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Comparative light and electron microscopic studies of dorsal skin melanophores of Indian toad, Bufo melanostictus
Journal of Microscopy and Ultrastructure 2 (2014) 230–235
Contents lists available at ScienceDirect
Journal of Microscopy and Ultrastructure journal homepage: www.elsevier.com/locate/jmau
Original Article
Comparative light and electron microscopic studies of dorsal skin melanophores of Indian toad, Bufo melanostictus Sharique A. Ali ∗ , Ishrat Naaz Post Graduate Department of Biotechnology, Saifia Science College, Bhopal 462001, India
a r t i c l e
i n f o
Article history: Received 9 April 2014 Received in revised form 26 June 2014 Accepted 6 July 2014 Available online 23 July 2014 Keywords: Toad melanophores Melanosomes Integumental pigmentation Anatomical significance
a b s t r a c t Indian toad, Bufo melanostictus is an amphibian, which is able to change the body color to adapt to its ambient need. This ability is due to specialized skin pigment cells known as melanophores which are excellent animal-cell models to study most of the physiological phenomenon related to color changes. In the present investigation morpho-anatomic details of dorsal skin melanophores of B. melanostictus were studied by means of light and electron microscope to establish their phylogenetic relevance with other vertebrates. Light microscopic observations revealed that toad skin contains dominantly present black pigment cells, the melanophores in its sub epidermis while dermal melanophores are rare. The ultrastructure of the melanophore is characterized by oval nucleus and numerous pigment granules, the melanosomes which remain scattered in the cytoplasm. Other sub-cellular organelles like mitochondria, well-developed tubular endoplasmic reticulum and Golgi vesicles were also found to be present in the remaining cytoplasmic area. Toad melanophore contains four distinct stages of melanosomes which are similar in development pattern to the mammalian melanocytes. These findings indicate that toad melanophores contain phylogenetically significant information of anatomical similarity with lower as well as higher vertebrates which can help to better understand the inter relationships between vertebrate pigment cells and their role in skin dysfunctions. © 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
1. Introduction Amphibians adapt readily to a light or dark colored backgrounds due to pigment movement in their chromatophores including the black-pigmented cells – melanophores. Melanophores are large, black, specialized dendritic cells which arise from neural crest and are widely distributed among the lower vertebrates [1–6]. Melanin pigments are tyrosine-based biopolymers synthesized and deposited within highly specialized subcellular organelles termed as melanosomes [7]. The expansion or contraction of melanin granules results in the darkening or lightening
∗ Corresponding author. Tel.: +91 9893015818. E-mail address: [email protected] (S.A. Ali).
of the skin which ultimately plays principal role in rapid color changes in the skin; restricted to fishes, amphibians and reptiles [8–10]. The melanophores have been used as unique cell models to study the underlying mechanism of integumental pigmentation. Pigment migration process in the melanophores are of a complex nature, and are regulated either by nervous or by endocrine stimulation or by both [2,3]. In response to the various stimuli, they rapidly move their pigment granules, the melanosomes to the cell center or periphery. Receptor systems participate in a very coordinated manner in mediation of these responses of color change [11]. Due to this dynamic property and availability of multiple receptor–ligand binding sites on melanophores, they are excellent targets for understanding the effects of various pharmacological agents [12–14].
http://dx.doi.org/10.1016/j.jmau.2014.07.002 2213-879X/© 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
S.A. Ali, I. Naaz / Journal of Microscopy and Ultrastructure 2 (2014) 230–235
However, ultrastructural details of lower vertebrate melanophores are still wanting. Unless the ultrastructural details of melanophores become clearly known, the cellular mechanism within these pigment cells leading to pigmentary changes or disorders is difficult to understand. Some ultrastructural investigations have been done by previous researchers on melanophores of various classes of vertebrates like teleosts and lung fishes [9,15,16], reptiles [17], larval amphibians [18] which have revealed remarkably consistent fine structural features of these melanosomes synthesizing and containing cells in order to explain their role in physiological color changes. However, the pattern of distribution and association of these cell types has been found to vary considerably in different vertebrates [15]. The mediation of melanophores for varied responses is species dependent and hence no definite conclusion has yet been reached, in spite of extensive work in chromatic science. Despite a large body of literature on melanophores, no report on fine structure of dermal skin melanophores of Indian toad, Bufo melanostictus has appeared to date. As toads are familiar vertebrates and share similar mechanism of skin pigmentation with human beings, they have outstanding potential as phylogenetical tools to demystify and better understand the role of pigment cell system and its evolution. Hence, the present study has been carried out to investigate in detail the fine structure of dorsal skin melanophores of B. melanostictus by means of light and transmission electron microscopy to provide some information to bridge the missing link in the chain of events of melanophore structure and function. 2. Materials and methods 2.1. In vitro dorsal skin preparation Irrespective of sex, adult B. melanostictus of 7 cm length and 30–50 g of body weight were collected from the local areas of Bhopal, Madhya Pradesh during the rainy season and kept in large aquaria with normal photoperiod i.e. 12 h of light–dark cycle. Prior to the experiments, B. melanostictus were allowed to acclimatize to laboratory conditions for two days. During the acclimatization period toads were regularly fed with live snail and small insects. Diseased, injured or lethargic B. melanostictus were removed and only active, uniformly sized B. melanostictus were used. For in vitro studies the dorsal skin was peeled away from decapitated toad and a series of nearly 1 mm × 1 mm diameter dorsal skin pieces were cut out with a sharpened steel scissor. The pieces were kept in 10 mL of Amphibian Ringer Saline (ARS), containing 111 mM of sodium chloride, potassium chloride 2 mM, calcium chloride 1 mM and sodium hydroxide 2 mM, in 100 mL of double distilled water at pH 7.4, in small Petri dishes and they were equilibrated in saline medium for 15–20 min with frequent stirring. The Ethical Committee for Animal Experimentation and Research, Saifia College of Science, Bhopal, India certified the use of animals (approval number SSC/06–06–22, dated 26.10.2006). The research work of the Institute is done in strict compliance with the Guidelines for Laboratory Animals in Medical Colleges (2001) of the Indian Council of Medical Research (ICMR), as well as with the Breeding of
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and Experiments on Animals Amendment Rules (2001) and the Prevention of Cruelty to Animal Act (1966). 2.2. Transmission electron microscopy Small pieces of dorsal skin of toad were cut and were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 12 h at 4 ◦ C. After washing in buffer, the skin pieces were post fixed in 1% OsO4 for 1 h at 4 ◦ C. The samples were dehydrated in an ascending grade of acetone, infiltrated and embedded in araldite CY 212 (TAAB, UK). Thick sections (1 m) were cut with an ultramicrotome, mounted on to glass slides, stained with aqueous toluidine blue and observed under a light microscope for gross observation of the area and quality of the tissue fixation. For electron microscope examination, thin sections of gray-silver color interference (70–80 nm) were cut and mounted onto 300 mesh-copper grids. Sections were stained with alcoholic uranyl acetate and alkaline lead citrate, washed gently with distilled water and observed under a Morgagni 268D transmission electron microscope (Fei Company, The Netherlands) at an operating voltage 80 kV. Images were digitally acquired by using a CCD camera (Megaview III, Fei Company) attached to the microscope. 3. Results 3.1. Light microscopic observations of dorsal skin melanophores of B. melanostictus Light microscopically in vertical sections of dorsal skin of B. melanostictus pigment cells were found in distinct layers of sub-epidermis (Fig. 1a and b). The dominating pigment cell of dorsal skin is the melanophore which plays principal role in physiological color changes. No “chromatophore unit” was observed due to the absence of other pigment cells. The darkness of the skin is due to the presence of melanophores in the sub-epidermis and dermis. The epidermis is thick and is composed of epidermal cells. Occasionally, epidermal glands occur interrupting the epidermal layers. Toad melanophores are organized in two distinct layers: one sub-epidermal melanophore layer which remains present closely below the epidermis while the other remains scattered in the loose dermis matrix of the skin. 3.2. Electron microscopic observations of dorsal skin melanophores of B. melanostictus observations of dorsal skin Ultrastructural melanophores of toad have revealed remarkably consistent fine structural features of these melanosome synthesizing cells. Sub epidermal melanophores contain oval nuclei which possesses a coarsely granulated nucleoplasm. The melanophores are surrounded by a thin single membrane corresponding to the plasma membrane of the cell. Oval shaped melanosomes were limited by unit membrane and were found to be scattered around the nucleus. Bundle of collagen fibers were also noticed (Fig. 1c). Besides melanosomes the cytoplasm furnished
232
S.A. Ali, I. Naaz / Journal of Microscopy and Ultrastructure 2 (2014) 230–235
Fig. 1. Light and electron microscope photomicrograph of dorsal skin of B. melanostictus in Amphibians Ringer Saline (ARS). (a and b) Light micrograph showing: The general organization of the toad skin and the localization of melanophores can be seen. epidermis (E), epidermal gland (G), sub-epidermis (S), dermis (D), melanophores (M) (arrow). 400×. (c) Transmission electron micrograph of vertical section of dorsal skin melanophores of toad: showing, oval nucleus (N) contains granulated nucleolus (n), surrounded by oval shaped premelanosomes (p) and melanosomes (m) scattered throughout cytoplasm. Endoplasmic reticulum (ER) remains scattered between melanosomes. 20,000×. (d) Showing: Low magnification electron micrograph of sub epidermal melanophores. They are actively engaged in the synthesis of melanosomes (m) of various stages of development. 4000×.
few mitochondria of tubular type and some vesicular components of the endoplasmic reticulum were noticed (Fig. 1c and d). Sub-epidermal melanophores contain oval shaped, unit membrane bound melanosomes of various stages of development. Immature, unmelanized premelanosome with intraluminal fibrils were observed. In premelanosomes of stage II complete intraluminal fibrils, stage III with pigment deposition at intraluminal fibrils was observed. Although
majority of granules of stage IV are highly electron dense as they are fully melanized therefore their internal structure is indistinguishable (Fig. 3a). On the other hand a number of granules exhibit a distinct premelanosomal structure which are electron-lucent pigment (Fig. 2a and b). Dermal melanophores occur between loose collagen fibers just beneath the sub-epidermis, also contains melanosomes with various degrees of pigmentation which are analogous to sub-epidermal melanosomes in shape and size (Fig. 3b).
S.A. Ali, I. Naaz / Journal of Microscopy and Ultrastructure 2 (2014) 230–235
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Fig. 2. Transmission electron micrograph of vertical section of a part of dorsal skin melanophores of Bufo melanostictus: (a) Showing, cytoplasm filled with melanosomes (m) of various stage of development, surrounded by endoplasmic reticulum (ER), vesicles (V) in the sub epidermis. 20,000×. (b) Showing, high magnification electron micrograph of electron-lucent premelanosomes (p) contains fibrillar matrix, electron-dense melanosomes (m) and vesicles (v). 40,000×.
Fig. 3. Transmission electron micrograph of vertical section of a part of dorsal skin melanophores of Bufo melanostictus showing: (a) Sub-epidermal melanosomes of various stages (I, II, III, IV) of development. 20,000×. (b) High magnification electron micrograph of dermal melanosomes (m) analogous to sub-epidermal melanosomes. 40,000×.
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4. Discussion Many vertebrate animals exhibit complex skin pigmentation patterns. According to Bagnara and Hadley [2] the distribution pattern of chromatophores is the main factor that determines the ultimate pigmentation pattern of a species. Many interpretations have been made to explain the location and mechanism of pigment translocation inside the melanophores of vertebrates to better understand the mysterious phenomenon of physiological color changes. However, it appears that even after occupying their final destinations, the melanophores retain a high degree of plasticity. In this series of events, the present investigation throws some light on the ultrastructure of dorsal skin melanophores of B. melanostictus, the common Indian toad. Adult B. melanostictus is an amphibian model which has a dominant black pigment cell system containing melanophores. It was observed light microscopically that the melanophores of this species are organized in two distinct layers: one sub-epidermal melanophores layer which remains present closely below the epidermis while the other layer of melanophores remains scattered in the loose dermal matrix of the skin. High resolving power of electron microscope confirmed the findings of the toad melanophores seen by light microscope. When compared with other vertebrates it was found that the dorsal skin epidermal melanophores of Indian toad, dominantly lie in the sub-epidermis. This situation is in contrast to that found in lungfishes i.e. Neoceratodus forsteri, Lepidosiren paradoxa, Protopterus sp., [15,16], larval Rana pipiens [19]. In these animal models the epidermal melanophores present in the intercellular spaces between level of epidermis while in more developed hierarchical forms like reptiles and mammals [20,21] in which the epidermal melanophores lie in the epidermis. Due to the lack of other pigment cells i.e. iridiophores or iridiophore–xanthophore no dermal chromatophore unit was observed in B. melanostictus in contrast to that described by Bagnara et al. [22] in Hyla cinerea, Agalychnis dachnicolo, etc. Ultrastructural observation of toad skin melanophores also revealed the presence of well marked prominent oval nucleus filled with granular nucleolus. We have found a thin, moderately electron-dense granular layer on inner surface of nuclear envelope which is similar in appearance as reported by Lamer and Chavin [23] in the integumental melanophores of the marine fish, Latimeria chalumnae. Cytoplasm of the toad melanophore was found to be filled with melanosomes of varying degree of pigmentation around nucleus. Remaining cytoplasmic areas contained mitochondria of tubular type, vacuolar endoplasmic reticulum, and Golgi apparatus. Stage I premelanosomes are spherical vesicles present near Golgi apparatus. These observations confirm the findings of Seiji et al. [24] who reported Golgi vesicles as possible precursor of melanosomes. We have also found that developing melanosomes as described by Fitzpatrick et al. [25] in mammalian melanocytes are present in various parts of the toad melanophores. Classical electron microscopy of human skin melanocytes describes four distinct stages (I, II, III and
IV) of melanosome development [26], a similar development progression has been found here. The sub-epidermal melanophores of Bufo were found to possess a large number of premelanosomes of stage II and III, suggesting that the process of active melanogenesis occurs in the sub-epidermis. Imaki and Chavin [15,16] reported that premelanosomes occurred abundantly in the sub-epidermal melanophores in comparison to dermal melanophores, however premelanosomes in both of these different layers share identical internal structure. Their ultrastructural observation agrees with our present study. We have also demonstrated that fully developed melanosomes of the stage IV are oval shaped and are frequently recognizable as being surrounded by a limiting membranous body which contains uniformly electron-dense matrix whereas the immature premelanosomes possess electron-lucent, fibrillar protein matrix in which melanin deposition takes place in later stage of development. The presence of typical pre-melanosomes and melanosomes showing progressive degrees of melanization in toad melanophores leads to the conclusion that the basic structure of the melanin granule and its mode of development is the same in higher vertebrates [27–29]. Toad skin melanophores anatomical structures appears to have a treasure of information, which have physiological links with the lung fish melanosomes as well as human melanosomes from phylogenetic point of view. The data of the present finding provides a foundation to better understand the morpho-anatomic and phylogenetic details of amphibian melanophores with human melanocytes which can account for the development of therapeutic agents against pigment disorders. Here, we conclude that ultrastructure of dorsal skin melanophores of B. melanostictus resembles the fine structural features of melanocytes of higher vertebrates including human beings as no significant differences were observed. These data taken in conjugation with earlier evidences regarding anatomy of pigment cells continue to suggest that a centrally occurring phenomenon of melanin biogenesis is associated with four distinct phases of melanosome development and translocation of these melanosomes intra-cellularly along with other pigment cells results in color change in vertebrates with common ancestral history. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements Thanks are due to M.P. Council of Science and Technology, Govt. of MP for granting a Research Project No. 5690/CST/BTAC/2012 to Dr. Sharique A. Ali. Authors are also thankful to the Secretary and Principal of Saifia Science College, Bhopal, for providing the necessary facilities. The authors wish to thank SAIF, AIIMS, New Delhi for sample preparation, providing technical assistance and images of TEM. References [1] Brouwer E, Van de Veerdonk FCG. Possible involvement of ␣ and  receptors in the natural colour change and the ␣-MSH-induced
S.A. Ali, I. Naaz / Journal of Microscopy and Ultrastructure 2 (2014) 230–235
[2] [3]
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[5]
[6]
[7]
[8] [9] [10] [11]
[12]
[13]
[14]
dispersion in Xenopus laevis in vivo. Eur J Pharmacol 1972;17: 234–9. Bagnara JT, Hadley ME. Chromatophores and colour change. Englewood Cliffs: Prentice Hall; 1973. p. 202. Fujii R, Miyashita Y, Fujii Y. Muscarinic cholinoreceptor mediate neutrally evoked pigment aggregation in glass catfish melanophores. J Neural Transm 1982;54:29–39. Aspengren S, Hedberg D, Skold HN, Wallin M. New insights into melanosomes transport in vertebrate pigment cell. Int Rev Cell Mol Biol 2009;272:245–302. Ali SA, Meitei KV. Nigella sativa seed extract and its bioactive compound thymoquinone: the new melanogens causing hyperpigmentation in the wall lizard melanophores. J Pharm Pharmacol 2011;63:741–6. Ali SA, Salim S, Sahni T, Peter J, Ali AS. Serotonin receptors as novel targets for optimizing skin pigmentary responses in Indian bullfrog Hoplobatrachus tigerinus. Br J Pharmacol 2012;165(5): 1515–25. Bagnara JT, Bareiter HJ, Matoltsy AG, Richards KS. Biology of the integument vertebrates, vol. 2. Berlin: Springer-Verlag; 1986. p. 136–49. Hadley ME, Quevedo WC. Role of epidermal melanocytes in adaptive color changes in amphibians. Adv Biol Skin 1967;8:337–59. Fujii R, Novales RR. Cellular aspects of the control of physiological color changes in fishes. Am Zool 1969;9:453–63. Wasmeire C, Hume AN, Bolasco G, Seabra MC. Melanosomes at a glance. J Cell Sci 2008;121:3995–9. Aspengren S, Hedberg D, Wallin M. Melanophores: a model system for neuronal transport and exocytosis? J Neurosci Res 2006;85(12):2591–600. Ali SA, Peter J, Ali AS. Histamine receptors in the skin melanophores of Indian Bull frog, Rana tigerina. Comp Biochem Physiol A 1998;121:229–34. Salim S, Ali SA. Vertebrate melanophores as potential model for drug discovery and development. Cell Mol Biol Lett 2011;16:162– 200. Ali SA, Galgut JM, Choudhary RK. On the novel action of melanolysis by leaf extract of Aloe vera and its active ingredient aloin, the potent depigmenting agent. Planta Med 2012;78:1–5.
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[15] Imaki H, Chavin W. Ultrastructure of the integumental melanophores of the Australian lungfish, Neoceratodus forsteri. Cell Tissue Res 1975;158:365–73. [16] Imaki H, Chavin W. Ultrastructure of the integumental melanophores of the South American lungfish (Lepidosiren paradoxa) and the African lungfish (Protopterus sp.). Cell Tissue Res 1975;158:375–89. [17] Roth SI, Jones WA. The ultrastructure and enzymatic activity of the boa constrictor (Constrictor constrictor) skin during the resting phase. J Ultrastruct Res 1967;18:304–23. [18] Wise G. Ultrastructure of amphibian melanophores after light–dark adaptation and hormonal treatment. J Ultrastruct Res 1969;27:472–85. [19] Jande SS. Fine structure of tadpole melanophores. Anat Rec 1966;154:533–44. [20] Bartley JA. M.A.T. thesis. Providence, RI: Brown University; 1966. [21] Charles A, Ingram TJ. Electron microscopic observations of the melanocytes of the human epidermis. J Biophys Biochem Cytol 1959;6:41–4. [22] Bagnara JT, Taylor JD, Hadley ME. The dermal chromatophore unit. J Cell Biol 1968;38:67–79. [23] Lamer HI, Chavin W. Ultrastructure of the integumental melanophores of the Coelacanth, Latimeria chalumnae. Cell Tissue Res 1975;163:383–94. [24] Seiji M, Fitzpatrick TB, Birbeck MSC. The melanosome: a distinctive subcellular particle of mammalian melanocytes and the site of melanogenesis. J Invest Dermatol 1961;36:243. [25] Fitzpatrick TB. Terminology of vertebrate melanin cells. Science 1965;152:88–9. [26] Wasmeier C, Hume AN, Bolasco G, Seabra MC. Melanosomes at a glance. J Cell Sci 2009;121:3995–9. [27] Drochmans P. Electron microscopic studies of epidermal melanocytes and the fine structure of melanin granules. J Biophys Biochem Cytol 1960;8:165–80. [28] Seiji M, Shimao K, Birbeck MSC, Fitzpatrick. Subcellular localization of melanin biosynthesis. Ann N Y Acad Sci 1963;100:497–533. [29] Jimbow K, Kutika A. Fine structure of pigment granules in the human hair bulb: ultrastructure of pigment granules. In: Kawamura T, Fitzpatrick TB, Seiji M, editors. Biology of normal and abnormal melanocytes. Baltimore: University Park Press; 1971. p. 171–93.
Contents lists available at ScienceDirect
Journal of Microscopy and Ultrastructure journal homepage: www.elsevier.com/locate/jmau
Original Article
Comparative light and electron microscopic studies of dorsal skin melanophores of Indian toad, Bufo melanostictus Sharique A. Ali ∗ , Ishrat Naaz Post Graduate Department of Biotechnology, Saifia Science College, Bhopal 462001, India
a r t i c l e
i n f o
Article history: Received 9 April 2014 Received in revised form 26 June 2014 Accepted 6 July 2014 Available online 23 July 2014 Keywords: Toad melanophores Melanosomes Integumental pigmentation Anatomical significance
a b s t r a c t Indian toad, Bufo melanostictus is an amphibian, which is able to change the body color to adapt to its ambient need. This ability is due to specialized skin pigment cells known as melanophores which are excellent animal-cell models to study most of the physiological phenomenon related to color changes. In the present investigation morpho-anatomic details of dorsal skin melanophores of B. melanostictus were studied by means of light and electron microscope to establish their phylogenetic relevance with other vertebrates. Light microscopic observations revealed that toad skin contains dominantly present black pigment cells, the melanophores in its sub epidermis while dermal melanophores are rare. The ultrastructure of the melanophore is characterized by oval nucleus and numerous pigment granules, the melanosomes which remain scattered in the cytoplasm. Other sub-cellular organelles like mitochondria, well-developed tubular endoplasmic reticulum and Golgi vesicles were also found to be present in the remaining cytoplasmic area. Toad melanophore contains four distinct stages of melanosomes which are similar in development pattern to the mammalian melanocytes. These findings indicate that toad melanophores contain phylogenetically significant information of anatomical similarity with lower as well as higher vertebrates which can help to better understand the inter relationships between vertebrate pigment cells and their role in skin dysfunctions. © 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
1. Introduction Amphibians adapt readily to a light or dark colored backgrounds due to pigment movement in their chromatophores including the black-pigmented cells – melanophores. Melanophores are large, black, specialized dendritic cells which arise from neural crest and are widely distributed among the lower vertebrates [1–6]. Melanin pigments are tyrosine-based biopolymers synthesized and deposited within highly specialized subcellular organelles termed as melanosomes [7]. The expansion or contraction of melanin granules results in the darkening or lightening
∗ Corresponding author. Tel.: +91 9893015818. E-mail address: [email protected] (S.A. Ali).
of the skin which ultimately plays principal role in rapid color changes in the skin; restricted to fishes, amphibians and reptiles [8–10]. The melanophores have been used as unique cell models to study the underlying mechanism of integumental pigmentation. Pigment migration process in the melanophores are of a complex nature, and are regulated either by nervous or by endocrine stimulation or by both [2,3]. In response to the various stimuli, they rapidly move their pigment granules, the melanosomes to the cell center or periphery. Receptor systems participate in a very coordinated manner in mediation of these responses of color change [11]. Due to this dynamic property and availability of multiple receptor–ligand binding sites on melanophores, they are excellent targets for understanding the effects of various pharmacological agents [12–14].
http://dx.doi.org/10.1016/j.jmau.2014.07.002 2213-879X/© 2014 Saudi Society of Microscopes. Published by Elsevier Ltd. All rights reserved.
S.A. Ali, I. Naaz / Journal of Microscopy and Ultrastructure 2 (2014) 230–235
However, ultrastructural details of lower vertebrate melanophores are still wanting. Unless the ultrastructural details of melanophores become clearly known, the cellular mechanism within these pigment cells leading to pigmentary changes or disorders is difficult to understand. Some ultrastructural investigations have been done by previous researchers on melanophores of various classes of vertebrates like teleosts and lung fishes [9,15,16], reptiles [17], larval amphibians [18] which have revealed remarkably consistent fine structural features of these melanosomes synthesizing and containing cells in order to explain their role in physiological color changes. However, the pattern of distribution and association of these cell types has been found to vary considerably in different vertebrates [15]. The mediation of melanophores for varied responses is species dependent and hence no definite conclusion has yet been reached, in spite of extensive work in chromatic science. Despite a large body of literature on melanophores, no report on fine structure of dermal skin melanophores of Indian toad, Bufo melanostictus has appeared to date. As toads are familiar vertebrates and share similar mechanism of skin pigmentation with human beings, they have outstanding potential as phylogenetical tools to demystify and better understand the role of pigment cell system and its evolution. Hence, the present study has been carried out to investigate in detail the fine structure of dorsal skin melanophores of B. melanostictus by means of light and transmission electron microscopy to provide some information to bridge the missing link in the chain of events of melanophore structure and function. 2. Materials and methods 2.1. In vitro dorsal skin preparation Irrespective of sex, adult B. melanostictus of 7 cm length and 30–50 g of body weight were collected from the local areas of Bhopal, Madhya Pradesh during the rainy season and kept in large aquaria with normal photoperiod i.e. 12 h of light–dark cycle. Prior to the experiments, B. melanostictus were allowed to acclimatize to laboratory conditions for two days. During the acclimatization period toads were regularly fed with live snail and small insects. Diseased, injured or lethargic B. melanostictus were removed and only active, uniformly sized B. melanostictus were used. For in vitro studies the dorsal skin was peeled away from decapitated toad and a series of nearly 1 mm × 1 mm diameter dorsal skin pieces were cut out with a sharpened steel scissor. The pieces were kept in 10 mL of Amphibian Ringer Saline (ARS), containing 111 mM of sodium chloride, potassium chloride 2 mM, calcium chloride 1 mM and sodium hydroxide 2 mM, in 100 mL of double distilled water at pH 7.4, in small Petri dishes and they were equilibrated in saline medium for 15–20 min with frequent stirring. The Ethical Committee for Animal Experimentation and Research, Saifia College of Science, Bhopal, India certified the use of animals (approval number SSC/06–06–22, dated 26.10.2006). The research work of the Institute is done in strict compliance with the Guidelines for Laboratory Animals in Medical Colleges (2001) of the Indian Council of Medical Research (ICMR), as well as with the Breeding of
231
and Experiments on Animals Amendment Rules (2001) and the Prevention of Cruelty to Animal Act (1966). 2.2. Transmission electron microscopy Small pieces of dorsal skin of toad were cut and were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 12 h at 4 ◦ C. After washing in buffer, the skin pieces were post fixed in 1% OsO4 for 1 h at 4 ◦ C. The samples were dehydrated in an ascending grade of acetone, infiltrated and embedded in araldite CY 212 (TAAB, UK). Thick sections (1 m) were cut with an ultramicrotome, mounted on to glass slides, stained with aqueous toluidine blue and observed under a light microscope for gross observation of the area and quality of the tissue fixation. For electron microscope examination, thin sections of gray-silver color interference (70–80 nm) were cut and mounted onto 300 mesh-copper grids. Sections were stained with alcoholic uranyl acetate and alkaline lead citrate, washed gently with distilled water and observed under a Morgagni 268D transmission electron microscope (Fei Company, The Netherlands) at an operating voltage 80 kV. Images were digitally acquired by using a CCD camera (Megaview III, Fei Company) attached to the microscope. 3. Results 3.1. Light microscopic observations of dorsal skin melanophores of B. melanostictus Light microscopically in vertical sections of dorsal skin of B. melanostictus pigment cells were found in distinct layers of sub-epidermis (Fig. 1a and b). The dominating pigment cell of dorsal skin is the melanophore which plays principal role in physiological color changes. No “chromatophore unit” was observed due to the absence of other pigment cells. The darkness of the skin is due to the presence of melanophores in the sub-epidermis and dermis. The epidermis is thick and is composed of epidermal cells. Occasionally, epidermal glands occur interrupting the epidermal layers. Toad melanophores are organized in two distinct layers: one sub-epidermal melanophore layer which remains present closely below the epidermis while the other remains scattered in the loose dermis matrix of the skin. 3.2. Electron microscopic observations of dorsal skin melanophores of B. melanostictus observations of dorsal skin Ultrastructural melanophores of toad have revealed remarkably consistent fine structural features of these melanosome synthesizing cells. Sub epidermal melanophores contain oval nuclei which possesses a coarsely granulated nucleoplasm. The melanophores are surrounded by a thin single membrane corresponding to the plasma membrane of the cell. Oval shaped melanosomes were limited by unit membrane and were found to be scattered around the nucleus. Bundle of collagen fibers were also noticed (Fig. 1c). Besides melanosomes the cytoplasm furnished
232
S.A. Ali, I. Naaz / Journal of Microscopy and Ultrastructure 2 (2014) 230–235
Fig. 1. Light and electron microscope photomicrograph of dorsal skin of B. melanostictus in Amphibians Ringer Saline (ARS). (a and b) Light micrograph showing: The general organization of the toad skin and the localization of melanophores can be seen. epidermis (E), epidermal gland (G), sub-epidermis (S), dermis (D), melanophores (M) (arrow). 400×. (c) Transmission electron micrograph of vertical section of dorsal skin melanophores of toad: showing, oval nucleus (N) contains granulated nucleolus (n), surrounded by oval shaped premelanosomes (p) and melanosomes (m) scattered throughout cytoplasm. Endoplasmic reticulum (ER) remains scattered between melanosomes. 20,000×. (d) Showing: Low magnification electron micrograph of sub epidermal melanophores. They are actively engaged in the synthesis of melanosomes (m) of various stages of development. 4000×.
few mitochondria of tubular type and some vesicular components of the endoplasmic reticulum were noticed (Fig. 1c and d). Sub-epidermal melanophores contain oval shaped, unit membrane bound melanosomes of various stages of development. Immature, unmelanized premelanosome with intraluminal fibrils were observed. In premelanosomes of stage II complete intraluminal fibrils, stage III with pigment deposition at intraluminal fibrils was observed. Although
majority of granules of stage IV are highly electron dense as they are fully melanized therefore their internal structure is indistinguishable (Fig. 3a). On the other hand a number of granules exhibit a distinct premelanosomal structure which are electron-lucent pigment (Fig. 2a and b). Dermal melanophores occur between loose collagen fibers just beneath the sub-epidermis, also contains melanosomes with various degrees of pigmentation which are analogous to sub-epidermal melanosomes in shape and size (Fig. 3b).
S.A. Ali, I. Naaz / Journal of Microscopy and Ultrastructure 2 (2014) 230–235
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Fig. 2. Transmission electron micrograph of vertical section of a part of dorsal skin melanophores of Bufo melanostictus: (a) Showing, cytoplasm filled with melanosomes (m) of various stage of development, surrounded by endoplasmic reticulum (ER), vesicles (V) in the sub epidermis. 20,000×. (b) Showing, high magnification electron micrograph of electron-lucent premelanosomes (p) contains fibrillar matrix, electron-dense melanosomes (m) and vesicles (v). 40,000×.
Fig. 3. Transmission electron micrograph of vertical section of a part of dorsal skin melanophores of Bufo melanostictus showing: (a) Sub-epidermal melanosomes of various stages (I, II, III, IV) of development. 20,000×. (b) High magnification electron micrograph of dermal melanosomes (m) analogous to sub-epidermal melanosomes. 40,000×.
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4. Discussion Many vertebrate animals exhibit complex skin pigmentation patterns. According to Bagnara and Hadley [2] the distribution pattern of chromatophores is the main factor that determines the ultimate pigmentation pattern of a species. Many interpretations have been made to explain the location and mechanism of pigment translocation inside the melanophores of vertebrates to better understand the mysterious phenomenon of physiological color changes. However, it appears that even after occupying their final destinations, the melanophores retain a high degree of plasticity. In this series of events, the present investigation throws some light on the ultrastructure of dorsal skin melanophores of B. melanostictus, the common Indian toad. Adult B. melanostictus is an amphibian model which has a dominant black pigment cell system containing melanophores. It was observed light microscopically that the melanophores of this species are organized in two distinct layers: one sub-epidermal melanophores layer which remains present closely below the epidermis while the other layer of melanophores remains scattered in the loose dermal matrix of the skin. High resolving power of electron microscope confirmed the findings of the toad melanophores seen by light microscope. When compared with other vertebrates it was found that the dorsal skin epidermal melanophores of Indian toad, dominantly lie in the sub-epidermis. This situation is in contrast to that found in lungfishes i.e. Neoceratodus forsteri, Lepidosiren paradoxa, Protopterus sp., [15,16], larval Rana pipiens [19]. In these animal models the epidermal melanophores present in the intercellular spaces between level of epidermis while in more developed hierarchical forms like reptiles and mammals [20,21] in which the epidermal melanophores lie in the epidermis. Due to the lack of other pigment cells i.e. iridiophores or iridiophore–xanthophore no dermal chromatophore unit was observed in B. melanostictus in contrast to that described by Bagnara et al. [22] in Hyla cinerea, Agalychnis dachnicolo, etc. Ultrastructural observation of toad skin melanophores also revealed the presence of well marked prominent oval nucleus filled with granular nucleolus. We have found a thin, moderately electron-dense granular layer on inner surface of nuclear envelope which is similar in appearance as reported by Lamer and Chavin [23] in the integumental melanophores of the marine fish, Latimeria chalumnae. Cytoplasm of the toad melanophore was found to be filled with melanosomes of varying degree of pigmentation around nucleus. Remaining cytoplasmic areas contained mitochondria of tubular type, vacuolar endoplasmic reticulum, and Golgi apparatus. Stage I premelanosomes are spherical vesicles present near Golgi apparatus. These observations confirm the findings of Seiji et al. [24] who reported Golgi vesicles as possible precursor of melanosomes. We have also found that developing melanosomes as described by Fitzpatrick et al. [25] in mammalian melanocytes are present in various parts of the toad melanophores. Classical electron microscopy of human skin melanocytes describes four distinct stages (I, II, III and
IV) of melanosome development [26], a similar development progression has been found here. The sub-epidermal melanophores of Bufo were found to possess a large number of premelanosomes of stage II and III, suggesting that the process of active melanogenesis occurs in the sub-epidermis. Imaki and Chavin [15,16] reported that premelanosomes occurred abundantly in the sub-epidermal melanophores in comparison to dermal melanophores, however premelanosomes in both of these different layers share identical internal structure. Their ultrastructural observation agrees with our present study. We have also demonstrated that fully developed melanosomes of the stage IV are oval shaped and are frequently recognizable as being surrounded by a limiting membranous body which contains uniformly electron-dense matrix whereas the immature premelanosomes possess electron-lucent, fibrillar protein matrix in which melanin deposition takes place in later stage of development. The presence of typical pre-melanosomes and melanosomes showing progressive degrees of melanization in toad melanophores leads to the conclusion that the basic structure of the melanin granule and its mode of development is the same in higher vertebrates [27–29]. Toad skin melanophores anatomical structures appears to have a treasure of information, which have physiological links with the lung fish melanosomes as well as human melanosomes from phylogenetic point of view. The data of the present finding provides a foundation to better understand the morpho-anatomic and phylogenetic details of amphibian melanophores with human melanocytes which can account for the development of therapeutic agents against pigment disorders. Here, we conclude that ultrastructure of dorsal skin melanophores of B. melanostictus resembles the fine structural features of melanocytes of higher vertebrates including human beings as no significant differences were observed. These data taken in conjugation with earlier evidences regarding anatomy of pigment cells continue to suggest that a centrally occurring phenomenon of melanin biogenesis is associated with four distinct phases of melanosome development and translocation of these melanosomes intra-cellularly along with other pigment cells results in color change in vertebrates with common ancestral history. Conflict of interest The authors have no conflict of interest to declare. Acknowledgements Thanks are due to M.P. Council of Science and Technology, Govt. of MP for granting a Research Project No. 5690/CST/BTAC/2012 to Dr. Sharique A. Ali. Authors are also thankful to the Secretary and Principal of Saifia Science College, Bhopal, for providing the necessary facilities. The authors wish to thank SAIF, AIIMS, New Delhi for sample preparation, providing technical assistance and images of TEM. References [1] Brouwer E, Van de Veerdonk FCG. Possible involvement of ␣ and  receptors in the natural colour change and the ␣-MSH-induced
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