Os genes dos receptores do hormônio luteinizante (LHR) e folículo estimulante (FSHR), conhecidos por sua influência na manifestação da puberdade, foram avaliados por análise PCR-RFLP em uma população de 370 novilhas de corte compostas, de diferentes composições raciais Europeu-Zebu. Os objetivos foram caracterizar geneticamente a população investigada, utilizando-se de freqüências genotípicas e alélicas e estimativas de variabilidade e diversidade gênica; avaliar o efeito dos marcadores sobre a precocidade sexual, caracterizada pela probabilidade de prenhez por ocasião da primeira estação de monta (PP), e estimar a proporção da variância genética total da variável PP atribuída aos marcadores investigados, bem como a herdabilidade da característica pelo método de Máxima Verossimilhança Restrita (REML) sob modelo animal e sob modelo touro. Contatou-se elevada freqüência de animais heterozigotos em quase todas as composições raciais investigadas, para ambos os genes, com um valor médio de heterozigosidade de 57%, resultados estes que refletem a elevada variabilidade genética desta população híbrida. As novilhas heterozigotas apresentaram maiores taxas de prenhez (67 e 66% respectivamente, para os genes do LHR e FSHR), entretanto não se constataram efeito dos polimorfismos RFLP LHR (P=0,9188) e FSHR (P=0,8831) sobre a manifestação deste evento. As estimativas de herdabilidade obtidas para a PP foram de 0,21 e 0,41, respectivamente sob modelo animal e sob modelo touro. A magnitude das estimativas dos componentes de (co)variância atribuídas aos efeitos dos marcadores se mostrou muito baixa, constatando-se a pequena contribuição destes marcadores na proporção da variância total da prenhez, indicando ser esta uma característica de herança poligênica. Os resultados aqui demonstrados indicam que a seleção de novilhas para a precocidade sexual, com base em sua informação genotípica para os marcadores RFLP LHR e FSHR, não se justifica em programas de melhoramento genético animal, sugerindo-se a investigação de outros genes igualmente importantes, envolvidos na manifestação deste evento. Contudo, os marcadores avaliados se mostraram informativos, sendo indicados em estudos de caracterização genética em outras populações bovinas. A elevada heterozigosidade verificada na população composta estudada viabiliza a exploração destes animais em cruzamentos / The luteinizing hormone receptor (LHR) and follicle-stimulating receptor (FSHR) genes, known for their influence on the onset of puberty were evaluated by PCR-RFLP analysis in a population of 370 European-Zebu composite beef heifers from different breed contributions. The objectives were to genetically characterize the investigated population using genotype and allelic frequencies values besides on variability and gene diversity estimates; to evaluate the effect of markers on sexual precocity, characterized as the probability of pregnancy during the first breeding season (PP); and to estimate the proportion of total genetic variance in the PP related to the investigated markers, as well as the heritability estimate for PP by the Restricted Maximum Likelihood (REML) method for animal and sire models. The high number of heterozygous animals observed in almost all breed compositions studied for both loci, with average heterozigosity values of 57%, showed the high genetic variability on this hybrid population. Higher pregnancy rates were observed in heterozygous heifers (67% and 66% for LHR and FSHR genes, respectively), however, no effect of RFLP polymorphism for LHR (P=0.9188) and FSHR (P=0.8831) on pregnancy rate was observed. The heritabily estimates for PP were 0.21 and 0.41, using the animal and sire model, respectively. The small magnitude of the (co)variance components estimates related to random effects of LHR and FSHR, showed their small contribution in the proportion of total variance of pregnancy, indicating a polygenic inheritance for this trait. The results found in this work indicate that selection of beef heifers in animal genetic breeding programs for sexual precocity based on the inclusion of genotype information on RFLP for LHR e FSHR markers is not recommended. The investigation of other important genes related to puberty onset in heifers is necessary. However, the evaluated markers were informative and may be indicated for genetic characterization studies in other bovine populations. The high heterozigosity observed on the studied composite population maximizes the exploration of these animals in crossbreeding

Os genes dos receptores do hormônio luteinizante (LHR) e folículo estimulante (FSHR), conhecidos por sua influência na manifestação da puberdade, foram avaliados por análise PCR-RFLP em uma população de 370 novilhas de corte compostas, de diferentes composições raciais Europeu-Zebu. Os objetivos foram caracterizar geneticamente a população investigada, utilizando-se de freqüências genotípicas e alélicas e estimativas de variabilidade e diversidade gênica; avaliar o efeito dos marcadores sobre a precocidade sexual, caracterizada pela probabilidade de prenhez por ocasião da primeira estação de monta (PP), e estimar a proporção da variância genética total da variável PP atribuída aos marcadores investigados, bem como a herdabilidade da característica pelo método de Máxima Verossimilhança Restrita (REML) sob modelo animal e sob modelo touro. Contatou-se elevada freqüência de animais heterozigotos em quase todas as composições raciais investigadas, para ambos os genes, com um valor médio de heterozigosidade de 57%, resultados estes que refletem a elevada variabilidade genética desta população híbrida. As novilhas heterozigotas apresentaram maiores taxas de prenhez (67 e 66% respectivamente, para os genes do LHR e FSHR), entretanto não se constataram efeito dos polimorfismos RFLP LHR (P=0,9188) e FSHR (P=0,8831) sobre a manifestação deste evento. As estimativas de herdabilidade obtidas para a PP foram de 0,21 e 0,41, respectivamente sob modelo animal e sob modelo touro. A magnitude das estimativas dos componentes de (co)variância atribuídas aos efeitos dos marcadores se mostrou muito baixa, constatando-se a pequena contribuição destes marcadores na proporção da variância total da prenhez, indicando ser esta uma característica de herança poligênica. Os resultados aqui demonstrados indicam que a seleção de novilhas para a precocidade sexual, com base em sua informação genotípica para os marcadores RFLP LHR e FSHR, não se justifica em programas de melhoramento genético animal, sugerindo-se a investigação de outros genes igualmente importantes, envolvidos na manifestação deste evento. Contudo, os marcadores avaliados se mostraram informativos, sendo indicados em estudos de caracterização genética em outras populações bovinas. A elevada heterozigosidade verificada na população composta estudada viabiliza a exploração destes animais em cruzamentos / The luteinizing hormone receptor (LHR) and follicle-stimulating receptor (FSHR) genes, known for their influence on the onset of puberty were evaluated by PCR-RFLP analysis in a population of 370 European-Zebu composite beef heifers from different breed contributions. The objectives were to genetically characterize the investigated population using genotype and allelic frequencies values besides on variability and gene diversity estimates; to evaluate the effect of markers on sexual precocity, characterized as the probability of pregnancy during the first breeding season (PP); and to estimate the proportion of total genetic variance in the PP related to the investigated markers, as well as the heritability estimate for PP by the Restricted Maximum Likelihood (REML) method for animal and sire models. The high number of heterozygous animals observed in almost all breed compositions studied for both loci, with average heterozigosity values of 57%, showed the high genetic variability on this hybrid population. Higher pregnancy rates were observed in heterozygous heifers (67% and 66% for LHR and FSHR genes, respectively), however, no effect of RFLP polymorphism for LHR (P=0.9188) and FSHR (P=0.8831) on pregnancy rate was observed. The heritabily estimates for PP were 0.21 and 0.41, using the animal and sire model, respectively. The small magnitude of the (co)variance components estimates related to random effects of LHR and FSHR, showed their small contribution in the proportion of total variance of pregnancy, indicating a polygenic inheritance for this trait. The results found in this work indicate that selection of beef heifers in animal genetic breeding programs for sexual precocity based on the inclusion of genotype information on RFLP for LHR e FSHR markers is not recommended. The investigation of other important genes related to puberty onset in heifers is necessary. However, the evaluated markers were informative and may be indicated for genetic characterization studies in other bovine populations. The high heterozigosity observed on the studied composite population maximizes the exploration of these animals in crossbreeding

<p>An up-regulation of gene expression of the gonadotropin receptors FSHR and LHR, and a down-regulation of anti-Müllerian hormone (AMH), in the gonads of early maturing male Atlantic salmon parr (<em>Salmo salar</em>) have been shown to take place during the process of sexual maturation. It has however not been determined if this happens prior to or after the onset of spermatogenesis. The aim of this study was to see if such an up-regulation of the gene expression of FSHR and LHR mRNA, and down-regulation of AMH in the gonad tissue could be seen prior to the onset of spermatogenesis in early maturing salmon. For this study gonad tissues were sampled before and after the onset of spermatogenesis. Small pieces of testis tissue were removed using surgery from individually tagged one year old male Atlantic salmon parr in April prior to the onset of spermatogenesis. The same salmon were sampled again in July and maturing and non-maturing fish could be distinguished by differences in GSI. Each tissue sample from April could be matched with the July sample from the same individual by pit-tag numbers. This made it possible to separate the April samples into a maturing and non-maturing group. Gene expression levels were analysed using real-time PCR. The findings of this study showed that all three genes were expressed in the gonads in April but no significant difference in expression levels between maturing and non-maturing salmon was seen for any of the genes, which indicate that no up- or down-regulations had taken place in early maturing fish at this time. In July however, total FSHR and LHR expression levels/testis were significantly higher in maturing salmon which is in accordance with previous studies. AMH expression levels/unit RNA in July were found to be on average 25 times higher in the non-maturing group. A 100-fold drop in AMH from April through July was seen in the maturing fish, while only a 4-fold drop was seen in the non-maturing group which may indicate that a down-regulation of AMH expression took place as spermatogenesis was initiated in the maturing males.</p> / <p>En uppreglering av genuttrycket av gonadotropinreceptorerna (FSHR och LHR) samt en nedreglering av anti-Müllerian hormon (AMH) har observerats i gonaderna under spermatogenesen hos tidigt mognande atlantlaxhanar (<em>Salmo salar</em>). Man har dock inte kunnat visa huruvida detta sker före eller efter spermatogenesen inletts. Syftet med denna studie var att undersöka om en uppreglering av FSHR och LHR samt en nedreglering av AMH kunde ses hos tidigt mognande laxar redan före spermatogenesen inletts. I den här studien användes gonadprover tagna före och efter spermatogenesen inletts. Små gonadvävnadsprover togs från individuellt märkta ettåriga atlantlaxhanar i april, före spermatogenesen inletts, genom ett operativt ingrepp. I juli togs sedan gonadprover från samma individer igen och mognande och icke-mognande individer kunde nu särskiljas genom deras GSI-värden. Varje gonadprov från april kunde matchas med proverna från juli för samtliga individer genom att jämföra pit-tag nummer och möjliggjorde att även aprilproverna kunde sorteras i en mognande och en icke-mognande grupp. Nivåerna av genuttryck av mRNA analyserades med real-time PCR. Resultaten från denna studie visade att alla tre generna var uttryckta i de omogna gonaderna i april men det var ingen signifikant skillnad mellan de mognande och icke-mognande fiskarna för någon av generna, vilket betyder att det ännu inte skett någon upp- eller nedreglering vid denna tidpunkt hos de tidigt mognande fiskarna. I juli var dock de totala FSHR och LHR mRNA nivåerna/testikel signifikant högre hos mognande fiskar vilket överrensstämmer med tidigare studier. AMH mRNA/enhet RNA var uttryckt 25 gånger högre i de omogna gonaderna jämfört med de mognande. Mellan april och juli föll AMH nivåerna hos mognande fiskar nästan 100 gånger, medan de hos de omogna minskade endast 4 gånger vilket kan indikera att en nedreglering av genuttrycket för AMH skett i de individer där spermatogenesen inletts.</p>

An up-regulation of gene expression of the gonadotropin receptors FSHR and LHR, and a down-regulation of anti-Müllerian hormone (AMH), in the gonads of early maturing male Atlantic salmon parr (Salmo salar) have been shown to take place during the process of sexual maturation. It has however not been determined if this happens prior to or after the onset of spermatogenesis. The aim of this study was to see if such an up-regulation of the gene expression of FSHR and LHR mRNA, and down-regulation of AMH in the gonad tissue could be seen prior to the onset of spermatogenesis in early maturing salmon. For this study gonad tissues were sampled before and after the onset of spermatogenesis. Small pieces of testis tissue were removed using surgery from individually tagged one year old male Atlantic salmon parr in April prior to the onset of spermatogenesis. The same salmon were sampled again in July and maturing and non-maturing fish could be distinguished by differences in GSI. Each tissue sample from April could be matched with the July sample from the same individual by pit-tag numbers. This made it possible to separate the April samples into a maturing and non-maturing group. Gene expression levels were analysed using real-time PCR. The findings of this study showed that all three genes were expressed in the gonads in April but no significant difference in expression levels between maturing and non-maturing salmon was seen for any of the genes, which indicate that no up- or down-regulations had taken place in early maturing fish at this time. In July however, total FSHR and LHR expression levels/testis were significantly higher in maturing salmon which is in accordance with previous studies. AMH expression levels/unit RNA in July were found to be on average 25 times higher in the non-maturing group. A 100-fold drop in AMH from April through July was seen in the maturing fish, while only a 4-fold drop was seen in the non-maturing group which may indicate that a down-regulation of AMH expression took place as spermatogenesis was initiated in the maturing males. / En uppreglering av genuttrycket av gonadotropinreceptorerna (FSHR och LHR) samt en nedreglering av anti-Müllerian hormon (AMH) har observerats i gonaderna under spermatogenesen hos tidigt mognande atlantlaxhanar (Salmo salar). Man har dock inte kunnat visa huruvida detta sker före eller efter spermatogenesen inletts. Syftet med denna studie var att undersöka om en uppreglering av FSHR och LHR samt en nedreglering av AMH kunde ses hos tidigt mognande laxar redan före spermatogenesen inletts. I den här studien användes gonadprover tagna före och efter spermatogenesen inletts. Små gonadvävnadsprover togs från individuellt märkta ettåriga atlantlaxhanar i april, före spermatogenesen inletts, genom ett operativt ingrepp. I juli togs sedan gonadprover från samma individer igen och mognande och icke-mognande individer kunde nu särskiljas genom deras GSI-värden. Varje gonadprov från april kunde matchas med proverna från juli för samtliga individer genom att jämföra pit-tag nummer och möjliggjorde att även aprilproverna kunde sorteras i en mognande och en icke-mognande grupp. Nivåerna av genuttryck av mRNA analyserades med real-time PCR. Resultaten från denna studie visade att alla tre generna var uttryckta i de omogna gonaderna i april men det var ingen signifikant skillnad mellan de mognande och icke-mognande fiskarna för någon av generna, vilket betyder att det ännu inte skett någon upp- eller nedreglering vid denna tidpunkt hos de tidigt mognande fiskarna. I juli var dock de totala FSHR och LHR mRNA nivåerna/testikel signifikant högre hos mognande fiskar vilket överrensstämmer med tidigare studier. AMH mRNA/enhet RNA var uttryckt 25 gånger högre i de omogna gonaderna jämfört med de mognande. Mellan april och juli föll AMH nivåerna hos mognande fiskar nästan 100 gånger, medan de hos de omogna minskade endast 4 gånger vilket kan indikera att en nedreglering av genuttrycket för AMH skett i de individer där spermatogenesen inletts.

Com o objetivo de semi-quantificar a expressão gênica para receptores de gonadotrofinas nas células de folículos dominantes em animais pré-púberes (bezerras) das raças Nelore e Marchigiana e novilhas púberes da raça Nelore, buscando diferenças que possibilitem a procura de respostas para a deficiência da competência de desenvolvimento dos oócitos de animais pré-púberes que permitam a utilização destes em programas de melhoramento genético animal, foram utilizados 8 animais, sendo três bezerras Nelore, três bezerras Marchigiana, com 7 meses de idade, e duas novilhas Nelore, com 25 meses de idade. Foi realizado acompanhamento da onda folicular através de ultra-sonografia por dois meses, e os animais foram abatidos no momento que o folículo dominante apresentava diâmetro máximo (8, 10 e 14 mm, respectivamente). Folículos dominantes foram dissecados e isolados, e foi feita extração de RNA total. Foram feitas quatro reações de síntese de cDNA a partir de cada amostra. Para análise da expressão gênica semi-quantitativa, foram realizadas reações de Duplex-PCR, utilizando-se dois oligonucleotídeos iniciadores em cada reação, um para gene controle, o gapdh, e outro para o gene alvo, receptor do hormônio folículo estimulante ou receptor do hormônio luteinizante. Estas reações foram realizadas em triplicata a partir de cada fonte de cDNA de cada amostra, e os produtos foram aplicados em gel de poliacrilamida, e fotografados. Os resultados foram obtidos a partir da razão entre a intensidade da banda do gene alvo e a intensidade da banda do gene controle, e os valores médios foram analisados estatisticamente. Foi encontrada menor expressão relativa do gene do receptor de LH em bezerras da raça Nelore quando comparadas com as novilhas da raça Nelore e as bezerras da raça Marchigiana. Foi detectada a presença de um splicing alternativo para o gene do LHr, que apresentou comportamento semelhante ao gene padrão. Não foi encontrada diferença significativa na expressão do gene do FSHr entre os grupos estudados. Os resultados sugerem uma maior importância do LH nos estágios finais de desenvolvimento folicular, demonstrado aqui pela menor expressão relativa de lhr nos folículos dominantes de bezerras Nelore em relação às novilhas púberes, que pode ser um dos motivos para o baixo potencial de desenvolvimento dos oócitos destes animais após a fertilização in vitro / With the objective to semiquantify the gene expression for gonadotrophins receptors in dominant follicles in Nelore and Marchigiana prepubertal heifers and Nelore adult cows, being searched differences that make possible the search of answers for the low developmental potential of oocytes from prepubertal heifers that allow the use of these in programs of animal genetic improvement, had been used 8 animals, being three Nelore heifers, three Marchigiana heifers, with 7 months of age, and two Nelore heifers, with 25 months of age. Accompaniment of the follicular wave was carried through daily ultrasonografic exams for two months, and the animals had been slaughtered at the moment that dominant follicle presented maximum diameter (8, 10 and 14 mm, respectively). Dominant follicles had been dissected and isolated, and extraction of total RNA was performed. Four reactions of synthesis of cDNA from each sample had been made. For analysis of the semi-quantitative gene expression, reactions of Duplex-PCR had been carried through, using themselves two initiating oligonucleotídeos in each reaction, one for housekeeping gene, gapdh, and another one for the target gene, fshr and lhr. These reactions had been carried through in third copy from each source of cDNA of each sample, and the products had been applied in poliacrilamida gel electrophoresis, and photographed. The results had been gotten from the reason enter the intensity of the band of the target gene and the intensity of the band of the housekeeping gene, and the average values had been analyzed in a statistical software. Lower relative expression of lhr in Nelore heifers was found when compared with the Nelore adult cows and the Marchigiana heifers. An alternative splicing for lhr was detected, that presented similar behavior to the gene standard. Significant difference in the expression of the gene of the FSHr between the studied groups was not found. The results suggest a importance of the LH in the final periods of follicular development, demonstrated for the lower relative expression of lhr in the dominant follicles of Nelore heifers in relation to the adult cows in this study, that can after be the one of the reasons for the low developmental capacity of oocytes of these animals after in vitro fertilization

Com o objetivo de semi-quantificar a expressão gênica para receptores de gonadotrofinas nas células de folículos dominantes em animais pré-púberes (bezerras) das raças Nelore e Marchigiana e novilhas púberes da raça Nelore, buscando diferenças que possibilitem a procura de respostas para a deficiência da competência de desenvolvimento dos oócitos de animais pré-púberes que permitam a utilização destes em programas de melhoramento genético animal, foram utilizados 8 animais, sendo três bezerras Nelore, três bezerras Marchigiana, com 7 meses de idade, e duas novilhas Nelore, com 25 meses de idade. Foi realizado acompanhamento da onda folicular através de ultra-sonografia por dois meses, e os animais foram abatidos no momento que o folículo dominante apresentava diâmetro máximo (8, 10 e 14 mm, respectivamente). Folículos dominantes foram dissecados e isolados, e foi feita extração de RNA total. Foram feitas quatro reações de síntese de cDNA a partir de cada amostra. Para análise da expressão gênica semi-quantitativa, foram realizadas reações de Duplex-PCR, utilizando-se dois oligonucleotídeos iniciadores em cada reação, um para gene controle, o gapdh, e outro para o gene alvo, receptor do hormônio folículo estimulante ou receptor do hormônio luteinizante. Estas reações foram realizadas em triplicata a partir de cada fonte de cDNA de cada amostra, e os produtos foram aplicados em gel de poliacrilamida, e fotografados. Os resultados foram obtidos a partir da razão entre a intensidade da banda do gene alvo e a intensidade da banda do gene controle, e os valores médios foram analisados estatisticamente. Foi encontrada menor expressão relativa do gene do receptor de LH em bezerras da raça Nelore quando comparadas com as novilhas da raça Nelore e as bezerras da raça Marchigiana. Foi detectada a presença de um splicing alternativo para o gene do LHr, que apresentou comportamento semelhante ao gene padrão. Não foi encontrada diferença significativa na expressão do gene do FSHr entre os grupos estudados. Os resultados sugerem uma maior importância do LH nos estágios finais de desenvolvimento folicular, demonstrado aqui pela menor expressão relativa de lhr nos folículos dominantes de bezerras Nelore em relação às novilhas púberes, que pode ser um dos motivos para o baixo potencial de desenvolvimento dos oócitos destes animais após a fertilização in vitro / With the objective to semiquantify the gene expression for gonadotrophins receptors in dominant follicles in Nelore and Marchigiana prepubertal heifers and Nelore adult cows, being searched differences that make possible the search of answers for the low developmental potential of oocytes from prepubertal heifers that allow the use of these in programs of animal genetic improvement, had been used 8 animals, being three Nelore heifers, three Marchigiana heifers, with 7 months of age, and two Nelore heifers, with 25 months of age. Accompaniment of the follicular wave was carried through daily ultrasonografic exams for two months, and the animals had been slaughtered at the moment that dominant follicle presented maximum diameter (8, 10 and 14 mm, respectively). Dominant follicles had been dissected and isolated, and extraction of total RNA was performed. Four reactions of synthesis of cDNA from each sample had been made. For analysis of the semi-quantitative gene expression, reactions of Duplex-PCR had been carried through, using themselves two initiating oligonucleotídeos in each reaction, one for housekeeping gene, gapdh, and another one for the target gene, fshr and lhr. These reactions had been carried through in third copy from each source of cDNA of each sample, and the products had been applied in poliacrilamida gel electrophoresis, and photographed. The results had been gotten from the reason enter the intensity of the band of the target gene and the intensity of the band of the housekeeping gene, and the average values had been analyzed in a statistical software. Lower relative expression of lhr in Nelore heifers was found when compared with the Nelore adult cows and the Marchigiana heifers. An alternative splicing for lhr was detected, that presented similar behavior to the gene standard. Significant difference in the expression of the gene of the FSHr between the studied groups was not found. The results suggest a importance of the LH in the final periods of follicular development, demonstrated for the lower relative expression of lhr in the dominant follicles of Nelore heifers in relation to the adult cows in this study, that can after be the one of the reasons for the low developmental capacity of oocytes of these animals after in vitro fertilization

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-02-17T22:37:29Z No. of bitstreams: 1 GabrielaLiberalinoLima_TESE.pdf: 6308180 bytes, checksum: b658a30fc2afc3a608b4b5b454c6d36a (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-02-19T22:18:54Z (GMT) No. of bitstreams: 1 GabrielaLiberalinoLima_TESE.pdf: 6308180 bytes, checksum: b658a30fc2afc3a608b4b5b454c6d36a (MD5) / Made available in DSpace on 2016-02-19T22:18:54Z (GMT). No. of bitstreams: 1 GabrielaLiberalinoLima_TESE.pdf: 6308180 bytes, checksum: b658a30fc2afc3a608b4b5b454c6d36a (MD5) Previous issue date: 2015-02-27 / O objetivo da presente tese foi utilizar a manipula??o de o?citos inclusos em fol?culos ovarianos pr? antrais (MOIFOPA) como ferramenta para o resgate e otimiza??o do uso de gametas femininos oriundos de esp?cies silvestres do bioma Caatinga. A tese foi dividida em quatro experimentos. No primeiro, foi realizada a estimativa e descri??o das caracter?sticas histol?gicas e ultraestruturas dos fol?culos pr? antrais (FOPA) de cutias (Dasyprocta leporina), nos quais foram estimados 4419.8 ? 532.26 e 5397.52 ? 574.91 fol?culos para os ov?rios direito e esquerdo, respectivamente, sendo a maioria (86,63%) pertencente a categoria de fol?culos primordiais (P<0,05). A maior parte da popula??o consiste de fol?culos morfologicamente normais (70,78%), apresentando n?cleo oocit?rio grande e central, com citoplasma uniforme. Na avalia??o ultraestrutural verificou-se a presen?a de um grande n?mero de mitoc?ndrias arredondadas e gotas de lip?dios. No segundo experimento, foi realizada a estimativa e a descri??o das caracter?sticas dos FOPA de pre?s (Galea spixii), bem como avalia??o do efeito da vitrifica??o em superf?cie s?lida (VSS) sobre a morfologia de FOPA in situ. O total de 416,0 ? 342,8 FOPA foi estimado por par de ov?rios e a presen?a de uma grande quantidade de fol?culos prim?rios foi evidenciada (P<0,05). A maior parte dos FOPA apresentou-se morfologicamente normal (94,6%), possuindo n?cleo oocit?rio contendo gr?nulos condensados de heterocromatina. Mitoc?ndrias com formato arredondado ou alongado representaram as organelas mais abundantes. Quanto a VSS, o protocolo utilizando o dimetilsulf?xido (DMSO) 3M possibilitou a preserva??o de 69,5% de FOPA morfologicamente normais, sendo evidenciado atrav?s da an?lise por microscopia de luz e eletr?nica de transmiss?o. No terceiro experimento, foi realizada a avalia??o do efeito da VSS sobre a morfologia e viabilidade de FOPA in situ de catetos (Pecari tajacu). N?o foram observadas diferen?as entre os tratamentos, onde o uso dos crioprotetores DMSO, etilenoglicol (EG) e dimetilformamida (DMF), independente da concentra??o utilizada (3 ou 6 M) promoveu a preserva??o da morfologia de mais de 70% dos FOPA. Quanto a viabilidade, os crioprotetores DMSO e EG, demonstraram melhor manuten??o da mesma. O quarto experimento teve por objetivo avaliar o efeito do ? MEM+ ou TCM199 associados ou n?o a 50 ng de FSHr sobre a morfologia, ativa??o e crescimento de FOPA de catetos, cultivados in vitro (CIV) durante 1 ou 7 dias e o efeito sobre a matriz extracelular (MEC). Ap?s 7 dias de CIV apenas o TCM199/FSH manteve a propor??o de FOPA intactos similar ao dia 1 (63,2%), contudo nenhuma diferen?a foi observada entre os tratamentos (P>0,05). Adicionalmente, um aumento na propor??o de FOPA em desenvolvimento foi verificada (P>0.05). Atrav?s da an?lise com Ag-NOR, observou-se que apenas o tratamento com TCM199/FSH manteve a propor??o de prolifera??o celular similar ao dia 1 (P>0.05). A colora??o com picrosirius red revelou que a MEC permaneceu intacta em todos os tratamentos (P>0.05). Assim, como conclus?o geral, o uso da MOIFOPA nas referidas esp?cies permitiu o conhecimento de aspectos relacionados a sua morfofisiologia reprodutiva, possibilitando tanto a conserva??o do material gen?tico destas esp?cies, com a possibilidade de forma??o de bancos de germoplasma, como a elucida??o de mecanismos relacionados a sobreviv?ncia e desenvolvimento dos FOPA in vitro. / The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the estimative and description of the agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural features, in which it was estimated 4419.8 ? 532.26 and 5397.52 ? 574.91 follicles for the right and left ovary, respectively, and the majority (86,63%) belonged to the primordial follicles category (P<0.05). Most of the population consists of morphologically normal follicles (70.78%), presenting a large and central nuclei and uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great number of round mitochondrias associated to lipid droplets. In the second experiment, it was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the in situ PF morphology. The total of 416.0 ? 342.8 PF was estimated for the ovary pair and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of the PF was morphologically normal (94.6%), in which the oocyte nuclei presented condensed granules of heterochromatin. Round or elongated shaped mitochondria constituted the most abundant organelles. In regard of the SSV, the protocol using the dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically normal PF, which was evidenced by the light and transmission electronic microscopy. At third experiment, the evaluation of the SSV procedure on the morphology and viability in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were observed among treatments, in which the use of DMSO, ethylene glycol (EG) and dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the morphology preservation of much than 70% of PF. Concerning the PF viability, the DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate the effect of ? MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology, activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however, no differences were observed among treatments (P>0.05). Also, an improvement of the proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was observed that only the treatment using TCM199/FSH promoted the maintenance of cell proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of MOEPF in the refereed species allowed the knowledge of aspects related to its reproductive morphology and physiology, enabling the germplasm conservation, with the possibility of germplasm bank formation, as the elucidation of mechanisms related to the PF survive and in vitro development.

Contexte : Le récepteur hormone folliculo-stimulante (FSHR) est exprimé par les cellules de l'endothélium vasculaire dans un large éventail de tumeurs humaines primaires. Notre but était d'évaluer l'intérêt de FSHR comme marqueur des vaisseaux sanguins tumoraux associés aux sarcomes, les sous-types moléculaires du cancer du sein et des métastases ainsi que comme biomarqueur prédictif de la réponse au traitement anti-angiogénique. Méthodes : Nous avons utilisé l'immunohistochimie comme technique de révélation. Ceci implique la production d'un anticorps monoclonal hautement spécifique anti-FSHR (produit chez la souris) et l'hybridation in situ pour détecter FSHR dans des échantillons de tissus provenant de patients atteints de sarcomes (308 patients), les sous-types moléculaires du cancer du sein (84 patients), et des métastases (203 patients). Pour évaluer FSHR comme marqueur prédictif du traitement anti-angiogénique du cancer du rein métastatique avec le sunitinib, nous avons utilisé la microscopie confocale à immunofluorescence. Nous avons également co-localiser FSHR avec le facteur von Willebrand, un marqueur des cellules endothéliales vasculaires (50 patients).RésultatsFSHR est exprimé dans les 11 sous-types de patients atteints de sarcomes ont analysés et dans 75% des tumeurs métastatiques examinées, ainsi que dans tous les sous-types moléculaires des cancers du sein. Dans cadre de l'étude du cancer du rein métastatique, le pourcentage de vaisseaux marqués FSHR était en moyenne cinq fois plus élevé pour les patients qui ont répondu au traitement par rapport au groupe stable et presque huit fois plus élevé que dans le groupe non-réponse (57%, 11% et 7 %, respectivement).ConclusionsNos résultats montrent que, en plus des cancers signalés précédemment, FSHR peut être considéré comme marqueur tumoral pour les sarcomes et les métastases. En outre, FSHR peut être utilisé, avec une sensibilité et une spécificité élevée, en tant que biomarqueur prédictif de la réponse au traitement par sunitinib des patients atteints de cancer du rein métastatique. / Background : Follicle Stimulating Hormone receptor (FSHR) is expressed by the vascular endothelium in a wide range of human primary tumors. Our purpose was to further evaluate FSHR as marker of tumor blood vessels associated with sarcomas, breast cancer molecular subtypes, and metastases as well as predictive biomarker of response to antiangiogenic treatment.MethodsWe used immunohistochemistry involving a highly specific mouse monoclonal anti-FSHR antibody and in situ hybridization to detect FSHR in tissue samples from patients with sarcomas (308 patients), breast cancer molecular subtypes (84 patients), and metastases (203 patients). To evaluate FSHR as predictive marker of antiangiogenic treatment of metastatic kidney cancer with sunitinib, we used immunofluorescence confocal microscopy to co-localize FSHR with von Willebrand factor, a marker of vascular endothelial cells (50 patients).ResultsFSHR is expressed in all 11 subtypes of sarcoma patients analysed, in 75% of metastatic tumors examined as well as in all different molecular subtypes of breast cancers. In metastatic kidney cancer patients the percentage of FSHR stained vessels was on average fivefold higher for the patients who responded to the treatment in comparison with the stable group and almost eightfold higher than in the non-responsive group (57%, 11%, and 7%, respectively).ConclusionsOur results suggest that, in addition to the cancers previously reported, FSHR can be considered as tumor marker for sarcomas and metastasis. Moreover, FSHR can be used, with high sensitivity and specificity, as predictive biomarker for the response to sunitinib treatment of patients with metastatic kidney cancer.

Introdução: Considerando a prevalência, a importância social dos tratamentos de alta complexidade de mulheres inférteis e o contexto atual da literatura que atribui relevância aos polimorfismos dos genes reguladores do desenvolvimento inicial e crescimento folicular, entre eles o FSHR, LH, LHR, AMH, AMHR, BMP15 e GDF9; torna-se imprescindível o melhor conhecimento e a quantificação desses fatores. Com isso poderemos individualizar e abordar de forma mais racional a investigação e o tratamento destas mulheres submetidas à fertilização in vitro (FIV). Objetivos: Avaliar se os polimorfismos dos genes do LH (Trp8Arg e Ile15Thr), AMH (Ile49Ser), BMP15 (673C/T, 9C/G, IVSI+905A/G), GDF9 (546G>A, 398C>G, 447C>T e 646G>A), e dos receptores do FSH (Ser680Asn), do LH (18isnILQ) e do AMH (Ile49Ser), estão relacionados a diferentes desfechos reprodutivos em pacientes submetidas à fertilização in vitro. Métodos: Realizamos dois estudos em mulheres submetidas à indução ovulatória para FIV: (1) um estudo caso-controle entre pacientes normo respondedoras e má respondedoras, (2) um estudo transversal em pacientes jovens submetidas à indução ovulatória para fertilização in vitro (FIV). Foi extraído DNA das pacientes submetidas à indução ovulatória para FIV a partir do sangue periférico para realização de polymerase chain reaction, com o objetivo de detectar os polimorfismos dos referidos genes e as respectivas relações com os resultados obtidos na estimulação ovariana, no Laboratório de Terapia Gênica do HCPA/UFRGS. Resultados: Foi evidenciado que a presença do polimorfismo 398C>G no gene GDF9 está associada à má resposta em pacientes inférteis submetidas à estimulação ovariana para fertilização in vitro (68% em má respondedoras versus 23% normo respondedoras, OR: 4.01, 95% IC:1.52-10.60). Além disso, o genótipo mutante para o polimorfismo G447C>T no gene do GDF9 foi encontrado em 50% nas pacientes má respondedoras versus 19% nas pacientes normo respondedoras (OR: 2.88, 95% IC:1.19-6.04), evidenciando uma forte associação destes polimorfismos com a má resposta ovariana à estimulação. Encontramos, também, que as mulheres portadoras do alelo mutante do gene 447C>T do GDF9 tiveram um número menor de folículos entre 12-14 mm no dia do hCG (1,62 versus 2,46, P = 0,007). As mulheres com o alelo mutante do gene do GDF9 398C>G tiveram um menor número de folículos maiores que 17 mm no dia do hCG (4,33 versus 6,49, P = 0,001), menor número de folículos entre 12 e 14 milímetros no dia do hCG (1,42 versus 2,25, P= 0,017), um menor número de folículos no dia do hCG (7,33 versus 10,11 versus, P = 0,007), e redução total de oócitos MII coletados (5,38 versus 8,84 P = 0,017). Conclusão: Concluímos que polimorfismos no gene do GDF9 têm uma influência significativa no desenvolvimento do oócito, uma vez que a presença dos alelos mutantes 447C>T e 398C>G diminui o número total de folículos maduros e o número total de oócitos coletados de tais pacientes, além deste último estar associado à má resposta ovariana em pacientes submetidas à indução da ovulação para fertilização in vitro. Isso mostra que este membro da família TGFβ além de atuar nas fases iniciais da foliculogênese também tem influência importante sobre a fase final do desenvolvimento do oócito. / Introduction: Given the prevalence, the social importance of high complexity treatments of infertile women and the current context of the literature assigns relevance to polymorphisms of genes regulating early follicle growth and development, including LH, AMH, BMP15, GDF9, FSHR, LHR and AMHR; become essential to better understanding and quantification of these factors. With this we can individualize and address more rationally research and treatment of these women undergoing IVF. Objectives: Evaluate the relationship of polymorphisms of LH (Trp8Arg andIle15Thr), AMH (Ile49Ser), BMP15 ( 673C/T, 9C/ G, IVSI+905A/ G) and GDF9 (546G>A, 398C>G, 447C>Tand646G>A) genes, and FSH (Ser680Asn), LH (18isnILQ) and AMH (Ile49Ser) receptors genes, related to different reproductive outcomes in patients undergoing IVF. Methods: Our study consisted of two phases: the first conducted a case-control study among patients with normal responders and poor responders, and the second a cross-sectional study in young patients undergoing ovulation induction for in vitro fertilization. DNA was extracted from peripheral blood for performing polymerase chain reaction (PCR) and analyzed at the Laboratory of Gene Therapy HCPA/UFRGS, with the objective of detecting polymorphisms of these genes and their relationships to the results obtained in ovarian stimulation. Results: It was shown that the presence of polymorphism 398C>G in GDF9 gene is associated with poor response in infertile patients undergoing controlled ovarian stimulation for in vitro fertilization (68% in poor responders versus 23% in normal responders). Furthermore, the genotype GDF9 447C>T mutant polymorphism was found in 50% and 19%, respectively, in poor and normal responders patients, showing a strong association with this polymorphism and a poor response in ovarian stimulation. Women carrying the GDF9 398C>G mutant allele had a smaller number of follicles between 12-14 mm on the day of r-hCG (1.62 vs. 2.46, respectively P=0.007). Women with GDF9 398C>G mutant allele had a smaller number of follicles larger than 17 mm on the r-hCG day (4.33 vs. 6.49, P=0.001), a smaller number of follicles between 12 and 14mm on the r-hCG day (1.42 vs. 2.25, P=0.017), a smaller number of follicles on the r-hCG day (7.33 vs. 10.11, P=0,007), and a reduced overall number of MII oocytes collected (5.38 vs. 8.84 ,P=0.017). Conclusion: We conclude that GDF9 polymorphisms in the gene have a significant influence on the development of the oocytes, since the presence of the mutant alleles 447C>T and 398C>G decreases the total number of mature follicles, total number of oocytes collected, and are associated a poor ovarian response in patients undergoing ovulation induction for in vitro fertilization. This shows that this member of the TGFβ family besides acting in the early stages of folliculogenesis also has important influence on the final stage of oocytes development.

The glycoprotein hormones, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) are heterodimeric proteins with an identical α-subunit associated noncovalently with the hormone specific β-subunit and play important roles in reproduction and overall physiology of the organism (Pierce & Parsons, 1981). The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have a large extracellular domain (ECD)comprising of 9-10 leucine rich repeats (LRR) followed by a flexible hinge region, a seven helical transmembrane domain (TMD) and a C terminal cytoplasmic tail (Vassart et al, 2004). Despite significant sequence and structural homologies observed between the ECDs of the receptors and the specific β-subunits of the hormones, the hormone-receptor pairs exhibit exquisite specificity with very low cross-reactivity with other members of the family. Several biochemical, immunological and molecular biological tools have been employed to elucidate the structure– function relationship of the hormones and their receptors. These studies also helped in deciphering some of the regions present in both the hormones and the receptors involved in maintaining the specificity of their interaction (Fan & Hendrickson, 2005b; Fox et al, 2001; Wu et al, 1994). However, the complete understanding of the hormone-receptor contact sites and mechanism of receptor activation are still an enigma. Understanding the molecular details of these phenomena can lead to the development of novel strategies of regulating hormone action. Binding of FSH to FSHR occurs in the large extracellular NH2-terminal domain where the participation of the LRRs (amino acids 18-259) is essential to determine the ligand selectivity (Dias & Van Roey, 2001; Fan & Hendrickson, 2005a; Szkudlinski et al, 2002). In fact, mutations in these regions lead to reduction in binding of the agonist to the receptor. It is not known how the signal from the large extracellular domain liganded complex is transmitted to the TMD (amino acids 367-695). It is envisioned that hormone binding to the LRRs leads to series of conformational changes leading activation of the TMD resulting in signal transduction. The recently reported crystal structure of the single chain form of FSH in complex with the leucine rich repeats of the FSHR (amino acids 1-268) (Fan & Hendrickson, 2005b), although provides detailed understanding of the molecular interactions of the LRRs with the hormone, fails to provide any insights into mechanism of receptor activation as the information regarding critical interaction of the hormone with TMD. This structure also did not provide any information on the role of the hinge region (amino acids 259-366) that connects the LRRs to the TMD in hormone binding and activation of the receptor. In the present study an attempt has been made to understand the role of the hinge region in hormone binding and signal transduction. The overall objective of the study is to elucidate the molecular details of the hormone receptor interactions, particularly FSH-FSHR interaction. Antibodies to glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone-receptor interactions and signal transduction. While the TSH receptor antibodies and their effects on the overall physiology have been well documented (Khoo & Bahn, 2007; Rapoport & McLachlan, 2007), reports of such antibodies against FSHR or LHR and their possible effects on the reproductive functions are not available. In the present study, effects of FSHR antibodies with different specificities on FSH-FSHR interactions have been investigated. Antibodies to different regions of rat FSHR, were raised and extensively characterized and their effects of FSH-FSHR interactions and signaling were investigated. It was found that a polyclonal antibody against the hinge of the receptor (RF2 antiserum, amino acids 218-336), while having no significant effect on hormone binding and response could stimulate the receptor by itself bypassing the hormone. This stimulation of FSHR was very specific as this antiserum could not stimulate LHR or TSHR and could be blocked by preincubating the antibody with the antigen. Through competition experiments with different synthetic peptides of human FSHR, a stretch of hinge region corresponding to amino acids 296-331 was identified as the site recognized by the stimulatory antibody. This antibody did not interfere in hormone binding and could also bind to the pre-formed hormone-receptor complex suggesting that the binding site of the antibody may not participate directly in hormone binding. Subsequently the antibody was extensively characterized for its effect of hormone receptor interactions (Chapter 2). Previous studies considered the hinge region to be an inert linker connecting the LRRs to the TMD, a structural entity without any known functional significance (Vlaeminck-Guillem et al, 2002). However, the data with RF2 antibody suggested a direct role of the hinge region in signal transduction. Therefore, a systematic study to dissect the role to hinge region in hormone binding and signal transduction was conducted. Several truncations, deletions, activating and inactivating point mutations in the FSHR were generated to understand the mechanism of receptor activation. Firstly, these mutant receptors were characterized for their ability to translocate to the cell surface when transfected in the cultured mammalian cells. Secondly, affinity of all the mutant receptors for the hormone was determined in order to understand the effect of mutations on hormone binding. Finally, the cAMP response of these mutant receptors to the hormone and the stimulatory antibody was investigated to understand the effects of mutations on signal transduction. The results are described in Chapter 3. The hormone binding analysis and the affinity measurement of the mutant receptors showed that the LRRs are involved in high affinity hormone binding while the hinge region may not contribute to the process. This is in agreement with the crystal structure data which showed that the hormone was bound to the truncated receptor fragment representing only the LRRs (Fan & Hendrickson, 2005b). These binding data also corroborated the earlier data indicating that the antibodies against the hinge region do not interfere in hormone-receptor interactions. Further, the analysis of different N-terminally truncated receptor mutants provided strong evidence indicating that the constraining intramolecular interactions between the extracellular and the transmembrane domains are required to maintain the FSHR in an inactive conformation in the absence of an agonist. The analysis of the constitutive basal activity of the mutant receptors in absence of hormone suggested that certain regions of the extracellular domain had an attenuating effect over the TMDs that prevented constitutive activation of the receptor. This was demonstrated by a marked increase in the basal constitutive activity of the receptor upon the complete removal of its extracellular domain. Detailed analysis of the mutants suggested that LRR portion does not contribute to this attenuating effect, but it is the hinge region that perhaps interacts with the TMDs and dampens its basal constitutive activity. This attenuating effect was further narrowed down to a small stretch of 35 amino acids (296-331) within the hinge region. It was striking that the similar stretch was identified as the binding site of the stimulatory receptor antibody. In pharmacology, an ‘inverse agonist’ is an agent which binds to the receptor and reverses the constitutive activity of receptors. Thus the hinge region of the receptor could be termed as a ‘tethered inverse agonist’ of the TMD, since it is covalently associated with the TMD and their interactions dampen the basal constitutive activity of the receptor. However, careful comparison of the activities of the mutants (receptors harboring deletions and gain-of-function mutations) with maximally stimulated wild-type FSHR indicated that these mutations of the receptor resulted only in partial activation of the serpentine domain suggesting that only the ECD in complex with the hormone is the full agonist of the receptor. Moreover, the hinge region stabilizes the TMD in an inactive conformation and the activating mutations disengage the inhibitory ECD–TMD interactions bringing about partial activation of the receptor. Most interestingly, the deletion of amino acids 296-331 from hFSHR resulted in no further response to the hormone indicating that this part of the receptor is also critical for hormonal activation, perhaps playing a dual role in the attenuation of the basal activity and a direct involvement in the hormonal activation of the receptor. Progressive sequential deletions of ten amino acids from 290 to 329 yielded similar results (high basal cAMP production with concomitant loss of hormone and antibody response) clearly demonstrating that the integrity of this region is absolutely essential for hormonal activation. In conclusion, the study provides a conclusive evidence to show that the hinge region of FSHR, although not involved in primary high affinity hormone binding, plays a critical role in the modulation of the receptor activity in absence, as well as, presence of the hormone. A large array of reproductive abnormalities is associated with malfunctioning of FSHR. To explore the possibility of using the stimulatory antibodies for therapeutic purpose, three inactivating mutations of hFSHR were analyzed. In corroboration with the earlier reports (Doherty et al, 2002; Touraine et al, 1999), the mutants A419T and L601V are incapable of transducing the signal, despite having adequate cell surface expression and wild type affinities for the hormone, mainly because of defective TMD. The RF2 antibody failed to elicit any response from these mutants suggesting that its ability to activate the receptor depends on the status of the TMD. Interestingly, the activating mutant D576G, which showed very high basal cAMP production, could be stimulated by both antibody and the hormone to the nearly wild type levels suggesting that in this mutant the interactions between the hinge region and TMD are similar to that of wild type and higher basal cAMP production could be due to different interactions of the TMD with the G-Proteins. Structure-function studies of glycoprotein hormones and their receptors have been hampered due to low levels of expression of the properly folded proteins in heterologous systems (Chazenbalk & Rapoport, 1995; Hong et al, 1999b; Peterson et al, 2000; Sharma & Catterall, 1995; Thomas & Segaloff, 1994). Previous studies from the laboratory have shown that the Pichiapastoris,which blends the advantages of both bacterial and mammalian expression systems, can be used to hyper-express biologically active hormones (Blanchard et al, 2008; Gadkari et al, 2003; Samaddar et al, 1997). In addition, the same expression system has been used to produce single chain hormone analogs (Roy et al, 2007; Setlur & Dighe, 2007). Further, methodologies for Pichiafermentation and purification of recombinant hormones from the fermentation media have been wellestablished in the laboratory. Chapter 4 describes the work carried out to express, purify and characterize a fully functional hFSHR extracellular domain. Thus a stage is now set to attempt structural studies with the receptor. The results are discussed at the end of each of these chapters and future directions have been discussed at the end of this thesis.