Walter C. Quevedo

The Developmental Fate of Melanocytes in Murine Skin

In the hair-covered general body skin of adult pigmented mice, melanogenically-active melanocytes are largely restricted to the bulbs of growing hair follicles. After cutaneous applications of appropriate chemical agents or exposure to ultraviolet light (UV), numerous melanotic melanocytes are found at the dermoepidermal junction of the interfollicular (superficial) epidermis and in the upper external root sheaths of hair follicles [6, 15 to 18]. These findings suggest that an extensive system of inactive “precursor” melanocytes forms an integral part of the epidermis in the hairy skin of adult mice. The melanocytes of this system although programmed to produce melanin granules and to discharge them into epidermal cells must await the application of an appropriate stimulus [13].

Formation, Chemical Composition and Function of Melanin Pigments

Pigments of the mammalian skin may be divided into four major classes; (a) brown-black pigments of melanin, (b) yellow pigments of carotinoid, (c) red pigments of oxygenated hemoglobulin and (d) blue pigments of reduced hemoglobulin. The major determinant of skin pigmentation is melanin. Pigmentation is related to the number, size, type, and distribution pattern of melanin-containing cytoplasmic particles, i.e. melanosomes (Seiji et al. 1961; Jimbow et al. 1976). The melanosomes are products of secretory cells, melanocytes, that are present in the basal layer of the epidermis. Melanocytes transfer their product, melanosomes, into keratinocytes, and they become distributed throughout the epidermis by the upward movement of basal keratinocytes. The symbiotic interaction of these different cells, i.e. melanocytes and the associated pool of keratinocytes, is called epidermal melanin unit (Fitzpatrick and Breathnach 1963) (Fig. 1, cf. Fig. 1, Chap.12,this Vol.).

Human skin color: Origin, variation and significance

Human evolution has been characterized by a marked reduction in body hair and the consequent increased importance of the naked pigmented epidermis as a screen against the harmful effects of solar radiation. Within the epidermis and hair follicles are melanin-containing melanosomes synthesized by melanocytes and transferred to surrounding keratinocytes. Human skin and hair color chiefly depend on the quantities as well as the qualities of melanosomes which are distributed throughout the keratinocytes of the epidermis and hair. Melanin synthesis requires the copper-containing enzyme tyrosinase which acts on tyrosine as its initial substrate. Eumelanin (black-brown) synthesis may be found at all pigmented sites in the body whereas pheomelanin (yellow-red) apparently is only produced within hair follicles. Albinism results when melanocytes of hair, skin and eyes either fail to synthesize melanin owing to a tyrosinase defect, or produce markedly reduced amounts of it for as yet little understood reasons. In general, the human melanin pigmentary system follows the general mammalian plan and shows few, if any, striking differences from those possessed by other primates. Accordingly, although many genes may influence skin and hair color in man as they do in other mammals such as the mouse, it is likely that ethnic variation in human skin pigmentation may involve as few as three to six gene pairs which interact during melanogenesis. Multiple alleles at each of the loci of the polygenic series permit the generation of a gradient of skin color which closely models the variation found on a worldwide basis among humans today. Solar radiation-induced somatic gene mutations within melanocytes may account for the origin of human freckles and spontaneous somatic mutations of some type for the documented reduction of functional epidermal melanocytes with advancing age. Although human cutaneous melanin pigmentation may have functions beyond photoprotection, many of the proposed adaptive functions of melanin are probably fanciful, for they result from dubious attempts to create too tight a link between natural selection as a perfecting force in human evolution and current human physiology.

Effect of Altered Copper Metabolism Induced by Mottled Alleles and Diet on Mouse Tyrosinase

Summary An analysis of tyrosinase activity in extracts of hair bulbs from mice bearing alleles at the mottled locus, Mobr (brindled), and Moblo (blotchy), indicates that reduced melanogenesis in mottled mice is associated with restricted availability of copper. Although tyrosinase activity in 35,000g supernatants of hair-bulb homogenates of mottled mice is reduced compared to that of nonmottled controls, addition of copper to the former but not the latter supernatants markedly increases tyrosinase activity as measured in polyacrylamide gel electropherograms (dopa as substrate) or by the Pomerantz radioassay (tyrosine as substrate). Comparable results were obtained when tyrosinase from dietarily copper-deficient hypopigmented mice was assayed by either method. In the electropherograms of dietarily and genetically copper-deficient hair-bulb supernatants, the enhancement of activity is selectively restricted to the T1 variety of tyrosinase. It is concluded that alleles at the mottled locus influence tyrosinase and consequently melanin pigmentation by acting on melanocytes to reduce copper levels below those necessary to produce a normal complement of functional tyrosinase molecules.

Action of Trypsin and Detergents on Tyrosinase of Normal and Malignant Melanocytes

Summary The T1 variety of tyrosinase is present in both particulate and soluble or readily solubilized forms in the pigmented hypodermis (hair bulbs) of C57BL mice and Harding-Passey mouse melanoma. Trypsin treatment of 35,000g supernatants containing the microsomal (small granule) fraction of gentle homogenates of hair bulbs and melanoma results in significantly increased T1 activity within polyacrylamide gels. Similar treatment of 100,000g supernatants results in a slight increase in T1 activity. Addition of Triton-X or DOC to 35,000g supernatants of hair bulb and melanoma homogenates followed by centrifugation at 100,000g results in a marked enhancement of T1 when the latter supernatants are treated with trypsin. In the absence of trypsin treatment, T1 activity is comparable to nondetergent-treated controls. A slow-moving dopa-reactive band (Ts) is found in electropherograms of the nontrypsinized 100,000g supernatants of detergent-treated 35,000g supernatants. It is absent in those treated with trypsin. The slow-moving enzyme appears to give rise to T1 molecules when eluted from acrylamide gels and even to a greater extent when elution is combined with trypsin treatment prior to reelectro-phoresis. In mammals, tyrosinase apparently is not derived by a proteolytic activation of protyrosinase.

Dopa oxidase in melanocytes of x-irradiated quiescent (telogen) hair follicles.

Summary “Dopa oxidase” activity was absent in melanocytes of telogen hair follicles of non-irradiated mice. Following partial-body exposure to 200 r week of X-radiation for 3 or 4 weeks, numerous dopa reactive dendritic cells were observed in the epithelial germ regions of irradiated quiescent hair follicles. Cells of the dermal papillae in irradiated and non-irradiated telogen hair follicles failed to exhibit “dopa oxidase” activity. The results thus support the view that melanocytes normally are restricted to the epithelial germ regions of hair follicles during telogen stage of hair growth cycle.

Ultrastructural observations on DMBA-induced dermal hyperpigmentation and blue nevus-like tumors in the Mongolian gerbil.

Summary Electron microscopic observations were made on the dermal hypepigmentation and blue nevus-like tumors induced in the dorsal trunk skin of Mongolian gerbils by DMBA. Hyperpigmented dermis was also examined in gerbils treated with DMBA and croton oil. The dermal hyperpigmentation was characterized by numerous melanin-bearing melanocytes and macrophages. Some of the dermal melanocytes were loaded with fully melanized (Stage IV) melanosomes, whereas others contained both mature melanosomes and immature ones (Stages I-III). Some dermal melanocytes displayed giant melanosomes (“mac-romelanosomes”) along with melanosomes of normal size. Finally, occasional dermal melanocytes exhibited typical Stages I-III melanosomes as well as round Stage I-like melanosomes in which melanin deposition occurred prematurely on the vesicular and filamentous matrices. The macrophages of hyperpigmented dermis contained groups of melanosomes segregated within membrane-limited secondary lysosomes. Paralleling the diversity in melanosome formation within the dermis, examination of DMBA-induced blue nevus-like tumors indicated that melanosomes were formed (1) in the standard manner with melanin deposited on a matrix of aligned filaments within a membrane-limited vacuole, and (2) by melanin deposition on a disorganized matrix of nonaligned filaments and/or mi-crovesicular bodies within a membrane-limited vacuole. Melanosomes formed via both pathways were subsequently grouped within autophagosomes and degraded therein.

Effect of Biotin Deficiency on Follicular Melanocytes of Mice

Summary Both yellow (Ay) and black (C57BL) mice produce grey hairs when weaned to a diet known to cause biotin deficiency. Histologically, the greyed hair follicles of biotin deficient mice bear a striking resemblance to the hair follicles of albino mice. The depigmented melanocytes of biotin deficient mice are similar in morphology to the follicular clear cells of the albino. Unlike clear cells, however, depigmented melanocytes react positively to tests for the presence of dopa oxidase.

Lactate dehydrogenase isozymes of mouse epidermis.

Five isozymes of LDH are demonstrable in the epidermis of the ear pinnae, hind feet, trunk dorsa, and tails of adult C57BL, C57HR, and C3HB mice by polyacrylamide gel electrophoresis. LDH-5 activity predominates in electropherograms. The ratio of LDH-1 to LDH-5 is greater in the epidermis of ear pinna and trunk dorsum than in that of tail and hind foot. The region-specific patterns of epidermal LDH isozymes are not correlated with melanin pigmentation or ‘hairiness’ of the skin.