Arch. Derm. Forsch. 252, 297--304 (1975) © by Springer-Verlag 1975
Fine Structure and X-Ray Microanalysis of Melanosomes in Pigmented Nevi and Melanomas Lenke Szekeres Department of Dermatology, Medical University of Szeged (Director: Prof. N. Simon), Hungary
Summary. Further finestructural characteristics of melanosomes in pigmented nevi and melanomas are described. The differences in the c~lcium, iron and sulphur content of melanosomes derived from melanocytes, nevocytes and malignant melanoma ceils are pointed out. Zusammen/assung. Weitere feinstrukturelle Eigensch~ften der Melanosomen yon pigmentierten Naevi und Melanomen werden beschrieben. Auf die Differenzen des Calcium-, Eisen- und Schwefel-Inhalt der Melanosomen yon Melunoeyten, Naevuszellen und malignen ~elanomzellen wird hingewiesen. Introduction Our present knowledge about the subcellular localisation of melanin biosynthesis has been developed b y Seiji et al. (1961, 1963), Seiji and Iwashita (1965) as well as b y Toda et al. (1968). Electron microscopical investigations revealed the structure of melanocy%es and melanosomes in normal (Birbeck, 1962, 1963; Birbeck et al., 1956; Charles and Ingrain, 1959; Drochmans, 1960, 1963; Fitzpatrick and Szabd, 1959), pathological (Birbeck et al., 1961 ; Braunsteiner etal., 1958; Drochmans, 1963 ; Jakubowicz et al., 1970; J i m b o w et al., 1972; Nakai and Shubik, 1964; Schreiner and Wolff, 1970 ; Toshima et al., 1968 ; Wellings and Siegel, 1959 ; Zelickson et al., 1967) and in experimental conditions (Hunter et al., 1970 ; Maul, 1969 ; Mishima et al., 1962). Fine structure of nevus cells has been investigated by Breathnach, 1964; Drochmans, t963; Gottlieb et al., 1965; Mishima, 1962, 1963; Tr6ger and Klingmiiller, 1966. Mishima (1962, 1965) found melanosomes of junctional nevus cells averaging up to 500 m~ × 150 m~ in size, while spherical granules of A- and B-type intradermal nevus cells have been established as the smallest of any melanosomes averaging up to 200 m~ in their maximal diameter in contrast with ripe melanosomes in normal h u m a n melanocytes which were approximately 800 m~ × 200 m~ in size with football or rod-like shape. Structural differences of melanosomes from different sources m a y refer to varied biosynthetic processes of the melanogenesis. Therefore, it seemed worthwhile to investigate beside the fine structural characteristics of nevocytic and melanomatie melanosomes their ion content and to make a comparision between the measurable ion content of nevocytic and melanocy~ic melanosomes on the one hand and t h a t of the nevocytic and melanomatic melanosomes on the other.
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Material and Method Five junction nevi, 25 compound nevi and 5 malignant melanomas were examined for the structure of melanosomes by traditional electron microscopy. Specimens from 5 melanomas, 5 compound nevi and pigmented normal human epidermis were prepared for X-ray microanalysis. Material was obtained by punch or in toto biopsies under local anaesthesia with Lidocain or originated from surgical removal of malignant melanomas. Specimens were prefixed in 2.50/0 glutaraldehyde solution buffered with eacodylate buffer (pH 7.4) for 2 hrs, washed in caeodylate buffer, fixed in 1°/0 osmium tetroxide for 1 hr, dehydrated in rising concentration of ethanol solution, transferred through two changes of propylene oxide and embedded in Durcupan M. Ultrathin sections were cut by a Reichert ultramicroteme and stained with uranyl acetate and lead citrate and viewed in a Jeol 100 B electron microscope operated at 80 kV. Semithin, approximately 2500 A thick sections (unstained, carbon coated) from fixed material on plastic greeds were used and subjected for X-ray analysis in a Jeol Jem 100 B electron microscope operated in the scanning transmission (STEM) mode. The established values of X-ray emission lines were taken from the "X-Ray Emission Wavelength and KeV Tables" (G. Gr. Johnson and E. W. White, Amer. Soc. for Testing and Materials Date Series DS 46, 1970).
Results Melanosomes i n j u n c t i o n n e v u s cells were f o u n d to be spherical or ovoid with a n average of 400 m ~ × 180 m~z in size. Melanosomes of i n t r a d e r m a l n e v u s cells (type A a n d B) were observed as spherical, sometimes polygonal organelles possessing two kinds of i n t e r n a l struct u r e : striated a n d s p o t t e d with the prevalence of the later. Their size varied depending on the m a t u r i n g processes. Young, spherical premelanosomes averaged u p to 5 0 - - 100 m ~ in diameter, while s p o t t e d v a r i a n t s were f o u n d to have diameters
Fig. 1. Melanosomes of an A-type nevus cell in different maturing phases. N Nucleus, G Golgi apparatus, pr premelanosomes, sp spotted melanosomes, st striated melanosomes, M mitochondrion, V vesicle. × 15000 × 1.8
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299
of 180--200 mb~, striated or fully melanized organdies averaged up to 200--220mb~ in diameter (Fig. 1). I n addition to spherical variants, polygonal (five- or sexangle) melanosomes were also observed averaging up to 280--290 mb~ in size. Their internal structure consisted of homogenous, dense material with an average of 150 mbL in diameter. Between the outer double-layered limiting membrane and tii e in~er melanized nucleus, an electron lucent halo (approximately 70 mbt wide) ~ a s observed (Fig.2). I n malignant melanomas both cigar-shaped and spherical melanosomes were found, nevertheless, spherical types prevailed in the examined material. Spherical melanosomes revealed variable internal structure (striated, spotted, mottled, vesiculated), degenerated forms resulting in myelin figures were often seen. The size of the spherical variants averaged up to 250--300 mb~ in diameter (Fig.3 and 4). X - R a y Microanalysis. The most intense calcium X-radiation of melanosomes from intradermal nevus cells had an energy of 3.69 KeV. A comparision has been made between the semiquantitative values of calcium content in nevocytic, melanocytic and inelanomatic melanosolnes. Naevocytic melanosomes revealed a relatively high calcium content, while normal h u m a n melanocytic melanosolnes similarly to the melanomatic ones were found to contain no significant amount of calcium.
Fig.2. Spherical and polygonal melanosomes in a B-type nevus cell. pr premelanosomes, sp spotted melanosomes, m fully melanized melanosomes, pm polygonal melanosomes, lm double layered limiting membrane of melanosome, / intracytoplasmie filaments × 30002 × 2
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Fig.3. Heterogenity of melanosomes in a melanoma cell. sr Striated organelles, sp spotted variants, v vesieulated melanosomes, m y myelin figures, r free ribosomes, R E R rough endoplasmic reticulum. × 10600 × 1.8
Fig. 4. Melanosomes of a malignant melanoma cell. st Striated organelles, m y myelin-figurelike variants, R E R rough endoplasmic reticulum, r free ribosomes × 35000 × 2
I r o n X - r a d i a t i o n of n e v o e y t i c m e l a n o s o m e s was f o u n d t o h a v e a n e n e r g y o f 6.4 K e V a n d t h e s e m i q u a n t i t a t i v e values o f t h e i r o n of n e v o c y t i c m e l a n o s o m e s in c o m p a r i s i o n w i t h m e l a n o c y t i c a n d m e l a n o m a t i c m e l a n o s o m e s were also rel a t i v e l y high. On t h e o t h e r h a n d , m e l a n o m a t i c m e l a n o s o m e s were f o u n d t o c o n t a i n a considerable a m o u n t of sulphur, t h e m o s t intense X - r a d i a t i o n of which h a d a n e n e r g y of 2.31 KeV. Melanocytic or n e v o c y t i e melauosomcs c o n t a i n e d far less a m o u n t of s u l p h u r as has been established from t h e c o m p a r a t i v e e s t a b l i s h m e n t (Table). O t h e r ions were n o t o b s e r v e d in considerable a m o u n t s or t h e i r p e a k s were covered b y t h e p e a k s of fixative (osmium, u r a n i u m , etc.).
301
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Table. The differences between the calcium, iron and sulphur contents of melanocytic; nevocytic and melanomatic melanosomes revealed by X-ray microanulysis. Each column represents the average of ten evaluations
I
0 [--7 ~11
S
Ca
0,5
Fe
1,0
naevocytic me[anosome me[anocytic melanosome me[anomatic me[anosome
Discussion In this present study differences between the structure of the melanosomes of junction, A- and B-type intradermal nevus cells have been confirmed. In addition, differences have also been established between the calcium, iron and sulphur content of melanosomes. Structural and analytical differences suggest deviations in the biosynthetic pathways operating in these organelles. It has been reported that naturally occurring melanin derived from different sources contained metal ions such as zinc, copper, cobalt and iron (Nicolaus, 1962). Manganese was found by Cotzias in a variety of pigmented tissues (Cotzias and Papavasiliou, 1964). Bruenger et al. (1967) investigated the incorporation of various metal ions into in vivo (B 16 melanoma tissue) and in vitro produced melanin and has established that metal ions and melanin can interact by ion exchange. The high calcium binding capacity of nevocytic melanosomes is interesting but can be interpreted with difficulty. Calcium is very important in synaptical transmission influencing the membran permeability for Na+ and K +. It seems probable that selective permeability of melanosomal membranes is also regulated by calcium. The iron binding capacity of nevocytic melanosomes was observed in every specimen in spite of the fact that examined materials originated from individuals with no red complexion. Flesch (1968) believed that iron-containing pigments are the major epidermal pigments in red species. It is interesting to note that neuromelanin and lipofuscin also demonstrate iron binding capacity, k~euromelanin invested with ferrous or cupric ions could be melanized with dopa or dopamine (Barden and Moses, 1970). Olivary lipofuscin from the rhesus inferior olive could also be invested with ferrous ions and pseudoperoxidatively melanized by dopa. In spite of the fact that neuromelanin, this end-product of eathecolamin metabolism of certain neural elements and melanin pigment appeared to be distinct substances not closely related chemically and structurally (Lillie and Yamada, 1960; Moses et al., 1966), a number of adrenergic material related structurally and chemically to L-tyrosine or L-Dopa, proved to be the precursor of melanin
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in the mammalian and human skin and hair (Harrison et al., 1974; Yu and van Scott, 1973). A neuronal pigment identical to neuromelanin of the substantia nigra and locus coeruleus also occurs in other sites within the h u m a n central and peripheral nervous system, including several brain stem nuclei, the trigeminal ganglion, other cranial sensory ganglia, dorsal root ganglia and symphathetic ganglia. Pigmented cells closely associated with cutaneous sensory and autonomic nerves have been demonstrated by K a w a m u r a et al. (1964), but the exact nature of these perineurial pigment zones in human skin has remained undetermined. Mishima (1967) considered melanosomes within Schwann cells, cndoneurium and perineurium of cutaneous nerves as the results of phagocytosis. Whereas, it seems probable t h a t perineural pigment zones as well as the pigment of nevus cells represent substances which are formed in certain parts of the nervous system of humans and higher primates by a special neural metabolism similar in certain regions that take their origin from the same genetical place, but different at the same time depending on the anatomical localisation. The morphological and analytical differences between melanocytic and nevoeyrie melanosomes m a y refer to a different biosynthetic pathway, by which melanin is formed in these organelles. The possible neural origin of nevus cells, the abundance of the nervous elements in nevi m a y suggest that nevocytie melanin is not " t r u e " melanin but a product of neural metabolism. The relatively high sulphur content found in melanosomes of malignant melanomas m a y be connected with the presence of 5-S-cysteinyl-Dopa, which was already demonstrated by Agrup et al. (1974) as well as b y Vogel et al. (1974) in melanoma tissue of Caucasian and Ugandan African patients. The role of 5-S-cysteinyl-Dopa in pathological melanin biosynthesis became evident recently. Unfortunately, we were not able to study .the most characteristic ion, copper. I n spite of the fact t h a t plastic greeds and copper-free intruments were used during the analysis, the evaluation of copper content in melanosomes from different sources proved not to be reliable. Many comparative morphological and analytical studies are needed for the final conclusion, but the observed differences both from morphological and analytical point of view m a y underline a possible difference in melanin biosynthesis of melanocytic, nevocytie and melanomatic melanosomes. I n adddition, new concepts of pigment genesis differing from the tyrosine-tyrosinase pathway m a y be fruitful in the better understanding of pigmentation of certain peripheral nervous elements or related tissues (e.g. nevi). Acknowledgements. The author is gratefully indebted to Dr. F. Jo6 for the possibilities of using the EDAX analyzer and would like to express her gratitude to Miss Elizabeth Szil£rd, Mr. Iv£n Paskuj and Mr. John Szeles for their skillful technical assistance. References
Agrup, G., Falck, B., Jacobsson, S., Rorsman, tt., Rosengren, A.-M., Rosengren, E.: 5-Seysteinyl-dop~ in melanomas of Caucasians. Acta derm.-venereol. (Stockh.) 54, 21--22 (1974) Birbeck, M. S. C. : Electron microscopy of melanoeytes. Brit. reed. Bull. 18, 220--222 (1962)
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Birbeck, M. S. C. : Electron microscopy of melanocytes: the fine structure of hair-bulb premelanosomes. Ann. N. Y. Aead. Sci. 100, 540--547 (1963) Birbeck, M. S. C., Breathnach, A. S., Everall, J. D. : An electron microscopic study of basal melanocytes and high-level clear cells (Langerhans cells) in vitiligo. J. invest. Derm. 87, 51--64 (196!) Birbeck, M. S. C., Mercer, E. H., Barnicot, N. A.: The structure and formation of pigment granules in human hair. Exp. Cell. Res. 1O, 505--514 (1956) Braunsteiner, H., Mlczoch, F., Pakesch, F. : Elektronenmikroskopische Untersuchungen fiber die Struktur yon intracellul~rem Melanin beim Melanoblastom. Klin. Wschr. 86, 262--263 (1958) Breathnach, A. S.: Electron microscopy of a small pigmented cutaneous lesion. J. invest. Derm. 42, 21--25 (1964) Breathnach, A. S., Fitzpatrick, T. B., Wyllie, L. M.-A.: Electron microscopy of melanocytes in human piebaldism. J. invest. Derm. 45, 28--37 (1965) Bruenger, F. W., Stover, B.J., Atherton, D. R.: The incorporation of various metal ions into in vivo and in vitro produced melanin. Radiat. Res. 82, 1--12 (1967) Charles, A., Ingram, J. T. : Electron microscope observations of the melanocyte of the human epidermis. J. biophys, biochem. Cytol. 6, 41--45 (1959) Cotzias, G. C., Papavasiliou, P. S.: Manganese in melanin. Nature (Lond.) 291, 1228--1229 (1964) Drochmans, P. : Electron microscopic studies of epidermal melanocytes, and the fine structure of melanin granules. J. biophys, biochem. Cytol. 8, 165--180 (1960) Drochmans, P. : Melanin granules: their fine structure, formation and degradation in normal and pathological tissues. Int. Rev. exp. Path. 2, 357--422 (1963) Fitzpatrick, T. B., SzabS, G. : The melanocyte: cytology and cytochemistry. J. invest. Derm. 82, 197--209 (1959) Flesch, P. : The epidermal iron pigments of red species. J. invest. Derm. 51, 337--343 (1968) Gottlieb, B., Brown, A. L., Winkelmann, R. K.: Fine structure of the nevus cell. Arch. Derm. 92, 81--90 (1965) Harrison, W. H., Gray, R.M., Solomon, L. M. : Incorporation of L-Dopa, L-~-methyldopa and DL-isoproterenol into guinea pig hair. Acta derm.-venereol. (Stockh.) 54, 249--253 (1974) Hunter, J. A. A., Mottaz, J. H., Zelickson, A. S. : Melanogenesis: ultrastructural histoehemieal observations on ultraviolet irradiated human melanocytes. J. invest. Derm. 54, 213--221 (1970) Jakubowicz, K., Dabrowski, J., Biczysko, W., Walski, M. : Ultrastruktur der Melanosomen im Melanomalignom. Derm. Mschr. 156, 299--307 (1970) Jimbow, K., Fitzpatrick, T. B., SzabS, G.: Mechanism of decreased pigmentation in tuberous sclerosis, nevus depigmentosus and piebaldism. J. invest. Derm. 58, 170--171 (1972) Kawamura, T., ~ishiyama, S., Ikeda, S., Tajima, K. : The human haarscheibe, its structure and function. J. invest. Derm. 42, 87--90 (1964) Lillie, R. D., Yamada, H. : Histochemical studies on the neuromelanins. Okajimas Folia anat. Jap. 86, 155--163 (1960) Maul, G. G.: Golgi-melanosome relationship in human melanoma in vitro. J. Ultrastruct. Res. 163--176 (1969) Mishima, Y. : Electron microscopy of melanin synthesis in intradermal nevus cells J. invest. Derm. 89, 369--372 (1962) Mishima, Y., Loud, A. V., Schaub, F. F., Jr. : Electron microscopy of "premelanin". J. invest. Derm. 89, 55--66 (1962) Mishima, Y., Schaub, F. F., Jr. : Origin of the nevus cell. Electron microscopy and induced melanin formation. X I L Int. Congr. of Dermatology. Edited by D. M. Pillsbury, C. S. Livingood, pp. 1588--1592. Amsterdam, l~ew York, Milan, Tokyo: Excerpta Med. Found. 1963 Mishima, Y.: Macromolecular changes in pigmentary disorders. Arch. Derm. 91, 519--557 (1965) Mishima, Y.: Electron microscopy of human cutaneous nerve pigmentation. Brit. J. Derm. 79, 611--616 (1967) 22 Arch.Derm. ]~orsch.~Bd. 252
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Moses, H. L., Ganote, C. E., Beaver, D. L., Schuffman, S. S. : Light and electron microscopic studies of pigment in human and rhesus monkey substantia nigra and locus coeruleus. Anat. Rec. 155, 167--184 (1966) Nakai, T., Shubik, P.: Eleetronmicroseopic radioautography: the melanosome as a site of melanogenesis in neoplastic melanocytes. J. invest. Derm. 48, 267--269 (1964) 1Xicolaus, R. A.: Biogenesis of melanins. Rass. IVied. Sper. Suppl. 1. 1962. cir. by Bruenger et aL Radiat. Res. 82, 1--12 (1967) Sehreiner, E., Wolff, K.: Die Ultrast~ktur des benignen juvenilen Melanoms. Arch. klin. exp. Derm. 287, 749--768 (1970) Seiji, M., Fitzpatrick, T. B., Birbeck, M. S.C.: The melanosome: a distinctive subeellular particle of mammalian melanocytes and the site of melanogenesis. J. invest. Derm. 86, 243--252 (1961) Seiji, M., Shimao, K., Birbeck, M. S. D., Fitzpatrick, T. B. : Subeellular localization of melanin biosynthesis. Ann. N. Y. Aead. Sci. 100, 497--533 (1963) Seiji, M., Iwashita, S. : Intracellular localization of tyrosinase and site of melanin formation in melanocyte. J. invest. Derm. 45, 305--314 (1965) Toda, K., Hori, ¥., Fitzpatrik, T. B. : Isolation of the intermediate "vesicles" during ontogeny of melanosomes in embryonic chick retinal pigment epithelium. Fed. Proc. 27, 722--728
(1968) Toshima, S., Moore, G. E., Sandberg, A. A. : Ultrastrudture of human melanoma in cell culture. Cancer 21, 202--216 (1968) TrSger, H., Klingmfiller, G. : Die Melaningranula im Naevuszellnaevus. Arch. klin. exp. Derm. 226, 1--12 (1966) Vogel, C. L., Dhru, D., Rorsman, H., Rosengren, A.-M., Rosengren, E. : Dopa and 5-S-cysteinyldopa in malignant melanoma in Ugandan Africans. Aeta derm.-venereol. (Stoekh.) 54, 19--20 (1974) Wetlings, S. R., Siegel, B. V. : Role of Golgi apparatus in the formation of melanin granules in human malignant melanoma. J. Ultrastruct. Res. 8, 147--154 (1959) Zelickson, A. S., Windhorst, D. B., White, J. G., Good, R. A. : The Chediak-Hig~shi syndrome: formation of giant melanosomes and the basis of hypopigmentation. J. invest. Derm. 49, 575--581 (1967) Yu, R. J., van Scott, E. J. : Substrate identity in mammalian melanogenesis. J. invest. Derm. 60, 234--237 (1973) Dr. Lenke Szekeres Department of Dermatology Medical University of Szeged P. O. Box 480 H-6701 Szeged Hungary
Fine Structure and X-Ray Microanalysis of Melanosomes in Pigmented Nevi and Melanomas Lenke Szekeres Department of Dermatology, Medical University of Szeged (Director: Prof. N. Simon), Hungary
Summary. Further finestructural characteristics of melanosomes in pigmented nevi and melanomas are described. The differences in the c~lcium, iron and sulphur content of melanosomes derived from melanocytes, nevocytes and malignant melanoma ceils are pointed out. Zusammen/assung. Weitere feinstrukturelle Eigensch~ften der Melanosomen yon pigmentierten Naevi und Melanomen werden beschrieben. Auf die Differenzen des Calcium-, Eisen- und Schwefel-Inhalt der Melanosomen yon Melunoeyten, Naevuszellen und malignen ~elanomzellen wird hingewiesen. Introduction Our present knowledge about the subcellular localisation of melanin biosynthesis has been developed b y Seiji et al. (1961, 1963), Seiji and Iwashita (1965) as well as b y Toda et al. (1968). Electron microscopical investigations revealed the structure of melanocy%es and melanosomes in normal (Birbeck, 1962, 1963; Birbeck et al., 1956; Charles and Ingrain, 1959; Drochmans, 1960, 1963; Fitzpatrick and Szabd, 1959), pathological (Birbeck et al., 1961 ; Braunsteiner etal., 1958; Drochmans, 1963 ; Jakubowicz et al., 1970; J i m b o w et al., 1972; Nakai and Shubik, 1964; Schreiner and Wolff, 1970 ; Toshima et al., 1968 ; Wellings and Siegel, 1959 ; Zelickson et al., 1967) and in experimental conditions (Hunter et al., 1970 ; Maul, 1969 ; Mishima et al., 1962). Fine structure of nevus cells has been investigated by Breathnach, 1964; Drochmans, t963; Gottlieb et al., 1965; Mishima, 1962, 1963; Tr6ger and Klingmiiller, 1966. Mishima (1962, 1965) found melanosomes of junctional nevus cells averaging up to 500 m~ × 150 m~ in size, while spherical granules of A- and B-type intradermal nevus cells have been established as the smallest of any melanosomes averaging up to 200 m~ in their maximal diameter in contrast with ripe melanosomes in normal h u m a n melanocytes which were approximately 800 m~ × 200 m~ in size with football or rod-like shape. Structural differences of melanosomes from different sources m a y refer to varied biosynthetic processes of the melanogenesis. Therefore, it seemed worthwhile to investigate beside the fine structural characteristics of nevocytic and melanomatie melanosomes their ion content and to make a comparision between the measurable ion content of nevocytic and melanocy~ic melanosomes on the one hand and t h a t of the nevocytic and melanomatic melanosomes on the other.
298
L. Szekeres
Material and Method Five junction nevi, 25 compound nevi and 5 malignant melanomas were examined for the structure of melanosomes by traditional electron microscopy. Specimens from 5 melanomas, 5 compound nevi and pigmented normal human epidermis were prepared for X-ray microanalysis. Material was obtained by punch or in toto biopsies under local anaesthesia with Lidocain or originated from surgical removal of malignant melanomas. Specimens were prefixed in 2.50/0 glutaraldehyde solution buffered with eacodylate buffer (pH 7.4) for 2 hrs, washed in caeodylate buffer, fixed in 1°/0 osmium tetroxide for 1 hr, dehydrated in rising concentration of ethanol solution, transferred through two changes of propylene oxide and embedded in Durcupan M. Ultrathin sections were cut by a Reichert ultramicroteme and stained with uranyl acetate and lead citrate and viewed in a Jeol 100 B electron microscope operated at 80 kV. Semithin, approximately 2500 A thick sections (unstained, carbon coated) from fixed material on plastic greeds were used and subjected for X-ray analysis in a Jeol Jem 100 B electron microscope operated in the scanning transmission (STEM) mode. The established values of X-ray emission lines were taken from the "X-Ray Emission Wavelength and KeV Tables" (G. Gr. Johnson and E. W. White, Amer. Soc. for Testing and Materials Date Series DS 46, 1970).
Results Melanosomes i n j u n c t i o n n e v u s cells were f o u n d to be spherical or ovoid with a n average of 400 m ~ × 180 m~z in size. Melanosomes of i n t r a d e r m a l n e v u s cells (type A a n d B) were observed as spherical, sometimes polygonal organelles possessing two kinds of i n t e r n a l struct u r e : striated a n d s p o t t e d with the prevalence of the later. Their size varied depending on the m a t u r i n g processes. Young, spherical premelanosomes averaged u p to 5 0 - - 100 m ~ in diameter, while s p o t t e d v a r i a n t s were f o u n d to have diameters
Fig. 1. Melanosomes of an A-type nevus cell in different maturing phases. N Nucleus, G Golgi apparatus, pr premelanosomes, sp spotted melanosomes, st striated melanosomes, M mitochondrion, V vesicle. × 15000 × 1.8
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299
of 180--200 mb~, striated or fully melanized organdies averaged up to 200--220mb~ in diameter (Fig. 1). I n addition to spherical variants, polygonal (five- or sexangle) melanosomes were also observed averaging up to 280--290 mb~ in size. Their internal structure consisted of homogenous, dense material with an average of 150 mbL in diameter. Between the outer double-layered limiting membrane and tii e in~er melanized nucleus, an electron lucent halo (approximately 70 mbt wide) ~ a s observed (Fig.2). I n malignant melanomas both cigar-shaped and spherical melanosomes were found, nevertheless, spherical types prevailed in the examined material. Spherical melanosomes revealed variable internal structure (striated, spotted, mottled, vesiculated), degenerated forms resulting in myelin figures were often seen. The size of the spherical variants averaged up to 250--300 mb~ in diameter (Fig.3 and 4). X - R a y Microanalysis. The most intense calcium X-radiation of melanosomes from intradermal nevus cells had an energy of 3.69 KeV. A comparision has been made between the semiquantitative values of calcium content in nevocytic, melanocytic and inelanomatic melanosolnes. Naevocytic melanosomes revealed a relatively high calcium content, while normal h u m a n melanocytic melanosolnes similarly to the melanomatic ones were found to contain no significant amount of calcium.
Fig.2. Spherical and polygonal melanosomes in a B-type nevus cell. pr premelanosomes, sp spotted melanosomes, m fully melanized melanosomes, pm polygonal melanosomes, lm double layered limiting membrane of melanosome, / intracytoplasmie filaments × 30002 × 2
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Fig.3. Heterogenity of melanosomes in a melanoma cell. sr Striated organelles, sp spotted variants, v vesieulated melanosomes, m y myelin figures, r free ribosomes, R E R rough endoplasmic reticulum. × 10600 × 1.8
Fig. 4. Melanosomes of a malignant melanoma cell. st Striated organelles, m y myelin-figurelike variants, R E R rough endoplasmic reticulum, r free ribosomes × 35000 × 2
I r o n X - r a d i a t i o n of n e v o e y t i c m e l a n o s o m e s was f o u n d t o h a v e a n e n e r g y o f 6.4 K e V a n d t h e s e m i q u a n t i t a t i v e values o f t h e i r o n of n e v o c y t i c m e l a n o s o m e s in c o m p a r i s i o n w i t h m e l a n o c y t i c a n d m e l a n o m a t i c m e l a n o s o m e s were also rel a t i v e l y high. On t h e o t h e r h a n d , m e l a n o m a t i c m e l a n o s o m e s were f o u n d t o c o n t a i n a considerable a m o u n t of sulphur, t h e m o s t intense X - r a d i a t i o n of which h a d a n e n e r g y of 2.31 KeV. Melanocytic or n e v o c y t i e melauosomcs c o n t a i n e d far less a m o u n t of s u l p h u r as has been established from t h e c o m p a r a t i v e e s t a b l i s h m e n t (Table). O t h e r ions were n o t o b s e r v e d in considerable a m o u n t s or t h e i r p e a k s were covered b y t h e p e a k s of fixative (osmium, u r a n i u m , etc.).
301
Fine Structure and X-Ray Microanalysis
Table. The differences between the calcium, iron and sulphur contents of melanocytic; nevocytic and melanomatic melanosomes revealed by X-ray microanulysis. Each column represents the average of ten evaluations
I
0 [--7 ~11
S
Ca
0,5
Fe
1,0
naevocytic me[anosome me[anocytic melanosome me[anomatic me[anosome
Discussion In this present study differences between the structure of the melanosomes of junction, A- and B-type intradermal nevus cells have been confirmed. In addition, differences have also been established between the calcium, iron and sulphur content of melanosomes. Structural and analytical differences suggest deviations in the biosynthetic pathways operating in these organelles. It has been reported that naturally occurring melanin derived from different sources contained metal ions such as zinc, copper, cobalt and iron (Nicolaus, 1962). Manganese was found by Cotzias in a variety of pigmented tissues (Cotzias and Papavasiliou, 1964). Bruenger et al. (1967) investigated the incorporation of various metal ions into in vivo (B 16 melanoma tissue) and in vitro produced melanin and has established that metal ions and melanin can interact by ion exchange. The high calcium binding capacity of nevocytic melanosomes is interesting but can be interpreted with difficulty. Calcium is very important in synaptical transmission influencing the membran permeability for Na+ and K +. It seems probable that selective permeability of melanosomal membranes is also regulated by calcium. The iron binding capacity of nevocytic melanosomes was observed in every specimen in spite of the fact that examined materials originated from individuals with no red complexion. Flesch (1968) believed that iron-containing pigments are the major epidermal pigments in red species. It is interesting to note that neuromelanin and lipofuscin also demonstrate iron binding capacity, k~euromelanin invested with ferrous or cupric ions could be melanized with dopa or dopamine (Barden and Moses, 1970). Olivary lipofuscin from the rhesus inferior olive could also be invested with ferrous ions and pseudoperoxidatively melanized by dopa. In spite of the fact that neuromelanin, this end-product of eathecolamin metabolism of certain neural elements and melanin pigment appeared to be distinct substances not closely related chemically and structurally (Lillie and Yamada, 1960; Moses et al., 1966), a number of adrenergic material related structurally and chemically to L-tyrosine or L-Dopa, proved to be the precursor of melanin
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in the mammalian and human skin and hair (Harrison et al., 1974; Yu and van Scott, 1973). A neuronal pigment identical to neuromelanin of the substantia nigra and locus coeruleus also occurs in other sites within the h u m a n central and peripheral nervous system, including several brain stem nuclei, the trigeminal ganglion, other cranial sensory ganglia, dorsal root ganglia and symphathetic ganglia. Pigmented cells closely associated with cutaneous sensory and autonomic nerves have been demonstrated by K a w a m u r a et al. (1964), but the exact nature of these perineurial pigment zones in human skin has remained undetermined. Mishima (1967) considered melanosomes within Schwann cells, cndoneurium and perineurium of cutaneous nerves as the results of phagocytosis. Whereas, it seems probable t h a t perineural pigment zones as well as the pigment of nevus cells represent substances which are formed in certain parts of the nervous system of humans and higher primates by a special neural metabolism similar in certain regions that take their origin from the same genetical place, but different at the same time depending on the anatomical localisation. The morphological and analytical differences between melanocytic and nevoeyrie melanosomes m a y refer to a different biosynthetic pathway, by which melanin is formed in these organelles. The possible neural origin of nevus cells, the abundance of the nervous elements in nevi m a y suggest that nevocytie melanin is not " t r u e " melanin but a product of neural metabolism. The relatively high sulphur content found in melanosomes of malignant melanomas m a y be connected with the presence of 5-S-cysteinyl-Dopa, which was already demonstrated by Agrup et al. (1974) as well as b y Vogel et al. (1974) in melanoma tissue of Caucasian and Ugandan African patients. The role of 5-S-cysteinyl-Dopa in pathological melanin biosynthesis became evident recently. Unfortunately, we were not able to study .the most characteristic ion, copper. I n spite of the fact t h a t plastic greeds and copper-free intruments were used during the analysis, the evaluation of copper content in melanosomes from different sources proved not to be reliable. Many comparative morphological and analytical studies are needed for the final conclusion, but the observed differences both from morphological and analytical point of view m a y underline a possible difference in melanin biosynthesis of melanocytic, nevocytie and melanomatic melanosomes. I n adddition, new concepts of pigment genesis differing from the tyrosine-tyrosinase pathway m a y be fruitful in the better understanding of pigmentation of certain peripheral nervous elements or related tissues (e.g. nevi). Acknowledgements. The author is gratefully indebted to Dr. F. Jo6 for the possibilities of using the EDAX analyzer and would like to express her gratitude to Miss Elizabeth Szil£rd, Mr. Iv£n Paskuj and Mr. John Szeles for their skillful technical assistance. References
Agrup, G., Falck, B., Jacobsson, S., Rorsman, tt., Rosengren, A.-M., Rosengren, E.: 5-Seysteinyl-dop~ in melanomas of Caucasians. Acta derm.-venereol. (Stockh.) 54, 21--22 (1974) Birbeck, M. S. C. : Electron microscopy of melanoeytes. Brit. reed. Bull. 18, 220--222 (1962)
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Birbeck, M. S. C. : Electron microscopy of melanocytes: the fine structure of hair-bulb premelanosomes. Ann. N. Y. Aead. Sci. 100, 540--547 (1963) Birbeck, M. S. C., Breathnach, A. S., Everall, J. D. : An electron microscopic study of basal melanocytes and high-level clear cells (Langerhans cells) in vitiligo. J. invest. Derm. 87, 51--64 (196!) Birbeck, M. S. C., Mercer, E. H., Barnicot, N. A.: The structure and formation of pigment granules in human hair. Exp. Cell. Res. 1O, 505--514 (1956) Braunsteiner, H., Mlczoch, F., Pakesch, F. : Elektronenmikroskopische Untersuchungen fiber die Struktur yon intracellul~rem Melanin beim Melanoblastom. Klin. Wschr. 86, 262--263 (1958) Breathnach, A. S.: Electron microscopy of a small pigmented cutaneous lesion. J. invest. Derm. 42, 21--25 (1964) Breathnach, A. S., Fitzpatrick, T. B., Wyllie, L. M.-A.: Electron microscopy of melanocytes in human piebaldism. J. invest. Derm. 45, 28--37 (1965) Bruenger, F. W., Stover, B.J., Atherton, D. R.: The incorporation of various metal ions into in vivo and in vitro produced melanin. Radiat. Res. 82, 1--12 (1967) Charles, A., Ingram, J. T. : Electron microscope observations of the melanocyte of the human epidermis. J. biophys, biochem. Cytol. 6, 41--45 (1959) Cotzias, G. C., Papavasiliou, P. S.: Manganese in melanin. Nature (Lond.) 291, 1228--1229 (1964) Drochmans, P. : Electron microscopic studies of epidermal melanocytes, and the fine structure of melanin granules. J. biophys, biochem. Cytol. 8, 165--180 (1960) Drochmans, P. : Melanin granules: their fine structure, formation and degradation in normal and pathological tissues. Int. Rev. exp. Path. 2, 357--422 (1963) Fitzpatrick, T. B., SzabS, G. : The melanocyte: cytology and cytochemistry. J. invest. Derm. 82, 197--209 (1959) Flesch, P. : The epidermal iron pigments of red species. J. invest. Derm. 51, 337--343 (1968) Gottlieb, B., Brown, A. L., Winkelmann, R. K.: Fine structure of the nevus cell. Arch. Derm. 92, 81--90 (1965) Harrison, W. H., Gray, R.M., Solomon, L. M. : Incorporation of L-Dopa, L-~-methyldopa and DL-isoproterenol into guinea pig hair. Acta derm.-venereol. (Stockh.) 54, 249--253 (1974) Hunter, J. A. A., Mottaz, J. H., Zelickson, A. S. : Melanogenesis: ultrastructural histoehemieal observations on ultraviolet irradiated human melanocytes. J. invest. Derm. 54, 213--221 (1970) Jakubowicz, K., Dabrowski, J., Biczysko, W., Walski, M. : Ultrastruktur der Melanosomen im Melanomalignom. Derm. Mschr. 156, 299--307 (1970) Jimbow, K., Fitzpatrick, T. B., SzabS, G.: Mechanism of decreased pigmentation in tuberous sclerosis, nevus depigmentosus and piebaldism. J. invest. Derm. 58, 170--171 (1972) Kawamura, T., ~ishiyama, S., Ikeda, S., Tajima, K. : The human haarscheibe, its structure and function. J. invest. Derm. 42, 87--90 (1964) Lillie, R. D., Yamada, H. : Histochemical studies on the neuromelanins. Okajimas Folia anat. Jap. 86, 155--163 (1960) Maul, G. G.: Golgi-melanosome relationship in human melanoma in vitro. J. Ultrastruct. Res. 163--176 (1969) Mishima, Y. : Electron microscopy of melanin synthesis in intradermal nevus cells J. invest. Derm. 89, 369--372 (1962) Mishima, Y., Loud, A. V., Schaub, F. F., Jr. : Electron microscopy of "premelanin". J. invest. Derm. 89, 55--66 (1962) Mishima, Y., Schaub, F. F., Jr. : Origin of the nevus cell. Electron microscopy and induced melanin formation. X I L Int. Congr. of Dermatology. Edited by D. M. Pillsbury, C. S. Livingood, pp. 1588--1592. Amsterdam, l~ew York, Milan, Tokyo: Excerpta Med. Found. 1963 Mishima, Y.: Macromolecular changes in pigmentary disorders. Arch. Derm. 91, 519--557 (1965) Mishima, Y.: Electron microscopy of human cutaneous nerve pigmentation. Brit. J. Derm. 79, 611--616 (1967) 22 Arch.Derm. ]~orsch.~Bd. 252
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Moses, H. L., Ganote, C. E., Beaver, D. L., Schuffman, S. S. : Light and electron microscopic studies of pigment in human and rhesus monkey substantia nigra and locus coeruleus. Anat. Rec. 155, 167--184 (1966) Nakai, T., Shubik, P.: Eleetronmicroseopic radioautography: the melanosome as a site of melanogenesis in neoplastic melanocytes. J. invest. Derm. 48, 267--269 (1964) 1Xicolaus, R. A.: Biogenesis of melanins. Rass. IVied. Sper. Suppl. 1. 1962. cir. by Bruenger et aL Radiat. Res. 82, 1--12 (1967) Sehreiner, E., Wolff, K.: Die Ultrast~ktur des benignen juvenilen Melanoms. Arch. klin. exp. Derm. 287, 749--768 (1970) Seiji, M., Fitzpatrick, T. B., Birbeck, M. S.C.: The melanosome: a distinctive subeellular particle of mammalian melanocytes and the site of melanogenesis. J. invest. Derm. 86, 243--252 (1961) Seiji, M., Shimao, K., Birbeck, M. S. D., Fitzpatrick, T. B. : Subeellular localization of melanin biosynthesis. Ann. N. Y. Aead. Sci. 100, 497--533 (1963) Seiji, M., Iwashita, S. : Intracellular localization of tyrosinase and site of melanin formation in melanocyte. J. invest. Derm. 45, 305--314 (1965) Toda, K., Hori, ¥., Fitzpatrik, T. B. : Isolation of the intermediate "vesicles" during ontogeny of melanosomes in embryonic chick retinal pigment epithelium. Fed. Proc. 27, 722--728
(1968) Toshima, S., Moore, G. E., Sandberg, A. A. : Ultrastrudture of human melanoma in cell culture. Cancer 21, 202--216 (1968) TrSger, H., Klingmfiller, G. : Die Melaningranula im Naevuszellnaevus. Arch. klin. exp. Derm. 226, 1--12 (1966) Vogel, C. L., Dhru, D., Rorsman, H., Rosengren, A.-M., Rosengren, E. : Dopa and 5-S-cysteinyldopa in malignant melanoma in Ugandan Africans. Aeta derm.-venereol. (Stoekh.) 54, 19--20 (1974) Wetlings, S. R., Siegel, B. V. : Role of Golgi apparatus in the formation of melanin granules in human malignant melanoma. J. Ultrastruct. Res. 8, 147--154 (1959) Zelickson, A. S., Windhorst, D. B., White, J. G., Good, R. A. : The Chediak-Hig~shi syndrome: formation of giant melanosomes and the basis of hypopigmentation. J. invest. Derm. 49, 575--581 (1967) Yu, R. J., van Scott, E. J. : Substrate identity in mammalian melanogenesis. J. invest. Derm. 60, 234--237 (1973) Dr. Lenke Szekeres Department of Dermatology Medical University of Szeged P. O. Box 480 H-6701 Szeged Hungary
