The seasonal reproduction of the three-spined stickleback is stimulated by long day photoperiod. As in other vertebrates, the reproductive system of stickleback is regulated by the brain-pituitary-gonadal (BPG) axis which is largely controlled by feedback effects. Both negative and positive feedback effects on the BPG axis have been found in fish. So far, the roles feedback effects on the BPG axis play in the photoperiodic regulation of seasonal reproduction are still unclear. This thesis focused on the photoperiodic regulation and gonadal feedback effects on the gene expressions of gonadotropin (GtH) and gonadotropin-releasing hormones (GnRH) in the brain and pituitary, and how gonadal feedback regulated the steroid homeostasis in stickleback.Both GnRH2 and GnRH3 mRNA was found in the hypothalamus. Higher expression levels of both GnRH2 and 3 in breeding than in post-breeding males suggested that they are both involved in seasonal reproduction. There was no evidence for a role of GnRH3, which may be the dominating form, in the photoperiodic control of reproduction. However, the polarity of the feedback effect on gnrh3 gene expression may turn from positive to be negative when the males went into post-breeding state. Tapeworm, Schistocephalus solidus, infection inhibited the reproduction of sticklebacks. However, the infection caused higher expression levels of both GnRHs and GtHs genes, which may be due to feedback effect on the BPG axis.Under short day, both lh-β and fsh-β were suppressed by low androgen levels. This negative feedback may inhibit maturation completely, unless a rise of androgens triggers positive feedback under long day. The change in feedback polarity may result in all or nothing maturation. Furthermore, the androgen inhibitory effect on lh-β and fsh-β under short day could be abolished by aromatase inhibitor, which means the estrogen may cause negative feedback in males under short day.There was no compensation effect on plasma androgen level in fully mature hemi-castrated fish. However, both testosterone and 11-ketoandrostenedione treatments increased plasma levels much less in sham-operated fish than in castrated ones, indicating that homeostatic mechanisms are nevertheless present. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows<strong>:  </strong>Paper 1: Submitted. Paper 3: Submitted. Paper 4: Submitted.</p>

In the present study our objectives were (1) to follow the temporal patterns of testicular LH and FSH receptor (LH-R and FSH-R) concentrations and affinity (Ka) during sexual maturation in bulls, to see if such patterns could explain the control of rapid testicular growth that occurs after 25 weeks of age, when serum gonadotropin concentrations are low; (2) to see if transformation growth factors (TGF- alpha and beta 1, 2 and 3) and interleukins (IL-1 and IL-6) are produced in the developing bovine testis and if their concentrations change during development; (3) to see if the onset of puberty could be hastened by treating bull calves subcutaneously (sc) with 3 mg of bLH (n=6) or 4 mg of bFSH (n=6) once every 2 days, from 4 to 8 weeks after birth. Mean LH-R concentrations decreased from 13 to 25 weeks of age and increased to 56 weeks of age (P<0.05). LH-RKa decreased from 9 to 17 weeks of age, increased to 29 weeks and declined to 33 weeks of age (P<0.05). FSH-R concentrations declined from 17 to 25 weeks of age then increased to 56 weeks of age (P<0.05). FSH-RKa increased from 17 to 25 weeks of age (P<0.05). Testicular TGF-alpha concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks and from 33 to 56 weeks of age (P<0.05). Testicular TGF-beta 1 concentrations decreased from 17 to 21 weeks of age, increased to 25 weeks and decreased from 25 to 29 weeks of age (P<0.05). Testicular TGF-beta 2 concentrations increased from 5 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks and decreased at 29 weeks of age (P<0.05). Testicular TGF-beta 3 concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks of age and from 25 to 29 weeks of age (P<0.05). Mean testicular IL-1 alpha concentrations decreased from 5 to 9 weeks of age and 13 to 21 weeks of age (P<0.01) while mean testicular IL-1 beta concentrations decreased from 13 to 17 weeks and 29 to 33 weeks of age (P<0.01). Mean IL-1 bioactivity increased from 13 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks, decreased to 29 weeks and decreased from 33 to 56 weeks of age (P<0.05). Mean testicular IL-6 concentrations decreased (P<0.05) from 9 to 13 weeks of age, increased (P<0.05) to 21 weeks, decreased (P<0.05) to 25 weeks of age, increased (P<0.05) to 29 weeks and decreased (P<0.01) to 56 weeks of age. <p>We concluded that high concentrations of gonadotropin receptors might be critical to initiate postnatal testis growth and support it after 25 weeks of age in the face of low serum gonadotropin concentrations. Testicular TGF-alpha concentrations were higher in calves than adults while concentrations of TGF-beta and IL-1 were higher in the early postnatal period than the peripubertal period. The changes in testicular concentrations of TGFs and ILs led us to suggest a possible local regulatory role in development. Testicular IL-6 concentrations were higher in prepubertal calves than adults. Treatment of bull calves with bFSH from 4 to 8 weeks of age increased testicular growth (SC), hastened the onset of puberty (SC ≥ 28 cm), and enhanced spermatogenesis.

A prolonged growth phase of the ovulatory follicle results in follicular aging. Whether follicular aging is detrimental or beneficial to oocyte competence is not fully known. The objective of this thesis is to investigate the effects of follicular aging on oocyte competence and granulosa cell gene expression in cattle. Four sets of experiments were designed to address the objective. The following hypotheses were tested during the course of these studies: 1) oocyte competence will improve by the longer growing phase but will be adversely affected by FSH starvation, 2) follicles that undergo superstimulation will have different gene expression than dominant follicles from a natural cycle, 3) extending the superstimulation protocol by 3 days will allow follicles to mature better and 4) markers of maturity, cellular health and survival will be turned off by FSH starvation. The objective of the first study (Chapter 3) was to determine the effects of extending the length of superstimulation and follicular aging on oocyte competence by in vitro embryo production. Multiple follicles were allowed to grow for 4 (Short FSH) or 7 days (Long FSH) under the treatment of 8 or 14 injections of FSH (at 12-hour intervals), respectively. Multiple follicles in the FSH starvation group were allowed to grow for 7 days but FSH was provided for only the first 4 days of superstimulation. Extending the duration of follicular growth by superstimulation resulted in a greater number of ≥9 mm follicles and in 2.5 more transferable embryos per animal (morulae+blastocysts) at Day 9 of in vitro embryo culture. The FSH starvation resulted in a greater proportion of poor quality oocytes lower cleavage rate and lower embryonic development. Microarray analysis was used to assess the effect of superstimulation (Chapter 4), follicular aging (Chapter 5) and FSH starvation (Chapter 6) on the gene expression profile of superstimulated granulosa cells. Gene expression of granulosa cells from the post-LH preovulatory dominant follicle was compared (Chapter 4) with those from follicles of the same status after a standard 4-day superstimulation (same protocol as Short FSH group from Chapter 3). A total of 190 genes were down-regulated and 280 genes were upregulated in the superstimulated group when compared with the reference (non-superstimulated control). Data analysis showed that superstimulated follicles are still in a growing phase compared to untreated dominant follicles (most of the upregulated genes are related to matrix remodeling due to tissue proliferation) and did not respond to LH properly (down regulation of LH gene markers). Four-day superstimulation also disturbed genes related to angiogenesis and activated oxidative stress response genes. Extending the superstimulation protocol (7 days; same protocol as Long FSH from Chapter 3) allowed more time for follicles to leave the growing stage and properly respond to LH surge (most of the upregulated genes in the Long FSH group are markers of post LH surge) when compared to the standard 4 day superstimulation protocol (Short FSH; reference group) (Chapter 5). Moreover, the follicles from Long FSH show proximity to ovulation. The continuous FSH support during the extended superstimulation protocol is crucial for follicular health since FSH starvation disturbed genes markers of oocyte quality and embryo development (Chapter 6). Granulosa cells that underwent FSH starvation do not respond to LH surge, which could be detrimental to ovulation (Chapter 6). Therefore, follicles from Short FSH are delayed in maturation and differentiation but the oocyte competence is not compromised. Extending superstimulation protocol by 3 d enhanced the ovarian response to FSH treatment and allowed more time for follicles to mature and properly respond to the LH stimulus. A period of FSH starvation after superstimulatory treatment compromised follicular health, ability to respond to LH and ovulate, oocyte quality and the fertilization process.

In the present study our objectives were (1) to follow the temporal patterns of testicular LH and FSH receptor (LH-R and FSH-R) concentrations and affinity (Ka) during sexual maturation in bulls, to see if such patterns could explain the control of rapid testicular growth that occurs after 25 weeks of age, when serum gonadotropin concentrations are low; (2) to see if transformation growth factors (TGF- alpha and beta 1, 2 and 3) and interleukins (IL-1 and IL-6) are produced in the developing bovine testis and if their concentrations change during development; (3) to see if the onset of puberty could be hastened by treating bull calves subcutaneously (sc) with 3 mg of bLH (n=6) or 4 mg of bFSH (n=6) once every 2 days, from 4 to 8 weeks after birth. Mean LH-R concentrations decreased from 13 to 25 weeks of age and increased to 56 weeks of age (P<0.05). LH-RKa decreased from 9 to 17 weeks of age, increased to 29 weeks and declined to 33 weeks of age (P<0.05). FSH-R concentrations declined from 17 to 25 weeks of age then increased to 56 weeks of age (P<0.05). FSH-RKa increased from 17 to 25 weeks of age (P<0.05). Testicular TGF-alpha concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks and from 33 to 56 weeks of age (P<0.05). Testicular TGF-beta 1 concentrations decreased from 17 to 21 weeks of age, increased to 25 weeks and decreased from 25 to 29 weeks of age (P<0.05). Testicular TGF-beta 2 concentrations increased from 5 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks and decreased at 29 weeks of age (P<0.05). Testicular TGF-beta 3 concentrations increased from 13 to 17 weeks of age, decreased to 21 weeks of age and from 25 to 29 weeks of age (P<0.05). Mean testicular IL-1 alpha concentrations decreased from 5 to 9 weeks of age and 13 to 21 weeks of age (P<0.01) while mean testicular IL-1 beta concentrations decreased from 13 to 17 weeks and 29 to 33 weeks of age (P<0.01). Mean IL-1 bioactivity increased from 13 to 17 weeks of age, decreased to 21 weeks, increased to 25 weeks, decreased to 29 weeks and decreased from 33 to 56 weeks of age (P<0.05). Mean testicular IL-6 concentrations decreased (P<0.05) from 9 to 13 weeks of age, increased (P<0.05) to 21 weeks, decreased (P<0.05) to 25 weeks of age, increased (P<0.05) to 29 weeks and decreased (P<0.01) to 56 weeks of age. <p>We concluded that high concentrations of gonadotropin receptors might be critical to initiate postnatal testis growth and support it after 25 weeks of age in the face of low serum gonadotropin concentrations. Testicular TGF-alpha concentrations were higher in calves than adults while concentrations of TGF-beta and IL-1 were higher in the early postnatal period than the peripubertal period. The changes in testicular concentrations of TGFs and ILs led us to suggest a possible local regulatory role in development. Testicular IL-6 concentrations were higher in prepubertal calves than adults. Treatment of bull calves with bFSH from 4 to 8 weeks of age increased testicular growth (SC), hastened the onset of puberty (SC ≥ 28 cm), and enhanced spermatogenesis.

Hemocyanin, a copper containing protein, is the respiratory protein of Crustacea. Here, 4 major hemocyanin molecules in the hemolymph of Scylla olivacea were purified and studied. Both two-hexamer (24S) and hexamer (16S) form of hemocyanin are present in all adult¡¦s hemolymph. In vitro, 24S hemocyanin dissociates into two 16S hemocyanin in the absence of calcium under high pH value (> 8.9). Little of 16S hemocyanin assembles spontaneously into 24S hemocyanin after calcium is added. Both 16S and 24S hemocyanins were purified from hemolymph, the 16S hemocyanin differs from the 24S hemocyanin in the presence of intersubunit disulfide bonds, it is inferred that not all native 16S hemocyanin dissociates from 24S hemocyanin. Besides the 24S and 16S hemocyanin, there are two massive proteins in the hemolymph of ovary-maturing female S. olivacea. One is female-specific hemocyanin (FSH), which occurs in the hemolymph of ovary-maturing females, but not in the hemolymph of juveniles of either sex or in adult males. FSH is confirmed as a hemocyanin due to its copper content and oxygen binding ability. FSH is also found in ovary, embryo and early-stage zoea, and is proposed to be an important hemocyanin that supplies enough oxygen for ovary-maturing female crab, egg, embryo and early-stage zoea. Another massive hemolymph protein is a non-respiratory protein (NRP) that is present in hemolymph of adults of both sex, the molecular mass of its subunits is similar to hemocyanin and FSH, and one of subunits can be slightly recognized by anti-FSH antibody. NRP is not a respiratory protein, there is neither copper containing nor absorbance of 340nm. FSH and NRP, especially NRP, contain more carbohydrates than the 16S hemocyanin.

Chez la vache le développement des techniques d’induction d’ovulations multiples et de production d’embryons est limité par la variabilité inter-individuelle de la réponse ovarienne au traitement de superovulation. Chez la femme, l’Hormone anti-Müllérienne (AMH) est le meilleur marqueur endocrinien de la réponse folliculaire au traitement de stimulation ovarienne. Les objectifs de mon travail étaient d’étudier l’AMH comme marqueur endocrinien de la réponse ovarienne à un traitement gonadotrope chez la vache et d’étudier la régulation de la production d’AMH par les cellules de granulosa. Nos résultats montrent que les concentrations plasmatiques d’AMH sont prédictives de la réponse ovulatoire à un traitement gonadotrope. Au cours du cycle, les concentrations d’AMH ont un profil en 2 temps : elles diminuent entre les chaleurs et J5 à J8, puis augmentent jusqu’aux chaleurs suivantes. Pour prédire efficacement la réponse ovulatoire, les concentrations d’AMH doivent être mesurées aux chaleurs ou après J12 du cycle. L’étude de la régulation de la production in vitro d’AMH par les cellules de granulosa montre que BMP stimule tandis que FSH inhibe la production d’AMH. / In the cow, the development of multiple ovulation and embryo transfer technologies is limited by the variability between animals in ovarian responses to superovulatory treatments. In the women, the anti-Mullerian Hormone (AMH) is the best marker of follicular response to a stimulatory treatment. The objectives of my work were to study AMH as an endocrine marker of the ovulatory response to a stimulatory treatment in the cow and to study the regulation of AMH production by granulosa cells. Our results show that plasma AMH concentrations can predict the ovulatory responses to a stimulatory treatment. During the estrous cycle, AMH concentrations have a dynamic profile in 2 steps: AMH concentrations decrease between estrus and D5 to D8 and then they increase until the next estrus. To predict in optimal conditions, AMH has to be measured at the estrus time or after D12 of the estrous cycle. In vitro, the study about regulation of AMH production by granulosa cells indicates that BMP enhances whereas FSH decreases AMH production.

La signalisation cellulaire induite par les récepteurs à sept domaines transmembranaires(R7TM) contrôle les principales fonctions physiologiques humaines. Ces R7TMs sont cibles de médicaments et initient de larges réseaux d'interactions. Nous avons modélisé dynamiquement les réseaux de signalisation du récepteur à l'hormone folliculo-stimulante(FSH) régulant la fonction de reproduction et du récepteur angiotensine, un R7TM modèle régulant la tension pour comprendre le fonctionnement de ces réseaux et prédire des données inaccessibles expérimentalement. Notre modélisation a utilisé des équations différentielles ordinaires, en assimilant une variable par espèce et un paramètre par constante cinétique. Les paramètres manquants ont été déterminés par optimisation paramétrique.Puis, nous avons développé un environnement afin de comparer plusieurs algorithmes d'optimisation et créer une nouvelle méthode hybride plus performante et adaptée à la paramétrisation des réseaux de signalisation. / Seven transmembrane receptor (7TMR) signaling controls the main human physiological functions. R7TMs are targeted by drugs and initiate large and complex networks responsible for physiological effects. In this thesis, we dynamically modelled the FSHR induced network that have critical role in reproduction and the angiotensin receptor induced network which regulates blood presure and is considered as a model 7TMR with the objective to understand their mechanisms of functionning and to predict experimentally unreachable data. Our modeling, based on ODE formalism, assumes each species as a variable and each kinetic rate as a parameter. Some parameters were unknown and requiered an adjustment. This led us to develop an environement allowing the comparaisonof existing adjustment methods and to create a novel and efficient hybrid method well-adapted to parametrization of signaling networks.

Orientador: José Buratini Júnior / Resumo: Na produção in vitro (IVP), trabalha-se com uma população de oócitos heterogênea em relação ao estágio da maturação nuclear que estes oócitos se encontram, mais especificamente o estágio de vesícula germinativa (GV), uma vez que estes são obtidos de folículos em diferentes estágios de desenvolvimento. Visto que essa heterogeneidade impacta nos resultados da IVP, torna-se necessário que os processos de seleção de oócitos e de maturação in vitro sejam adequados e articulados, para que ocorra o desenvolvimento da competência oocitária para subsequente desenvolvimento. Neste estudo, objetivou-se avaliar a população de ovócitos obtida de folículos antrais grandes, testando a hipótese de que folículos dominantes saudáveis conteriam oócitos com grau intermediário de compactação da cromatina (oócitos em GV2). Em seguida, avaliou-se a população de oócitos obtida em dia aleatório do ciclo estral após OPU e testou-se o efeito de protocolo de sincronização combinando aspiração de folículos e tratamento com FSH para homogeneizar a população e controlar a qualidade dos oócitos. Os resultados sugerem que folículos dominantes saudáveis são predominantemente compostos por oócitos com níveis intermediários de compactação da cromatina e que protocolos de sincronização de aspiração do folículo combinadas ao tratamento com FSH podem ser úteis para controlar a qualidade do oócito para OPU / IVP. / Abstract: In vitro production (IVP), a heterogeneous oocyte population is employed in relation to the stage of nuclear maturation that these oocytes are found, more specifically the germinal vesicle (GV) stage, once they are obtained from follicles in different stages of development. Since this heterogeneity impacts the results of IVP, it is necessary that the processes of oocyte selection and in vitro maturation are adequate and articulated, so that occurs development of oocyte competence for subsequent development. The objective of this study was to evaluate the oocyte population obtained from large antral follicles, testing the hypothesis that healthy dominant follicles would contain oocytes with an intermediate degree of chromatin compaction (GV2 oocytes). Then we evaluated the population of oocytes obtained at random day of the estrous cycle after OPU and tested the synchronization protocol combining follicle aspiration and FSH treatment to homogenize the population and control the quality of oocytes. The results suggest that healthy dominant follicles are predominantly composed of oocytes with intermediate levels of chromatin compaction and that follicle aspiration synchronization protocols combined with FSH treatment may be useful to control oocyte quality for OPU / IVP. / Mestre

A falência ovariana prematura (FOP) é uma patologia rara, definida como a falência da função ovariana antes dos 40 anos de idade, causando amenorréia, hipogonadismo e níveis elevados de gonadotrofinas. Na maioria dos casos, apresenta-se na forma esporádica, pois apenas 5% apresentam história familial. Com relativa freqüência, a causa etiológica não é obtida, sendo então denominada de idiopática. Entre as causas conhecidas estão as alterações dos genes ligados ao cromossomo X e cromossomos autossômicos, doenças autoimunes, alterações tóxicas e iatrogênicas. Vários estudos têm sugerido que a FOP possa ser uma desordem genética, sendo o gene do receptor do FSH (FSHR) considerado um dos principais genes candidato. A primeira mutação inativadora do gene do receptor do FSH (FSHR) foi descrita por Aittomaki et al., 1995, em mulheres de famílias finlandesas que apresentavam amenorréia primária e uma mutação em ponto C566T no exon 7. Posteriormente, outras mutações inativadoras foram descritas: Ile169Thr e Arg573Cys (BEAU et al.,1998), Asp224Val e Leu601Val (TOURAINE et al.,1999), Ala419Thr (DOHERTY et al., 2002), Pro348Arg (ALLEN et al., 2003) e Pro519Thr (MEDURI et al., 2003). Por outro lado, duas variantes polimórficas, Ala307Thr e Ser307Asn, também, foram identificadas em pacientes com FOP (DA FONTE KOHEK et al., 1998, SUNDBLAD et al., 2004), embora uma relação com o fenótipo não tenha sido sistematicamente investigada.Assim, estudou-se uma coorte de 39 mulheres com FOP (casos esporádicos e familiais) que estão em acompanhamento na Unidade de Endocrinologia Ginecológica, Serviço de Endocrinologia do Hospital de Clínicas de Porto Alegre, com o objetivo de determinar a presença de mutações e/ou polimorfismos no gene do FSHR e verificar se estão associadas com o fenótipo clínico. Para este fim foram realizados 2 trabalhos, o primeiro analisando 36 casos com FOP esporádica e incluindo 2 pacientes probantes de 2 familias com FOP. O segundo estudo descrevendo o genótipo e fenótipo de 5 pacientes oriundas de 2 famílias com FOP. Variáveis clínicas e hormonais foram determinadas de todas pacientes. O DNA foi isolado de leucócitos periféricos. Os exons 6, 7, 9 e 10 do gene do FSHR foram analisados pela reação de polimerização em cadeia (PCR), seguidos por análise de restrição enzimática, eletroforese em gel com gradiente de desnaturação (DGGE) e seqüenciamento direto. Também foram medidos o volume uterino, espessura endometrial e volume ovariano porultrassonografia pélvica transvaginal. Embora não se tenha encontrado nenhuma mutação no gene do FSHR, identificou-se uma alta prevalência dos polimorfismos Ala307Thr e Ser680Asn, que se encontram em desequilíbrio de ligação. Não foram observadas associações entre a presença destes polimorfismos com os níveis séricos de FSH, LH, estradiol, bem como com volume ovariano e presença de folículos. No entanto, as pacientes com FOP esporádicas, com o polimorfismo Ala307Thr, apresentaram a última menstruação mais precocemente (A: idade=33.3 ± 7.1 anos vs. T: 28.6 ± 11.4 anos, p=0.04). A genotipagem dos casos de FOP familial evidenciou a presença dos 2 polimorfismos do gene do FSHR nas 5 pacientes e o fenótipo foi semelhante ao apresentado pelas mulheres com FOP esporádica. Em conclusão, a presença do polimorfismo Ala307Thr pode estar associada com um início mais precoce das manifestações clínicas em pacientes com FOP. Entretanto, estudos longitudinais são necessários para confirmar os resultados do presente estudo. / Premature ovarian failure (POF) is a rare pathology, defined as the failure of ovarian function before age 40, causing amenorrhea, hypogonadism and high gonadotropin levels. In most cases, premature ovarian failure is sporadic and only 5% of the affected individuals have a family history. Relatively often, the etiological cause is not determined and POF is thus labeled idiopathic. Among the known causes are alterations associated with the X chromosome and autosomal chromosomes, autoimmune diseases, and toxic and iatrogenic alterations. Several studies have suggested that POF may be a genetic disorder, the FSH receptor (FSHR) gene being considered one of the main candidate genes.The first inactivating mutation in the FSHR gene was described by Aittomaki et al., in 1995, in women of Finnish families with primary amenorrhea and a C566T point mutation in exon 7. Other inactivating mutations have since then been described: Ile169Thr and Arg573Cys (BEAU et al., 1998), Asp224Val and Leu601 Val (TOURAINE et al., 1999), Ala419Thr (DOHERTY et al., 2002), Pro348Arg (ALLEN et al., 2003), and Pro519Thr (MEDURI et al., 2003). On the other hand, two polymorphic variants, Ala307Thr and Ser307Asn, were also identified in POF patients (DA FONTE KOHEK et al., 1998, SUNDBLAD et al., 2004), although a relation with the phenotype has not been systematically investigated. Thus, a cohort was studied comprising 39 women with POF (sporadic and familial cases) being followed at the Gynecological Endocrinology Unit, Service of Endocrinology, Hospital de Clínicas de Porto Alegre, in order to determine the presence of mutations and/or polymorphisms in the FSHR gene and to ascertain if these are associated with the clinical phenotype. For this purpose, 2 studies were conducted, the first assessing 36 cases with sporadic POF, including 2 patients from 2 families with POF and the second describing the genotype and phenotype of 5 patients from 2 families with POF. Clinical and hormonal variables were determined for all patients. The DNA was isolated from peripheral leukocytes. Exons 6, 7, 9 and 10 of the FSHR gene were analyzed by polymerase chain reaction (PCR), followed by restriction enzyme analysis, denaturating gradient gel electrophoresis (DGGE), and direct sequencing. Also, uterine size, endometrial thickness and ovarian size were measured by transvaginal pelvic ultrasonography. Although no mutation of the FSHR gene was found, a high prevalence for Ala307Thr and Ser680Asn polymorphisms was found, that are in linkagedisequilibrium. No association was observed between the presence of these polymorphisms and the serum levels of FSH, LH and estradiol, as well as ovarian size and presence of follicles. However, patients with sporadic POF, presenting Ala307Thr polymorphism, had their latest menses earlier (A: age=33.3 ± 7.1 years vs. T: 28.6 ± 11.4 years, p = 0.04). The genotyping of cases with familial-related POF showed the presence of 2 polymorphisms in the FSHR gene in 5 patients, and the phenotype was similar to that presented by women with sporadic POF. In conclusion, the presence of Ala307Thr polymorphism may be associated with an earlier onset of clinical manifestations in POF patients. However, longitudinal studies are needed to confirm the results of the present study.

Protocolos de sucesso para a maturação in vitro de oócitos de peixe são importantes, uma vez que é necessário para garantir uma fertilização bem sucedida, formação do zigoto, crescimento do embrião e seu completo desenvolvimento. Em algumas espécies, a eficiência deste processo ainda é muito baixa ou restrita a poucas substâncias que podem ser utilizadas. Assim, pesquisou-se a utilização de hormônios alternativos ao protocolo já existente para maturação in vitro de ovócitos de zebrafish. O objetivo foi avaliar a eficiência do extrato de hipófise de carpa (EHC), dos hormônios folículo estimulante (FSH) e luteinizante (LH) para fazer a maturação dos ovócitos estádio III de zebrafish. Os oócitos estádio III foram colocados em meio de cultivo Leibovitz modificado, suplementado com soro fetal bovino e adicionado o hormônio correspondente a seu tratamento (T1-controle; T2-16 μg/ml de EHC; T3- 32 μg/ml de EHC; T4- 48 μg/ml de EHC; T5- 64 μg/ml de EHC; T6- 80 μg/ml de EHC; T7- 0,5 μg/ml de FSH; T8- 0,5 μg/ml de LH e T9- 0,5 μg/ml de FSH e 0,5 μg/ml de LH). A taxa de maturação foi avaliada através da visualização da quebra da vesícula germinal (GVBD). Em todos os tratamentos houve maturação, embora o EHC tenha demonstrado taxas de maturação muito baixas (T2= 12,8%; T3=24,8%; T4=27%; T5=22,7%; T6=9,7%) e inferiores em relação a maior eficiência dos hormônios gonadotrópicos (T7=16%; T8=35%; T9=50%). Além disso foi possível verificar a viabilidade dos oócito através da fertilização in vitro do melhor tratamento (T9) com uma taxa de eclosão e desenvolvimento em larva de 60%. Os resultados da maturação in vitro utilizando estes indutores hormonais em oócitos estádio III de zebrafish mostraram-se promissores, e reforçam as perspectivas para o aprimoramento e uso desta técnica para produção in vitro de embriões viáveis. / Successful protocols for maturation of oocytes are important, as it is necessary for ensuring successful fertilization, zygote formation, embryo growth and full development. In some species the efficiency of in vitro maturation is still very low or is still restricted to a little amount of substances which can be used for the matter. Thus, we studied the use of alternative hormones to the existing protocol for in vitro maturation of zebrafish oocytes. The aim of this study was to evaluate the efficiency of the use of carp pituitary extract (CPE), the follicle stimulating hormone (FSH) and luteinizing hormone (LH) to oocyte maturation stage III of zebrafish. Oocytes stage III were placed in modified Leibovitz culture medium, suplemented with fetal bovine serum and added to the correnponding hormone treatment (T1-control; T2-16 g / ml of CHE; T3 32 g / ml of CHE, T4 - 48 g / ml of CHE; T5- 64 g / ml of CHE; T6- 80 g / ml of CHE; T7- 0.5 g / ml of FSH, T8 0.5 mg / ml of LH and T9- 0.5 g / ml of FSH and 0.5 mg / ml LH). The maturation rate was assessed by the germinal vesicle break down (GVBD). In all cases there was maturation, though the EHC has demonstrated fairly low maturation rate (T2= 12,8%; T3=24,8%; T4=27%; T5=22,7%; T6=9,7%) and lower in relation of the high efficiency presented by the gonadotropic hormones (T7=16%; T8=35%; T9=50%). In addition it was possible to verify the viability of the oocyte through IVF of the best treatment (T9) with a result of 60% of hatching and larvae development rate. The results of maturation in turn using this hormones in stage III oocytes of zebrafish proved promising, and enhance the prospects for improvement and use of this technique for in vitro production of viable embryos.