /?-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity. Cell Tiss. Res. 161, FITZPATRICK, T. B., HORI, Y., TODA, K. & SEIJI, M. (1969). Melanin 1969: some definitions and problems. Jap. J. Derm. Ser. B 79, GESCHWIND, T. L, HUSEBY, R. A. & NISHIOKA, R. (1972). The effect of melanocyte-stimulating hormone on coat color in the mouse. Recent Prog. Norm. Res. 28, HIROBE, T. & TAKEUCHI, T. (1976). Induction of melanogenesis in vitro in the epidermal melanoblasts of new-born mouse skin. In Vitro (in press). JOHNSON, G. S. & PASTAN, I. (1972). N 6,O 2 '-dibutyryl adenosine 3',5'-monophosphate induces pigment production in melanoma cells. Nature New Biol. 237, KnANO, Y. (1973). Stimulation of dendritogenesis in human melanocytes by dibutyryl adenosine 3',5'-cyclic monophosphate in vitro. Arch. Derm. Forsch. 248, KITANO, Y. (1974). Response of melanocytes to cyclic AMP. Jap. J. Derm. 84, KREIDER, J. W., ROSENTHAL, M. & LENGLE, N. (1973). Cyclic adenosine 3',5'-monophosphate in the control of melanoma cell replication and differentiation. /. natn. Cancer Inst. 50, LERNER, A. B. & MCGUIRE, J. S. (1961). Effect of alpha- and beta-melanocyte stimulating hormone on the skin color of man. Nature, Lond. 189, LERNER, A. B., SHIZUME, K. & BUNDING, I. (1954). The mechanism of endocrine control of melanin pigmentation. /. clin. Endocr. Metab. 14, MISHIMA, Y. (1960). New technic for comprehensive demonstration of melanin, promelanin, and tyrosinase sites. /. invest. Derm. 34, MISHIMA, Y. (1964). Electron microscopic cytochemistry of melanosomes and mitochondria. /. Histochem. Cytochem. 12, MISHIMA, Y. & LOUD, A. V. (1963). The ultrastructure of unmelanized pigment cells in induced melanogenesis. Ann. N. Y. Acad. Sci. 100,

12 90 T. HIROBE AND T. TAKEUCHI MISHIMA, Y., LOUD, A. V. & SCHAUB, F. F. (1962). Electron microscopy of premelanin. /. invest. Derm. 39, NAKANE, P. K. (1975). Cell differentiation in rat pituitary gland. Symposia Cell Biol. 27, NOVALES, R. R. & DAVIS, W. J. (1967). Melanin dispersing effect of adenosine 3',5'-monophosphate on amphibian melanophores. Endocrinology 81, NOVALES, R. R. & FUJII, R. (1970). A melanin dispersing effect of cyclic adenosine monophosphate on Fundulus melanophores /. cell. Physiol. 75, PAWELEK, J., SANSONE, M., MOROWITZ, J., MOELLMANN, G. & GODAWSKA, E. (1974). Genetic control of melanization: isolation and analysis of amelanotic variants from cultured melanotic melanoma cells Proc. natn. Acad. Sci. U.S.A. 71, RAWLES, M. E. (1940). The development of melanophoies from embryonic mouse tissues grown in the coelom of chick embryos. Proc. natn. Acad. Sci. U.S.A. 26, RAWLES, M. E. (1947). Origin of pigment cells from the neural crest in the mouse embryo. Physiol. Zool. 20, RAWLES, M. E. (1948). Origin of melanophores and their role in development of color patterns in vertebrates. Physiol. Rev. 28, REYNOLDS, J. (1954). The epidermal melanocytes of mice. /. Anat. 88, ROVEE, D. T. & REAMS, W. M. (1964). An experimental and descriptive analysis of melanocyte population in the venter of the PET mouse. Anat. Rec. 149, SETALO, G. & NAKANE, P. K. (1972). Studies on the functional differentiation of cells in fetal anterior pituitary glands of rats with peroxidase-labeled antibody method. Anat. Rec. 172, SHIZUME, K., LERNER, A. B. & FITZPATRICK, T. B. (1954). In vitro bioassay for the melanocyte stimulating hormone. Endocrinology 54, SNELL, R. S. (1962). Effect of melanocyte stimulating hormone of the pituitary on melanocytes and melanin in the skin of guinea-pigs. /. Endocrinol. 25, TAKEUCHI, T. (1968). Genetic analysis of a factor regulating melanogenesis in the mouse melanocyte. Jap. J. Genet. 43, WONG, G. & PAWELEK, J. (1973). Control of phenotypic expression of cultured melanoma cells by melanocyte stimulating hormones. Nature New Biol. 241, WONG, G., PAWELEK, J., SANSONE, M. & MOROWITZ, J. (1974). Response of mouse melanoma cells to melanocyte stimulating hormone. Nature, Lond. 248, WONG, G. & PAWELEK, J. (1975). Melanocyte stimulating hormone promotes activation of pre-existing tyrosinase molecules in Cloudman S91 melanoma cells. Nature, Lond. 255, WRIGHT, M. R. & LERNER, A. B. (1960). On the movement of pigment granules in frog melanocyte. Endocrinology 66, (Received 18 June 1976; revised 29 September 1976)