Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oocyte quality, I have compared oocytes and cumulus cells of different qualities at a molecular level. I present in this thesis the pathways and molecules that may determine the developmental competence of oocyte as well as candidate molecular markers of oocyte and embryo quality. A cDNA microarray analysis was performed, comparing the transcriptomes of rhesus monkey MII oocytes of different qualities, high quality VVM oocytes and poor quality IVM oocytes. A small set of 59 Oocyte quality plays a critical role in establishment of pregnancies, embryo development, implantation and the health of offspring. The oocyte provides maternal factors necessary for the initial development of its embryo during the period of transcriptional silence. Despite the consistent increase in number of couples seeking assisted reproductive treatments, oocyte quality still remains as an obstacle in successful fertility treatments and the mechanisms governing the quality of oocyte are poorly understood. Among various factors that may potentially affect the quality of oocyte, the acquisition of oocyte developmental competence seems to mainly occur during the final stage of oocyte maturation. The correct temporal regulation of series of molecular events and the proper exchange of signals with surrounding follicular environment during this critical period will ensure the developmental competence of oocyte and its subsequent embryo. In order to identify molecular factors affecting oo was identified as differentially expressed between the two types of oocytes. These mRNAs are involved in steroid metabolism, cell-cell interactions, cellular homeostasis, cell adhesion, mRNA stability and translation. In addition, the overexpression of several imprinted genes in IVM oocytes were detected, indicating a possible loss of correct epigenetic programming during IVM. These results indicate that normal oocyte-somatic cell interactions may be disrupted during IVM and the interruptions of these interactions during the final phase of oocyte maturation may be the prime cause of reduced developmental competence of IVM oocytes. To elucidate oocyte quality factors linked to the cumulus cell phenotype, the transcriptomes of two types of rhesus monkey cumulus cells, IVM and VVM, were compared using a cDNA microarray analysis. In contrast to a relatively small difference between IVM and VVM oocytes, a large number of genes were differentially expressed between IVM and VVM rhesus cumulus cells. Moreover, a much larger number of differential mRNA expressions were observed comparing the transitions from pre-maturation cumulus cells to the IVM and VVM cumulus cells. The results from these array comparisons indicated that the cumulus cells may fail to achieve successfully normal gene regulation during IVM and thus make a remarkable amount of changes in gene expression to compensate for the loss. Numerous genes involved in lipid metabolism are incorrectly regulated during IVM, and the synthesis of sex hormones appears not suppressed during IVM. In addition, a panel of 24 cumulus cell markers of oocyte quality was identified. Genetic effects on oocyte quality were explored by comparing transcriptomes of oocytes obtained from two different inbred mouse strains, B6 and D2, and F1 hybrid. A clustering analysis and statistical tests showed that the transcriptome of F1 oocytes is more similar to the B6 transcriptome than to the D2 at both GV and MII stages. Also, comparison analyses of GV stage oocyte transcriptomes with MII oocyte transcriptomes from three different mouse strains indicated that the number of overdominance genes at the MII stage is bigger than the number of overdominance genes at the GV stage. In order to investigate how the genes gain the overdominance during the GV to MII transition, overdominance genes were categorized according to their mRNA expression patterns at GV and MII stages. The results showed that more than 80% of overdominance genes belong to one of the four major transition groups. The further evaluation of changes in array intensities from GV to MII stage transition revealed that F1 oocytes and inbred strain oocytes differentially regulate the mRNA abundance during oocyte maturation and that the differential regulation of mRNA abundance by the F1 genotype is responsible for the increase of the number of overdominance genes during maturation from GV stage to MII stage. A mRNA sequence analysis indicated that the gain of overdominant low in F1 mRNA expression pattern during maturation may be regulated by 3'UTR motif elements. The number of dominance genes also increase during GV to MII transition. At both GV and MII stages, there are more genes with B6 dominant mRNA expression pattern than those with D2 dominance pattern. Lipid metabolism, small molecule biochemistry and cell death are biofunctions overrepresented in both dominance and overdominance genes. In addition, `blebbing' was identified as a biofunction significantly downregulated in the F1 and B6 MII eggs, indicating that the cellular function may be involved in oocyte maturation. / Molecular Biology and Genetics / Accompanied by one .pdf file: YLEE_SupplementalTables.pdf

Made available in DSpace on 2016-01-26T12:51:34Z (GMT). No. of bitstreams: 1 wagnervicensoto_dissert_parte1.pdf: 574820 bytes, checksum: 52b81a5827b47b0dd96036bb137bd216 (MD5) Previous issue date: 2004-06-01 / The oocyte donation and reception program is a technique in which female gametes from a woman (donor) are donated to other woman (recipient) in order to be fertilized with the respective recipient s husband spermatozoa. The present study analyzed fourteen patients who underwent 21 cycles of egg reception by this assisted reproductive medical technique at the Reproductive Medicine Institute (RMI) of São José do Rio Preto-SP, in the period from January 1998 to December 2002. The recipients age ranged between 29 to 49 years; the mean age 40 years. Ten patients (71.5%) did not report previous gestation, therefore considered women with primary infertility. In order to perform the indications to the oocyte reception we classified five patients (35.7%) as having premature menopause, five (35.7%) as ovarian failure, three (21.5%) as physiological menopause, and one (7.1%) as unsuccessful responder to previous treatments. Only six patients (42.9%) had not undergone previous infertility treatments. In 92.9% of the patients, the assisted reproductive technique used was the Intracytoplasmic Sperm Injection (ICSI). The number of embryos transferred per cycle was between two and four. A total of 21 cycles were performed with a rate of 52.4% of gestation per cycle and 71.5% gestation per patient. The rate of multiple gestations was 27.3%. Regarding the gestation evolutions, it was observed a rate of 36.4% of miscarriage and 63.6% of ongoing pregnancies, in which 9.1% had premature delivery, and 54.5% had full term delivery without intercurrences. The rate of home baby was 50%. The oocyte donation and reception program showed to be a successful technique, with excellent results, providing a feasible and ethic way of getting pregnant some selected patients who have otherwise been considered as having an infertility reserved diagnostic. / O programa de doação-recepção de oócítos é técnica pela qual os gametas femininos (oócitos) de uma mulher (doadora) são doados a outra (receptora) para que sejam fertilizados com espermatozóides dos respectivos maridos. Este estudo analisou quatorze pacientes submetidas a 21 ciclos de ovorecepção por técnica de reprodução medicamente assistida no Instituto de Medicina Reprodutiva (IMR) de São José do Rio Preto-SP, no período de janeiro de 1998 a dezembro de 2002. A idade das pacientes receptoras variou de 29 a 49 anos com média de 40 anos. Dez (71,5%) pacientes não referiram gestação anterior, sendo consideradas como infertilidade primária. Como indicações para realização de recepção de oócitos classificamos cinco (35,7%) pacientes como menopausa precoce, cinco (35,7%) como falência ovaríana, três (21,5%) como menopausa fisiológica e uma (7,1 %) como má respondedora. Apenas seis (42,9%) pacientes não haviam sido submetidas a tratamento anterior para infertilidade. Em 92,9% das pacientes foi utilizado a injeção intra-citoplasmática de espermatozóide (lCSl) como técnica de fertilização assistida. Foram transferidos por ciclo entre dois e quatro embriões. Dos 21 ciclos realizados obteve-se taxa de 52,4% de gestação por ciclo e de 71,5% de gestação por paciente. A taxa de gestação múltipla foi de 27,3%. Em relação à evolução das gestações observou-se taxa de abortamento de 36,4% e evolução da gestações em 63,6%, com 9,1% de parto prematuro e 54,5% gestações a termo sem íntercorrêncías. A taxa de "bebê em casa" foi de 50%. O programa de doação-recepção de oócitos mostrou-se técnica de excelentes resultados, representando uma forma viável e ética de se obter gestação em pacientes selecionadas que antes tinham diagnóstico reservado de infertilidade.

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.

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.

Mammalian oocytes undergoing meiotic maturation are transcriptionally silent and gene expression is therefore regulated at the level of translation. One of the well established mechanisms employed in translational regulation of maternal mRNAs in oocytes is cytoplasmic polyadenylation. This process is generally controlled by phosphorylation and activation of cytoplasmic polyadenylation element binding protein (CPEB). The aim of this thesis is to determine the role of mitogen-activated protein kinase (MAPK) in regulation of CPEB-mediated cytoplasmic polyadenylation in maturing mouse and porcine oocytes. For this purpose, MAPK activity was inhibited using its specific inhibitor, GDC-0994 and the effect of MAPK inhibition on cyclin B1 mRNA polyadenylation was monitored. In mouse oocytes, MAPK inhibition impaired neither cyclin B1 mRNA polyadenylation nor its translation and MAPK is thus unlikely to be involved in regulation of cytoplasmic polyadenylation in this species. Based on the results of experiments performed using porcine oocytes, the possible role of MAPK in CPEB-mediated cytoplasmic polyadenylation can neither be confirmed nor ruled out. Keywords: cytoplasmic polyadenylation, mouse oocyte, porcine oocyte, mitogen-activated protein kinase (MAPK), cyclin B1, GDC-0994 inhibitor

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.

Centrosomes are cellular organelles present in most animal cells, and are formed of two main components: the centrioles and the pericentriolar material (PCM). Centrosomes perform a variety of functions: they are the main microtubule organising centre in the cell, and are important localisation hubs for kinases involved in regulating the cell cycle. Hundreds of proteins are thought to localise to centrosomes, but work in the last decade has narrowed down this list to a handful of proteins that are thought to be essential for centrosome structure and function in Drosophila. Asl, Ana2, DSas-4, DSas-6 and Sak have been identified as essential components for centriole duplication, while Cnn and DSpd-2 are thought to be important in establishing the PCM. However, the relative position of these 7 components in the pathway of centrosome assembly in Drosophila embryos remains elusive, as a genetics analysis of this process is hampered by the absence of centrioles in most mutant embryos for these proteins. In this thesis I elucidate the pathway of centrosome assembly in Drosophila by using SAPs (DSas-6/Ana2 particles that form in Drosophila unfertilised eggs upon moderate expression of DSas-6 and Ana2) as proxy models of centrosomes. I show SAPs are very similar to centrosomes in composition and dynamics but unlike centrosomes are able to form even in the absence of some of the essential centriolar components. SAP analysis in the absence of each of the main centrosome components reveals that: Sak is not required for the recruitment of downstream components; DSas-4 is necessary for Ana2 and DSas-6 to interact; Asl is the most upstream component of the PCM recruitment pathway, followed by DSpd-2; it is likely that there is an additional PCM recruitment pathway. I then take advantage of some of these results to examine how centrosome formation is potentiated after egg activation. My work allows me to propose an improved description of the pathway of centrosome formation in Drosophila.

The trade-off between gametes and soma is central to life-history evolution. Oosorption has been proposed as a mechanism that can mediate this trade-off. When conditions are not conducive to successful reproduction, females are expected to be able to recoup nutrients from unfertilized oocytes and reinvest them into the somatic processes that promote survival and hence future reproduction. Although positive correlations between oocyte degradation and lifespan have been documented in oviparous insects, the adaptive significance of this process in species with more complex reproductive biology has not been explored. Oocyte degradation via apoptosis (programmed cell death) occurs in response to enforced virginity in females of the ovoviviparous cockroach, Nauphoeta cinerea. Observed apoptosis may represent oosorption, however, an alternative but not mutually exclusive argument is that oocyte apoptosis may represent oocyte ageing and clearance in order to maintain reproductive synchrony. The aim of this thesis was to test the hypothesis that the function of oocyte apoptosis is oosorption in N. cinerea. I found that in addition to enforced virginity, starvation induces oocyte apoptosis. However, the life history outcome following one form of stress is the opposite of the other. Hence, the functional role of oocyte apoptosis appears to be different depending on whether apoptosis is induced through starvation or age. Following a period of starvation-induced apoptosis females exhibit the increase in survival and future reproduction predicted by oosorption. Whereas, following a period of age-induced apoptosis females suffer fecundity and longevity cuts. However, age-induced apoptosis does not appear to simply be cellular ageing and clearance. In conjugation with age-induced apoptosis, ovariole number declines whilst the size of surviving oocytes increases. Hence, it appears that resources from sacrificed oocytes are being recycled into the survivors, and that this reinvestment in current reproduction trade-offs with future reproductive capacity. My thesis shows the importance of studying proximal mechanisms alongside more traditional measures of life history, as the relationship between isolated biological levels is not always clear.

Le diabète insipide néphrogénique (DIN) autosomal peut être causé par les mutations du gène codant pour le canal à eau aquaporine-2 (AQP2). Un modèle couramment utilisé pour l’étude des protéines membranaires telle l’AQP2 est l’expression hétérologue dans les ovocytes de Xenopus laevis. Malheureusement, les techniques déjà existantes de purification de membranes plasmiques sont soit trop longues, trop difficiles ou demandent trop de matériel, ne permettent pas l’analyse adéquate du ciblage des formes sauvage comme mutantes, un élément crucial de ce type d’étude. Nous avons donc dans un premier temps mis au point une technique rapide et efficace de purification de membranes plasmiques qui combine la digestion partielle de la membrane vitelline, sa polymérisation à la membrane plasmique suivi de centrifugations à basse vitesse pour récolter les membranes purifiées. Nous avons utilisé cette technique dans l’étude de deux nouveaux cas familiaux de patients hétérozygotes possédant les mutations V24A et R187C dans un cas et K228E et R187C dans le second cas. Pour chaque mutation, nous avons analysé autant les éléments de fonctionnalité que les paramètres d’expression des protéines mutantes. Les expériences de perméabilité membranaire démontrent que les ovocytes exprimant AQP2-V24A (Pf = 16.3 ± 3.5 x 10-4 cm/s, 10 ng) et AQP2- K228E (Pf = 19.9 ± 7.0 x 10-4 cm/s, 10 ng) ont des activités similaires à celle exprimant la forme native (Pf = 14.4 ± 5.5 x 10-4 cm/s, 1 ng), tandis que AQP2- R187C (Pf = 2.6 ± 0.6 x 10-4 cm/s, 10 ng) ne semble avoir aucune activité comme ce qui est observé chez les ovocytes non-injectés (Pf = 2.8 ± 1.0 x 10-4 cm/s). Les études de co-expression ont démontré un effet d’additivité lorsque AQP2-V24A et -K228E sont injectées avec la forme native et un effet s’apparentant à la dominance négative lorsque AQP2-R187C est injecté avec la forme native, avec AQP2-V24A ou avec –K228E. Les résultats obtenus par immunobuvardage représente bien ce qui a été démontré précédemment, on remarque la présence des mutations K228E, V24A et la forme sauvage à la membrane plasmique, contrairement à la mutation R187C. Cependant, lorsque les mutations sont exprimées dans des cellules mIMCD-3, il n’y a qu’une faible expression à la membrane de la forme –K228E et une absence totale des formes –V24A et –R187C à la membrane plasmique, contrairement à la forme native. Les résultats de nos études démontrent que tout dépendant du système d’expression les formes –K228E et –V24A peuvent être utiles dans l’étude des problèmes d’adressage à la membrane à l’aide de chaperonne chimique. De plus, la forme –R187C démontre des difficultés d’adressage qui devront être étudiées afin de mieux comprendre la synthèse des formes natives. / The autosomal nephrogenic diabetes insipidus (NDI) is caused by mutations of the gene coding for the water channel aquaporine-2 (AQP2). An oftenly used model for the study of membrane proteins such as AQP2 is the heterogenous expression in Xenopus laevis oocytes. Unfortunately, the existing techniques of plasma membranes purification are either too long, too difficult or require too much material, which does not allow adequate analysis of targeting of the native and mutants forms, which is crucial for this type of study. We developed a fast and effective plasma membrane purification technique which combines partial digestion of the vitellin membrane, its polymerization with the plasma membrane followed by a serie of low speed centrifugations to collect the purified membranes. We used this technique to study of two new family cases of heterozygote patients carrying the V24A and R187C mutations in a case and K228E and R187C in the second case. For each mutation, we analyzed the functionality and the parameters of expression of the mutant proteins. The membrane permeability experiments show that the oocytes expressing AQP2- V24A (Pf = 16.3 ± 3.5 x 10-4 cm/s, 10 ng) and AQP2-K228E (Pf = 19.9 ± 7.0 x 10-4 cm/s, 10 ng) have similar activities to the oocytes expressing the native form (Pf = 14.4 ± 5.5 x 10-4 cm/s, 1 ng), while AQP2-R187C (Pf = 2.6 ± 0.6 x 10-4 cm/s, 10 ng) doesn’t seem to have any activity like the un-injected oocytes (Pf = 2.8 ± 1.0 x 10-4 cm/s). The coexpression studies showed an additive effect when AQP2-V24A and -K228E are injected with the native form and an effect being associated with negative dominance when AQP2-R187C was injected with AQP2-V24A, -K228E and the native form. Western blot results confirmed what was observed in the functionality studies. However, when the mutations were expressed in mIMCD-3 cells, there was a slight expression of the K228E mutation to the plasma membrane and a total absence of the mutations –V24A and R187C at the plasma membrane. The results of our studies showed that depending on the expression system the mutations –K228E and -V24A can be used in targeting studies using chemical chaperones.

Implantation requires complex molecular and cellular events involving coagulation, angiogenesis and immunological processes that need to be well regulated for a pregnancy to establish and progress normally.  The overall aim of this thesis was to study different models associated with atypical angiogenesis, impaired implantation and/or placentation, such as recurrent miscarriage (RM), oocyte donation (OD) and preeclampsia. Histidine-rich glycoprotein (HRG), a serum protein with angiogenic potential has been previously shown to have an impact on implantation and fertility.  In two retrospective case-control studies, women suffering from RM (Study I) and gestational hypertensive disorders (GHD) (Study IV) have been compared to healthy control women, regarding carriership of HRG genotypes (HRG A1042G and C633T SNP, respectively).  According to the findings of this thesis, heterozygous carriers of the HRG A1042G SNP suffer from RM more seldom than homozygous carriers (Study I).  Additionally, the presence of the HRG 633T allele was associated with increased odds of GHD (GHD IV).  Studies II and III comprised a national cohort of relatively young women with optimal health status conceiving singletons with donated oocytes versus autologous oocytes (spontaneously or via IVF).  We explored differences in various obstetric (Study II) and neonatal (Study III) outcomes from the Swedish Medical Birth Register.  Women conceiving with donated oocytes had a higher risk of GHD, induction of labor and cesarean section, as well as postpartum hemorrhage and retained placenta, when compared to autologously conceiving women.  OD infants had higher odds of prematurity and lower birthweight and length when born preterm, compared to neonates from autologous oocytes.  With regard to the indication of OD treatment, higher intervention but neverthelss favourable neonatal outcomes were observed in women with diminished ovarian reserve; the risk of GHD did not differ among OD recipients after adjustment. In conclusion, HRG genetic variation appears to contribute to placental dysfunction disorders.  HRG is potential biomarker that may contribute in the prediction of the individual susceptibility for RM and GHD.  Regarding OD in Sweden, the recipients-despite being of optimal age and health status- need careful preconceptional counselling and closer prenatal monitoring, mainly due to increased prevalence of hypertensive disorders and prematurity.