O nascimento de gêmeos na espécie humana é rodeado por muitos mistérios e, há bastante tempo, desperta a curiosidade tanto leiga quanto científica. Os estudos com gêmeos vêm contribuindo para o entendimento de diversas áreas da biologia humana; porém, aspectos epidemiológicos e etiológicos relacionados aos nascimentos gemelares em si permanecem pouco elucidados. Para além de mera curiosidade, pesquisas nessa área podem ajudar a compreender mecanismos relacionados à reprodução em geral, além de serem importantes em um contexto de saúde pública, já que a gestação gemelar representa riscos adicionais à mãe e à prole. Em países industrializados observa-se um aumento acentuado nas taxas gemelares (TGs) que parece estar relacionado ao aumento da idade materna na primeira gravidez e tecnologias de Reprodução Medicamente Assistida (RMA). No Brasil, os aspectos geográficos e sociodemográficos dos nascimentos gemelares ainda não foram profundamente estudados. Assim, este estudo objetivou responder a três questões principais relacionadas aos nascimentos de gêmeos: “Quantos?” “Onde? e “Como?”, isto é, considerando aspectos epidemiológicos e etiológicos. Para o estudo epidemiológico, desenhamos um estudo de base de dados populacional e investigamos as TGs em duas dimensões: espacial e temporal. Para isso, utilizamos dados provenientes do Sistema de Informações sobre Nascidos Vivos (SINASC) e analisamos mais de 41 milhões de nascimentos que ocorreram em todos os 5.565 municípios brasileiros entre 2001 e 2014. Encontramos uma TG nacional média de 9,41 por 1.000 nascimentos e o modelo de análise de séries temporais revelou tendência de aumento global ao longo do período estudo, mas com notáveis diferenças regionais. De fato, resultados da “Análise de Cluster e Outlier” (Anselin Local Moran’s I) revelaram concentração de municípios com altas TGs em uma área que vai do sul do Nordeste brasileiro até o Rio Grande do Sul (Global Moran Index = 0.062, P < 0.001). Além disso, encontramos correlação positiva entre o Índice de Desenvolvimento Humano (IDH) local e as TGs em diferentes cenários, sugerindo que o IDH pode ser um importante indicador de RMA no Brasil. Nossas análises também revelaram aumento de 26.42% na TG entre as mulheres com mais de 44 anos durante o período estudado. A tendência de aumento temporal encontrada para algumas regiões do país está de acordo com o que é observado em outros países industrializados, enquanto que a análise geográfica revelou duas situações bem distintas dentro do Brasil. Por sua vez, os 9 estudos acerca da etiologia dos nascimentos de gêmeos foram concentrados em investigar polimorfismos de nucleotídeo único (SNPs) em mães de Cândido Godói (CG), uma pequena cidade do Sul do Brasil conhecida como a “Cidade dos Gêmeos”. Este título é devido à alta TG observada no município e à recorrência do traço nas famílias locais, com forte descendência europeia. Nós desenhamos um estudo caso-controle e genotipamos sete SNPs relacionados à foliculogênese (rs6166:C>T em FSHR, rs11031006:G>A próximo a FSHB, e rs17293443:T>C in SMAD3) ou a gestações de sucesso (rs1801131:T>G e rs1801133:G>A em MTHFR, rs2010963:C>G em VEGFA, e rs1800629:G>A em TNF) em 44 mães de gêmeos (casos) e 102 mães de filhos únicos (controles), todas residentes de CG. Para todos os SNPs, a distribuição das frequências alélicas e genotípicas foi similar entre casos e controles. Diferentes combinações de alelos de risco e análises haplotípicas também foram homogeneamente distribuídas entre ambos os grupos. Assim, estes resultados sugerem uma ausência de associação entre os nascimentos gemelares em CG e sete SNPs relacionados à foliculogênese ou a gestações de sucesso, mas é possível que outras variantes genéticas ligadas a ambos os processos possam estar envolvidas neste fenômeno que possui uma base genética subjacente. / The birth of twins in human species is surrounded by many mysteries and it has long aroused both lay and scientific curiosity. Studies with twins have contributed to the understanding of several areas of human biology; however, the epidemiological and etiological aspects related to twin births by themselves remain unclear. Beyond mere curiosity, research in this field may help us to understand the mechanisms related to reproduction in general, and it is important in a public health context since a twin pregnancy represents additional risks to the mother and offspring. Some countries have reported a striking increase in twinning rates (TRs), which seems to be related mainly to delayed childbearing and to Medically-Assisted Reproduction (MAR) technologies. In Brazil, the epidemiological scenario of twin births has not been studied while considering its territorial and sociodemographic magnitude. Therefore, this study aimed to answer three main questions related to twin births: "How many?", “Where” and "How?", that is, we investigated twinning in light of epidemiology and etiology. For the epidemiological study, we carried out a population-based study, investigating twin births in two dimensions: spatial and temporal. For that, we used data from Brazil’s Live Birth Information System (SINASC), and we analyzed over 41 million births that occurred in all 5,565 Brazilian municipalities between 2001 and 2014. We found an average TR of 9.41 per 1,000, and a first-order autoregressive model of time-series analysis revealed a global upward trend over time, but with important regional differences. In fact, a Cluster and Outlier Analysis (Anselin Local Moran’s I) was performed and identified clusters of high TR in an area stretching from the south of Brazil’s Northeast Region to the South Region (Global Moran Index = 0.062, P < 0.001). Furthermore, we found a positive correlation between the local Human Development Index (HDI) and TRs in different scenarios, suggesting that the HDI may be an important proxy indicator of MAR in Brazil. We also found a sharp increase (26.42%) in TR in women aged over 44 years. The upward temporal trend in TRs is in line with recent observations from other countries, whereas the spatial analysis revealed two very different realities within our country. In turn, studies on the etiology of twin births were focused on single nucleotide polymorphisms (SNPs) in mothers from Cândido Godói (CG), a small southern Brazilian city known as the "Twin’s Town”. This title is due to the high TR attributed to the municipality and to the recurrence of the twin trait through the 11 local families, who have a strong European descent. We performed a case-control study and genotyped seven SNPs related to folliculogenesis (rs6166:C>T in FSHR, rs11031006:G>A near FSHB, and rs17293443:T>C in SMAD3) and to a successful pregnancy (rs1801131:T>G and rs1801133:G>A in MTHFR, rs2010963:C>G in VEGFA, and rs1800629:G>A in TNF) in 44 mothers of twins (cases) and 102 mothers of singletons (controls), all of them from CG. For all SNPs, the distributions of the genotypic and allelic frequencies were similar between the cases and controls. Different combinations of risk alleles and haplotypic analyses were also homogeneously distributed between both groups. Thus, these results suggest a lack of association between twin births in CG and seven SNPs related to folliculogenesis or successful pregnancies, but it is possible that other genetic variants linked to both processes may be involved in this phenomenon, which has an underlying genetic basis.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23570号 / 医博第4784号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 近藤 玄, 教授 滝田 順子, 教授 篠原 隆司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L’hormone anti-Müllerienne (AMH), une glycopréotéine exclusivement produite par les cellules de la granulosa (CG) des follicules ovariens de la femme, est un marqueur unique de du statut folliculaire ovarien. Contrairement à l’inhibine B, l’estradiol et la FSH, l’AMH est produite par un large éventail de follicules allant des follicules primaires aux follicules à petit antrum. Cependant, les mécanismes précis régulant la production d’AMH par les CG restent mal connus. Nous avons montré que la sélection folliculaire précoce au cours de la phase de transition lutéo-folliculaire, un phénomène fréquemment retrouvé chez les femmes ayant un vieillissement ovarien, caractérisé par la présence d’au moins un follicule surdéveloppé au cours de la phase folliculaire précoce, n’altérait pas la puissance de la relation entre le compte folliculaire antral et les concentrations sériques d’AMH. En revanche, cette situation perturbait significativement celle entre le nombre de follicules antraux et les taux sériques de FSH, d’inhibine B et d’estradiol. Nous avons par la suite mis en évidence, en utilisant un nouvel outil, nommé Follicular Output RaTe (FORT), que le pourcentage de follicules qui répondent effectivement à la FSH exogène en atteignant la maturation pré-ovulatoire, était négativement et indépendamment lié aux taux sériques d’AMH, ce qui va dans le sens de l’hypothèse d’un effet inhibiteur de l’AMH sur la sensibilité des follicules à la FSH. Ensuite, nous avons regardé si la production d’AMH par ovaire et par follicule était altérée chez les femmes n’ayant plus qu’un seul ovaire suite à une ovariecomie unilatérale. En effet, tout indique que chez ces femmes, des réarrangements majeurs de la folliculogenèse sont mis en place pour maintenir une fonction ovarienne malgré la perte brutale d’une partie du pool folliculaire. Ainsi, par une analyse extensive et comparative de la folliculogenèse utilisant des marqueurs hormono-folliculiares, nous n’avons pu mettre aucune modification significative chez les femmes avec un ovaire unique, comparativement aux contrôles. A l’aide du modèle précédemment utilisé, nous avons constaté une augmentation de la sensibilité des follicules antraux à la FSH exogène, évaluée par le FORT, chez des femmes avec un seul ovaire, comparativement aux femmes avec 2 ovaires. Ces résultats supportent l’hypothèse d’une augmentation de la sensibilité folliculaire à la FSH, qui pourrait faire partie des possibles mécanismes compensatoires en jeu dans le maintien d’une folliculogenèse efficace chez les femmes ayant eu une ovariectomie unilatérale.Finalement, à l’aide de 2 approches complémentaires, in vitro and in vivo, nous avons montré que la FSH et l’AMPc stimulaient la transcription de l’AMH, et que la LH avait un effet additif. Nous avons montré que les gonadotrophines et l’AMPc agissaient à travers la protéine kinase A et la P38 MAP Kinase, impliquant notamment les facteurs de transcription GATA binding factor-4 et le steroidogenic factor-1. Par ailleurs, nous avons également mis en évidence que l’expression d’AMH pouvait être régulée de manière différentielle par l’estradiol, en fonction du type de récepteur aux estrogènes exprimés par les CG. Ainsi, la chute d’expression de l’AMH au sein des CG des follicules matures, qui expriment essentiellement ERβ, est probablement liée à un effet de l’estradiol. En résumé, ces travaux de thèse ont permis d’apporter de nouvelles données sur la régulation de la croissance folliculaire et sur la production d’AMH chez la femme. / Anti-Müllerian hormone (AMH), a glycoprotein that is exclusively produced by the granulosa cells (GC) of ovarian follicles in the adult female, is a unique biomarker of ovarian follicular status. In contrast with inhibin B, estradiol and FSH, AMH is produced in a wide range of follicles that goes from the primary to the small antral stages of folliculogenesis. However, the precise mechanisms that drive AMH expression by GC remain poorly understood.We showed that untimely and/or accelerated antral follicle growth during the luteal–follicular transition, a phenomenon that is frequent in ovarian-aged women and that is characterized by the presence of at least one overdeveloped antral follicle during the first days of the follicular phase does not alter the strength of the relationship between antral follicle count and serum AMH levels but does affect the relationship between serum FSH, inhibin B and estradiol levels and the number of antral follicles. The heftiness of AMH in relation to advanced antral follicle growth provides a further explanation for the reported stronger association between serum AMH levels and antral follicle counts as compared with the other hormonal markers of the ovarian fertility status. We subsequently demonstrated, using an innovative tool, the Follicular Output RaTe (FORT), that the percentage of follicles that effectively respond to exogenous FSH by reaching pre-ovulatory maturation is negatively and independently related to serum AMH levels, which is in keeping with the hypothesis that AMH inhibits follicle sensitivity to FSH. Given this hypothesis, we wondered if per-ovary and per-follicle AMH production could be altered in patients having a single ovary as a result of unilateral oophorectomy. Indeed, all indicate that major rearrangements of folliculogenesis occur to preserve and maintain ovarian function despite the abrupt halving of follicular stockpile in these patients. We performed an extensive and comparative evaluation of the folliculogenesis using homono-follicular markers failing to show major changes in unilaterally oophorectomized when compared with control women. Using the same model, we demonstrated an increased antral follicle responsiveness to exogenous FSH, as assessed by FORT, in normo-ovulating unilaterally oophorectomized women undergoing controlled ovarian hyperstimulation. These results support the hypothesis that increased FSH sensitivity ranks among the possible compensating mechanisms at stake in the maintenance of successful folliculogenesis after unilateral oophorectomy.Finally, using complementary approaches, in vitro and in vivo, we showed that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by GC. We also demonstrated that AMH expression can be differentially regulated by estradiol depending on the estradiol receptors by GC. Therefore the decrease in AMH expression by GC of mature follicles, which mainly express ERβ, is likely due to the effect of estradiol.In short, this Ph.D. work offers new insight into the regulation of the follicular growth and AMH production in woman.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-05-08T15:13:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Cristina_Haas.pdf: 1093993 bytes, checksum: 98c20b25abd988ea189ec880c59e615c (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-05-09T10:51:23Z (GMT) No. of bitstreams: 2 Dissertacao_Cristina_Haas.pdf: 1093993 bytes, checksum: 98c20b25abd988ea189ec880c59e615c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-09T10:51:23Z (GMT). No. of bitstreams: 2 Dissertacao_Cristina_Haas.pdf: 1093993 bytes, checksum: 98c20b25abd988ea189ec880c59e615c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-10-02 / A foliculogênese culmina com a ovulação sendo que, em espécies monovulares, apenas um folículo é selecionado, enquanto que os demais entram em processo de atresia. Uma vez que todos os folículos encontram-se sob o mesmo ambiente endócrino, fica evidente que fatores produzidos no ovário são reguladores do crescimento e da diferenciação. Neste contexto, destacam-se fatores pertencentes à superfamília dos fatores de crescimento transformantes beta (TGF-β) e seus receptores. Apesar da grande relevância, a real função dos TGF-β na fisiologia ovariana ainda não foi elucidada. Desta forma, o presente estudo teve por objetivo avançar no entendimento da função e regulação de fatores locais no ovário bovino, avaliando a função do fator de crescimento e diferenciação 9 (GDF9) no crescimento folicular final. Além disso, objetivou-se caracterizar a expressão de membros da família TGF-β, em especial as proteínas morfogenéticas ósseas (BMPs) e seus receptores, na luteinização e ovulação em bovinos. Em um primeiro experimento, a injeção intrafolicular de GDF9 em folículos dominantes (9±0,2 mm de diâmetro), nas concentrações finais de 100 ou 1000 ng/mL, não teve efeito sobre o crescimento folicular final, estro e ovulação (P>0,05). No segundo estudo, utilizando células da granulosa provenientes de folículos pré-ovulatórios (>12 mm), coletados in vivo em diferentes momentos (0, 3, 6, 12 e 24 h) após aplicação i.m de GnRH, a expressão de RNAm das BMPs 1 e 4 não foi regulada. Por outro lado, a expressão de BMP2 aumentou significativamente 3 e 6 h após GnRH (P<0,05). A expressão dos receptores BMPR2 e TGFBR1 aumentou significativamente 12 h após tratamento, enquanto que os receptores BMPR-1A e -1B foram mais expressos 0 e 12 h após GnRH. Os níveis de progesterona no fluído folicular decresceram linearmente com o aumento da expressão de BMPR-1A (R2=0,49) e -1B (R2=0,5). O aumento na expressão de TGFBR1 determinou uma diminuição quadrática nos níveis de progesterona (R2=0,45). O aumento na expressão de BMP2 (R2=0,4) e BMPR1A (R2=0,28) determinou um aumento na concentração de estradiol, apresentando efeito linear e quadrático, respectivamente. Por outro lado, os níveis de estrógeno decresceram linearmente com o aumento na expressão de BMPR2 (R2=0,31). Os dados sugerem que a elevação nos níveis de GDF9 em folículos diferenciados não altera o destino dos mesmos. A expressão da BMP2 está associada a maiores níveis de estradiol, enquanto que a expressão dos receptores de BMPs sugere uma inibição no processo de luteinização. A elucidação do papel dos fatores locais na fisiologia ovariana contribui com o entendimento de patologias reprodutivas e desenvolvimento de tecnologias com fins de contracepção ou aumento da taxa ovulatória. / Antral follicle development culminates with ovulation and, in monovulatory species, only one follicle is selected to grow whereas all the others undergo atresia. During these processes, all the follicles are under the same endocrine environment, indicating the participation of locally produced factors in the regulation of follicular growth and differentiation. In this context, the involvement of transforming growth factors-beta (TGF-β) family members and their receptors is highlighted. Despite their high relevance, the real functions of TGF-β proteins in ovarian physiology is still poorly understood. In this regard, the aim of this study was to gain insight into the function and regulation of ovarian factors, investigating the function of growth and differentiation factor 9 (GDF9) during dominant follicle growth. Furthermore, we tested the hypothesis that TGF-β members, especially bone morphogenetic proteins (BMPs) and their receptors are regulated during luteinization and ovulation processes in cattle. In a first experiment, the intrafollicular injection of GDF9 in dominant follicles (9±0.2 mm diameter) at final concentrations of 100 and 1000ng/mL did not affect follicular growth, estrus manifestation and ovulation (P>0.05). In a second study, using granulosa cells from preovulatory follicles (>12mm), collected in vivo at different time-points (0, 3, 6, 12 and 24 h) after GnRH treatment, mRNA expression of BMPs 1 and 4 was not regulated. By the other size, BMP2 expression was significantly upregulated at 3 and 6 h (P<0.05). The mRNA expression of BMPR2 and TGFBR1 increased 12 h after GnRH treatment (P<0.05), whereas the receptors BMPR-1A and -1B were more expressed at the moment (0 h) and 12 h after GnRH injection. Follicular fluid progesterone concentrations decreased linearly with increased BMPR-1A (R2=0.49) and -1B (R2=0.5) mRNA expression. The increase in TGFBR1 had a negative quadratic effect on progesterone concentration (R2=0.45). The increase in BMP2 (R2=0.4) and BMPR1A (R2=0.28) determined an increase in estradiol, having linear and quadratic effects, respectively. By the other side, follicular fluid estradiol concentrations decreased linearly with increased BMPR2 (R2=0.31) expression. Collectively, the results suggest that increasing the levels of GDF9 in differentiated follicles does not affect their fate. BMP2 expression is associates to high levels of estradiol in follicular fluid, whereas BMPRs expression suggests an inhibitory effect on luteinization. Elucidating the functions of local factors in ovarian physiology would allow to better understand pathological processes and the development of technologies for contraception or, paradoxally, to increase ovulation rates.

The follicle and its enclosed oocyte share intimate and critical communication that regulates folliculogenesis and produces a mature oocyte. Protein and RNA accumulated in the oocyte during oogenesis control fertilization and direct embryonic development until the embryonic genome activates. Most knowledge of mammalian oocyte biology has been derived from eutherian species. Marsupials deserve more detailed studies because they have a distinct reproductive biology that offers a unique perspective from which to consider mammalian reproduction. The oocyte biology of the tammar wallaby, Macropus eugenii, is the focus of research in this thesis. Cold storage, a simple method for transporting ovarian tissue, was evaluated using histological techniques and follicle culture to assess the structure and function of tammar ovarian tissue. In vitro techniques were used to examine and compare: -folliculogenesis in prepubertal and adult animals, - fertilization of in vivo and in vitro matured oocytes, - and embryo development in follicular and tubal oocytes. / Tammar ovaries were placed in cold storage (PBS at 4?C) for 24 or 48 hours. Necrotic changes were minimal in ovarian follicles after cold storage and preantral follicles isolated from ovarian tissue after cold storage grew by similar amounts as non-stored follicles when cultured for 4 days in vitro. Although the general morphology and growth of follicles are unaffected after cold storage for up to 48 hours, the viability of the oocyte is of prime importance. The next important stages of this study were to develop in vitro techniques for follicle culture and for oocyte maturation and fertilization for future assessment of oocytes after cold storage. / A defined (serum-free) culture system was developed to grow isolated preantral follicles from prepubertal and adult tammars. FSH promoted follicle growth and antrum formation in follicles from prepubertal tammars. Although FSH promoted growth in follicles from adult tammars, other factors present in serum were required for antrum formation. Therefore, once the hypothalmo-pituitary-gonadal axis is mature, hormones and growth factors modify the mechanism of antrum formation. Only follicles that developed an antrum in the presence of serum had granulosa and theca layers that had appropriately differentiated. While FSH stimulates follicle growth in vitro, more complex conditions are required to promote granulosa and theca differentiation. / Intra-cytoplasmic sperm injection (ICSI) was successfully used to compare fertilization of in vivo and in vitro matured oocytes as well as the development of mature oocytes collected from the ovary (surrounded by zona pellucida) or from the oviduct (surrounded by zona pellucida and mucoid coat). In vitro matured oocytes proceeded though the early stages of fertilization (e.g. sperm nuclear decondensation, pronuclear formation), but not syngamy. After sperm injection, in vivo matured oocytes cleaved as far as the 8-cell stage. Oocytes do not lose their ability to fertilize after acquisition of the mucoid coat, since tubal oocytes cleaved as far as the 8-cell stage after sperm injection. Follicular oocytes develop as far as the 5-cell stage after sperm injection, but embryos had a large cleavage cavity that hindered cell-cell contact. While the mucoid coat is not required for cleavage, it is important for appropriate cell-cell interaction and normal early development of the embryo. / This, the most detailed in vitro study of marsupial oocyte biology, has shown that there are many similarities in the biology of marsupial and eutherian oocytes but that the unique biology of marsupials offers a significant perspective on mammalian reproduction. This work also lays the foundation for the effective use of assisted reproductive techniques for conservation of Australia’s unique mammalian fauna.

The follicle and its enclosed oocyte share intimate and critical communication that regulates folliculogenesis and produces a mature oocyte. Protein and RNA accumulated in the oocyte during oogenesis control fertilization and direct embryonic development until the embryonic genome activates. Most knowledge of mammalian oocyte biology has been derived from eutherian species. Marsupials deserve more detailed studies because they have a distinct reproductive biology that offers a unique perspective from which to consider mammalian reproduction. The oocyte biology of the tammar wallaby, Macropus eugenii, is the focus of research in this thesis. Cold storage, a simple method for transporting ovarian tissue, was evaluated using histological techniques and follicle culture to assess the structure and function of tammar ovarian tissue. In vitro techniques were used to examine and compare: -folliculogenesis in prepubertal and adult animals, - fertilization of in vivo and in vitro matured oocytes, - and embryo development in follicular and tubal oocytes. / Tammar ovaries were placed in cold storage (PBS at 4?C) for 24 or 48 hours. Necrotic changes were minimal in ovarian follicles after cold storage and preantral follicles isolated from ovarian tissue after cold storage grew by similar amounts as non-stored follicles when cultured for 4 days in vitro. Although the general morphology and growth of follicles are unaffected after cold storage for up to 48 hours, the viability of the oocyte is of prime importance. The next important stages of this study were to develop in vitro techniques for follicle culture and for oocyte maturation and fertilization for future assessment of oocytes after cold storage. / A defined (serum-free) culture system was developed to grow isolated preantral follicles from prepubertal and adult tammars. FSH promoted follicle growth and antrum formation in follicles from prepubertal tammars. Although FSH promoted growth in follicles from adult tammars, other factors present in serum were required for antrum formation. Therefore, once the hypothalmo-pituitary-gonadal axis is mature, hormones and growth factors modify the mechanism of antrum formation. Only follicles that developed an antrum in the presence of serum had granulosa and theca layers that had appropriately differentiated. While FSH stimulates follicle growth in vitro, more complex conditions are required to promote granulosa and theca differentiation. / Intra-cytoplasmic sperm injection (ICSI) was successfully used to compare fertilization of in vivo and in vitro matured oocytes as well as the development of mature oocytes collected from the ovary (surrounded by zona pellucida) or from the oviduct (surrounded by zona pellucida and mucoid coat). In vitro matured oocytes proceeded though the early stages of fertilization (e.g. sperm nuclear decondensation, pronuclear formation), but not syngamy. After sperm injection, in vivo matured oocytes cleaved as far as the 8-cell stage. Oocytes do not lose their ability to fertilize after acquisition of the mucoid coat, since tubal oocytes cleaved as far as the 8-cell stage after sperm injection. Follicular oocytes develop as far as the 5-cell stage after sperm injection, but embryos had a large cleavage cavity that hindered cell-cell contact. While the mucoid coat is not required for cleavage, it is important for appropriate cell-cell interaction and normal early development of the embryo. / This, the most detailed in vitro study of marsupial oocyte biology, has shown that there are many similarities in the biology of marsupial and eutherian oocytes but that the unique biology of marsupials offers a significant perspective on mammalian reproduction. This work also lays the foundation for the effective use of assisted reproductive techniques for conservation of Australia’s unique mammalian fauna.

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa. / Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis.

A infertilidade é uma condição clínica que acomete até 15% dos casais em idade reprodutiva. Como forma de tratamento para essa parcela da população a Medicina Reprodutiva dispõe de várias técnicas de Reprodução Assistida que visam auxiliar o casal na obtenção da gestação. O primeiro passo para obtenção de sucesso na Medicina Reprodutiva é o correto diagnóstico da infertilidade e, do ponto de vista didático, as causas de infertilidade podem ser classificadas em: feminina, masculina, mista ou desconhecida. A causa feminina de infertilidade apresenta como importante fator o ovariano, representado, principalmente, por alterações no processo de maturação do oócito e/ou no processo de foliculogênese. Com relação à maturação do oócito, um dos possíveis tratamentos oferecidos pela Medicina Reprodutiva é a aplicação da técnica de maturação in vitro (MIV). Infelizmente a MIV não apresenta resultados animadores, e um dos motivos é a falta de um método adequado de seleção dos gametas de humanos que podem ser destinados à ela. No entanto, várias técnicas de seleção de oóticos já foram descritas para espécies animais; dentre elas destaca-se a coloração dos complexos cumuli-oócitos com o corante Azul Cresil Brilhante (BCB). A sua aplicação na espécie humana permanece com ressalvas, uma vez que a comunidade científica preocupa-se com os possíveis efeitos tóxicos dessa substância. Nesta Tese, conseguimos demonstrar, através da avaliação de ensaios de viabilidade e de proliferação celular, e da expressão gênica e proteica, a inocuidade dessa substância para o modelo de cultura primária de células foliculares ovarianas luteinizadas, indicando que o protocolo de coloração com BCB é seguro para a espécie humana. Adicionalmente, conseguimos caracterizar e padronizar o cultivo dessas células, que são amplamente utilizadas em estudos na área da Medicina Reprodutiva. Com relação ao outro fator ovariano de infertilidade (o processo de foliculogênese), sabe-se que a depleção acelerada dos folículos ovarianos pode provocar falência ovariana precoce (FOP), uma condição clínica que acomete até 1% das mulheres em idade reprodutiva e leva à infertilidade. Uma das causas da FOP é a alteração no gene Fragile X Mental Retardation 1 (FMR1) e consequentemente em sua proteína (FMRP). Muito do conhecimento do controle dessa proteína provem de experimentos em neurônios, onde o seu controle já foi elucidado. Nesta Tese, conseguimos demonstrar que o controle dessa proteína nas células ovarianas de humanos é similar ao controle que ocorre nos neurônios, envolvendo a via de sinalização S6K. / Infertility is a clinical condition that affects up to 15% of couples of reproductive age. Reproductive medicine applies many assisted reproductive technologies (ARTs) in order to help them to achieve pregnancy. The first step for infertility treatment success is the correct infertility diagnosis, which is divided into four categories: female, male, mixed or unknown. Female infertility is chiefly represented by ovarian dysfunctions, which are generally related to oocyte maturation and/or folliculogenesis. With regard to oocyte maturation, in vitro oocyte maturation (IVM) is one ART that can be applied but, unfortunately, nowadays it does not present suitable results, since we do not have an effective method of selecting competent human oocytes. On the other hand, in animal reproduction brilliant cresyl blue (BCB) staining has already been described as an appropriate method for oocyte selection. However its clinical applicability to humans is some away off, owing to concerns about its safety. In this thesis we have demonstrated (through many cellular viability and proliferation assays and gene and protein expression), that BCB staining, applying the correct protocol, seems to be safe for use in humans (using the primary culture of human ovarian follicular cells as an experimental model). Additionally, we have characterized and standardized the primary culture of human ovarian follicular cells, and this experimental model is widely applied in reproductive medicine experiments. With regard to folliculogenesis, it is well known that premature ovarian failure/insufficiency (POF/POI) is one condition that can be caused by follicle depletion (when folliculogenesis occurs too fast). This condition affects about 1% of females of reproductive age, causing infertility. Genetic alterations, such as in the fragile X mental retardation 1 (FMR1) gene and its protein FMRP, are considered as one cause of POF/POI. Most of our knowledge about FMRP cellular control comes from studies on neurons, and in these cells we have a clue about its control. In this thesis, we have shown that FMRP control on human granulosa cells is similar to its control on neurons, and this involves the S6K pathway.

Les mammifères femelles naissent avec un très grand nombre de follicules ovariens primordiaux (104-106); par contre, la grande majorité (99%) de ces follicules n’atteignent jamais la maturité et subissent l’atrésie, principalement par l’apoptose des cellules de la granulosa. Notre laboratoire a démontré que les hyaluronidases des mammifères induisent l’apoptose des cellules de la granulosa et sont impliquées dans l’atrésie des follicules mais que cet effet apoptotique ne serait pas dû à leur activité enzymatique. Notre modèle propose que les hyaluronidases aient un rôle dans les follicules non destinés à ovuler. Le but de la présente étude est d’évaluer la folliculogénèse et la fertilité des souris déficientes de ces enzymes. Les résultats montrent que la délétion de Hyal-3 ne semble pas affecter la fonction ovarienne des souris mais qu’il pourrait y avoir un effet compensatoire par Hyal-1 chez les souris déficientes de Hyal-3 étant donné que son expression est augmentée chez ces souris. La délétion de Hyal-1 a pour effet d’augmenter le nombre des follicules primordiaux, primaires et secondaires, particulièrement chez les souris de bas âge, et de diminuer le niveau d’apoptose des cellules de la granulosa. Afin d’évaluer la fonction de Hyal-1, -2 et -3 sans effet compensatoire entre elles, nous avons voulu créer une souris déficiente des ces 3 hyaluronidases spécifiquement dans les gonades en utilisant le système Cre/loxP. Un vecteur contenant la séquence Cre sous le contrôle du promoteur de Inhibin-α, qui conduit l’expression des gènes en aval chez les cellules somatiques des gonades, a été construit avec succès. En conclusion, cette étude nous révèle que Hyal-3 ne semble pas affecter la fonction ovarienne mais que la délétion de Hyal-1 augmente la folliculogénèse et diminue l’apoptose des cellules de la granulosa. / Female mammals are born with a large number of ovarian primordial follicles, though the vast majority of these never reach the preovulatory stage and undergo atresia, mainly through granulosa cell apoptosis. Our laboratory has established that mammalian hyaluronidases induce apoptosis of ovarian granulosa cells and that they are involved in follicular atresia but that their apoptotic effect is not due to their enzymatic activity. Our model suggests that mammalian hyaluronidases might have a role in follicles not destined to ovulate. The aim of this study was to evaluate the folliculogenesis and fertility of mice devoid of these enzymes. Our results showed that Hyal-3 KO mice have normal folliculogenesis, which could be explained by a compensatory effect of Hyal-1 since its expression is upregulated in these mice. In contrast, Hyal-1 KO mice had increased numbers of primordial, primary and secondary follicles, particularly in young mice, and lower levels of granulosa cell apoptosis. In order to investigate the effect of the three hyaluronidases, Hyal-1, -2 and -3, without a compensatory effect by one another, we decided to create a transgenic mouse deficient in all these three hyaluronidases but only in the gonads by using the Cre/loxP system. We successfully created a plasmid containing the Cre sequence under the control of Inhibin-α promoter, which conducts gene expression in somatic cells of the gonads. In conclusion, the present work demonstrates that Hyal-3 does not have any effect on ovarian function, but that deletion of Hyal-1 in mice promotes increased folliculogenesis and lowers granulosa cell apoptosis.

A infertilidade é uma condição clínica que acomete até 15% dos casais em idade reprodutiva. Como forma de tratamento para essa parcela da população a Medicina Reprodutiva dispõe de várias técnicas de Reprodução Assistida que visam auxiliar o casal na obtenção da gestação. O primeiro passo para obtenção de sucesso na Medicina Reprodutiva é o correto diagnóstico da infertilidade e, do ponto de vista didático, as causas de infertilidade podem ser classificadas em: feminina, masculina, mista ou desconhecida. A causa feminina de infertilidade apresenta como importante fator o ovariano, representado, principalmente, por alterações no processo de maturação do oócito e/ou no processo de foliculogênese. Com relação à maturação do oócito, um dos possíveis tratamentos oferecidos pela Medicina Reprodutiva é a aplicação da técnica de maturação in vitro (MIV). Infelizmente a MIV não apresenta resultados animadores, e um dos motivos é a falta de um método adequado de seleção dos gametas de humanos que podem ser destinados à ela. No entanto, várias técnicas de seleção de oóticos já foram descritas para espécies animais; dentre elas destaca-se a coloração dos complexos cumuli-oócitos com o corante Azul Cresil Brilhante (BCB). A sua aplicação na espécie humana permanece com ressalvas, uma vez que a comunidade científica preocupa-se com os possíveis efeitos tóxicos dessa substância. Nesta Tese, conseguimos demonstrar, através da avaliação de ensaios de viabilidade e de proliferação celular, e da expressão gênica e proteica, a inocuidade dessa substância para o modelo de cultura primária de células foliculares ovarianas luteinizadas, indicando que o protocolo de coloração com BCB é seguro para a espécie humana. Adicionalmente, conseguimos caracterizar e padronizar o cultivo dessas células, que são amplamente utilizadas em estudos na área da Medicina Reprodutiva. Com relação ao outro fator ovariano de infertilidade (o processo de foliculogênese), sabe-se que a depleção acelerada dos folículos ovarianos pode provocar falência ovariana precoce (FOP), uma condição clínica que acomete até 1% das mulheres em idade reprodutiva e leva à infertilidade. Uma das causas da FOP é a alteração no gene Fragile X Mental Retardation 1 (FMR1) e consequentemente em sua proteína (FMRP). Muito do conhecimento do controle dessa proteína provem de experimentos em neurônios, onde o seu controle já foi elucidado. Nesta Tese, conseguimos demonstrar que o controle dessa proteína nas células ovarianas de humanos é similar ao controle que ocorre nos neurônios, envolvendo a via de sinalização S6K. / Infertility is a clinical condition that affects up to 15% of couples of reproductive age. Reproductive medicine applies many assisted reproductive technologies (ARTs) in order to help them to achieve pregnancy. The first step for infertility treatment success is the correct infertility diagnosis, which is divided into four categories: female, male, mixed or unknown. Female infertility is chiefly represented by ovarian dysfunctions, which are generally related to oocyte maturation and/or folliculogenesis. With regard to oocyte maturation, in vitro oocyte maturation (IVM) is one ART that can be applied but, unfortunately, nowadays it does not present suitable results, since we do not have an effective method of selecting competent human oocytes. On the other hand, in animal reproduction brilliant cresyl blue (BCB) staining has already been described as an appropriate method for oocyte selection. However its clinical applicability to humans is some away off, owing to concerns about its safety. In this thesis we have demonstrated (through many cellular viability and proliferation assays and gene and protein expression), that BCB staining, applying the correct protocol, seems to be safe for use in humans (using the primary culture of human ovarian follicular cells as an experimental model). Additionally, we have characterized and standardized the primary culture of human ovarian follicular cells, and this experimental model is widely applied in reproductive medicine experiments. With regard to folliculogenesis, it is well known that premature ovarian failure/insufficiency (POF/POI) is one condition that can be caused by follicle depletion (when folliculogenesis occurs too fast). This condition affects about 1% of females of reproductive age, causing infertility. Genetic alterations, such as in the fragile X mental retardation 1 (FMR1) gene and its protein FMRP, are considered as one cause of POF/POI. Most of our knowledge about FMRP cellular control comes from studies on neurons, and in these cells we have a clue about its control. In this thesis, we have shown that FMRP control on human granulosa cells is similar to its control on neurons, and this involves the S6K pathway.