Made available in DSpace on 2014-08-20T14:38:04Z (GMT). No. of bitstreams: 1 tese_fabiana_moreira.pdf: 1442913 bytes, checksum: 613ed46b361d675bfb046a7b74462171 (MD5) Previous issue date: 2011-11-22 / Leptin is a multifunctional peptidic hormone, primarily produced by the adipose tissue, which has receptors (Ob-Rs) in reproductive organs, hypothalamus and hypophysis, acting in the appetite regulation and energetic expenditure, potentially influencing the expression of the reproductive function, at puberty and lactation. Reproductive failure are the main causes for female culling, representing 30% to 40%,of the total female culling. Thus, post-mortem evaluation of the genital organs at slaughter may have an important diagnostic value, helping on the identification of the stage of female`s estrous cycle and aiding the interpretation of reproductive abnormalities. Its long form receptor (OBR-b) performs signal transduction in several cell types via mitogen- activated protein kinases (MAPK) cascade, such as ERK 1/2 and p38. The first stage of this thesis is aimed to evaluate the presence of leptin and MPAK in oocites of pubertal sows and prepubertal gilts. Ovaries from 10 pubertal sows and 10 prepubertal gilts were collected at a slaughterhouse. Slides were analyzed for the presence of oocites included in primordial/primary (OIPF), secondary (OISF) and tertiary follicles (OITF). Immunohistochemistry (IHC) was performed with polyclonal antibodies anti-leptin and anti-phospho-MAPK (ERK 1/2 and p38). The second stage involved 28 pubertal sows and the analyses were performed in hypothalamic neurons, endometrial glands and oocytes. For IHC, the antibodies were polyclonal anti-leptin and anti-leptin receptor. Responses were compared with reproductive data registered for each female. All images in both stages were analyzed by 16-Bit Histogram application from Image J® software. Immunolabeling for leptin and activated ERK 1/2 MAPK were more intense in sows oocytes (p ≤ 0.05), while the immunolabeling for activated p38 MAPK was more intense in gilts oocytes (p = 0.05). In sows, OIPF were more intensely marked for leptin and p38 MAPK (p <0.05), on the other hand, for gilts there was no difference in the marking of follicular oocytes from the analyzed categories. Hypothalamic neurons were more intensely marked in the sows culled by reproductive problems (p <0.05). The hypothalamus, uterus and oocytes of luteal or follicular phase sows showed more intense immunolabeling for leptin and OBR-b than the females with cystic ovaries. The immunolabeling for leptin was more pronounced in the hypothalamus and uterus of sows from parity order 2 group (PO 2-4) (p <0.05). These results demonstrate that leptin may be a marker for oocyte competence. Also, cyclic females having PO 2-4 presented more intense leptin immunolabeling in the hypothalamus and uterus and for OBR-b in the oocyte and uterus. The OBR-b immunolabeling was more pronounced in primiparous and multiparous females than for nulliparous. Therefore, immunolabeling for leptin and OBR-b can be a marker for reproductive performance in swine females. / A leptina é um hormônio peptídico multifuncional, produzido primariamente pelo tecido adiposo, com receptores (Ob-Rs) presentes em órgãos reprodutivos, hipotálamo e hipófise, que atua na regulação do apetite e no gasto energético, com potencial influência sobre a expressão da função reprodutiva, na puberdade e após a lactação. Desordens reprodutivas são as causas mais comuns, atribuídas ao total dos descartes, entre 30 e 40%. Considerando a alta proporção de descartes por estes problemas, a avaliação dos órgãos genitais no abate tem importante valor diagnóstico, auxiliando na identificação do estágio do ciclo estral da fêmea e na interpretação de possíveis anormalidades reprodutivas. O seu receptor de forma longa (OBR-b) realiza transdução de sinal em diversos tipos celulares via cascata das proteínas quinases ativadas por mitógenos (MAPK), tais como ERK 1/2 e p38. A primeira etapa do trabalho teve por objetivo avaliar a presença da leptina, e das MAPK nos oócitos de fêmeas suínas púberes e pré-púberes. Envolveu ovários de 10 fêmeas pré-púberes e 10 púberes coletados no abate e lâminas foram analisadas de oócitos inclusos em folículos primordiais/primários (OIFP), secundários (OIFS) e terciários (OIFT). Para a técnica de imuno-histoquímica (IHQ) os anticorpos policlonais instilados foram anti-leptina, anti-phospho- MAPK (ERK 1/2 e p38). A segunda etapa envolveu 28 porcas púberes descartadas, das quais foram analisados os neurônios do hipotálamo, glândulas endometriais e oócitos. Para a IHQ foram utilizados anticorpos policlonais anti-leptina e anti-receptor da leptina e objetivou comparar as respostas com os dados produtivos e reprodutivos a partir do registro de cada fêmea. Em ambas as etapas de trabalho foram utilizadas as modas obtidas pelo aplicativo 16 Bit-Histograma do software Image J®. A imunomarcação da leptina e a presença de ERK 1/2 ativada MAPK foram mais intensas em oócitos de porcas (p≤0,05), enquanto que a marcação para p38 MAPK ativada foi mais intensa em oócitos de leitoas (p=0,05). Em porcas, os OIFP estavam mais intensamente marcados para a leptina e p38 MAPK (p<0,05), mas para leitoas não foi observada diferença na marcação dos oócitos independente do estádio folicular. No hipotálamo a imunomarcação para a leptina foi mais intensa nas porcas descartadas por problemas reprodutivos (p<0,05). O hipotálamo, útero e oócitos das porcas que estavam na fase lútea ou fase folicular apresentaram imunomarcação para leptina e OBR-b mais acentuada que naquelas que possuíam cistos ovarianos. A imunomarcação para a leptina foi mais acentuada no hipotálamo e útero daquelas porcas do grupo ordem de parto 2 (OP 2-4) (p<0,05). Estes resultados demonstram que a leptina pode ser um dos marcadores para competência oocitária. Além disso, pode também ser observado que fêmeas cíclicas com OP 2-4 partos apresentam imunomarcação mais intensa para leptina no hipotálamo e útero, e OBR-b nos oócitos e útero. O OBR-b obteve marcação mais acentuada em fêmeas primíparas e multíparas, que nulíparas. Portanto, a imunomarcação para leptina e OBR-b podem ser um marcador de sinalização reprodutivo em fêmeas suínas.

Depuis quelques années, une biopsie testiculaire suivie d’une congélation du tissu testiculaire est proposée aux enfants atteints de cancer avant introduction d’un traitement gonadotoxique. Cette procédure de préservation de la fertilité est proposée avec l’espoir qu’une méthode de restauration de la fertilité soit développée. Le tissu testiculaire décongelé pourrait ainsi être utilisé afin d’effectuer une maturation in vitro, évitant la réintroduction de cellules tumorales, pour produire des spermatozoïdes. Ce travail de thèse a consisté, dans un premier temps, à évaluer la mise en place de la méthylation de l’ADN au sein du tissu testiculaire prépubère de souris au cours de la spermatogenèse in vitro. La culture de tissu testiculaire frais ou décongelé de souris prépubère permet le maintien des niveaux d’expression des ADN méthyltransférases 1 et 3a dans les spermatogonies et les spermatocytes. De plus, la méthylation de l’ADN est retrouvée jusque dans les spermatozoïdes produits in vitro. Par la suite, la qualité nucléaire des spermatozoïdes ainsi obtenus a été analysée. La culture de tissu testiculaire n’a pas d’impact sur le taux d’aneuploïdie, la condensation de la chromatine et la fragmentation de l’ADN spermatique. Cependant, la congélation suivie par la culture organotypique augmente la proportion de spermatozoïdes avec un ADN oxydé. Enfin, la fonctionnalité des spermatozoïdes produits in vitro a été analysée par micro-injection ovocytaire et la dynamique de différentes marques épigénétiques a été étudiée au cours du développement préimplantatoire. Les taux de développement embryonnaire sont diminués par l’utilisation de spermatozoïdes produits in vitro. Les niveaux des histones H3K4me3, H3K27me3 et H3K9ac sont peu modifiés dans les embryons issus de spermatozoïdes générés in vitro alors que la méthylation et déméthylation de l’ADN sont plus impactées. La production de spermatozoïdes après culture de tissu prépubère frais ou décongelé dans le modèle murin a permis de mettre en évidence que cette procédure n’est pas sans impact sur l’embryon précoce bien que la qualité des spermatozoïdes produits soit peu altérée. / In recent years, testicular biopsy followed by the freezing of testicular tissue has been proposed to children with cancer before the introduction of a gonadotoxic treatment. This fertility preservation procedure is offered with the hope that a fertility restoration method will be developed. The thawed testicular tissue could thus be used to perform in vitro maturation, avoiding the reintroduction of tumor cells, to produce spermatozoa. This thesis work first consisted in assessing the establishment of DNA methylation in mouse prepubertal testicular tissue during in vitro spermatogenesis. The culture of fresh or thawed mouse testicular testicular tissue allows the expression levels of DNA methyltransferases 1 and 3a to be maintained in spermatogonia and spermatocytes. In addition, DNA methylation is found even in in vitro produced spermatozoa. The nuclear quality of these spermatozoa was then analyzed. The culture of testicular tissue has no impact on sperm aneuploidy rate, chromatin condensation and DNA fragmentation. However, freezing followed by organotypic culture increases the proportion of spermatozoa with oxidized DNA. Finally, the functionality of in vitro produced spermatozoa was analyzed by oocyte microinjection and the dynamics of different epigenetic marks was studied during preimplantation development. Embryo developmental rates are decreased when using in vitro produced spermatozoa. The levels of H3K4me3, H3K27me3 and H3K9ac are slightly modified in embryos derived from spermatozoa generated in vitro whereas DNA methylation and demethylation are more affected. The production of spermatozoa after culture of fresh or thawed prepubertal tissue in the mouse model has shown that this procedure is not without impact on the early embryo, although the quality of the spermatozoa produced is relatively unaltered.

In der Präadoleszenz verbessert sportliches Training die Muskelkraftkapazität, die Belege für Auswirkungen auf Muskel- und Sehnenplastizität sind jedoch begrenzt. Jedoch könnte es, ähnlich wie bei jugendlichen Sportler:innen, bei präadoleszenten Athlet:innen zu Dysbalancen zwischen Muskelkraft und Sehnensteifigkeit kommen, insbesondere bei häufigen plyometrischen Belastungen. Dadurch wird die Sehne stärker mechanisch beansprucht, was zu einem erhöhten Verletzungsrisiko der Sehne führen kann. Aufgrund des Forschungsmangels über die Auswirkungen sportlichen Trainings auf die Muskel- und Sehnenplastizität in der Präadoleszenz untersucht die vorliegende Arbeit die Entwicklung der morphologischen und mechanischen Eigenschaften der Plantarflexoren und der Achillessehne bei präadoleszenten Athlet:innen mittels Ultraschall- und Dynamometrie. Es wurden zwei Vergleichsstudien zwischen Turner:innen mit mehrjähriger Trainingserfahrung und untrainierten Personen ähnlichen Alters durchgeführt. Die Studien zeigten eine höhere Muskelkraft der Plantarflexoren bei den Athlet:innen, jedoch keine Anpassungen der Achillessehnensteifigkeit, was zu einem höheren Anteil von Athlet:innen mit stark erhöhter Sehnendehnung führte. Zudem gab es keine Hinweise auf eine trainingsbedingte Muskelhypertrophie der Plantarflexoren. Die Auswirkungen von Training und Reifung auf die Muskel-Sehnen-Einheit wurden zusätzlich über ein Jahr untersucht. Die Muskelkraft der Athlet:innen entwickelte sich im Vergleich zu Nichtathlet:innen ähnlich, war jedoch durch größere Schwankungen und eine unausgewogene Adaptation zwischen Muskelkraft und Sehnensteifigkeit gekennzeichnet. Dies führte zu einer höheren Häufigkeit von Sportler:innen mit erhöhter Sehnendehnung und größeren Schwankungen der Sehnendehnung über die Zeit. Die Ergebnisse deuten auf eine erhöhte mechanische Beanspruchung der Sehne bei präadoleszenten Athlet:innen hin, was Auswirkungen auf das Risiko von Überlastungsbeschwerden der Sehne haben könnte. / In preadolescence, physical exercise and athletic training improve muscle strength capacity, yet the evidence on the effects of long-term athletic training on muscle-tendon plasticity is limited. Similar to adolescent athletes, preadolescents might develop imbalances between muscle strength and tendon stiffness, particularly with high-frequency plyometric loading. This imbalance exposes the tendon to an increased mechanical demand, potentially leading to an increased risk of tendon injury. Despite limited evidence on preadolescence research about the effects of athletic training on muscle-tendon plasticity, the thesis investigates the development of morphological and mechanical properties of the plantar flexors and Achilles tendon in preadolescent athletes via ultrasonography and dynamometry procedures. Two comparison studies were initially performed between artistic gymnastic athletes, with extensive training history, against untrained peers of similar age. The studies demonstrated higher plantar flexor muscle strength in the athletes, yet the gymnastic-specific training did not cause adaptations of Achilles tendon stiffness, resulting in a higher percentage of athletes with high-level tendon strain. Moreover, there was no evidence of training-induced muscle hypertrophy in the plantar flexors. The effects of athletic training and maturation on the triceps surae muscle-tendon unit were investigated over one year during preadolescence. The development of muscle strength in athletes, even though it was similar compared to non-athletes, was characterised by significantly greater fluctuations and a non-uniform adaptation between muscle strength and tendon stiffness. These two facts together resulted in a higher frequency of athletes with a high level of tendon strain and greater strain fluctuations over time. The findings indicate an increased mechanical demand for the tendon in preadolescent athletes that could have implications for the risk of tendon overuse injury.