No / Prostaglandins are potent mediators of the inflam-matory response and are also involved in cancer development. In this study, we show that human melanocytes and FM55 melanoma cells express cyclooxygenase-1 and -2 (COX-1 and-2) and thus have the capability to produce prostaglandins. TheFM55 cells produced predominantly PGE2and PGF2a, whereas the HaCaT keratinocyte cell line produced mainly PGE2. The anti-inflammatory peptide, a-melanocyte stimulating hormone(a-MSH), reduced prostaglandin production in FM55 and HaCaT cells and reversed the effect of the pro-inflammatory cytokine TNF-a in the former. These results indicate that melanocytes produce prostaglandins and that a-MSH, by inhibiting this response, may play an important role in regulating inflammatory responses in the skin.

No / Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) are produced in the skin under the influence of UV radiation. These compounds are highly reactive and can induce DNA lesions in epidermal cells. Melanin is considered to protect human skin against DNA damage by absorbing UV radiation. We have investigated whether melanin can, in addition, offer protection against the effects of H2O2 in human melanocytes and HaCaT keratinocytes. In the present study, it was shown that 40 and 100 μM H2O2 increased the number of DNA strand breaks as measured using the comet assay, in melanocytes of Caucasian origin. In melanocytes of the same origin in which melanin levels were increased by culturing in presence of 10 mM NH4Cl and elevated l-tyrosine, H2O2-induced DNA damage was reduced compared to that in control melanocytes. Similarly, HaCaT cells that were loaded with melanin were better protected against H2O2-induced DNA strand breaks than control HaCaT cells. These protective effects of melanin were mimicked by the intracellular Ca2+-chelator BAPTA. Thus, BAPTA reduced the level of H2O2-induced DNA strand breaks in melanocytes. Like BAPTA, melanin is known to be a potent chelator of Ca2+ and this was confirmed in the present study. It was shown that melanin levels in melanocytic cells correlated directly with intracellular Ca2+ binding capacity and, in addition, correlated inversely with H2O2-induced increases in intracellular Ca2+. Our results show that melanin may have an important role in regulating intracellular Ca2+ homeostasis and it is suggested that melanin protects against H2O2-induced DNA strand breaks in both melanocytes and keratinocytes and through its ability to bind Ca2+.

Yes / Humans are social animals that communicate disproportionately via potent genetic signals imbued in the skin and hair, including racial, ethnic, health, gender, and age status. For the vast majority of us, age-related hair pigment loss becomes the inescapable signal of our disappearing youth. The hair follicle (HF) pigmentary unit is a wonderful tissue for studying mechanisms generally regulating aging, often before this becomes evident elsewhere in the body. Given that follicular melanocytes (unlike those in the epidermis) are regulated by the hair growth cycle, this cycle is likely to impact the process of aging in the HF pigmentary unit. The formal identification of melanocyte stem cells in the mouse skin has spurred a flurry of reports on the potential involvement of melanocyte stem cell depletion in hair graying (i.e., canities). Caution is recommended, however, against simple extrapolation of murine data to humans. Regardless, hair graying in both species is likely to involve an age-related imbalance in the tissue's oxidative stress handling that will impact not only melanogenesis but also melanocyte stem cell and melanocyte homeostasis and survival. There is some emerging evidence that the HF pigmentary unit may have regenerative potential, even after it has begun to produce white hair fibers. It may therefore be feasible to develop strategies to modulate some aging-associated changes to maintain melanin production for longer.

Vitiligo is a skin disease with 2 % prevalence in a worldwide population. It is characterised by loss or decrease in activity of epidermal melanocytes, which lead to skin and hair depigmentation. It has negative impact on psyche, social relationships of patients and reduces the protection of the organism against UV radiation. One of the treatment methods is autologous transplantation of melanocytes or suspension of melanocytes with keratinocytes. Use of the biocompatible membrane, which allows the cultivation of these cells with resulting transplantation on the depigmented lesion, could improve treatment and make it more efficient. The main goal of this work was to create the biocompatible membrane from nanofiber layers of polyvinylalcohol (PVA) which can stand as a carrier for cell transplants in vitiligo therapy. PVA scaffolds were prepared by electrostatic spinning and later on modified by cold methane plasma (CH4) for lowering their hydrofility. Samples of modified nanofiber carriers were analysed according to their physical and chemical characteristics (visualization fiber morphology by SEM, XPS and surface Zeta potential analysis and contact angle). Consequently, adhesion, proliferation and metabolic activity of cultivating mice cell lines of melanocytes (Melan-a) and keratinocytes (XB2) were examined...

Le vitiligo est une dermatose inflammatoire caractérisée par une perte progressive des mélanocytes de la lame basale de l’épiderme. Cette pathologie reste à ce jour orpheline de traitement efficace. Toutefois, les mécanismes qui sont liés à la perte des mélanocytes restent débattus et impliquent un détachement des mélanocytes de la lame basale ou leur mort cellulaire par apoptose. Nous avons montré au sein de notre équipe l’implication des lymphocytes T CD8+ effecteurs mémoires dans la pathogénie du vitiligo. Ces populations produisent des niveaux élevés de deux cytokines inflammatoires qui sont, le TNF-α (tumor necrosis factor) et l’IFN-γ (interféron γ), suggérant ainsi un rôle majeur de ces deux cytokines dans la pathogénie du vitiligo. L’objectif de mon projet de thèse est d’étudier les mécanismes immunologiques impliqués dans la perte du mélanocyte au cours du vitiligo. Ainsi, nous avons observé dans les zones péri-lésionnelles de vitiligo ou lésionnelles de psoriasis, une localisation suprabasale des mélanocytes et une anomalie de l’expression de la E-cadhérine dans l’épiderme, protéine majeure impliquée dans l’attachement des mélanocytes. Nous avons également montré l’absence de la mort cellulaire par apoptose des mélanocytes au niveau cutané chez les patients atteints de vitiligo et/ou de psoriasis. En se basant sur ces observations, nous nous sommes donc intéressés à évaluer les effets combinés du TNF-α et de l’IFN-γ sur l’adhésion des mélanocytes. Nos résultats ont montré que les deux cytokines combinés diminuent l’expression du gène codant la Ecadhérine et entrainent probablement la redistribution de cette protéine. De plus, nous avons observé que ces deux cytokines en combinaison altèrent l’expression de la E-cadhérine dans un modèle d’épidermes reconstruits pigmentés in vitro. Cette altération était associée à une augmentation des niveaux de la E-cadhérine soluble (sE-cad) au niveau des surnageants de culture. D’une manière intéressante, nous avons montré que ces deux cytokines induisent l’expression kératinocytaire de la métalloprotéase 9 (MMP9) dont l’action est connue pour cliver la E-cadhérine sous sa forme soluble, participant ainsi au détachement des mélanocytes. Des taux élevés de MMP9, mais également de la sE-cad sont retrouvés dans le sérum des patients atteints de vitiligo. L’inhibition de la MMP9 dans des modèles in vitro et in vivo empêche les effets combinés du TNF- α et de l’IFN-γ sur le détachement des mélanocytes permettant leur stabilisation à la lame basale. Par ailleurs, comme nous avons montré que la survie des mélanocytes n’était pas altérée dans le vitiligo, nous nous sommes intéressés à évaluer l’action combinée du TNF-α et de l’IFN-γ sur la fonction, le phénotype des mélanocytes et leur production des médiateurs inflammatoires. Nous avons montré que ces deux cytokines en combinaison inhibent l’expression des gènes mélanocytaires (MITF, TYR, DCT) et favorisent l’induction des chimiokines CXCL9 et CXCL10, de la cytokine inflammatoire TNF-α et de la molécule d’adhésion ICAM-1, suggérant un rôle majeur des mélanocytes dans la promotion de l’inflammation. Enfin, considérant que la voie de signalisation du récepteur de l’IFN-γ est dépendante de la voie JAK/STAT, nous avons étudié l’impact de l’inhibition de cette voie dans les effets induits dans nos modèles, et avons montré au niveau de l’expression des gènes, une amélioration des gènes associés à la fonction mélanocytaire et une inhibition de ceux associés à l’inflammation. L’ensemble de nos résultats mettent en évidence un nouveau mécanisme pour expliquer la perte des mélanocytes et identifie MMP9 ainsi que les inhibiteurs de JAK comme des cibles thérapeutiques prometteuses permettant ainsi de mieux comprendre les mécanismes physiopathologiques au cours du vitiligo et d’établir un lien direct entre immunité, facteurs solubles inflammatoires et perte des mélanocytes au cours du vitiligo. / Vitiligo is a chronic inflammatory skin disorder characterized by a progressive loss of melanocytes. This stigmatizing disease has a major social impact and no real effective therapies have been reported so far. However, the mechanisms leading to melanocyte disappearance remain debated and include melanocyte detachment and/or death. The role of the immune response has now been well described, implying CD8+ effector memory T cells that produce high levels of inflammatory cytokines as TNF-α (tumor necrosis factor) and IFN-γ(interferon γ), suggesting the involvement of these two cytokines in the pathogenesis of vitiligo. Thus, the aim of this project is to study the interplay between the inflammatory response characterizing vitiligo disease and melanocyte loss. We first observed that melanocytes are located in suprabasal layers of the epidermis in perilesional skin of vitiligo and lesional skin of psoriasis patients, which was associated with an altered expression of E-cadherin, a major protein involved in melanocyte attachment to the basal membrane. Such suprabasal melanocytes did not undergo apoptosis. Based on these observations, we next investigated the combined effects of TNF-α and IFN-γ on melanocyte adhesion. We showed that these two cytokines decrease E cadherin gene expression and probably induce a redistribution of E-cadherin. In addition, these two cytokines in combination altered the expression of E-cadherin in reconstructed human pigmented epidermis in vitro. This finding was associated with increased levels of soluble E-cadherin in culture supernatants. Furthermore, TNF-α and IFN-γ induced the production of matrix metalloproteinase 9 (MMP9) by keratinocytes, leading to the cleavage of the E-cadherin. Inhibition of MMP9 prevents the combined effects of TNF-α and IFN-γ on melanocyte detachment and led to their stabilization to the basal membrane of epidermis in vitro and in vivo models. Since we demonstrated that melanocyte survival is not impaired in vitiligo, we assessed the impact of these two cytokines on melanocyte function, phenotype and inflammation. We demonstrate that the combination of TNF-α and IFN-γ inhibits the expression of genes involved in melanocyte function (MITF, TYR, DCT) and promote the induction of the chemokine ligands CXCL9 and CXCL10, the inflammatory cytokine TNF-α and adhesion molecule ICAM-1, suggesting an important role of melanocytes in the promotion of inflammation. Lastly, considering that the signaling pathway of IFN-γ involves activation of the JAK / STAT pathway, we studied the impact of the inhibition of that pathway in our models. Our results show that the JAK inhibition suppressed the effects of TNF-α and IFN-γ on melanocyte function, on the release of pro-inflammatory mediators and led to the melanocyte stabilization to the basal membrane of epidermis. All of our results highlight a new mechanism to explain the loss of melanocytes and identify MMP9 and JAKs as promising therapeutic targets to better understand the physiopathological mechanisms during vitiligo and establish a direct link between immunity, soluble factors. Inflammation and loss of melanocytes during vitiligo.

Les mélanocytes, cellules responsables de la pigmentation de la peau et des poils, protègent les cellules des stress environnementaux, en particulier des rayonnements ultra-violets (UV) présents à la surface de la Terre. Les UV induisent des dommages moléculaires et régulent de nombreuses voies de signalisation en aval de MC1R, MAPK, PI3K, ou PKC. A court terme, les UV peuvent induire la mélanogenèse et à long terme participent à la mélanomagenèse. Dicer, protéine clef de la maturation des microARN, est régulée par différents stress. La protéine multifonctionnelle β-caténine est impliquée dans le développement des mélanocytes. Ces deux protéines participent à la régulation fine de l'expression génique. L'objectif de cette thèse est de mettre en évidence le rôle et la régulation de Dicer dans le lignage mélanocytaire dans des conditions normales et de stress (UVB). Dans une première partie, nous nous sommes intéressés au rôle de Dicer dans la pigmentation et sa régulation dans le lignage mélanocytaire. Nous avons montré, in vivo dans un modèle murin, que Dicer est nécessaire à la fois à la mise en place du lignage mélanocytaire et au fonctionnement de ce lignage chez l'adulte. L'absence de Dicer dans le lignage mélanocytaire affecte la localisation des mélanocytes de la papille dermique du follicule pileux et empêche la pigmentation du poil. In vitro, la transcription de Dicer est régulée par différentes voies, en particulier par les protéines PI3K, RSK, GSK3β et β-caténine. L'activité répressive de β-caténine sur la transcription de Dicer est dépendante de sites LEF/TCF. Dans une deuxième partie, nous nous sommes intéressés à l'implication de Dicer, en relation avec β-caténine, dans la réponse aux UVB. Nous avons mis en évidence in vivo et in vitro la relocalisation nucléaire et l'activation transcriptionnelle de β-caténine induites par les UV. Tout comme β-caténine, les UVB répriment la migration des mélanocytes in vitro. Nous avons montré in vitro que les UVB répriment l'expression de Dicer et que cette répression est dépendante de sites de fixation de facteurs de transcription, dont LEF/TCF, présents dans la région promotrice de Dicer. Une diminution de Dicer participe à la protection des mélanocytes contre les UVB. Ce travail de thèse a donc permis de montrer le rôle de Dicer dans la pigmentation adulte et de mettre en évidence des voies de régulation de l'expression de Dicer dans les mélanocytes non stressés et dans les mélanocytes soumis à un stress UVB. / Melanocytes, cells responsible for pigmentation of the skin and hair, protect cells from environmental stress, especially ultra-violet radiations (UV) present on Earth floor. UV induce molecular damages and regulate many signaling pathways downstream of MC1R, MAPK, PI3K, or PKC. In the short term, UV can increase melanogenesis and in the long term, participate in melanomagenesis. Dicer, a key protein involved in microRNA maturation, is regulated by different types of stress. The multifunctional protein β-catenin is implicated in melanocyte development. These two proteins participate in fine regulation of gene expression. The goal of this thesis is to highlight the role and regulation of Dicer in the melanocyte lineage in normal and UVB stress conditions. In the first part, we focused on the role of Dicer in pigmentation and its regulation in the melanocyte lineage. We showed that, in a mouse model in vivo, Dicer is necessary for both establishment of melanocyte lineage and proper function of this lineage in adults. The lack of Dicer in the melanocyte lineage affects localization of melanocytes in the dermal papilla of hair follicles, preventing hair pigmentation. In vitro, Dicer transcription is regulated by different pathways, including PI3K, RSK, GSK3β and β-catenin. LEF/TCF sites mediate the repressive activity of β-catenin on Dicer transcription. In the second part, we focused on the implication of Dicer, in connection with β-catenin, in the response to UVB by melanocytes. We showed the nuclear relocalization and transcriptional activation of β-catenin induced by UV both in vivo and in vitro. Like β-catenin, UVB represses melanocyte migration in vitro. We showed in vitro that UVB represses Dicer expression and that this repression is dependent on transcription factors binding sites in the Dicer promoter region including LEF/TCF. Decreased level of Dicer participates in protection of melanocytes against UVB. This thesis work allowed us to show the role of Dicer in adult pigmentation and to highlight signaling pathways implicated in Dicer expression regulation in non-stressed melanocytes and in UVB-stressed melanocytes.

Greenhill et al. (2011) developed a gene regulatory network of the main genes and interactions known to play a role in melanocyte biology, and generated a mathematical model to describe the behaviour of this complex network using semi quantitative data (ISH expression data). In this project we sought to collect expression data from four genes of the melanocyte GRN (sox10, kit, mitfa and dct) to develop a quantitative model that is able to describe the data more accurately. Moreover, we intended to identify more genes that are part of the melanocyte development process to be incorporated to the GRN. We analysed microarray data that compared differentially expressed genes between sox10 mutant and wild type embryos and validated five genes with a key role in melanocyte biology as downregulated in mitfa mutant embryos, which are downstream of mitfa in the GRN. We suggest that kit plays the role of factor Y in the Greenhill et al. (2011) GRN: Mitfa drives kit expression, and kit expression is transiently driven by Sox10 at early stages of development. As part of the feedback loop, kit seems to drive and maintain mitfa expression, however this needs to be validated. Finally we developed an experimental set up to obtain an estimate of gene expression per melanocyte from sox10, kit, mitfa and dct, using both qPCR and ISH cell count measurements. With this estimate we performed a parameter optimisation procedure, and found a set of parameters for the mathematical model that predicted the experimental data very accurately. The new model suggests that low expression values of sox10 are sufficient to drive mitfa expression in high levels. It also predicts that high expression of sox9b is needed to achieve the high expression levels of dct seen in the data, although these predictions need to be experimentally tested. This study represents the first attempt to obtain fine-scale gene expression data from melanocytes for the development of a quantitative mathematical model in zebrafish.

Made available in DSpace on 2014-06-11T19:30:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-06-01Bitstream added on 2014-06-13T19:01:01Z : No. of bitstreams: 1 zieri_r_dr_sjrp.pdf: 889017 bytes, checksum: 170c2d62d9c91d29ca0d8ba71a162b3b (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os vertebrados ectotérmicos apresentam um desenvolvido sistema de células com pigmentos melânicos em seu citoplasma, localizado em diversos órgãos e membranas. A dispersão dos grãos de melanina dentro dos melanócitos responde fortemente à exposição ao hormônio estimulante de melanócitos (MSH) e aos hormônios sexuais, promovendo alteração na coloração. Este trabalho buscou avaliar o efeito dos esteróides cipinato de testosterona, 17 β-estradiol e do MSH sobre a pigmentação hepática e testicular de Eupemphix nattereri, mediante análise histológica, e ultra-estrutural. Foram coletados 60 machos adultos na região de São José do Rio Preto, SP. Para cada experimento, dez machos adultos receberam doses subcutâneas contendo 0,5 mg/kg de cipionato de testosterona e 0,5 mg/kg de 17 β-estradiol, dissolvidos em óleo vegetal e, 2,5x10-7 mmol/10g de α-MSH dissolvido em PBS, durante 7 dias, a cada 48h. O grupo controle recebeu apenas óleo vegetal ou PBS. Grupos com cinco animais foram eutanasiados 24h após do término do tratamento, e outros cinco, após 15 dias. Foram realizados estudos morfológicos utilizando microscopia de luz e microscopia eletrônica de transmissão. No tecido hepático foram encontradas células de Kupffer. No interstício testicular observou-se melanócitos, com inúmeros melanossomos. Para o grupo tratado com cipionato de testosterona, foi observado um aumento de aproximadamente 2x na área ocupada pelas células pigmentadas no fígado e 4x nos testículos mediante analise a curto prazo. Para o mesmo tratamento, foi observado aumento de 1,8x no fígado dos animais eutanasiados após 15 dias. Tal resposta pode ser decorrente do aumento da atividade melanossintética, do aumento do número de células, ou ambos. Os animais tratados com 17 β-estradiol e α- MSH não apresentaram alterações na pigmentação hepática e testicular mediante... / Ectothermic vertebrates have a well-developed system of melanin-containing cells, which are distributed throughout several organs and tissues. The dispersion of melanin-containing granules within melanocytes responds to prolonged exposure to high concentration of MSH and sexual hormones inducing color change. The present study aimed to characterize morphological and stereological patterns of pigmented cells in liver and testis of the Eupemphix nattereri under effect of testosterone cypionate, 17 β-estradiol and α-MSH. Sixty adult males were collected in São José do Rio Preto, São Paulo State, Brazil. For each experiment, ten adult males were treated with the following hormones injected in the dorsal lymph sac during seven days: (1) 5 mg/kg dose of testosterone cypionate or (2) 5 mg/kg dose of 17 β-estradiol (dissolved in vegetal oil) or (3) 2,5x10-7 mmol/10g of α-MSH (dissolved in PBS). The control group received only vegetal oil or PBS solution at the same hormonal concentration. Groups of five animals were euthanatized after 24h and the other five, after 15 days. Morphological studies with light and transmission electron microscopy were carried out in the groups. In the hepatic tissue were found Kupffer cells, which are characterized by multivesicle bodies in the cytoplasm and large amount of melanosomes. In the testis, melanocytes-like cells are present in the interstitium. They presented large and irregular aspect and a great amount of intensely pigmented cytoplasm. For the groups treated with testosterone cypionate an increase of approximately 2x in the occupied area by the pigmented cells in the liver and 4x in the testis was observed in the treatment group against the control. The observed response can be due to increase of melanosynthesis Introdução 3 Zieri et al., 2010 activity, increase in the number of pigmented cells, or both. Moreover, for the same treatment... (Complete abstract click electronic access below)

Les mélanocytes sont des cellules productrices de mélanines, à l’origine de la teinte de la peau, des yeux et des cheveux. Elles dérivent d'une population multipotente appelée cellules de la crête neurale, qui génère entre autres également tout le système nerveux périphérique. Une prolifération accrue des précurseurs des mélanocytes durant le développement entraine chez l’homme l’apparition d’un nævus mélanocytaire congénital (NMC). Cette prolifération est due à une mutation somatique au sein d'un de ces précurseurs, dans des gènes de la voie de signalisation des MAP-Kinases, NRAS ou BRAF. Les plus grandes formes, couvrant des parties entières du corps, sont syndromiques. Ils peuvent associer des mélanocytoses, des malformations ou tumeurs cérébrales ou méningées, parfois épileptogènes, ainsi qu'un risque autour de 5% de dégénérer en mélanome, dans un des sites atteints. Durant ma thèse j’ai exploré des modèles murins où les protéines NRAS ou BRAF constitutivement actives sont exprimées très tôt au cours de l’embryogenèse, dans les cellules de la crête neurale. Les embryons mutants BrafV600E connaissent une létalité embryonnaire, probablement due à une superposition de défauts vasculaires et cérébraux. En revanche, les souris NrasG12D sont viables,présentent des mélanocytoses extracutanées dans des sites divers, ainsi qu’une hyperpigmentation cutanée, visible en postnatal. Cette hyperpigmentation est associée à une augmentation de la densité folliculaire, ainsi qu’à un dérèglement du cycle du follicule pileux. Des cultures de cellules de crête neurale murines, BrafV600E ou NrasG12D et contrôles, ont permis d’élucider sur le plan moléculaire les effets de telles mutations. / Melanocytes are the vertebrate cells that produce melanin, conferring color on skin, hair and eyes. They arise from a multipotent embryonic cell population called the neural crest, which also gives rise to the peripheral nervous system of the body and many other cell types. Abnormal proliferation of melanocyte precursors before birth can lead to human congenital melanocytic nevus (CMN). CMN are caused by prenatal somatic mutations in the NRAS or BRAF genes of the MAP-Kinase pathway, in one of these precursors. The largest CMN, covering entire segments of the body or head, are syndromic. They are sometimes associated with epileptogenic brain or meningeal malformations, tumors or melanocytosis, and they present a risk of about 5% in all these sites of becoming pediatric malignant melanoma. During my thesis, I explored mouse models expressing constitutively activated NRAS or BRAF proteins in neural crest cell lineages, from very early in embryogenesis. BrafV600E mutant embryos are embryonic lethal at mid-gestation, probably due to coinciding vascular and brain defects. In contrast, NrasG12D mice are viable, present extracutaneous melanocytosis in various sites as well as postnatal hyperpigmentation of the skin. This is associated with increased hair follicle density, and a deregulated hair cycle. Cell culture of mutant or wildtype mouse neural crest cells of both genotypes has permitted the comparison and discovery of molecular differences introduced by these mutations.

Resumo: Os vertebrados ectotérmicos apresentam um desenvolvido sistema de células com pigmentos melânicos em seu citoplasma, localizado em diversos órgãos e membranas. A dispersão dos grãos de melanina dentro dos melanócitos responde fortemente à exposição ao hormônio estimulante de melanócitos (MSH) e aos hormônios sexuais, promovendo alteração na coloração. Este trabalho buscou avaliar o efeito dos esteróides cipinato de testosterona, 17 β-estradiol e do MSH sobre a pigmentação hepática e testicular de Eupemphix nattereri, mediante análise histológica, e ultra-estrutural. Foram coletados 60 machos adultos na região de São José do Rio Preto, SP. Para cada experimento, dez machos adultos receberam doses subcutâneas contendo 0,5 mg/kg de cipionato de testosterona e 0,5 mg/kg de 17 β-estradiol, dissolvidos em óleo vegetal e, 2,5x10-7 mmol/10g de α-MSH dissolvido em PBS, durante 7 dias, a cada 48h. O grupo controle recebeu apenas óleo vegetal ou PBS. Grupos com cinco animais foram eutanasiados 24h após do término do tratamento, e outros cinco, após 15 dias. Foram realizados estudos morfológicos utilizando microscopia de luz e microscopia eletrônica de transmissão. No tecido hepático foram encontradas células de Kupffer. No interstício testicular observou-se melanócitos, com inúmeros melanossomos. Para o grupo tratado com cipionato de testosterona, foi observado um aumento de aproximadamente 2x na área ocupada pelas células pigmentadas no fígado e 4x nos testículos mediante analise a curto prazo. Para o mesmo tratamento, foi observado aumento de 1,8x no fígado dos animais eutanasiados após 15 dias. Tal resposta pode ser decorrente do aumento da atividade melanossintética, do aumento do número de células, ou ambos. Os animais tratados com 17 β-estradiol e α- MSH não apresentaram alterações na pigmentação hepática e testicular mediante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Ectothermic vertebrates have a well-developed system of melanin-containing cells, which are distributed throughout several organs and tissues. The dispersion of melanin-containing granules within melanocytes responds to prolonged exposure to high concentration of MSH and sexual hormones inducing color change. The present study aimed to characterize morphological and stereological patterns of pigmented cells in liver and testis of the Eupemphix nattereri under effect of testosterone cypionate, 17 β-estradiol and α-MSH. Sixty adult males were collected in São José do Rio Preto, São Paulo State, Brazil. For each experiment, ten adult males were treated with the following hormones injected in the dorsal lymph sac during seven days: (1) 5 mg/kg dose of testosterone cypionate or (2) 5 mg/kg dose of 17 β-estradiol (dissolved in vegetal oil) or (3) 2,5x10-7 mmol/10g of α-MSH (dissolved in PBS). The control group received only vegetal oil or PBS solution at the same hormonal concentration. Groups of five animals were euthanatized after 24h and the other five, after 15 days. Morphological studies with light and transmission electron microscopy were carried out in the groups. In the hepatic tissue were found Kupffer cells, which are characterized by multivesicle bodies in the cytoplasm and large amount of melanosomes. In the testis, melanocytes-like cells are present in the interstitium. They presented large and irregular aspect and a great amount of intensely pigmented cytoplasm. For the groups treated with testosterone cypionate an increase of approximately 2x in the occupied area by the pigmented cells in the liver and 4x in the testis was observed in the treatment group against the control. The observed response can be due to increase of melanosynthesis Introdução 3 Zieri et al., 2010 activity, increase in the number of pigmented cells, or both. Moreover, for the same treatment... (Complete abstract click electronic access below) / Orientador: Classius de Oliveira / Coorientador: Sebastião Roberto Taboga / Banca: Rejane Maira Góes / Banca: Patrícia Simone Leite Vilamaior / Banca: Selma Maria Almeida Santos / Banca: Wagner José Fávaro / Doutor