Sex determination is a key developmental process in several species regulated by sexrelated transcription factors. In many species a gene called doublesex and mab-3 related transcription factor 1 (dmrt1), plays an important role in sexual differentiation. I used African clawed frogs (Xenopus) to examine function of dmrt1 in two species: a diploid species, X. tropicalis, and an allotetraploid species, X. laevis. In both species, dmrt1 is an autosomal gene; Xenopus tropicalis has one copy of dmrt1 and X. laevis has two homeologous copies that by definition are derived from whole genome duplication: dmrt1.L and dmrt1.S in X. laevis. We generated knockouts of each of these genes to further examine their function in sexual differentiation. Histological examination showed testicular dysgenesis in X. tropicalis dmrt1 and X. laevis dmrt1.L null males whereas dmrt1.S null males presented no obvious difference in sperm density compared to wildtype males. X. tropicalis dmrt1 and X. laevis dmrt1.L null females were found to completely lack reproductive organs and are infertile whereas dmrt1.S null females appeared unaffected. The contrasting results between dmrt1.L and dmrt1.S in X. laevis provides evidence of both neofunctionalization and subfunctionalization following gene duplication and suggest that gene duplication is a major contributor to evolutionary change. Additional investigation of the transcriptome of these frogs and the role of dmrt1 in the secondary sex characteristic vocalization provides further evidence of the role of dmrt1 in development. Comprehensively, this investigation provides further knowledge of the role of dmrt1 and homeologs of this gene in sexual differentiation and introduces a novel aspect of this gene in female development. Future efforts are focused on generating double knockouts for dmrt1.L and dmrt1.S, further examining the role of dmrt1.S in somatic cell function and developing additional mutant lines in other Xenopus for comparative analysis. / Thesis / Master of Science (MSc) / In many species sexual differentiation is a crucial developmental event. Surprisingly, however, the systems orchestrating sexual differentiation are highly variable among species. The doublesex and mab-3 related transcription factor 1 (dmrt1) gene plays a role in sexual differentiation in many groups, but its specific roles in this process are incompletely characterized and potentially diverse. We used genetic engineering in two species of African clawed frog (Xenopus) to disable function of dmrt1 in order to explore effects on gonadal development and the development of secondary sex characteristics. We found that dmrt1 is required for normal ovary or testis development in both Xenopus species, and that functional divergence occurred following duplication of dmrt1 by whole genome duplication. Taken together, these findings identify previously uncharacterized roles of dmrt1 in Xenopus and provide evidence of dynamic functional evolution of this important gene.

A asma é uma doença crônica inflamatória das vias aéreas, de importância mundial pela sua elevada prevalência, com considerável impacto na qualidade de vida dos pacientes. Dentre os diferentes fenótipos de asma, a asma alérgica constitui a maioria dos casos. Dentre os diferentes desencadeantes de exacerbações na asma, os aeroalérgenos têm papel de destaque, sendo os ácaros o grupo mais importante e prevalente. Entre os ácaros, os mais relevantes para estudo são o Dermatophagoides pteronyssinus (Der p) e a Blomia tropicalis (Blo t), com importância significativa nos países tropicais. Pacientes podem ser sensibilizados a esses dois ácaros ou a um deles somente, mas a positividade do teste in vivo ou in vitro não significa reatividade clínica e que haja concordância com o alérgeno relevante para o desencadeamento dos sintomas. A reatividade cruzada entre esses dois ácaros vem sendo estudada. In vivo, há poucos trabalhos demonstrando a reatividade cruzada entre Der p e Blo t e analisando a correlação entre os testes cutâneos e os de provocação brônquica. O objetivo desse estudo é avaliar a reatividade clínica em pacientes com asma sensibilizados a ambos os ácaros, Der p e Blo t. Para isso, pacientes com asma do Ambulatório do Serviço de Imunologia Clínica e Alergia do HC-FMUSP foram selecionados. Broncoprovocações específicas foram realizadas com os dois ácaros, em diferentes momentos. Dezoito pacientes com asma foram selecionados, e trinta e seis provocações foram realizadas. Todas as provocações com o ácaro Der p foram positivas. Em relação à Blo t, o resultado foi positivo em 93% dos testes. No presente estudo, houve concordância na maioria dos casos entre a presença de sensibilização aos ácaros estudados e a positividade na boncoprovocação. Portanto, as sensibilizações aos ácaros identificados no teste cutâneo são relevantes para o desencadeamento dos sintomas de asma nesses pacientes / Asthma is a chronic inflammatory disease of the airways of global importance due to its high prevalence, with considerable impact on quality of life of patients. Among the different phenotypes of asthma, allergic asthma constitutes the majority of cases. Among the different triggers of asthma exacerbations, aeroallergens have the leading role, being mites group more important and prevalent. Among the mites, the most relevant to the study are Dermatophagoides pteronyssinus (Der p) and Blomia tropicalis (Blo t), with significant importance in tropical countries. Patients can be sensitive to these two mites or only one of them, but the positivity of test in vivo or in vitro clinical reactivity and does not mean that there is agreement with the relevant allergen for the triggering of symptoms. The cross-reactivity between these two mites has been studied. In vivo, there are few studies demonstrating the cross-reactivity between Der p and Blo t analyzing the correlation between skin tests and bronchial provocation. The aim of this study is to evaluate the clinical reactivity in patients with asthma sensitized to both house dust mites, Der p and Blo t. For this reason, patients with asthma, Ambulatory Service of Clinical Immunology and Allergy HCFMUSP were selected. Specific bronchial provocation tests were performed with both mites at different times. Eighteen patients with asthma were selected, and thirty-six provocations were performed. All provocations with the mite Der p were positive. Regarding Blo t, the result was positive in 93% of tests. In the present study, there was agreement in most cases between the presence of sensitization to mites and positivity in bronchial provocation test. Therefore, sensitization to mites in skin test identified is relevant to the triggering of asthma symptoms in these patients

A asma é uma doença crônica inflamatória das vias aéreas, de importância mundial pela sua elevada prevalência, com considerável impacto na qualidade de vida dos pacientes. Dentre os diferentes fenótipos de asma, a asma alérgica constitui a maioria dos casos. Dentre os diferentes desencadeantes de exacerbações na asma, os aeroalérgenos têm papel de destaque, sendo os ácaros o grupo mais importante e prevalente. Entre os ácaros, os mais relevantes para estudo são o Dermatophagoides pteronyssinus (Der p) e a Blomia tropicalis (Blo t), com importância significativa nos países tropicais. Pacientes podem ser sensibilizados a esses dois ácaros ou a um deles somente, mas a positividade do teste in vivo ou in vitro não significa reatividade clínica e que haja concordância com o alérgeno relevante para o desencadeamento dos sintomas. A reatividade cruzada entre esses dois ácaros vem sendo estudada. In vivo, há poucos trabalhos demonstrando a reatividade cruzada entre Der p e Blo t e analisando a correlação entre os testes cutâneos e os de provocação brônquica. O objetivo desse estudo é avaliar a reatividade clínica em pacientes com asma sensibilizados a ambos os ácaros, Der p e Blo t. Para isso, pacientes com asma do Ambulatório do Serviço de Imunologia Clínica e Alergia do HC-FMUSP foram selecionados. Broncoprovocações específicas foram realizadas com os dois ácaros, em diferentes momentos. Dezoito pacientes com asma foram selecionados, e trinta e seis provocações foram realizadas. Todas as provocações com o ácaro Der p foram positivas. Em relação à Blo t, o resultado foi positivo em 93% dos testes. No presente estudo, houve concordância na maioria dos casos entre a presença de sensibilização aos ácaros estudados e a positividade na boncoprovocação. Portanto, as sensibilizações aos ácaros identificados no teste cutâneo são relevantes para o desencadeamento dos sintomas de asma nesses pacientes / Asthma is a chronic inflammatory disease of the airways of global importance due to its high prevalence, with considerable impact on quality of life of patients. Among the different phenotypes of asthma, allergic asthma constitutes the majority of cases. Among the different triggers of asthma exacerbations, aeroallergens have the leading role, being mites group more important and prevalent. Among the mites, the most relevant to the study are Dermatophagoides pteronyssinus (Der p) and Blomia tropicalis (Blo t), with significant importance in tropical countries. Patients can be sensitive to these two mites or only one of them, but the positivity of test in vivo or in vitro clinical reactivity and does not mean that there is agreement with the relevant allergen for the triggering of symptoms. The cross-reactivity between these two mites has been studied. In vivo, there are few studies demonstrating the cross-reactivity between Der p and Blo t analyzing the correlation between skin tests and bronchial provocation. The aim of this study is to evaluate the clinical reactivity in patients with asthma sensitized to both house dust mites, Der p and Blo t. For this reason, patients with asthma, Ambulatory Service of Clinical Immunology and Allergy HCFMUSP were selected. Specific bronchial provocation tests were performed with both mites at different times. Eighteen patients with asthma were selected, and thirty-six provocations were performed. All provocations with the mite Der p were positive. Regarding Blo t, the result was positive in 93% of tests. In the present study, there was agreement in most cases between the presence of sensitization to mites and positivity in bronchial provocation test. Therefore, sensitization to mites in skin test identified is relevant to the triggering of asthma symptoms in these patients

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-10-10T13:46:10Z No. of bitstreams: 1 ingridpimentelcunhamagalhaesdesouzalima.pdf: 1571962 bytes, checksum: 521511b66ed89adf27b01a59f98c7cfc (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-10-16T13:34:14Z (GMT) No. of bitstreams: 1 ingridpimentelcunhamagalhaesdesouzalima.pdf: 1571962 bytes, checksum: 521511b66ed89adf27b01a59f98c7cfc (MD5) / Made available in DSpace on 2018-10-16T13:34:14Z (GMT). No. of bitstreams: 1 ingridpimentelcunhamagalhaesdesouzalima.pdf: 1571962 bytes, checksum: 521511b66ed89adf27b01a59f98c7cfc (MD5) Previous issue date: 2018-08-24 / O objetivo do presente estudo é avaliar a positividade do teste cutâneo de contato alérgico de leitura tardia para Dermatophagoides pteronyssinus, Dermatophagoides farinae e Blomia tropicalis em pacientes com doenças respiratórias como rinite alérgica e/ou asma com ou sem dermatite atópica. A maioria dos trabalhos existentes nessa área de pesquisa se refere ao emprego desse método diagnóstico na dermatite atópica, mas, neste estudo, a amostra é composta, principalmente, de pacientes com doenças respiratórias. Blomia tropicalis é um ácaro muito incidente no Brasil e só há dois trabalhos que envolvem o emprego deste ácaro da poeira domiciliar típico de países de clima tropical. Os pacientes foram selecionados pela história clínica e foram divididos em dois grupos: I- pacientes com doenças respiratórias, como asma e/ou rinite alérgica, com dermatite atópica e II- pacientes somente com doenças respiratórias. Foi realizado teste cutâneo de leitura imediata e teste de cutâneo de contato alérgico de leitura tardia para os três ácaros no mesmo dia. O teste de contato alérgico foi retirado em 48 horas. A análise estatísitica foi realizada em porcentagens e a tabela 1 apresenta as variáveis por sexo e por grupo estudado. Setenta e quatro pacientes, com idades de 2 a 60 anos, foram incluídos neste estudo; 16 no grupo I e 58 no grupo II. Considerando o teste cutâneo de leitura imediata, o ácaro mais prevalente foi o Dermatophagoides pteronyssinus, seguido pelo Dermatophagoides farinae e Blomia tropicalis. Em relação ao teste cutâneo de contato alérgico de leitura tardia, o ácaro que induziu maior positividade foi o Dermatophagoides farinae (78,4%), seguido pelo Dermatophagoides pteronyssinus (77%) e Blomia tropicalis (52,7%). Comparando o teste cutâneo de leitura imediata com o teste cutâneo de contato alérgico de leitura tardia, 53 pacientes (71,6%) foram positivos para ambos os testes, e 30 (56,6%) foram positivos ao mesmo ácaro. Foram identificados seis pacientes (8%) que tinham história clinica positiva para alergia e só apresentavam positividade no teste cutâneo de contato alérgico de leitura tardia. Estes resultados sugerem que o teste cutâneo de contato alérgico de leitura tardia é relevante e deve ser considerado como um teste diagnóstico adicional, em pacientes com história clínica positiva para doenças respiratórias, com teste cutâneo de leitura imediata negativa. / The aim of this study is to evaluate the positivity rates of atopy patch tests for Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis in patients with respiratory diseases such as asthma and allergic rhinitis with or without atopic dermatitis. Most studies have been performed with atopic dermatitis patients, but in our study, most of the patients had respiratory conditions. Blomia tropicalis is a mite that is prevalent in tropical areas, such as Brazil, and only two publications include these three mites, which are present in Brazil. The patients’ clinical histories were collected, and the patients were subjected to skin prick and patch tests with the three different house dust mites on the same day. The patch tests were examined 48 hours later, and then, the patients were divided into two groups: I- patients with respiratory diseases, such as asthma and/or rhinitis, and atopic dermatitis and II-patients with only respiratory diseases. The statistical analysis results are presented as percentages, and Table 1 presents the variables by gender and groups studied. A total of 74 patients ranging in age from 2 to 60 years old were included in this study; 16 patients were included in group I and 58 were included in group II. In the skin prick tests, the most prevalent mite that evoked a reaction was Dermatophagoides pterronyssinus, followed by Dermatophagoides farinae and Blomia tropicalis. Regarding the atopy patch tests, the mite that most frequently induced a positive reaction was Dermatophagoides farinae (78.4%), followed by Dermatophagoides pteronyssinus (77%) and Blomia tropicalis (52.7%). A comparison between the skin prick and atopy patch tests revealed that 53 patients (71.6%) were positive on both tests, and 30 (56.6%) patients were positivite for the same mite. We found six patients (8%) who had a positive clinical history of allergy and only exhibited positivity on the atopy patch test. These results suggest that the mite atopy patch test is relevant and should be considered as an additional test for patients with clinical histories of allergic respiratory disease who have negative prick test results.

Choisya ternata est une plante ornementale souvent touchée par la maladie de la pourriture racinaire provoquée par Phytophthora. Cette maladie peut induire de pertes allant jusqu’à 80 %, ce qui implique l’utilisation intensive de produits phytosanitaires. Afin de limiter l’utilisation de ces produits toxiques, une meilleure connaissance des acteurs de la défense des plantes est nécessaire. Pour cela, les objectifs de cette thèse sont I) d’identifier les espèces du genre Phytophthora pathogènes de C. ternata, II) d’étudier des acteurs de défense au niveau racinaire et III) de développer une approche de lutte alternative aux phytosanitaires. L’identification des espèces de Phytophthora par l’amplification et le séquençage de la région ITS, montre la présence de Phytophthora parasitica dans la production de C. ternata en pépinière. Ils mettent aussi en évidence, pour la première fois en France et chez Choisya, la présence de Phytophthora tropicalis. Deux cultivars de C. ternata sont utilisés pour étudier les acteurs de la défense racinaire contre P. parasitica, Aztec pearl (moins sensible à la pourriture racinaire) et Goldfinger (plus sensible). Les arabinogalactane-protéines (AGPs) sont des glycomolécules de la paroi cellulaire qui constitue une barrière physique face aux pathogènes. Des études ont montré le rôle des AGPs dans l’interaction plante-pathogène et plus particulièrement avec les oomycètes. Toutefois, le rôle des AGPs racinaires de Choisya dans l’interaction avec P. parasitica n’est pas étudié. Nos résultats montrent des différences biochimiques au niveau de la composition monosaccharidique des AGPs racinaires d’Aztec pearl en comparaison avec les AGPs de feuilles de ce cultivar et les AGPs de feuilles et de racines de Goldfinger. Contrairement aux autres fractions, ces AGPs n’ont pas augmenté la croissance du mycélium de P. parasitica. Ces résultats suggèrent que l’oomycète n’est pas capable de dégrader cette fraction pour l’utiliser comme une source d’énergie. Pendant l’infection de la plante par P. parasitica, ces AGPs peuvent freiner la pénétration du pathogène, et par conséquent diminuer la sévérité de la maladie. Les plantes ont aussi développé des molécules chimiques nommées métabolites secondaires (MS) capables de les protéger contre leurs agresseurs. Des études ont montré que la partie foliaire de Choisya est riche en MS tels que les alcaloïdes dont plusieurseffets pharmacologiques sont connus. Cependant, leur composition racinaire et leur rôle dans la protection de la plante contre P. parasitica ne sont pas élucidés. Nos résultats montrent que les racines des deux cultivars sont riches en alcaloïdes furoquinoliques. Certains alcaloïdes sont présents en plus grande quantité chez Aztec pearl, mais suite à l’inoculation de zoospores de P. parasitica cette différence n’est plus détectable. De plus, l’extrait contenant les alcaloïdes totaux d’Aztec pearl ont été capables d’inhiber la croissance du mycélium de l’oomycète, contrairement à l’extrait issu de Goldfinger. Ces résultats montrent le rôle potentiel des alcaloïdes furoquinoliques dans la protection de la plante contre cet oomycète. / Choisya ternata is an ornamental plant that suffers from root rot disease due to Phytophthora. This disease can lead to severe production losses (up to 80 %), which require intensive use of phytosanitary products. A better understanding of plants defenses in required in order to reduce the use of these products. Therefore, the objectives of this thesis are I) identifying Phytophthora spp. pathogens of C. ternata, II) studying roots defense actors and III) developing an alternative control approach. Phytophthora spp. identification by ITS region amplification and sequencing highlighted the presence of Phytophthora parasitica on Choisya. Furthermore, Phytophthora tropicalis was identified for the first time in France and on Choisya culture. Two C. ternata cultivars were used to study the plants root defense actors against P. parasitica, Aztec pearl (less susceptible to root rot) and Goldfinger (more susceptible to root rot). Arabinogalactan proteins (AGPs) are glycomolecules of the cell wall which constitutes a physical barrier to pathogens. Studies showed the role of AGPs in the plant-pathogen interaction and more specifically in the case of oomycetes. However, Choisya root AGPs role in the interaction with P. parasitica is not studied. Our results showed biochemical differences in the monosaccharide composition of Aztec pearl root AGPs and the other AGPs fractions (Aztec pearl leaves and Goldfinger roots and leaves). Contrary to other fractions Aztec pearl root AGPs did not increase P. parasitica mycelium growth. These results suggest that oomycete was not able to degrade this fraction and use it as energy source. When P. parasitica infects the plant, these AGPs may be able to slow the infection and reduce disease severity. Plants have also developed chemical molecules known as secondary metabolites (SM) capable of protecting them against attackers. Studies showed that C. ternata leaves are rich with SM as alkaloids that have many pharmacological activities. Nevertheless, roots alkaloids composition and role in plant protection are not studied. Our results showed that roots of the two cultivars are riche with furoquinoline alkaloids. Some these alkaloids were more concentrated in Aztec pearl. But after inoculation with P. parasitica zoospores, the difference was not detectable anymore. Moreover, the total alkaloids extract of Aztec pearl inhibited P. parasitica mycelium growth, unlike the extract of Goldfinger. Another strategy of plants protection is the use of beneficial soil microorganisms that limits pathogens development and stimulate plant defenses. These microorganisms are known as biological control agents (BCA) and are sometimes used in horticulture as an alternative control strategy. In our study, treatments of C. ternata by different BCA were evaluated by a developed real time PCR (qPCR) targeting ypt1 and by symptoms annotation. This evaluation showed that combined treatments by Glomus intraradices with Gliocladium catenulatum and G. intraradices with Trichoderma atroviridae (respectively mycorrhizal fungi and filamentous fungi) offer a better protection against P. parasitica.

Amphibian populations are declining world-wide and one of the suggested reasons is environmental pollutants. Studies of long-term effects on the reproductive system in frogs following larval exposure to environmental pollutants are scarce. It is therefore important to develop methods to study developmental reproductive toxicity in amphibians. In this thesis the usefulness of Xenopus tropicalis (the West African clawed frog) as a model species for a test system was investigated. Effects on the reproductive system after larval exposure to the pharmaceuticals ethynylestradiol (EE2) and clotrimazole were evaluated. The susceptibility to EE2 exposure was compared between the model species and a wild species, the European common frog (Rana temporaria). Larval exposure to EE2 caused female-biased sex ratios in both examined frog species, indicating male-to-female sex-reversal. In adult Xenopus tropicalis, male frogs that were not sex-reversed had reduced fertility and decreased amount of mature spermatozoa in the seminiferous tubules. The proportion of frogs with ovaries but lacking oviducts increased with increasing EE2-concentrations. A female frog without oviducts is sterile. The development of ovaries in sex-reversed male frogs was implied to be similar to control females. The combination of a reduced number of males, due to sex-reversal, and impaired fertility could have severe effects on frog populations. Larval exposure to clotrimazole modulated aromatase activity in gonads and brain in Xenopus tropicalis. Brain aromatase activity was decreased at the time for gonadal differentiation and gonadal aromatase activity was increased at metamorphosis. The findings in this thesis indicate that reproduction in wild frogs might be impaired by estrogenic compounds in the environment. The results combined with the short generation time supports the use of Xenopus tropicalis as a model species when evaluating long term effects of endocrine disruptors on the reproductive system in amphibians.

Testicular stem cells (TSCs) are relatively accessible potential source of pluripotent cells, which are particularly important for their application in regenerative medicine. Xenopus tropicalis is a useful model organism to study the migration and differentiation potential of stem cells. This amphibian is characteristic by outer fecundation and embryonic development of a great amount of embryos after fertilization. Oocytes and embryos are large enough (about 1 mm) to be suitable for micromanipulation micromanipulations. Laboratory of Developmental Biology, Faculty of Science, Charles University in Prague succeeded in the establishment of a mixed cell culture of TSCs growing on feeder layer of pre- Sertoli cells. This culture was derived from the testes of juvenile Xenopus tropicalis male. In the study of their differentiation potential it was found, that leukemia inhibitory factor (LIF) is the decisive factor allowing rapid proliferation of stem cells and their forming into characteristic colonies. This protein is produced by both types of cells which are present in the culture. The mouse LIF has the same positive effect on the proliferative potential of stem cells, which points at the evolutionary conservation of metabolic pathways associated with the maintenance of the stemness. RT-PCR analysis...

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.

It is generally thought that in amphibians, thyroid hormones (THs) regulate metamorphosis, while sex steroids (estrogens and androgens) regulate gonadal differentiation. However, inhibition of TH synthesis in frogs alters gonadal differentiation, suggesting instead that these two endocrine axes interact during development. Specifically, THs may be involved in male development, while estrogens may inhibit tadpole metamorphosis. However, we do not currently know the mechanisms that account for these interactions, let alone how such mechanisms may differ between species. To develop and test new hypotheses on the roles of sex steroids and THs, I first examined transcriptional profiles (mRNA) of enzymes and receptors related to sex steroids and THs during embryogenesis and metamorphosis in Silurana tropicalis. Tadpoles were exposed to either an estrogen synthesis inhibitor (fadrozole) or TH (triiodothyronine, T3) during early larval or tadpole development. Acute exposures of S. tropicalis to fadrozole or T3 during early development resulted in increased expression of androgen- and TH-related genes in whole body larvae, while chronic exposure to fadrozole during metamorphosis affected gonadal differentiation but did not affect tadpole development. On the other hand, acute exposure to T3 during metamorphosis increased the expression of androgen-related transcripts both in the brain and gonad. In S. tropicalis, the results suggested that cross-talk is primarily in one direction (i.e., effect of THs on the reproductive axis) with a strong relationship between TH and androgen status. Lastly, I established developmental transcript profiles and investigated T3 regulation of brain and gonad transcripts in Engystomops pustulosus. I then compared these results with S. tropicalis and an earlier study in Lithobates pipiens. While each species developed with similar profiles, they differed in their response to T3. Exposure to T3 resulted in either an increase in androgen-related genes (S. tropicalis) or a decrease in estrogen-related genes (E. pustulosus and L. pipiens). In conclusion, these data demonstrated that cross-talk mechanisms differ among these three evolutionary separate species, but in all cases, T3 appears to affect the balance of sex steroids, stimulating the androgen system and providing potential mechanisms of the masculinising effects of THs. These results will contribute to understanding the mechanisms of hormone interactions and their evolutionary basis in frogs.