Short-term effects of α-melanocyte-stimulating hormone in three distinct melanin-pigmented cell types of Anura.

Ectothermic animals present melanin-containing cells in their integument and viscera. Besides cutaneous melanophores, amphibians have melanomacrophages in the hepatic parenchyma and melanocytes in the viscera, which are also present in their testicular stroma. The native melanocyte stimulating hormone (α-MSH) is the main hormone that modulates the color change in melanophores. However, we still know too little about how the α-MSH acts in vivo on visceral melanin-containing cells. In this study, we collected 30 adult males of Physalaemus nattereri (Anura, Leptodactylidae) to evaluate the short-term effects of α-MSH on melanophores, melanocytes and melanomacrophages under light microscopy. For this, we injected 0.05 ml of a single intraperitoneal dose containing 2.5x10-7 mmol/10g of α-MSH, diluted in ringer solution, in five experimental groups with five individuals each one. The different groups were analyzed after 1, 3, 6, 12 and 24h. The control group with five other individuals received only 0.05 ml of ringer solution. The skin pigmentation increased quickly after animals received the hormone α-MSH with the consequent darkening of the body (body darkness). Melanophores, melanocytes and melanomacrophages responded similarly to the test, with an increase in the area containing melanin. However, melanophores and melanomacrophages reached their darkest pigmentation in a shorter period of time in comparison to the testicular melanocytes, probably due to specific metabolic characteristics of each organ. Thus, we verified that the three types of cells, although present in different organs, are responsive to the native hormone α-MSH, which enables us to treat them as a pigmentary system.

Cardiac biomarkers as sensitive tools to evaluate the impact of xenobiotics on amphibians: the effects of anionic surfactant linear alkylbenzene sulfonate (LAS).

Amphibian populations have been experiencing a drastic decline worldwide. Aquatic contaminants are among the main factors responsible for this decline, especially in the aquatic environment. The linear alkylbenzene sulfonate (LAS) is of particular concern, since it represents 84% of the anionic surfactants' trade. In Brazil, the maximal LAS concentration allowed in fresh waters is 0.5mgL-1, but its potential harmful effects in amphibians remain unknown. Therefore, this study aimed to analyze the effects of a sublethal concentration of LAS (0.5mgL-1) for 96h on sensitive cardiac biomarkers of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802). For this, we measured the activity level (AL - % of animals), in situ heart rate (fH - bpm), relative ventricular mass (RVM - % of body mass), in vitro myocardial contractility and cardiac histology of the ventricles. Tadpoles' AL and fH decreased in LAS group. In contrast, the RVM increased, as a result of a hypertrophy of the myocardium, which was corroborated by the enlargement of the nuclear measures and the increase of myocytes' diameters. These cellular effects resulted in an elevation of the in vitro contractile force of ventricle strips. Acceleration in the contraction (TPT - ms) also occurred, although no alterations in the time to relaxation (THR -ms) were observed. Therefore, it can be concluded that even when exposed to an environmentally safe concentration, this surfactant promotes several alterations in the cardiac function of bullfrog tadpoles that can impair their development, making them more susceptible to predators and less competitive in terms of reproduction success. Thus, LAS concentrations that are considered safe by Brazilian by regulatory agencies must be revised in order to minimize a drastic impact over amphibian populations. This study demonstrates the relevance of employing cardiac biomarkers at different levels (e.g., morphological, physiological and cellular) to evaluate effects of xenobiotics in tadpoles.

The pioneering use of ISSR (Inter Simple Sequence Repeat) in Neotropical anurans: preliminary assessment of genetic diversity in populations of Physalaemus cuvieri (Amphibia, Leiuperidae).

The greatest diversity of anurans in the world is in Brazil and one of the major challenges is to reconcile the accelerated economic development with strategies that aim to maintain this diversity in forest fragments, often representing ESUs of some biomes. This study aimed to obtain data that will support conservation projects through the pioneering use of ISSR analysis in Neotropical anurans, estimating the intra- and interpopulation genetic diversity of four populations of P. cuvieri (Paraná and São Paulo regions). Of the 65 loci scored 58 were polymorphic, with 0.797 intrapopulation variation and 0.203 interpopulation variation. The index of interpopulation genetic differentiation (FST) proved to be high among the population of Marmeleiro-PR and the three populations of SP (FST > 0.288); genetic dissimilarity was related to the geographical distance. The ISSR proved to be efficient and useful molecular markers in comparison with other markers most widely used for preliminary diagnosis of genetic diversity in populations of amphibians, and could be applied as a tool for future conservation projects, since they could identify potential ESUs and influence decisions on the preservation of fragments.

Anatomy of the male reproductive system of Phrynops geoffroanus (Testudines: Chelidae) / Anatomia do aparelho reprodutor masculino de Phrynops geoffroanus (Testudines: Chelidae)

The reproductive system of male Phrynops geoffroanus adults is macroscopically described and the variation in testicular biometry is evaluated. A pair of oval testes is connected by the efferent ductules to the epididymis, which continue as deferent ducts, which emerge in the penis. The volume of the gonads showed the highest averages during spring and summer months. GSI varied significantly throughout the year, with the highest averages observed in the months that correspond to the end of spring and the beginning of summer, when the reproduction of the species takes place, and the lowest averages were seen in winter, suggesting a cyclical testicular activity.

O sistema reprodutor de machos adultos de Phrynops geoffroanus é descrito macroscopicamente e a variação da biometria testicular é avaliada. Um par de testículos ovais está conectado pelos dúctulos eferentes aos epidídimos, que se continuam como ductos deferentes, e que por sua vez desembocam no pênis. O volume das gônadas apresentou as maiores médias durante os meses de primavera e verão. O IGS variou significativamente ao longo do ano, com maiores médias observadas nos meses que correspondem ao final da primavera e início do verão, quando ocorre a reprodução da espécie, e as menores médias durante o inverno, sugerindo uma atividade testicular cíclica.

Morphological changes of the epididymis and description of the excurrent ducts of Phrynops geoffroanus (Testudines: Chelidae) during the reproductive cycle.

The seminal ducts (efferent ductule, epididymis, and deferent duct) in adults of Phrynops geoffroanus were examined using light microscopy. A series of tubules (efferent ductules) connect the testes to the epididymides. The efferent ductules are formed by a rete of small tubules of varying diameters, with simple columnar epithelium formed by the ciliated cells, nonciliated cells, and few basal cells. The epididymis is a simple, long and highly convoluted tubule that receives the efferent ductules throughout its extension. It is covered by a pseudostratified columnar epithelium with three cellular types: the principal cells, which are the most abundant, basal cells, and a small narrow cell. The histological differences in the epididymis region (cranial, medial, and caudal), as well as the differences in the epithelium throughout the reproductive cycle, are discussed. The deferent ducts consist of a low pseudostratified epithelium with two cellular types: the principal and basal cells. During the months analyzed, spermatozoa were stored in the epididymis, and deferent ducts were found.

Nucleolar cycle and chromatoid body formation: is there a relationship between these two processes during spermatogenesis of Dendropsophus minutus (Amphibia, Anura)?

The goals of this study were to monitor the nucleolar material distribution during Dendropsophus minutus spermatogenesis using cytological and cytochemical techniques and ultrastructural analysis, as well as to compare the nucleolar material distribution to the formation of the chromatoid body (CB) in the germ epithelium of this amphibian species. Nucleolar fragmentation occurred during the pachytene of prophase I and nucleolus reorganization occurred in the early spermatid nucleus. The area of the spermatogonia nucleolus was significantly larger than that of the earlier spermatid nucleolus. Ultrastructural analysis showed an accumulation of nuages in the spermatogonia cytoplasm, which form the CB before nucleolar fragmentation. The CB was observed in association with mitochondrial clusters in the cytoplasm of primary spermatocytes, as well as in those of earlier spermatids. In conclusion, the nucleolus seems to be related to CB formation during spermatogenesis of D. minutus, because, at the moment of nucleolus fragmentation in the primary spermatocytes, the CB area reaches a considerable size and is able to execute its important functions during spermatogenesis. The reorganized nucleolus of the earlier spermatids has a smaller area due to several factors, among them the probable migration of nucleolar fragments from the nucleus to the cytoplasm, and plays a part in the CB chemical composition.

Variações anatômicas no aparelho reprodutor masculino de Chaunus ornatus (Wied-Neuwied, 1821) (Anura, Bufonidae) / Anatomical variations in the male reproductive system of Chaunus ornatus (Wied-Neuwied, 1821) (Anura, Bufonidae)

Este trabalho descreve variações morfológicas nos órgãos e estruturas do aparelho reprodutor masculino em Chaunus ornatus, analisados independentemente das alterações promotoras de variações estruturais. Foram utilizados sete exemplares adultos da espécie, coletados no Município de Botucatu (São Paulo, Brazil) na época de atividade reprodutiva. Os espécimes foram dissecados e medidas do comprimento rostro-cloacal, análises anatômicas descritivas e biométricas foram obtidas. Três destes animais foram encaminhados à rotina histológica para a análise da arquitetura testicular e esquematizada em papel vegetal com caneta nanquim. Análises estatísticas (média, desvio-padrão, teste de Shapiro-Wilk, teste t de Student e Regressão Linear Simples) foram empregadas para interpretação dos resultados. Os testículos de C. ornatus são cilíndricos com coloração branco-leitosa e possuem uma estrutura ovariana rudimentar, o órgão de Bidder, em sintopia com a extremidade testicular cranial. O aparelho reprodutor masculino de C. ornatus apresenta conspícuas variações anatômicas intra e interindividuais, especialmente no que concerne à forma e tamanho dos testículos e dos órgãos de Bidder, os quais não apresentaram relação com o tamanho do indivíduo. Quanto à localização, os testículos dispõem-se ventralmente aos rins, mas ocorrem em alturas diferentes, acompanhando diferenças atribuídas à própria posição dos rins. Nos corpos adiposos abdominais, a disposição e número de prolongamentos que o constitui é variável entre os membros do par, e conseqüentemente entre indivíduos. Histologicamente não foram observadas diferenças quanto ao arranjo do epitélio germinativo na constituição do lóculo seminífero, portanto estas características parecem seguir o padrão da descrição para os demais anuros. A heterogeneidade morfológica quanto às variações anatômicas nessa espécie teve uma constatação pouco significativa. Assim, outros estudos ainda são necessários para o...

This work describes morphologic variations in the organs and structures of the male reproductive system in Chaunus ornatus, analyzed independently from the alterations that promote structural variations. Seven adult samples were used, they were collected in Botucatu (São Paulo State, Brazil) during reproductive activity period. The specimens were dissected and rostrum-cloacal length measures were taken. Descriptive and biometrics anatomical analyses were also obtained. Three of these animals were sent to histological routine for the analysis of the testicular architecture, where it was schematized in vegetable paper with pen Indian ink. Statistical analyses (mean, standart deviation, Shapiro-Wilk test, Student's t and Simple Lineal Regression) were used for interpretation of the results. The testes of C. ornatus are cylindrical with white coloration and they possess a rudimentary ovarian structure, the Bidder's organ, in the testicular cranial extremity. The male reproductive system of C. ornatus presents conspicuous anatomical variations intra and interindividuals, especially to the form and size of the testes and of the Bidder's organ, which did not present relationship with the size of individual. As for the location, the testes are disposed ventral to the kidneys, but they are in different levels, accompanying differences attributed to the kidneys levels. The disposition and number of extensions in the abdominal fatty bodies is variable among the pair's members, and consequently among individuals. Histologically differences regarding the germ epithelium arrangement were not observed. The germ epithelium is arranged in seminiferous locule, therefore these characteristics seem to follow the pattern of the description for the other anurans. The morphologic heterogeneity for anatomical variations in that species were few significant. Thus, other studies are still necessary for the establishment of morphologic relations and its phylogenetic...

Morphological observations on the testes of physalaemus cuvieri: Amphibia, Anura

Generalmente, los testículos de anuros constituyen órganos ovoides, pares, formados por masa de estructuras seminíferas, envueltas en una capa de tejido conjuntivo fibroso, que contiene un epitelio germinativo provisto de tipo celulares característicos. En este trabajo se describe la anatomía y la arquitectura histológica de los testículos, así como también la organización y morfología de las células germinativas. Se utilizaron cinco ejemplares machos de la especie Physalaemus cuvieri (Leptodactylidae), proveniente de la zona de Botucatu (Säo Paulo, Brasil). Tras el análisis macroscópico y la obtención de los fragmentos testiculares, el material se sometió a las técnicas histológicas rutinarias para su inclusión en parafina y coloración con hematoxilina/eosina. Se constató como rara peculiaridad la presencia de numerosas células con pigmento distribuidas de forma aleatoria en la túnica albugínea e intersticio testicular, las cuales otorgan al testículo una coloración marrón oscura. En el tejido germinativo las espermatogonias I son las mayores células espermatogenéticas. En la secuencia de la diferenciación y proliferación celulares, se siguen los demás tipos de células (espermatogonias II, espermatocitos I y II, espermátidas I y II, y espermatozoides) que presentan una organización cística, es decir, grupos de células se asocian a las células de Sertoli, formando los cistos espermatogenéticos o espermatocistos. Se diferenciaron e identificaron las células del linaje espermatogenético, según la morfología de las células y del propio cisto