Cell Tiss. Res. 185, 331-337 (1977)
Cell and Tissue Research 9 by Springer-Verlag 1977
Ultrastructural Study on Hepatic Melanin in Xenopus laevis Koichi N o d a Laboratory of Electron Microscopy, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Takashi Nomaguchi Department of Biology Yasukazu Tanaka Department of Pathology
Summary. The livers of Xenopus laevis, grouped by chronological age (0.5, 2 and 3 yrs), were studied electron microscopically. Ultrastructurally most of the melanin granules in the mature female liver showed art internal structure similar to the melanin granules of the oocytes. The hepatic melanin granules of immature females and of all males were pleomorphic and failed to show the characteristic internal structure similar to those of the oocytes. The oocyte is the probable source of most of the hepatic melanin of the mature female. Key words: Ultrastructure - Hepatic melanin - Sex - Age - Xenopus laevis.
Accumulation of significant amounts of melanin granules in hepatic macrophages of both female Ambystoma mexicanum (Andrew, 1971) and female Xenopus laevis (Tanaka, et al., 1974) has been reported. The phenomenon is related to maturation and sex difference in these species (Andrew, 1971 ; Tanaka, et al., 1974), and appears to be of interest for research in aging. For a further morphological understanding of the hepatic melanin of Xenopus laevis, an electron microscopic analysis has been performed. This paper describes the presence of two types of melanin granules, in the liver of Xenopus one resembling those of the oocyte and the other those of cutaneous melanin. The former type was noted exclusively in the liver of the mature female toads.
Materials and Methods Three each of mature females and males(2 or 3 yrs) and fourjuvenile toads (0.5yr) were examined. The inbred animals were maintained in the temperature controlled water pool (22~C) of the Zoological Institute, Faculty of Science, Hokkaido University,Japan. Twicea week they were fed slices of fresh or stored pork livers.
Send offprint requeststo: Dr. Koichi Noda, Laboratory of Electron MicroscopyTokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo-173,Japan
332
K. Noda et al.
After anesthesia by MS 222, tissue was excisedfrom the liver, spleen, ovary and skin and fixed with 1.0 % glutaraldehyde in 0.1 M phosphate buffer for 2 h at room temperature. The tissues were treated with 1.0 % osmic acid for 2 h at 0-4~C and, after dehydration with ethanol, embeddedin Epoxy resins. Thin sectionswere cut on an LKB Ultratome III with diamond knivesand stained with uranyl and lead. Examination was made on a Hitachi HU-12A electron microscope. Results In ultrastructure, the pigment-bearing cells of both liver and spleen were indistinguishable regardless of differences in sex and in age. Thus the major description is confined to those of the liver. The ultrastructural characteristics of the pigment-bearing cells were comparable to those of hepatic macrophages except for the presence of a large number of cytoplasmic granules, the majority of which showed positive histochemical reactions characteristic of melanin (Tanaka, et al., 1974). In the immature females and in all males, the pigment-bearing cells were found diffusely distributed in the sinusoids and the subcapsular area of the liver, and near or around the hepatic vein (Tanaka et al., 1974). In the mature females they tended to aggregate and occasionally formed large nodular foci (Tanaka et al., 1974). The melanin granules in these pigment-bearing cells were surrounded by a m e m b r a n e as described previously (Balinsky and Devis, 1963). Their shape and electron density in immature females and in all males differed from those of mature females. In the former group, most of them were either slightly elongated or oval and homogeneously electron dense (Figs. 1, 2) and coexsisted in the pigment cell cytoplasm with granules of irregular contour and variable electron opacity (Fig. 1). Some of these showed a form transitional to larger cytoplasmic granules known as phagosomes or residual lysosomes (de Duve, 1969). Lamellar structures were frequently noted in larger compound granules. In contrast to the variegated morphology of the melanin granules in the former group, those in the hepatic pigment-bearing cells of mature females were spherical and fairly uniform in size (0.5-0.7 g in diameter) (Fig. 3). A few with irregular contours seen in the hepatic macrophages of male animals were also noted (Fig. 3). The spherical melanin granules consisted of an electron-dense outer shell of 0.05-0.131a thick and an internal core of less electron-dense matrix containing scattered dense speckles of 100-170 N in diameter(Fig. 4). The ultrastructural characteristics of these spherical granules of the mature female hepatic pigment-bearing cells conformed to those of melanin granules of oocytes as described in the following section. Only oocytes 800 la or more in diameter were studied (Fig. 5) as no incomplete melanin granules probably exsist in these cells (Balinsky and Devis, 1963). Most of the pigment granules in such oocytes ranged from 0.5-0.7g in diameter, far exceeding the value presented for mature melanin by previous investigators (Balinsky and Devis, 1963). The morphological characteristics of these granules were the same as those of the pigment granules found in female hepatic macrophages and also as those described by others (Balinsky and Devis, 1963; Eppig, 1970). They were absent from male hepatic macrophages. The melanin granules of skin pigment cells were of the homogeneous type found in the livers of both sexes (Fig. 6) and their ultrastructural details in other animals
Fig. 1. In parts of three pigment cells in the liver of a 2-year male two types of granules are distinguished: (1) a round to ovoid, highly electron-opaque type of relatively uniform size (large arrows), (2) a less electron-opaque type with significant size variation (double arrows). The latter type contained lamellar structures (double arrows) and electron opaque subunits resembling granules of the former type. There is a morphological transition between these two types suggesting that the granules with a variegated appearance represent different stages in the digestion of phagocytized melanin, x 12,000 Fig. 2. A higher magnification of the melanin granules from the same sample as shown in Fig. 1. Note that the uniformly opaque appearence of the melanin granules is occasionally interrupted by tiny vesicles, x 43,000
Fig. 3. A part of a pigment cell in the liver of a 2-year female showing a large number of pigment granules o f uniform size. As in the male macrophages, these granules may be classified into two types but those of the highly electron opaque type are exceedingly dominant. • 12,000 Fig. 4. A higher magnification of some melanin granules shown in Fig. 3. They possess a characteristic structure consisting of an electron-dense outer shell and an internal core of less electron-dense matrix containing scattered dense speckles. • 43,000
Fig. 5. The cortex of an oocyte 800 bt in diameter. Three types of granules are distinguished; (1) small, round, highly electron opaque pigment granules near the surface (small arrows), (2) round, less electron opaque lipid granules (large arrow), and (3) large, high electron opaque yolk platelets (asterisk). x 4700. The inset shows details of melanin granules in the oocyte. Note the presence of the internal structure similar to the previous illustration (Fig. 4). • 50,000 Fig. 6. Parts of two skin pigment cells showing a large number of pigment granules with a homogeneous content, x 8000. The inset shows the skin melanin granules at a higher magnification. Note the absence of the internal structure noted in the oocyte melanin (Fig. 5). x 50,000
336
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may be referred elsewhere (Szab6, 1969). Because they were of less importance for the purpose of the present study, the details of their organization in the tissue are not illustrated.
Discussion The ultrastructural characteristics of the melanin granules of hepatic pigmentbearing cells in female Xenopus laevis vary with maturation. In the liver of mature females, two types of melanin granules were ultrastructurally distinguishable. Most o f melanin granules showed the internal core characteristic of those in well developed amphibian oocytes (Balinsky and Devis, 1963; Eppig, 1970). The remainder of the granules showed no internal core and resembled those of the skin. The latter type was found in the liver of both sexes at various stages of maturation. The observations suggests that the melanin granules with cores occur only in the liver of mature females. To explain this interesting results, we hypothesize as follows. A large amount of melanin granules was accumulated in mature female Xenopus liver (Tanaka et al., 1974). Since melanin-bearing cells in the liver were non-melanopoietic macrophages the source of melanin granules in the liver must be organs other than the liver. The skin and ovary are the most probable sites of extensive melanin production. However, melanin granules in the skin could be distinguished ultrastructurally from most of melanin granules in the liver of mature female by the absence o f the internal core and did not show any notable difference attributable to age or sex. The internal core observed in the melanin granules of female liver resembles those of the granules of well developed Xenopus oocytes. This ultrastructural similarity of melanin granules between the liver and the ovary suggests an ovarian origin of mature females hepatic melanin. The absence of the internal core in hepatic melanin granules of immature females favors this assumption. We suggest that the hepatic melanin granules with the internal core deposited exclusively in mature female Xenopus liver were actually deriven from the ovary by the following process. In the mature ovary of Xenopus laevis, all stages of oocyte maturation are present at one time. The ovary also contains over-ripe eggs which undergo a degeneration process. This process is probably precipitated by an invasion of phagocytes which carry the debris away from the ovary. A large number of pigment-bearing cells is frequently noted in ovarian stroma (Tanaka et al., 1974); the phenomenon could be the end phase of auto-degradation of the over-ripe eggs. As has been suggested by Jordan (1925), the pigment or pigment-bearing cells of the ovary may be carried via blood stream to the parenchymatous organs where active phagocytosis occurs. The melanin granules are not digested by available enzymes of the Xenopus liver. As a result, the melanin granules are gradually accumulated in the liver and the amounts of the hepatic melanin granules in the female Xenopus may be used as an indicator of age in this species.
Acknowledgements: We are indebted to Dr. M. Wakahara, ZoologicalInstitute, Faculty of Science, Hokkaido University, Sapporo for furnishing the Xenopuslaevis used in this investigation. Skillful technical assistance of Mrs. C. Kanai, Laboratory of Electron microscopy, Tokyo Metropolitan
Hepatic Melanin in Xenopus
337
Institute of Gerontology, Tokyo, in preparation of the electron microscopic specimens is grateful acknowledged. We appreciate Mr. H. Gotoh, Department of Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, for preparation of the histological sections.
References Andrew, W.: The anatomy of aging in man and animals. New York-London: Grune & Stratton 1971 Balinsky, B.I., Devis, R.J.: Origin and differentiation of cytoplasmic structures in the oocyte of Xenopus laevis. Acta embryol, morph, exp. 6, 55-108 (1963) De Duve, C.: The lysosome in retrospect. In: Lysosomes in biology and pathology (J.T. Dingle and H.B. Fell, eds.), Vol. 1, pp. 3-40. Amsterdam-London: North-Holland Publishing Company 1969 Eppig, J.J., Jr.: Melanogenesis in amphibians. III. The buoyant density of oocyte and larval Xenopus laevis melanosomes and the isolation of oocyte melanosome from the eyes of PTU-treated larvae. J. exp. Zool. 175, 467-475 (1970) Jordan, H.E.: A source of origin of pigmented leukocytes in amphibia. Anat. Rec. 29, 387 (1925) Szab6, G.: The biology of the pigment cell. In: The biological basis of medicine (E.E. Bittar and N. Bittar, eds.), pp. 59-91. New York: Academic Press 1969 Tanaka, Y., Noda, K., Nomaguchi, T., Yamagishi, H.: Hepatic melanosis and ageing in amphibia. A preliminary observation on two species of anura Xenopus laevis and Rana nigromaculata. Exp. Geront. 9, 263-268 (1974)
Accepted September 12, 1977
Cell and Tissue Research 9 by Springer-Verlag 1977
Ultrastructural Study on Hepatic Melanin in Xenopus laevis Koichi N o d a Laboratory of Electron Microscopy, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Takashi Nomaguchi Department of Biology Yasukazu Tanaka Department of Pathology
Summary. The livers of Xenopus laevis, grouped by chronological age (0.5, 2 and 3 yrs), were studied electron microscopically. Ultrastructurally most of the melanin granules in the mature female liver showed art internal structure similar to the melanin granules of the oocytes. The hepatic melanin granules of immature females and of all males were pleomorphic and failed to show the characteristic internal structure similar to those of the oocytes. The oocyte is the probable source of most of the hepatic melanin of the mature female. Key words: Ultrastructure - Hepatic melanin - Sex - Age - Xenopus laevis.
Accumulation of significant amounts of melanin granules in hepatic macrophages of both female Ambystoma mexicanum (Andrew, 1971) and female Xenopus laevis (Tanaka, et al., 1974) has been reported. The phenomenon is related to maturation and sex difference in these species (Andrew, 1971 ; Tanaka, et al., 1974), and appears to be of interest for research in aging. For a further morphological understanding of the hepatic melanin of Xenopus laevis, an electron microscopic analysis has been performed. This paper describes the presence of two types of melanin granules, in the liver of Xenopus one resembling those of the oocyte and the other those of cutaneous melanin. The former type was noted exclusively in the liver of the mature female toads.
Materials and Methods Three each of mature females and males(2 or 3 yrs) and fourjuvenile toads (0.5yr) were examined. The inbred animals were maintained in the temperature controlled water pool (22~C) of the Zoological Institute, Faculty of Science, Hokkaido University,Japan. Twicea week they were fed slices of fresh or stored pork livers.
Send offprint requeststo: Dr. Koichi Noda, Laboratory of Electron MicroscopyTokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo-173,Japan
332
K. Noda et al.
After anesthesia by MS 222, tissue was excisedfrom the liver, spleen, ovary and skin and fixed with 1.0 % glutaraldehyde in 0.1 M phosphate buffer for 2 h at room temperature. The tissues were treated with 1.0 % osmic acid for 2 h at 0-4~C and, after dehydration with ethanol, embeddedin Epoxy resins. Thin sectionswere cut on an LKB Ultratome III with diamond knivesand stained with uranyl and lead. Examination was made on a Hitachi HU-12A electron microscope. Results In ultrastructure, the pigment-bearing cells of both liver and spleen were indistinguishable regardless of differences in sex and in age. Thus the major description is confined to those of the liver. The ultrastructural characteristics of the pigment-bearing cells were comparable to those of hepatic macrophages except for the presence of a large number of cytoplasmic granules, the majority of which showed positive histochemical reactions characteristic of melanin (Tanaka, et al., 1974). In the immature females and in all males, the pigment-bearing cells were found diffusely distributed in the sinusoids and the subcapsular area of the liver, and near or around the hepatic vein (Tanaka et al., 1974). In the mature females they tended to aggregate and occasionally formed large nodular foci (Tanaka et al., 1974). The melanin granules in these pigment-bearing cells were surrounded by a m e m b r a n e as described previously (Balinsky and Devis, 1963). Their shape and electron density in immature females and in all males differed from those of mature females. In the former group, most of them were either slightly elongated or oval and homogeneously electron dense (Figs. 1, 2) and coexsisted in the pigment cell cytoplasm with granules of irregular contour and variable electron opacity (Fig. 1). Some of these showed a form transitional to larger cytoplasmic granules known as phagosomes or residual lysosomes (de Duve, 1969). Lamellar structures were frequently noted in larger compound granules. In contrast to the variegated morphology of the melanin granules in the former group, those in the hepatic pigment-bearing cells of mature females were spherical and fairly uniform in size (0.5-0.7 g in diameter) (Fig. 3). A few with irregular contours seen in the hepatic macrophages of male animals were also noted (Fig. 3). The spherical melanin granules consisted of an electron-dense outer shell of 0.05-0.131a thick and an internal core of less electron-dense matrix containing scattered dense speckles of 100-170 N in diameter(Fig. 4). The ultrastructural characteristics of these spherical granules of the mature female hepatic pigment-bearing cells conformed to those of melanin granules of oocytes as described in the following section. Only oocytes 800 la or more in diameter were studied (Fig. 5) as no incomplete melanin granules probably exsist in these cells (Balinsky and Devis, 1963). Most of the pigment granules in such oocytes ranged from 0.5-0.7g in diameter, far exceeding the value presented for mature melanin by previous investigators (Balinsky and Devis, 1963). The morphological characteristics of these granules were the same as those of the pigment granules found in female hepatic macrophages and also as those described by others (Balinsky and Devis, 1963; Eppig, 1970). They were absent from male hepatic macrophages. The melanin granules of skin pigment cells were of the homogeneous type found in the livers of both sexes (Fig. 6) and their ultrastructural details in other animals
Fig. 1. In parts of three pigment cells in the liver of a 2-year male two types of granules are distinguished: (1) a round to ovoid, highly electron-opaque type of relatively uniform size (large arrows), (2) a less electron-opaque type with significant size variation (double arrows). The latter type contained lamellar structures (double arrows) and electron opaque subunits resembling granules of the former type. There is a morphological transition between these two types suggesting that the granules with a variegated appearance represent different stages in the digestion of phagocytized melanin, x 12,000 Fig. 2. A higher magnification of the melanin granules from the same sample as shown in Fig. 1. Note that the uniformly opaque appearence of the melanin granules is occasionally interrupted by tiny vesicles, x 43,000
Fig. 3. A part of a pigment cell in the liver of a 2-year female showing a large number of pigment granules o f uniform size. As in the male macrophages, these granules may be classified into two types but those of the highly electron opaque type are exceedingly dominant. • 12,000 Fig. 4. A higher magnification of some melanin granules shown in Fig. 3. They possess a characteristic structure consisting of an electron-dense outer shell and an internal core of less electron-dense matrix containing scattered dense speckles. • 43,000
Fig. 5. The cortex of an oocyte 800 bt in diameter. Three types of granules are distinguished; (1) small, round, highly electron opaque pigment granules near the surface (small arrows), (2) round, less electron opaque lipid granules (large arrow), and (3) large, high electron opaque yolk platelets (asterisk). x 4700. The inset shows details of melanin granules in the oocyte. Note the presence of the internal structure similar to the previous illustration (Fig. 4). • 50,000 Fig. 6. Parts of two skin pigment cells showing a large number of pigment granules with a homogeneous content, x 8000. The inset shows the skin melanin granules at a higher magnification. Note the absence of the internal structure noted in the oocyte melanin (Fig. 5). x 50,000
336
K. Noda et al.
may be referred elsewhere (Szab6, 1969). Because they were of less importance for the purpose of the present study, the details of their organization in the tissue are not illustrated.
Discussion The ultrastructural characteristics of the melanin granules of hepatic pigmentbearing cells in female Xenopus laevis vary with maturation. In the liver of mature females, two types of melanin granules were ultrastructurally distinguishable. Most o f melanin granules showed the internal core characteristic of those in well developed amphibian oocytes (Balinsky and Devis, 1963; Eppig, 1970). The remainder of the granules showed no internal core and resembled those of the skin. The latter type was found in the liver of both sexes at various stages of maturation. The observations suggests that the melanin granules with cores occur only in the liver of mature females. To explain this interesting results, we hypothesize as follows. A large amount of melanin granules was accumulated in mature female Xenopus liver (Tanaka et al., 1974). Since melanin-bearing cells in the liver were non-melanopoietic macrophages the source of melanin granules in the liver must be organs other than the liver. The skin and ovary are the most probable sites of extensive melanin production. However, melanin granules in the skin could be distinguished ultrastructurally from most of melanin granules in the liver of mature female by the absence o f the internal core and did not show any notable difference attributable to age or sex. The internal core observed in the melanin granules of female liver resembles those of the granules of well developed Xenopus oocytes. This ultrastructural similarity of melanin granules between the liver and the ovary suggests an ovarian origin of mature females hepatic melanin. The absence of the internal core in hepatic melanin granules of immature females favors this assumption. We suggest that the hepatic melanin granules with the internal core deposited exclusively in mature female Xenopus liver were actually deriven from the ovary by the following process. In the mature ovary of Xenopus laevis, all stages of oocyte maturation are present at one time. The ovary also contains over-ripe eggs which undergo a degeneration process. This process is probably precipitated by an invasion of phagocytes which carry the debris away from the ovary. A large number of pigment-bearing cells is frequently noted in ovarian stroma (Tanaka et al., 1974); the phenomenon could be the end phase of auto-degradation of the over-ripe eggs. As has been suggested by Jordan (1925), the pigment or pigment-bearing cells of the ovary may be carried via blood stream to the parenchymatous organs where active phagocytosis occurs. The melanin granules are not digested by available enzymes of the Xenopus liver. As a result, the melanin granules are gradually accumulated in the liver and the amounts of the hepatic melanin granules in the female Xenopus may be used as an indicator of age in this species.
Acknowledgements: We are indebted to Dr. M. Wakahara, ZoologicalInstitute, Faculty of Science, Hokkaido University, Sapporo for furnishing the Xenopuslaevis used in this investigation. Skillful technical assistance of Mrs. C. Kanai, Laboratory of Electron microscopy, Tokyo Metropolitan
Hepatic Melanin in Xenopus
337
Institute of Gerontology, Tokyo, in preparation of the electron microscopic specimens is grateful acknowledged. We appreciate Mr. H. Gotoh, Department of Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, for preparation of the histological sections.
References Andrew, W.: The anatomy of aging in man and animals. New York-London: Grune & Stratton 1971 Balinsky, B.I., Devis, R.J.: Origin and differentiation of cytoplasmic structures in the oocyte of Xenopus laevis. Acta embryol, morph, exp. 6, 55-108 (1963) De Duve, C.: The lysosome in retrospect. In: Lysosomes in biology and pathology (J.T. Dingle and H.B. Fell, eds.), Vol. 1, pp. 3-40. Amsterdam-London: North-Holland Publishing Company 1969 Eppig, J.J., Jr.: Melanogenesis in amphibians. III. The buoyant density of oocyte and larval Xenopus laevis melanosomes and the isolation of oocyte melanosome from the eyes of PTU-treated larvae. J. exp. Zool. 175, 467-475 (1970) Jordan, H.E.: A source of origin of pigmented leukocytes in amphibia. Anat. Rec. 29, 387 (1925) Szab6, G.: The biology of the pigment cell. In: The biological basis of medicine (E.E. Bittar and N. Bittar, eds.), pp. 59-91. New York: Academic Press 1969 Tanaka, Y., Noda, K., Nomaguchi, T., Yamagishi, H.: Hepatic melanosis and ageing in amphibia. A preliminary observation on two species of anura Xenopus laevis and Rana nigromaculata. Exp. Geront. 9, 263-268 (1974)
Accepted September 12, 1977
