O presente trabalho teve por objetivo investigar a variação na secreção de LH em resposta ao tratamento com neurotransmissores, na presença ou não de esteróides gonadais e desta forma gerar informações sobre os mecanismos neuroendócrinos envolvidos na maturação sexual de novilhas da raça Nelore. A concentração de LH foi quantificada por radioimunoensaio e as novilhas ovariectomizadas (OVX) apresentaram maior concentração basal de LH (P≤0,05), que as novilhas inteiras (INT). A administração de um antagonista gabaérgico (picrotoxina, 0,18 mg/kg, iv, amostras 15 min por 10 h) aos 8, 10, 14 e 17 meses de idade, aumentou (P≤0,05) a concentração média e área total de secreção de LH nas novilhas INT tratadas aos 14 meses, a área total dos picos e área do maior pico de secreção de LH foi maior (P≤0,05) nas novilhas OVX aos 14 e 17 meses de idade . A administração de um antagonista dopaminérgico (sulpiride, 0,59 mg/kg, sc, amostras 15 min por 10 h) aos 8, 12 e 16 meses de idade diminuiu (P≤0,05) a secreção de LH (concentração média, área total, área total dos picos e área do maior pico secreção de LH) nas novilhas OVX aos 8 meses de idade. A administração de um estimulador alfa-adrenérgico (clonidina, 10 µg/kg, iv, amostras 15 min por 4 h) aos 8, 12 e 15 meses de idade, diminuiu (P≤0,05) o número de picos nas novilhas OVX com 8 meses de idade. A administração do 17β-estradiol (2 µg/kg, iv, amostra 15 min por 3 h, 1 h por 7 h e 3 h por 22 h) aos 10, 13 e 17 meses de idade diminuiu a diferença (P≥0,05) entre os grupos OVX e INT em relação ao número de picos, área total de picos, área do maior pico e tempo necessário para acontecer o maior pico. Foi avaliada a secreção de LH da desmama à primeira ovulação em novilhas INT e OVX. A concentração de LH aumentou durante a maturação sexual, tanto nas novilhas INT quanto nas OVX. O número de picos de secreção de LH e amplitude máxima de secreção de LH foi maior (P≤0,05) nas novilhas OVX com o aproximar da primeira ovulação. Os resultados indicam uma diminuição da sensibilidade do hipotálamo aos esteróides gonadais durante o processo de maturação sexual nas novilhas da raça Nelore e a participação alternada de neurotransmissores, inibindo e estimulando a secreção de LH. Concluímos que, em novilhas pré-púberes da raça Nelore o desenvolvimento da retroalimentação positiva aos esteróides gonadais no hipotálamo foi importante para aumentar a secreção de LH antes da primeira ovulação, com a participação de neurotransmissores estimulando ou inibindo a secreção de LH. / The variation on LH secretion after neurotransmitter administration, on the presence or absence of gonadal steroid, was investigated, generating information about the mechanisms evolved on sexual maturation in Nellore heifers. LH concentration was quantified by RIA. As expected ovariectomized heifers higher basal LH concentration (P≤0,05) than intact heifers. The picrotoxin administration (GABA antagonist, 0,18 mg/kg, iv, samples 15 min for 10 h) at 8, 10, 14 and 17 months of age increased (P≤0,05) average concentration and total secretion area on intact treated heifers at 14 months and peak total area and area of highest peal on ovariectomized heifers at 14 and 17 months of age. The dopaminergic antagonist (sulpiride, 0.59 mg/kg, sc, sample 15 min for 10 h) administrated at 8, 12 and 16 months of age decreased (P≤0,05) LH secretion (average levels, total peak area and area of the highest secretion peak) on ovariectomized heifers at 8 months of age. The administration of an alfa-adrenérgic stimulatory (clonidine 10 µg/kg, iv, samples 15 min for 4 h) at 8, 12 and 15 months of age decreased (P≤0,05) decreased the number of peaks at 8 months of age. 17β-estradiol administration (2 µg/kg, iv, samples every 15 min for 3 h, every 1 h for 7 h and every 3 h for 22 h) decreased the differences between ovariectomized and intact heifers on number of peaks, total peak area, highest peak area and time to highest peak occurrence. The LH secretion from weaning to first ovulation in non treated intact and ovariectomized heifers was also evaluated. LH concentration increase during sexual maturation in both ovariectomized and intact heifers. The number of peaks, and maximum LH secretion amplitude was higher in ovariectomized heifer closest to first ovulation, when compared to intact heifers. The results suggested a decrease on hipothalamus sensitivity to gonadal steroid during the sexual maturation in Nelore heifer associated with neurotransmitter participation either stimulating or inhibiting LH secretion. It was possible to conclude that the decrease of negative feedback associated with the increase on positive feedback of gonadal steroids over hipothalamus was necessary to increase LH secretion before first ovulation, that was associated with neurotransmitter participation on LH secretion.

O Arapaima gigas, conhecido popularmente como pirarucu é uma espécie de peixe pertencente à ordem dos Osteoglossiformes, nativo da Bacia Amazônica e autóctone da Bacia de São Francisco e do Nordeste. É considerado um dos maiores peixes de água doce do mundo, chegando, na fase adulta, a três metros de comprimento e mais de 200 kg de peso, possuindo, portanto, uma grande importância para a alimentação e o comércio da região. Infelizmente esta espécie pertence à lista de animais sobre explorados do IBAMA, também em perigo de extinção, devido especialmente à pesca predatória e à sua dificuldade reprodutiva em cativeiro. Por estas razões, desenvolvemos o presente trabalho de clonagem e caracterização de um de seus hormônios da reprodução (gonadotrofinas), em particular o hormônio luteinizante (LH). Esta glicoproteína é constituída por duas subunidades ligadas de forma não covalente: a subunidade α (GTHα) comum também ao hormônio folículo estimulante (FSH) e a subunidade β, que confere a especificidade de sua ação biológica. Tanto o cDNA do ag-GTHα quanto aquele do ag-LHβ foram sintetizados pela reação de transcriptase reversa (RT) e pela reação de cadeia de polimerase (PCR) utilizando vários primers, a partir do RNA total obtido das glândulas hipofisárias de A.gigas. O cDNA de GTHα apresentou um comprimento total de 767 pb incluindo uma cadeia poli-A de 20 adeninas. Foi identificada uma região codificante (ORF) de 348 pb iniciando com o primeiro códon (ATG) na posição 58 e o códon de parada (stop) na posição 403. O sinal de poliadenilação (ATTAAA) foi localizado 18 pb antes da cauda poli-A. A região codificante traduz um peptídeo de 115 aminoácidos, com um sítio de clivagem do peptídeo sinalizador situado entre o aminoácido 24 e 25. A proteína apresenta portanto um suposto peptídeo sinal de 24 aminoácidos e um peptídeo maduro de 91 aminoácidos, que quando alinhado com outras espécies de peixes, mostra a conservação de 10 resíduos de cisteína, 3 prolinas e dois potenciais sítios de glicosilação entre os aminoáciodos 51-53 (NIT) e os aminoáciodos 77-79 (NHT). O cDNA de ag-LHβ apresenta um comprimento total de 711 pb, incluindo uma cadeia poli-A de 18 adeninas. Foi identificada uma região codificante (ORF) de 426 pb, iniciando com primeiro códon (ATG) na posição 47 e terminando na posição 469. O sinal de poliadenilação (AATAAA) foi localizado 18 pb antes da cadeia poli-A. A região codificante traduz um peptídeo sinalizador situado entre o aminoácido 24 e 25. Com isso, temos um peptídeo sinalizador de 24 e um peptídeo maduro de 117 aminoácidos que apresenta a conservação de 12 resíduos de cisteína, 6 prolinas e um sítio potencial de N-glicosilação identificado entre os aminoácidos 10-12 (NQT), enquanto um segundo possível sítio de N-glicosilação (alterado para QTT), entre os aminoácidos 27-29, foi perdido. Como na subunidade GTHα, a maior porcentagem de identidade de LHβ foi com os Cypriniformes (75.6%) enquanto a menor foi com os Gadiformes (53.8%). A análise filogenética realizada utilizando as sequências de FSHβ e LHβ de 41 espécies de peixes, incluido o A.gigas, confirmou os dados publicados relativos à subunidade GTHα, posicionando o A.gigas como grupo irmão dos Clupeocephala e os Elopomorpha (Anguilliformes) como grupo mais basal entre os teleósteos . / Arapaima gigas, popularly known as pirarucu, is a species of fish that belongs to the order of Osteoglossiformes, originating from the Amazon, São Francisco river basin and the North East of Brazil. It is considered one of the largest fresh water fishes in the world, reaching when adult three meters in length and more than 200 kg in weight. It is therefore very important for food and for the regional industry. Unfortunately, this species belongs to the list of overexploited animals from IBAMA and is in danger of disappearing due to fishing exploitation and to its reproductive difficulties, especially in captivity. For these reasons, we developed this project for the cloning and characterization of one of its hormones of reproduction (gonadotropins), namely luteinizing hormone (LH). This glycoprotein is formed by two subunits non-covalently bound: the α subunit (GTHα), in common with follicle-stimulating hormone (FSH) and the β subunit, that provides the specificity of its biological action. Both cDNAs of ag-GTHα and of ag-LHβ have been synthesized via reverse transcriptase (RT) and polymerase chain reaction (PCR) utilizing several primers, starting from total RNA extracted from A. gigas pituitary glands. The cDNA of ag-GTHα showed a total lenght of 767 bp, including a poli-A tail with 20 adenines. A coding reagion (ORF) of 348 bp, was also identified, starting from the first codon (ATG) at position 58, with the stop codon at position 403. The polyadenylation signal (ATTAAA) was identified 18 bp before the poly-A tail. This coding sequence translates a 115 amino acid peptide showing a signal-peptide cleavage site between amino acid 24 and 25. It has therefore a putative signal peptide with 24 and a mature peptide with 91 amino acids that, when aligned with other species of fish, presents 10 conserved residues of cysteine, 3 of proline and two potential glycosylation sites at amino acids 51-53 (NIT) and amino acids 77-79 (NHT). The cDNA of ag-LHβ has instead a total length of 711 bp, including a poly-A tail of 18 adenines. A coding region of 426 bp was identified, starting with the first codon (ATG) at position 47 and having the stop codon at position 469. The polyadenylation signal (AATAAA) was found 18 bp before the poly-A tail. The coding region translates a signal-peptide located between amino acid 24 and 25. It has a signal peptide with 24 and a mature peptide with 117 amino acids that presents 12 conserved residues of cysteine, 6 of proline and a potential N-glycosylation site at amino acid 10-12 (NQT), while a second possible N-glycosilation site at amino acid 27-29 (altered into QTT), was last due to the substitution of an asparagine with a glutamine. As for the case of ag-GTHα, the highest percent of identity was found with Cypriniformes (75.6%), while the lowest was with Gadiformes (53.8%). The phylogenetic analysis carried out with cDNA sequences of LHβ and FSHβ of 41 different fish species, confirmed previous published data concerning ag-GTHα, locating A.gigas as the sister group of Clupeocephala and the Elopomorpha (Anguilliformes) as the most basal group of all living teleosts.

O presente trabalho teve por objetivo investigar a variação na secreção de LH em resposta ao tratamento com neurotransmissores, na presença ou não de esteróides gonadais e desta forma gerar informações sobre os mecanismos neuroendócrinos envolvidos na maturação sexual de novilhas da raça Nelore. A concentração de LH foi quantificada por radioimunoensaio e as novilhas ovariectomizadas (OVX) apresentaram maior concentração basal de LH (P≤0,05), que as novilhas inteiras (INT). A administração de um antagonista gabaérgico (picrotoxina, 0,18 mg/kg, iv, amostras 15 min por 10 h) aos 8, 10, 14 e 17 meses de idade, aumentou (P≤0,05) a concentração média e área total de secreção de LH nas novilhas INT tratadas aos 14 meses, a área total dos picos e área do maior pico de secreção de LH foi maior (P≤0,05) nas novilhas OVX aos 14 e 17 meses de idade . A administração de um antagonista dopaminérgico (sulpiride, 0,59 mg/kg, sc, amostras 15 min por 10 h) aos 8, 12 e 16 meses de idade diminuiu (P≤0,05) a secreção de LH (concentração média, área total, área total dos picos e área do maior pico secreção de LH) nas novilhas OVX aos 8 meses de idade. A administração de um estimulador alfa-adrenérgico (clonidina, 10 µg/kg, iv, amostras 15 min por 4 h) aos 8, 12 e 15 meses de idade, diminuiu (P≤0,05) o número de picos nas novilhas OVX com 8 meses de idade. A administração do 17β-estradiol (2 µg/kg, iv, amostra 15 min por 3 h, 1 h por 7 h e 3 h por 22 h) aos 10, 13 e 17 meses de idade diminuiu a diferença (P≥0,05) entre os grupos OVX e INT em relação ao número de picos, área total de picos, área do maior pico e tempo necessário para acontecer o maior pico. Foi avaliada a secreção de LH da desmama à primeira ovulação em novilhas INT e OVX. A concentração de LH aumentou durante a maturação sexual, tanto nas novilhas INT quanto nas OVX. O número de picos de secreção de LH e amplitude máxima de secreção de LH foi maior (P≤0,05) nas novilhas OVX com o aproximar da primeira ovulação. Os resultados indicam uma diminuição da sensibilidade do hipotálamo aos esteróides gonadais durante o processo de maturação sexual nas novilhas da raça Nelore e a participação alternada de neurotransmissores, inibindo e estimulando a secreção de LH. Concluímos que, em novilhas pré-púberes da raça Nelore o desenvolvimento da retroalimentação positiva aos esteróides gonadais no hipotálamo foi importante para aumentar a secreção de LH antes da primeira ovulação, com a participação de neurotransmissores estimulando ou inibindo a secreção de LH. / The variation on LH secretion after neurotransmitter administration, on the presence or absence of gonadal steroid, was investigated, generating information about the mechanisms evolved on sexual maturation in Nellore heifers. LH concentration was quantified by RIA. As expected ovariectomized heifers higher basal LH concentration (P≤0,05) than intact heifers. The picrotoxin administration (GABA antagonist, 0,18 mg/kg, iv, samples 15 min for 10 h) at 8, 10, 14 and 17 months of age increased (P≤0,05) average concentration and total secretion area on intact treated heifers at 14 months and peak total area and area of highest peal on ovariectomized heifers at 14 and 17 months of age. The dopaminergic antagonist (sulpiride, 0.59 mg/kg, sc, sample 15 min for 10 h) administrated at 8, 12 and 16 months of age decreased (P≤0,05) LH secretion (average levels, total peak area and area of the highest secretion peak) on ovariectomized heifers at 8 months of age. The administration of an alfa-adrenérgic stimulatory (clonidine 10 µg/kg, iv, samples 15 min for 4 h) at 8, 12 and 15 months of age decreased (P≤0,05) decreased the number of peaks at 8 months of age. 17β-estradiol administration (2 µg/kg, iv, samples every 15 min for 3 h, every 1 h for 7 h and every 3 h for 22 h) decreased the differences between ovariectomized and intact heifers on number of peaks, total peak area, highest peak area and time to highest peak occurrence. The LH secretion from weaning to first ovulation in non treated intact and ovariectomized heifers was also evaluated. LH concentration increase during sexual maturation in both ovariectomized and intact heifers. The number of peaks, and maximum LH secretion amplitude was higher in ovariectomized heifer closest to first ovulation, when compared to intact heifers. The results suggested a decrease on hipothalamus sensitivity to gonadal steroid during the sexual maturation in Nelore heifer associated with neurotransmitter participation either stimulating or inhibiting LH secretion. It was possible to conclude that the decrease of negative feedback associated with the increase on positive feedback of gonadal steroids over hipothalamus was necessary to increase LH secretion before first ovulation, that was associated with neurotransmitter participation on LH secretion.

O Arapaima gigas, conhecido popularmente como pirarucu é uma espécie de peixe pertencente à ordem dos Osteoglossiformes, nativo da Bacia Amazônica e autóctone da Bacia de São Francisco e do Nordeste. É considerado um dos maiores peixes de água doce do mundo, chegando, na fase adulta, a três metros de comprimento e mais de 200 kg de peso, possuindo, portanto, uma grande importância para a alimentação e o comércio da região. Infelizmente esta espécie pertence à lista de animais sobre explorados do IBAMA, também em perigo de extinção, devido especialmente à pesca predatória e à sua dificuldade reprodutiva em cativeiro. Por estas razões, desenvolvemos o presente trabalho de clonagem e caracterização de um de seus hormônios da reprodução (gonadotrofinas), em particular o hormônio luteinizante (LH). Esta glicoproteína é constituída por duas subunidades ligadas de forma não covalente: a subunidade α (GTHα) comum também ao hormônio folículo estimulante (FSH) e a subunidade β, que confere a especificidade de sua ação biológica. Tanto o cDNA do ag-GTHα quanto aquele do ag-LHβ foram sintetizados pela reação de transcriptase reversa (RT) e pela reação de cadeia de polimerase (PCR) utilizando vários primers, a partir do RNA total obtido das glândulas hipofisárias de A.gigas. O cDNA de GTHα apresentou um comprimento total de 767 pb incluindo uma cadeia poli-A de 20 adeninas. Foi identificada uma região codificante (ORF) de 348 pb iniciando com o primeiro códon (ATG) na posição 58 e o códon de parada (stop) na posição 403. O sinal de poliadenilação (ATTAAA) foi localizado 18 pb antes da cauda poli-A. A região codificante traduz um peptídeo de 115 aminoácidos, com um sítio de clivagem do peptídeo sinalizador situado entre o aminoácido 24 e 25. A proteína apresenta portanto um suposto peptídeo sinal de 24 aminoácidos e um peptídeo maduro de 91 aminoácidos, que quando alinhado com outras espécies de peixes, mostra a conservação de 10 resíduos de cisteína, 3 prolinas e dois potenciais sítios de glicosilação entre os aminoáciodos 51-53 (NIT) e os aminoáciodos 77-79 (NHT). O cDNA de ag-LHβ apresenta um comprimento total de 711 pb, incluindo uma cadeia poli-A de 18 adeninas. Foi identificada uma região codificante (ORF) de 426 pb, iniciando com primeiro códon (ATG) na posição 47 e terminando na posição 469. O sinal de poliadenilação (AATAAA) foi localizado 18 pb antes da cadeia poli-A. A região codificante traduz um peptídeo sinalizador situado entre o aminoácido 24 e 25. Com isso, temos um peptídeo sinalizador de 24 e um peptídeo maduro de 117 aminoácidos que apresenta a conservação de 12 resíduos de cisteína, 6 prolinas e um sítio potencial de N-glicosilação identificado entre os aminoácidos 10-12 (NQT), enquanto um segundo possível sítio de N-glicosilação (alterado para QTT), entre os aminoácidos 27-29, foi perdido. Como na subunidade GTHα, a maior porcentagem de identidade de LHβ foi com os Cypriniformes (75.6%) enquanto a menor foi com os Gadiformes (53.8%). A análise filogenética realizada utilizando as sequências de FSHβ e LHβ de 41 espécies de peixes, incluido o A.gigas, confirmou os dados publicados relativos à subunidade GTHα, posicionando o A.gigas como grupo irmão dos Clupeocephala e os Elopomorpha (Anguilliformes) como grupo mais basal entre os teleósteos . / Arapaima gigas, popularly known as pirarucu, is a species of fish that belongs to the order of Osteoglossiformes, originating from the Amazon, São Francisco river basin and the North East of Brazil. It is considered one of the largest fresh water fishes in the world, reaching when adult three meters in length and more than 200 kg in weight. It is therefore very important for food and for the regional industry. Unfortunately, this species belongs to the list of overexploited animals from IBAMA and is in danger of disappearing due to fishing exploitation and to its reproductive difficulties, especially in captivity. For these reasons, we developed this project for the cloning and characterization of one of its hormones of reproduction (gonadotropins), namely luteinizing hormone (LH). This glycoprotein is formed by two subunits non-covalently bound: the α subunit (GTHα), in common with follicle-stimulating hormone (FSH) and the β subunit, that provides the specificity of its biological action. Both cDNAs of ag-GTHα and of ag-LHβ have been synthesized via reverse transcriptase (RT) and polymerase chain reaction (PCR) utilizing several primers, starting from total RNA extracted from A. gigas pituitary glands. The cDNA of ag-GTHα showed a total lenght of 767 bp, including a poli-A tail with 20 adenines. A coding reagion (ORF) of 348 bp, was also identified, starting from the first codon (ATG) at position 58, with the stop codon at position 403. The polyadenylation signal (ATTAAA) was identified 18 bp before the poly-A tail. This coding sequence translates a 115 amino acid peptide showing a signal-peptide cleavage site between amino acid 24 and 25. It has therefore a putative signal peptide with 24 and a mature peptide with 91 amino acids that, when aligned with other species of fish, presents 10 conserved residues of cysteine, 3 of proline and two potential glycosylation sites at amino acids 51-53 (NIT) and amino acids 77-79 (NHT). The cDNA of ag-LHβ has instead a total length of 711 bp, including a poly-A tail of 18 adenines. A coding region of 426 bp was identified, starting with the first codon (ATG) at position 47 and having the stop codon at position 469. The polyadenylation signal (AATAAA) was found 18 bp before the poly-A tail. The coding region translates a signal-peptide located between amino acid 24 and 25. It has a signal peptide with 24 and a mature peptide with 117 amino acids that presents 12 conserved residues of cysteine, 6 of proline and a potential N-glycosylation site at amino acid 10-12 (NQT), while a second possible N-glycosilation site at amino acid 27-29 (altered into QTT), was last due to the substitution of an asparagine with a glutamine. As for the case of ag-GTHα, the highest percent of identity was found with Cypriniformes (75.6%), while the lowest was with Gadiformes (53.8%). The phylogenetic analysis carried out with cDNA sequences of LHβ and FSHβ of 41 different fish species, confirmed previous published data concerning ag-GTHα, locating A.gigas as the sister group of Clupeocephala and the Elopomorpha (Anguilliformes) as the most basal group of all living teleosts.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-10-13T19:41:46Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTACAO GIOVANNA GUSMÃO.pdf: 640329 bytes, checksum: eb48b6d00fc9a30b18bc1c3414978fc5 (MD5) / Made available in DSpace on 2016-10-13T19:41:46Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTACAO GIOVANNA GUSMÃO.pdf: 640329 bytes, checksum: eb48b6d00fc9a30b18bc1c3414978fc5 (MD5) Previous issue date: 1998-11-13 / Os hormônios tireoidianos são essenciais para um crescimento e desenvolvimento normal pós natal. Várias investigações têm tentado estabilizar o papel específico dos hormônios tireoidianos nas funções e desenvolvimento do trato reprodutor masculino. Os hormônios tireoidianos possivelmente exercem uma ação recíproca entre o esteróide testicular e as células de Sertoli durante o período prematuro. O presente estudo tentou avaliar o efeito da tiroxina sobre os níveis de testosterona no soro e no fluido intersticial testicular (FIT) em ratos machos. O hipertireoidismo foi induzido com a tiroxina (20 g\kg) em ratos Wistar machos com 22 dias de idade, pesando 80g por um período de 5,10, 15 e 20 dias de tratamento. Os grupos controles receberam solução salina 0,1 ml/100g do peso corporal, como veículo. Após cada período de tratamento, os animais foram pesados e sacrificados, para obtenção do sangue, e os testículos foram pesados, para coleta do fluido intersticial testicular (FIT). Os nossos resultados mostram que a tiroxina em ratos sexualmente prematuros, promove um aumento de testosterona, tempdependente. Observa-se também, que a nível testicular, no FIT, os níveis de testosterona encontram-se elevados. A tiroxina promoveu um aumento do peso corporal e testicular, modificando a função metabólica. Estudos evidenciaram que os receptores de LH e hCG é regulado em homogeneizado celular de testículo em ratos tratados com tiroxina por 20 dias. A capacidade de ligação específica foi obtida pela motodologia envolvendo a saturação de receptores do LH e hCG, aumentando a concentração de LH marcado e hCG não marcado. Outros resultados mostram que a tiroxina é capaz de alterar os níveis de receptores de LH de alta afinidade presentes em homogeneizado testicular, promovendo uma redução da capacidade de ligação específica (Bmax), obtido através da Análise de Scatchard. Conclui-se que a tiroxina pode acelerar a formação de testosterona no lumen intersticial no processo de maturação sexual, modificando possivelmente o mecanismo intracelular das células testiculares, e que a capacidade de ligação do LH e hCG é alterada após o tratamento com tiroxina. / Thyroid hormones are essential for normal pos-natal growth and development. Several investigators have attempted to establish a specific role of thyroid hormones in reproductive tract growth and function. The thyroid hormones possibly exert a reciprocal action between the testicular steroid and the Sertoli cells during the early period. The present study tried to evaluate the effect of the thyroxine over the serum levels of the testosterone and in the interstitial fluid of the testis in male rats. The hyperthyroidism was induced with the thyroxine (20 g\kg), In Wistar male rats 22 days of age, weighing 80kg for a period of 5, 10, 15 and 20 days. The control group received 0,9% saline solution as vehicle. After each treatment period, the animals were weighty and killed to obtainment of blood, and the testis was weighty to collect of testicular interstitial fluid (TIF). These results show that the thyroxin in premature rats, promote an increase of testosterone, dependent time. It is observed either an increase on testosterone level in the TIF. The thyroxin promoted an increase the corporeal and testicular weighty, capable in modify a metabolic function. In the previos study the regulation of LH and hCG receptors by homogenized testicular cells of the 20 days treatment. The specific binding capacity was also obtained by LH and hCG receptors with increasing concentration LH labelled and hCG no labelled. The Scatchard analysis of the saturad curve has show that a affinity constant (ka) is around 108 M-1. This result demonstred that the control present more binding specific that thyroxine wich is a maximum induction of hCG receptor. These results indicate that the thyroxine is able to accelerate the formation the interstitial lumen in the process of sexual maturation, altering possibly the intra-cell mechanism of testicular cells, and the LH and hCG binding capacity is changed after the thyroxine treatment.

G protein-coupled receptors (GPCRs) are a large family of trans-membrane receptors that transmit signals from extracellular stimuli to target intracellular signal transduction pathways. The gonadotropin-releasing hormone receptor (GnRH-R) is a GPCR which binds the decapeptide GnRH. In the pituitary gonadotrope, GnRH stimulates gonadotropin (LH and FSH) biosynthesis and secretion to regulate reproduction. GnRH and the GnRH-Rs are also present in many extra-pituitary tissues, although their role at these sites remains largely undetermined. GnRH-Rs are known to recruit a diverse array of signalling pathway mediators in different cell-types. These include; Gq/11-PLCβ-IP3/DAG-Ca2+/PKC signalling, monomeric G-proteins and integrins to mediate cell adhesion and migration, the activation of the major members of the mitogen-activated protein kinase (MAPK) super-family (extracellular signal-regulated kinase (ERK), c-Jun N-terminal Kinase (JNK) and p38MAPK), and β-catenin and other mediators of the canonical Wnt signalling pathway. This thesis describes the regulation of Forkhead Box O (FOXO) transcription factors by GnRH. The mammalian FOXO transcription factors, FOXO1, FOXO3a and FOXO4, are emerging as an important family of proteins that modulate the expression of genes involved in cell-cycle regulation, induction of apoptosis, DNA damage repair and response to oxidative stress. In this thesis, emphasis is placed on delineating the novel role of FOXO transcription factors in mediating two important and widely-researched areas of GnRH biology. Firstly, the role of FOXO transcription factors in mediating cell-growth inhibition in response to GnRH treatment is assessed in a heterologous HEK293/GnRH-R expressing cell line. Secondly, the role of transcription factors in regulating luteinising hormone-β (LHβ)-subunit expression is investigated in the LβT2 gonadotrope cell line. Activation of the GnRH-R can inhibit cell proliferation and induce apoptosis in certain tumour-derived cell lines. Several studies have reported that these events can occur as a result of changes in the expression profiles of specific cell-cycle regulatory and apoptotic genes, many of which are FOXO-target genes, including GADD45, FasL, p21Cip1 and p27Kip1. In this thesis, a role for FOXOs in targeting the expression of several of these genes in response to GnRH is assessed, highlighting a specific role for FOXO3a in mediating GADD45 and FasL expression. The signalling mechanisms through which FOXO3a regulates GADD45 expression in response to GnRH is also described. Finally, a stable FOXO3a-knock-down cell line was generated in order to further examine FOXO3a involvement in GnRH-induced cell-growth inhibition. GnRH is an essential regulator of the reproductive process by stimulating the synthesis of LH and FSH in pituitary gonadotropes, thereby regulating gametogenesis and steroidogenesis. Diverse signalling pathways have been reported to regulate LHβ-subunit expression in response to GnRH, including the ERK/JNK/p38MAPK cascades and factors such as Egr1, SF1 and β-catenin. In the second part of this thesis, the role of FOXOs in regulating LHβ-subunit expression in response to GnRH is described. The data presented suggests that GnRH can regulate LHβ-subunit expression through both indirect and direct FOXO3a-mediated mechanisms. Firstly, FOXO3a was found to regulate Egr1 expression to indirectly target LHβ-promoter activity. Secondly, a role for β-catenin as a FOXO3a co-factor to directly regulate LHβ-subunit expression, together with Egr1 and SF1, is also proposed. FOXO3a expression and sub-cellular localisation was assessed and demonstrated in LβT2 cells and in adult human male pituitary sections. The research presented in this thesis adds to the diversity of signalling pathways and mediators that GnRH can target in different cellular backgrounds in order to mediate a variety of cellular processes. The antiproliferative and apoptotic effects of GnRH on tumour-derived cell lines are well-documented, and this research highlights a novel role for FOXO3a in mediating these events. The regulation of gonadotropin synthesis remains an important topic of research, and the novel implication of FOXO3a in mediating LHβ-subunit expression adds further complexity to gonadotrope physiology.

Luteinising hormone (LH) is a crucial regulator of ovarian follicle maturation, ovulation and luteinisation. Development of healthy follicles and fertile ovulation can only occur within a specific range of circulating LH concentrations, with differing upper and lower limits depending on the stage of the oestrous cycle. The objective of the three studies in this thesis was to investigate the effects of both physiological and non-physiological circulating LH levels on equine follicular maturity by examining ovulatory and steroidogenic capacity, gene expression profiles and miRNA expression in ovulatory-size follicles at various stages of the oestrous cycle and/or in response to supplementation with LH. The aim of the first study was to investigate the hypothesis that deficient circulating LH is a primary cause for the inability of equine follicles to ovulate during the physiological anovulatory season. A LH-rich equine pituitary fraction (eLH) given twice daily to early transitional mares did not restore steroidogenic capacity of the ovulatory-size follicle or advance the onset of the natural breeding season; however, it significantly stimulated follicular growth to a level similar to that occurring during the normal oestrous cycle. The results demonstrated that a deficiency in LH is critically involved in reduced follicle growth during the anovulatory season. The second study examined the effects of elevated circulating LH levels early during follicle development on follicle maturation and ovulatory ability in cycling mares, with the hypothesis that excessive LH would disrupt ovulation and produce haemorrhagic anovulatory follicles (HAFs). Treatment with eLH or a luteolytic dose of prostaglandin F2α (to stimulate an increase in endogenous levels of LH) did not have any effects on follicle growth or ovulation, but did impair follicular production of androstenedione and insulin-like growth factor 1 (IGF1), suggesting a deleterious effect of high LH on follicle and oocyte maturation. The third study examined the expression of different follicular factors associated with follicle maturation as well as microRNAs (miRNAs) in ovulatory-size follicles naturally developing under different LH milieus (oestrus, dioestrus and spring transitional period). Progesterone and IGF1 were significantly reduced in follicles developing in a low LH environment (dioestrus and transition). All four miRNAs measured, miR-378, miR-542, miR-202 and miR-21 were found at higher levels in subordinate follicles than in preovulatory follicles during oestrus. In addition miR- 202 and miR-21 were significantly increased in transitional follicles relative to oestrous follicles. The results of this study indicate that follicles developing during both the spring transitional and dioestrous periods are developmentally immature and suggested potential important roles of miRNAs in follicle maturation in the horse. In summary, although LH is a key factor promoting follicular growth, it is by itself not sufficient to restore steroidogenic activity in transitional follicles. Elevated LH levels during follicle development do not disrupt ovulation, but induce changes in follicular fluid factors related to follicle maturation and oocyte quality. Follicles developing under different LH milieus show altered miRNA expression, suggesting an important role of miRNAs in follicle maturation.

The objective was to investigate the effects of progesterone (P4) on luteinizing hormone (LH) release, follicle development, and oocyte competence in cattle. We tested the general hypotheses that: 1) The suppressive effect of P4 on gonadotrophin releasing hormone (GnRH)-mediated LH release can be overcome by increasing GnRH dose or pre-treatment with estradiol (E2); and 2) a shorter period of P4 exposure during the growing phase of the ovulatory follicle improves oocyte competence and fertility after fixed-time artificial insemination or superstimulation in cattle. <p>In the first experiment, heifers (n=22) were treated with 100 or 200 µg of GnRH or pretreated with E2 prior to administration of GnRH during high or low circulating P4 concentrations to characterize LH release (Chapter 2). Increasing the dose of GnRH did not alter LH secretion; however, E2 pretreatment overcame the suppressive effect of high P4 on LH secretion. Cattle with lower (n=11) P4 concentrations had higher circulating LH concentrations than those with higher P4 concentrations (n=11), and tended to have higher ovulation rates. <p>Two experiments were conducted to determine the effect of the duration of P4 exposure during the ovulatory wave on fertility followed fixed-time artificial insemination or superstimulation. In the first experiment (Chapter 3), the dominant follicle was allowed to grow for 3 days (n=181) or 6 days (n=184). Six days of growth resulted in a larger dominant follicle, but in both groups, ovulatory follicles had similar capacities to ovulate and establish pregnancy. In the second experiment (Chapter 4), multiple follicles were allowed to grow for 3 or 6 days by 8 or 14 injections of FSH (at 12-hour intervals). There was no difference between groups for ovulation rate or total ova/embryo recovery rate. Although the 3-day group had higher embryo quality at slaughter (4 days after insemination), further development (7, 9, and 10 days after insemination) did not differ among groups. The effect of FSH starvation following 4 days of FSH treatment (Chapter 4) resulted in loss of ovulatory capability. Overall, a shorter duration of P4 exposure during ovulatory follicle growth did not improve fertility after fixed-time AI or oocyte competence after superstimulation.

The objective was to investigate the effects of progesterone (P4) on luteinizing hormone (LH) release, follicle development, and oocyte competence in cattle. We tested the general hypotheses that: 1) The suppressive effect of P4 on gonadotrophin releasing hormone (GnRH)-mediated LH release can be overcome by increasing GnRH dose or pre-treatment with estradiol (E2); and 2) a shorter period of P4 exposure during the growing phase of the ovulatory follicle improves oocyte competence and fertility after fixed-time artificial insemination or superstimulation in cattle. <p>In the first experiment, heifers (n=22) were treated with 100 or 200 µg of GnRH or pretreated with E2 prior to administration of GnRH during high or low circulating P4 concentrations to characterize LH release (Chapter 2). Increasing the dose of GnRH did not alter LH secretion; however, E2 pretreatment overcame the suppressive effect of high P4 on LH secretion. Cattle with lower (n=11) P4 concentrations had higher circulating LH concentrations than those with higher P4 concentrations (n=11), and tended to have higher ovulation rates. <p>Two experiments were conducted to determine the effect of the duration of P4 exposure during the ovulatory wave on fertility followed fixed-time artificial insemination or superstimulation. In the first experiment (Chapter 3), the dominant follicle was allowed to grow for 3 days (n=181) or 6 days (n=184). Six days of growth resulted in a larger dominant follicle, but in both groups, ovulatory follicles had similar capacities to ovulate and establish pregnancy. In the second experiment (Chapter 4), multiple follicles were allowed to grow for 3 or 6 days by 8 or 14 injections of FSH (at 12-hour intervals). There was no difference between groups for ovulation rate or total ova/embryo recovery rate. Although the 3-day group had higher embryo quality at slaughter (4 days after insemination), further development (7, 9, and 10 days after insemination) did not differ among groups. The effect of FSH starvation following 4 days of FSH treatment (Chapter 4) resulted in loss of ovulatory capability. Overall, a shorter duration of P4 exposure during ovulatory follicle growth did not improve fertility after fixed-time AI or oocyte competence after superstimulation.

The Luteinizing hormone receptor (LHR) belongs to the G protein-coupled receptor family, asdo the other glycoprotein hormone receptors for FSH, TSH, and CG. The LHR comprises twohalves of ~350 amino acids: an extracellular hormone binding exodomain and a seventransmembrane-spanning endodomain responsible for signal generation. Hormone binds to theexodomain with high affinity, and the resulting conformational changes in thehormone/exodomain complex modulate the endodomain to generate hormone signals. Hormonebinding to an LHR produces hormonal signals (cis-activation), but it is not known whether aliganded LHR could activate other unoccupied LHRs (trans-activation). The LHR activates bothadenylyl cyclase and phospholipase C??. This dissertation shows that trans-activation of the LHRleads to the activation of adenylyl cyclase to induce cAMP but not to the activation ofphospholipase C?? to induce the inositol phosphate signaling. Trans-activation offers amechanism of signal amplification at the receptor level and also provides a mechanism ofmultiple signal generation for a liganded LHR to cis-activate phospholipase C?? and transactivateadenylyl cyclase. Also coexpression of Gi2 with a constitutively activating LHR(Asp578Gly), the most common mutation of male-limited precocious puberty, shows that Gi2could completely inhibit cAMP induction by the LHR mutant. Experiments using the carboxylterminal region of G protein ?? subunits demonstrate that LHR has overlapping binding sites forG?? subunits Gs and Gi2.