A NEW APPROACH FOR THE UNDERSTANDING OF MELANOCYTES: STEREOLOGICAL QUANTIFICATION OF MELANIZATION BY IMAGE ANALYSIS
Eric Donois and Jean-Etienne Surleve-Bazeille
Département de Biologie Cellulaire et Microscopie Electronique, Université Bordeaux I
351, cours de la Libération, 33405 Talence CEDEX
Tel: 56 84 63 88 / Fax: 56 84 66 70
Pigmentation of human skin results from the transport of melanized melanosomes (MMs) in melanocytes dendrites which developpement is controled by keratinocytes [1,2], their transfer to the surrounding undifferentiated keratinocytes according to a mechanism still debated today [3, 4, 5, 6], and finally from the more or less efficient degradation of melanosomes in keratinocytes  which ascend in the epidermis to finish their differentiation as corneocytes fated to desquamation.
The macroscopic cutaneous degree of pigmentation depends on several factors playing a critical role at various levels in this process: phototype (heredity, race), solar exposition, hormonal background [8, 9], etc. However, the initial requirement for variations of skin pigmentation is the potential variations of melanin biosynthesis by melanocytes themselves, which modulation may be reproduced in vitro by direct action of artificial ultra violet radiations [10,11,12].
Routine techniques for the quantification of melanin pigments are used for a long time. Spectrophotometrical dosages are the most commonly utilized and consist in the comparison of the optical densities obtained at 415 nm after alkaline extraction, with those of standards (e.g. techniques used in [10,1,13]); Nonetheless, natural (from Sepia officinalis), synthetic (chemical oxidation of L-tyrosine) or biosynthetic (oxidation of L-tyrosine or L-DOPA by purified tyrosinase) eumelanin may be used as standard .
Physically deposited melanins to be measured are extracted by alkaline solutions, which may induce alterations in the polymer with release of colorless precursors  and may be influenced by the percentage of pheomelanic and eumelanic monomers in the case of mixed type melanin .
More recently, High Performance Liquid Chromatography (HPLC) procedures have been ellaborated [16,17] and now permit to estimate pheomelanin and eumelanin contents separately, which allows to understand how certain bioactive molecules change the pheomelanin to eumelanin ratio, e.g. a-MSH in mammals [18, 19, 20]. Nevertheless, HPLC methods or like spectrophotometrical methods require to be applied, a great number of melanocytes.
With the aim to understand the fine mechanisms of stimulation and inhibition of pigment production at the organite level, we developped in our laboratory a quantitative method based on the analysis of melanocytes images obtained by transmission electron microscopy [21, 22].
Melanin profiles are automatically extracted from melanocytes images with the aid of an image analysis system which generates melanization primary data. Those primary data are introduced in stereological formula, stereology being the body of mathematical methods relating three-dimensional parameters defining the structure to two-dimensional measurements obtainable on sections of the structure [23,24,25]. Those formulae enables us to estimate various meaningful melanization parameters. Thus, we are able to estimate concomitantly the melanocyte volume, the melanin volume density in the cytoplasm, the mean melanin volume per melanocyte, the numerical density of MMs in the cytoplasm and the mean number of MMs per melanocyte.
Additionally, two parameters, the mean melanin volume per MM and an indicator of melanosomal maturation, i.e. NN(mp,m), the mean number of detected melanin particles per MM , can be calculated in order to quantitatively appraise the level of melanosomal maturation.
Only two studies on the melanocyte have used morphometrical techniques, including one relating to the manual morphometrical measurement of melanosomes observed by transmission electron microscopy  and the other, dealing with a quantitative image analysis of Fontana Masson-stained 1 µm plastic sections of human skin samples observed by light microscopy .
Our stereological computerized tool permits to quantify different aspects of the intracytoplasmic melanization, which have never been performed at the ultrastructural level with automatic methods, and never approached from a stereological angle.
Works are carried out at the present time in our laboratory, with the aim to validate this original method by comparing morphometrical data and results obtained from a classical melanin assay .
Moreover, the Inazu's alkali elution method, which permits to dissolve pheomelanin on ultrathin sections with an acceptable specificity , is actually applied to our quantitative method in order to measure pheomelanin and eumelanin like only HPLC methods allow.
At least, the last appreciable advantage of our method is that it requires a low number of pigment cells. As a consequence, more complex experiments with human epidermal melanocytes cultured physiologically might be perfomed with small experimental units of short term cultures.
In conclusion, this original approach, by using new type of data will contribute to a better understanding of intracytoplasmic events underlying stimulation or repression of human melanogenesis by physical or chemical agents or in pathological conditions.
1. Gordon PR, Mansur CP, Gilchrest BA: Regulation of human melanocyte
growth, dendricity, and melanization by keratinocyte derived factors. J Invest
Dermatol. 92:565-572, 1989.
2. Lacour J-P, Gordon PR, Eller M, Bhawan J, Gilchrest BA: Cytoskeletal events underlying dendrite formation by cultured pigment cells. J Cell Physiol. 151: 287-299, 1992.
3. Topol BM, Haimes HB, Dubertret L, Bell E: Transfer of melanosomes in a skin equivalent model in vitro. J Invest Dermatol. 87:642-647, 1986.
4. Wolff K, Jimbow K, Fitzpatrick TB: Experimental pigment donation in vivo. J Ultrastruct Res. 47:400-419, 1974.
5. Wolff K, Konrad K: Phagocytosis of latex beads by epidermal keratinocytes in vivo. J Ultrastruct Res. 39:262-280, 1972.
6. Yamamoto O, Bhawan J: Three modes of melanosome transfers in caucasian facial skin: hypothesis based on ultrastructural study. Pigment Cell Res. 7:158-159, 1994.
7. Jimbow K, Quevedo WC, Fitzpatrick TB, Szabo G: Some aspects of melanin biology:1950-1975. J Invest Dermatol. 67:72-89, 1976.
8. Fuller BB, Rungta D, Iozumi K, Hoganson GE, Corn TD, Cao VA, Ramadan ST, Owens KC: Hormonal regulation of melanogenesis in mouse melanoma and in human melanocytes. Ann N Y Acad Sci. 680:302-319, 1993
9. Jee SH, Lee SY, Chiu HC, Chang CC, Chen TJ: Effects of estrogen receptor in normal human melanocytes. Biochem Biophys Res Com. 199:l407-1412, 1994.
10. Friedmann PS, Gilchrest BA: Ultraviolet radiation directly induces pigment formation by cultured human melanocytes. J Cell Physiol. 133:88-94, 1987.
11. Gilchrest BA, Zhai S, Eller MS, Yarosh DB, Yaar M: Treatment of human melanocytes and S91 melanoma cells with the DNA Repair Enzyme T4 Endonuclease V enhances melanogenesis after ultraviolet irradiation. J Invest Dermatol. 101:666-672, 1993.
12. Ramirez-Bosca A, Bernd A, Werner R, Dold K, Holzmann H: Effect of the dose of ultraviolet radiation on the pigment formation by human melanocytes in vitro. Arch Dermatol Res. 284:358-362, 1992.
13. Naeyaert JM, Eller M, Gordon PR, Park H-Y, Gichrest BA: Pigment content of cultured human melanocytes does not correlate with tyrosinase message level. Br J Dermatol. 125:297-303, 1991.
14. Zeise L, Murr BL, Chedekel MR: Melanin standart method: Particle description. Pigment Cell Res. 5:132-142, 1992.
15. Prota G: Recent advance in the chemistry of melanogenesis in mammals. J Invest Dermatol. 75:122-127, 1980.
16. Ito S: High-Performance Liquid Chromatography (HPLC) analysis of eu- and pheomelanin in melanogenesis control. J Invest Dermatol. 100:166S-171S, 1993.
17. Ito S, Jimbow K: Quantitative analysis of eumelanin and pheomelanin in hair and melanomas. J Invest Dermatol. 80:268-272, 1983.
18. Burchill SA, Ito S, Thody AJ: Effects of melanocyte-stimulating hormone on tyrosinase expression and melanin synthesis in hair follicular melanocytes of the mouse. J Endocrinol. 137:189-195, 1993.
19. Granholm NH, Van Amerongen AW: Effects of exogenous MSH on the transformation from phaeo- to eumelanogenesis within C57BL/6J-Ay/a hairbulb melanocytes. J Invest Dermatol. 96:78-84, 1991.
20. Hunt G, Kyne S, Wakamatsu K, Ito S, Thody AJ: Nle4DPhe7 a-Melanocyte-Stimulating Hormone increases the Eumelanin: pheomelanin ratio in cultured human melanocytes. J Invest Dermatol. 104:83-85, 1995.
21. Donois E, Surlève-Bazeille J-E, Taïeb A: Quantification de la synthese mélanique de mélanocytes humains en culture. Mise au point d'un programme d'analyse d'image en microscopie électronique à transmission. Congrès Annuel de Recherche Dermatologique (CARD) Société de Recherche Dermatologique (SRD), 1993.
22. Donois E, Surlève-Bazeille J-E, Taïeb A: Quantitative Stereological Image Analysis of Human Cultured Melanocytes Observed by Transmission Electron Microscopy. Fifth Meeting of the European Society for Pigment Cell Research (ESPCR) Melanoma Research. 4:18, 1994.
23. Bolender RP: Biological stereology: history, present state, future directions. Microsc Res Tech. 21:255-261, 1992.
24. Weibel ER: Practical methods for biological morphometry. In: Parsons DF (ed.). Stereological methods, vol. 1. Academic press, London, 1979.
25. Weibel ER: Measuring through the microscope: development and evolution of stereological methods. J Microsc. 155:393-403, 1989.
26. Donois E, Surlève-Bazeille J-E, Taïeb A: Quantitative Stereological Image Analysis of Human Cultured Melanocytes Observed by Transmission Electron Microscopy. J Invest Dermatol. submitted for publication, 1995.
27. Bhawam J, Gonzales-serva A, Nehal K, Labadie R, Lufrano L, Thorne G, Gilchrest BA: Effects of tretinoin on photodamaged skin. A histologic study. Arch Dermatol. 127:666-672, 1991.
28. Friedmann PS, Wren F, Buffey J, Macneil S: a-MSH causes a small rise in cAMP but has no effect on basal or ultraviolet-stimulated melanogenesis in human melanocytes. Br J Dermatol. 123:145-151, 1990.
29. Inazu M, Mishima Y: Detection of eumelanogenic and pheomelanogenic melanosomes in the same normal human melanocyte. J Invest Dermatol. 100:172S-175S, 1993.