Orientador: Paulo Cesar Rodrigues Palma / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-03T23:20:40Z (GMT). No. of bitstreams: 1 Fraga_Rogeriode_M.pdf: 4171010 bytes, checksum: 3066bffb1257256bad02bd638f431b10 (MD5) Previous issue date: 2003 / Resumo: Introdução: Os experimentos conduzidos em animais até o presente para investigar o papel dos hormônios sexuais femininos na estrutura da parede vesical têm apresentado resultados contraditórios. Neste contexto, a despeito dos resultados apresentados na literatura acerca dos colágenos intersticiais, evidencia-se a necessidade de compreensão da estrutura da membrana basal urotelial e seu principal componente, o colágeno IV. A membrana basal está envolvida em uma grande quantidade de funções, como, por exemplo, prover suporte mecânico ao epitélio, promover um elo de fixação entre as células e a capacidade de ultrafiltração de íons e moléculas. Materiais e Métodos: Este estudo foi conduzido utilizando-se 40 ratas Wistar ( 3 meses de idade) divididas em 4 grupos de 10. Grupo 1: Controle; Grupo 2: submetido à ooforectomia bilateral e após 4 semanas iniciada reposição sub-cutânea diária de 17 f3-estradiol durante 12 semanas; Grupo 3: procedimento SHAM e após 4 semanas iniciada reposição diária sub--cutânea de óleo de sésamo por 12 semanas e Grupo 4: submetidas à ooforectomia bilateral e sacrificadas após 12 semanas sem reposição hormonal Foi utilizada imunohistoquímica para coloração das lâminas com anticorpo polic1onal anti-colágeno IV. A aferição da densidade volumétrica do colágeno IV na membrana basal foi efetuada através da estereologia utilizando-se o sistema - teste M42. Resultados: As ratas do Grupo IV, submetidas à ooforectomia sem reposição hormonal, apresentaram uma densidade volumétrica (concentração) de fibras de colágeno IV maior que os outros grupos. Através do teste de Newman-Keuls, a significância estatística apresentou p< 0.001. Nos outros parâmetros analisados não houve diferença. Conclusões: A ooforectomia bilateral , em ratas , induziu a uma maior densidade volumétrica do Colágeno IV da Membrana Basal vesicaL A administração de estradiol no grupo castrado apresentou diferença significativa nos parâmetros estereológicos analisados, em relação aos animais castrados sem reposição / Abstract: Introduction: Experiments conducted on animals to investigate the effect of ovarian hormones on bladder structure have presented contradictory results. The author quantified the type IV collagen fibers in the basement membrane of bladder wall of ovariectomized rats with and without estradiol rep1acement. The type IV collagen is found in basement membranes and its functions is to provide physicaI support to structures, cell attachment and filtration. Materiais and Methods: This study was conducted on 40 Wistar rats ( 3 months old ). Group 1: remained intact; Group 2: underwent bilateral ovariectomy and afier 4 weeks daily rep1acement of 17 6-estradiol for 12 weeks; Group 3: sham operated and afier 4 weeks daily replacement of sesame oil for 12 weeks and Group 4: underwent bilateral ovariectomy and were sacrified after 12 weeks. It was used imunohistochemistry evaluation using Type-4-Collagen polyc1onaI antibody to stain the fibers on parafin rat bladder sections. The M-42 stereologycal grid system was used to ana1yze the fibers. Results: Ovariectomy had an increase effect on the volumetric density of the type IV collagen fibers in the basement membrane of rats bladder wall. Estradiol rep1acement in castrated animals demonstrated a significative difference in the stereological parameters when compared to the castrated group without hormonal replacement. Conclusion: Surgical castration performed on rats induced an increasing on volumetric density of the type IV collagen fibers in the basement membrane of the rats bladder wall and the estradiol treatment had a significant effect keeping a low volumetric density of type IV collagen fibers in the basement membrane of rats bladder wall / Mestrado / Cirurgia / Mestre em Cirurgia

We have examined the effect of recent onset diabetes on several aspects of hepatic microsomal metabolism in both chemically-induced and spontaneously BB (Bio Breeding) diabetic male and female Wistar rats. Experiments were performed either 4 days post-streptozotocin injection or 4 days after withdrawal of insulin (BB rats). Differential alterations of the diabetic state on hepatic microsomal enzyme activities were observed. Female diabetic rats exhibited no change in benzo[a]pyrene hydroxylase activity, a decrease in testosterone A⁴ hydrogenase, and an increase in aniline hydroxylase. On the other hand, male diabetic rats demonstrated a decrease in hepatic benzo[a]pyrene hydroxylase activity, no change in testosterone A⁴ hydrogenase, and an increase in aniline hydroxylase. Insulin treatment reversed these effects. Benzo[a]pyrene hydroxylase kinetic studies did not reveal marked differences between control and diabetic rats. There were no marked differences between the chemically-induced and genetic models of diabetes with respect to the metabolism studies. Serum testosterone levels were significantly lower than control in BB diabetic males, whereas no change was apparent in female diabetics. Serum insulin determinations suggested that the BB diabetic animals we examined were not severely diabetic although they did exhibit hyperglycemia. Electrophoresis of hepatic microsomal proteins indicated that spontaneous diabetes of short duration altered the protein distribution in the cytochrome R450 region. Two [³H]-estradiol binding sites were detected in rat liver cytosol by Scatchard analysis with a ligand concentration range of 0.05 to 200 nM. The high affinity site, which was specific for estrogens, exhibited a K[sub=d] of ~10⁻¹⁰M and a capacity of ~100 fmol/mg protein in the 50% ammonium sulfate fraction. Unexpectedly, the data suggested that the capacity of this site was greater in males than in females. The moderate affinity binding site exhibited a k[sub=d] of ~10⁻⁷M and a capacity of M0 pmol/mg protein in the whole cytosol fraction. Binding at this site was markedly pH dependent. Both estradiol and dihydrotestosterone competed for binding to this site. A sex difference existed for moderate affinity binding because it was present only in males. We obtained unexpected results in binding studies conducted on a relatively small number of BB diabetic rats. In diabetic males, the capacity of the high affinity site was reduced to 50% of control, whereas the reduction in moderate affinity binding was not nearly so marked. Additional studies using a larger sample size and more sophisticated data analysis are required to verify these results. We concluded that alterations in sex dependent drug metabolism evident in streptozotocin-induced diabetic rats were also seen in the spontaneously diabetic rat model, and were accompanied by changes in the relative disposition of electrophoretically separable microsomal proteins. Changes in circulating androgen levels were also found in BB diabetic males, along with changes in the capacities of certain hepatic steroid binding sites. It is not yet possible to establish mechanistic relationships between these [³H]-estradiol binding sites and modulation of hepatic drug metabolism. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2016. / Made available in DSpace on 2016-09-20T04:18:54Z (GMT). No. of bitstreams: 1 341413.pdf: 2146207 bytes, checksum: 2fb763d41efb6d909a15589c629f8694 (MD5) Previous issue date: 2016 / A leptina é um hormônio produzido pelo tecido adiposo, participa da regulação da homeostase energética e pode atuar em diversos processos fisiológicos no organismo. A leptina atua via receptor transmembrana e acessa diversas vias de transcrição no núcleo celular, que podem estimular efeitos mitogênicos no microambiente da mama. Por esta razão, a leptina, produzida proporcionalmente ao tecido adiposo, relaciona a obesidade ao câncer de mama. No entanto, a leptina é parte de uma rede de sinalização e pode interagir com diversos componentes do microambiente, tal como o estradiol, outro hormônio com atividade mitogênica. Portanto, o objetivo deste trabalho foi avaliar o efeito em diferentes concentrações de leptina, estradiol e na combinação leptina-estradiol nas linhagens celulares de câncer de mama: a MCF-7 (estrógeno-sensível), MDA-MB-231 (insensível ao estrógeno) e em co-cultura com linhagem murina de câncer de mama 4T1 incubada com adipócitos. A leptina 100 ng/mL e a combinação leptina-estradiol 100 ng-pg/mL estimularam a proliferação na linhagem MCF-7. A leptina estimulou a migração na MDA-MB-231 incubada na concentração de 1000 ng/mL de leptina, mas não afetou o ciclo celular em nenhuma das linhagens. Na co-cultura entre a linhagem 4T1 e adipócitos incubados com leptina 100 ng/mL, estradiol 100 pg/mL e a combinação leptina-estradiol nas respectivas concentrações, foi observado tendência no aumento da expressão de IL-6 e IGF-1. Em ensaio para avaliar o efeito da viabilidade celular em cultura de MCF-7 e MDA-MB-231 incubadas com doxorrubicina, foi observado que os hormônios, nas concentrações supracitadas, reduziram o valor da CC50 da doxorrubicina. Contrariamente, na linhagem MCF-7, incubada nas mesmas condições da MDA-MB-231, porém com o tamoxifeno, fármaco inibidor do receptor de estradiol, os hormônios aumentaram a CC50 do tamoxifeno. Portanto, em conjunto, os resultados sugerem que a leptina desempenha papel importante na proliferação e viabilidade de células de câncer de mama, reforçando estudos prévios que apontam a existência de uma possível correlação entre obesidade e o câncer de mama.<br> / Abstract : Leptin is hormone produced by adipose tissue and plays an important role in the balance of energy homeostasis. Leptin acts through its receptor to control many routes of transcription in the cell nucleus, which induces proliferation, migration, adhesion and cell cycle regulation. For this reason, obesity can be associated with breast cancer, because leptin is proportionally produced by adipose tissue. However, leptin integrates a web of signalization which molds the microenvironment of the breast, and interacts with many others mitogenic compounds, as estrogen, a hormone with proliferative action in the context of the breast cancer. Under these circumstances, the aim of this work is evaluate the effects of leptin, estradiol and their combination in diferents concentrations in the breast cancer cell lines: MCF-7 (estrogen-sensitive cell), MDA-MB-231 (estrogen-insensitive cell) and coculture with 4T1 and adipocytes. MCF-7 cell line incubated, with leptin 100 ng/mL and the combination of leptin-estradiol 100 ng-pg/mL, was induced to proliferate in 48 hours. As for to the MDA-MB-231, which was induced to migrate after incubation with leptin 1000 ng/mL for 48 hours. Leptin, estradiol and the combination of leptin-estradiol did not change the cell cycle of both cell lines. The 4T1 cell line in coculture with adipocyte incubated with leptin 100 ng/mL, estradiol 100 pg/mL and the combination of leptin-estradiol in the respective concentration reveled a tendency to increase the expression of IL-6 and IGF-1. In the viability assay with MCF-7 and MDA-MB-231 treated with doxorubicin, was observed that, leptin 100 ng/mL, estradiol 100 pg/mL and the combination of leptin-estradiol in the respective concentration, decreased the IC50 compared to control. In contrast, the cell line MCF-7 incubated in the same conditions with hormones, but with tamoxifen, showed an increase in the IC50 compared to control. In conclusion, the data suggest a proliferation and viability role of leptin in the breast cancer cells, supporting previous studies in the literature which demonstrated the existence of a possible correlation between obesity and breast cancer.

In addition to their conventional role as hormones, studies have shown that steroids can act as pheromones in mammals. Emphasis has been placed on evaluating the physiological and behavioural effects of male, urinary 17β- estradiol (E2) exposure in pheromone phenomena including the prevention of embryo implantation and induced precocious puberty in females. Steroids have also been observed to transfer between female mice, leading to changes in the duration of their estrous cycle. Progesterone (P4), a crucial female sex steroid, promotes pro-social sexual reproductive behaviour and the growth of the endometrium in preparation for ovum implantation. Few studies have investigated the effects of P4 in a pheromonal context. Big brown bats (Eptesicus fuscus) are ideal models for pheromone research because they are evolutionarily distinct from rodents, live in highly social and sexually-competitive harems, and are regularly exposed to conspecific secretions in the close confines of their roost. Experimental analysis revealed absorption of tritium-labeled progesterone (3H-P4) (10 μCi) 1 h after cutaneous and intranasal application to adult females. Additionally, radioactivity was observed in mature female bats caged for 48 h with an adult female conspecific that had been intraperitoneally-injected with 3H- P4 (50 μCi). Using the same paradigm, 3H-E2 transfer was not observed between females. Enzyme-linked immunosorbent assays revealed measurable levels of unconjugated P4 and E2 present in the urine of female bats, suggesting urine as one likely vector for P4 transfer. Given corroborative findings in mice, progesterone transfer during cohabitation is likely a mammalian-wide phenomenon that could have evolved to prime conspecifics—and more specifically kin—for sexual reproduction. / Thesis / Master of Science (MSc) / Steroid molecules are conventionally assumed to act solely within the individual that produced them; however, recent experiments have demonstrated that the sex steroid 17β-Estradiol (E2) can be excreted in the urine of adult male mice and taken up into the neural, reproductive, and peripheral tissues of cohabitating females. This exogenous E2 can result in changes to female physiology and behaviour. Our lab has observed E2 to transfer between male and female captive big brown bats during the mating season. The current project aimed to determine whether E2 transfers between captive cohabitating female bats. We also examined whether progesterone (P4), an important female sex steroid involved in the preparation and maintenance of pregnancy, transfers between female bats. We determined that P4 reliably transfers between female bats, but E2 does not. Bioactive E2 and P4 were measured in the urine of non- pregnant female bats and propose urine as one likely vector of P4 transfer between cohabitating individuals.

One of the defining characteristics of the brain is its plasticity, which is the ability to alter and reorganize neuronal circuits. The brain is constantly being shaped and moulded by the external world through endogenous factors like neurotransmitters, growth factors and circulating hormones. 17β-estradiol, which is the most potent estrogen among the group of ovarian steroid hormones, has widespread effects throughout the central nervous system. Apart from its actions on regions of the brain concerned with reproduction, estradiol has profound effects on brain areas not classically associated with reproductive function like cerebral cortex, midbrain, brainstem, hippocampus and spinal cord. This enables the hormone to influence learning and memory, emotions, affective state, cognition, motor coordination and pain sensitivity. Estradiol exerts these effects by regulating gene expression via intracellular estrogen receptors. In addition to this, the hormone interacts with receptors at the cell membrane to rapidly alter the electrical activity of neurons and astrocytes, and regulate second messenger systems. The aim of this study was to investigate the cellular and functional effects of estradiol on neuronal networks and on signaling between neurons and astrocytes in primary mixed hippocampal cultures. Estradiol is proconvulsant; it increases neuronal excitability and decreases the threshold for seizures. This property of estradiol is instrumental in precipitating catamenial seizures in women with epilepsy. These are epileptic seizures influenced by cyclical hormone changes and occur in over one-third to half of women with epilepsy. In the first part of the work, the effects of 24-hour estradiol treatment on hippocampal neurons were investigated using fluorescence imaging and electrophysiological techniques. Further, the ability of gabapentin, an antiepileptic drug sometimes used to treat hormone sensitive seizures, to counteract the effects of estradiol was studied. Synaptic vesicles were labeled by uptake of FM 1-43, and high K+- triggered exocytotic release was monitored by fluorescence imaging. The reduction in intensity of FM 1-43 fluorescence, which is a measure of vesicular release, was enhanced by estradiol, suggesting that estradiol upregulates the exocytotic machinery. The high K+-evoked intracellular Ca2+ rise in neurons, studied by loading the neurons with the Ca2+ indicator dye fluo-3 AM, was potentiated following estradiol treatment. Electrophysiological recordings from neurons following estradiol treatment showed an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a larger number of mEPSC events with a predominant NMDA component. Many of the estradiol-induced excitatory effects on the neuronal network were abolished by incubating the cultures with a combination of estradiol and gabapentin suggesting a mechanism of action for the drug in the treatment of hormone sensitive seizures. Glial cells were once regarded as passive, supportive elements in the nervous system. This view of glial cells has drastically changed over the past decade and it is now known that glial cells are dynamic signaling elements in the brain. In view of the emerging importance of glia in the physiology of the nervous system and accumulating evidence of direct effects of steroid hormones on these cells, the subsequent part of the work delves into the consequences of 24-hour estradiol treatment on astrocytes and neuron-to-astrocyte signaling. Estrogen receptors have been described on both neurons and astrocytes in the hippocampus suggesting a complex interplay between the two cell types in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration, ([Ca2+]i). Astrocyte ([Ca2+]i) transients can modulate neuronal activity, indicating a bi-directional form of communication between neurons and astrocytes. Using simultaneous electrophysiology and calcium imaging techniques, neuronal activity-evoked ([Ca2+]i) changes in fluo-3 AM loaded astrocytes were monitored. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca2+]i) elevations in adjacent astrocytes which could be blocked by 200 µM MCPG and also 1 µM TTX. Comparison of astrocytic ([Ca2+]i) transients in control and estradiol treated cultures revealed that the amplitude of the ([Ca2+]i) transient, the number of responsive astrocytes and the ([Ca2+]i) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca2+]i) rise in astrocytes in response to local application of the metabotropic glutamate receptor agonist t-ACPD was attenuated in estradiol treated cultures suggesting functional changes in the astrocyte metabotropic glutamate receptor following 24-hour treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca2+]i) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling. The acute effects of estradiol on astrocyte-to-astrocyte and astrocyte-to-neuron signaling have been addressed in the next part of the study. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes, and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. In addition to its rapid actions on neuronal electrical activity, estradiol can rapidly alter astrocyte ([Ca2+]i) levels through a plasma membrane-associated estrogen receptor. The functional consequences of acute estradiol treatment (5 min) on astrocyte-astrocyte and astrocyte-neuron communication were investigated using calcium imaging and electrophysiological techniques. Mechanical stimulation of an astrocyte evoked a ([Ca2+]i) rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a ([Ca2+]i) wave. Following acute treatment with estradiol, the amplitude of the ([Ca2+]i) elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the ([Ca2+]i) rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, t-ACPD. Mechanical stimulation of astrocytes induced ([Ca2+]i) elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated ([Ca2+]i) rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol mediated hormonal control.

The present study has established that physiological concentrations of estradiol can modulate stress-induced increases in plasma epinephrine (EPI). In anesthetized female rats, insulin-induced hypoglycemia (0.25 U/kg) increased plasma EPI concentration to a significantly greater extent in 14-day ovariectomized (OVEX) rats compared to sham-operated controls. In 17-estradiol (E2)-replaced OVEX rats, the hypoglycemia-induced rise in plasma EPI was significantly reduced compared to OVEX rats. This suppression was due to both decreased adrenal medullary output and increased clearance of EPI. Adrenal venous EPI concentration was significantly reduced in OVEX+E2 rats, suggesting that EPI secretion from the adrenalmedulla was decreased by E2 replacement. The underlying mechanism(s) of this apparent E2-mediated reduction in secretion could not be established since 1) the expression levels of the biosynthetic enzymes tyrosine hydroxylase and phenylethanolamine N-methyltransferase were not affected in OVEX+E2 rats, suggesting that EPI biosynthesis is similar in these and OVEX rats; and 2) agonist-induced increases in intracellular CaP2+P were identical in isolated adrenal medullary chromaffin cells exposed to E2 (10 nM) or vehicle for 48 hr, suggesting that stimulus secretion coupling is unaffected by E2 treatment. In contrast, plasma clearance of EPI was significantly increased in OVEX+E2 rats. Although 48 hr exposure to E2 had no effect on intracellular signaling in chromaffin cells, acute (3 min) exposure to micromolar concentrations of E2 dose-dependently and reversibly inhibited agonist-induced CaP 2+Ptransients. Consistent with this observation, acute (30 min) infusions of E2 also significantly reduced the insulin-induced increase in plasma EPI in OVEX rats. These data demonstrate that physiological levels of circulating E2 can modulate hypoglycemia-induced increases in plasma EPI. This effect appears to be mediated by the steroids influence on adrenal medullary EPI output and plasma EPI clearance; however the mechanism(s) underlying these E2-mediated modulations remain undetermined. This study has also established that acute exposure to supra-physiological levels of E2 can suppress hypoglycemia-induced increases in plasma EPI, due at least in part to inhibition of stimulus-secretion coupling.

No description available.

El presente estudio tuvo como objetivo determinar el nivel sérico de la hormona estradiol en alpacas hembras adultas al momento del primer servicio postparto y su influencia sobre la tasa de concepción. Con esta finalidad, se utilizaron 85 alpacas hembras adultas, vacías y sin cría, las cuales fueron servidas por machos enteros de fertilidad comprobada, previa evaluación de conducta de receptividad frente al macho. Se tomaron muestras de sangre por punción de la vena yugular, luego fueron centrifugadas a 3000 rpm por 10 minutos y los sueros obtenidos fueron mantenidos en congelación a –20 ºC hasta su análisis. Posteriormente, para efecto de determinar la tasa de concepción se utilizó la técnica de ecografía a los 25-27 días postcopula, mediante un ecógrafo ALOKA SSD5OO y un transductor de 7.5MHz para detectar animales gestantes en base a la observación de una estructura anecoica en el útero correspondiente a la vesícula embrionaria y la del embrión en sí. La determinación de estradiol fue realizada mediante la técnica de radioinmunoensayo (RIA) para humanos. La media general de la concentración de estradiol fue de 0.78 ± 0.39 pg/ml. Los valores encontrados fueron divididos en dos grupos, según el nivel de estradiol: Grupo A (nivel basal de estradiol menor 0.5 pg/ml) y Grupo B (Nivel de estradiol > 0,5 pg/ml). Los resultados obtenidos permiten evidenciar niveles bajos de estradiol en alpacas. La concentración de estradiol no fue afectada por la categorización de los animales con preñadas o vacías. Las hembras preñadas presentaron una media más alta de nivel de estradiol (0,85 ± 0.59 pg/ml) que las hembras vacias (0,68 ± 0.47 pg/ml), pero esta diferencia no fue estadísticamente significativa (p > 0.05). / --- The objective of this study was to determine the serum oestradiol level in female adult alpacas during the first mating post-partum and its influence on the conception rate. For this purpose 85 non-pregnant and non-lactating female adult alpacas were used. Animals were mated with male adults and of proved fertility. Previously to mating, females were also evaluated by sexual receptivity to the male. Blood samples were taken by jugular venipunction to obtain serum. The samples were centrifuged by 10 minutes at 3000 rpm and serum was maintained in freezing to -20ºC, until their analysis. Subsequently, we did ultrasound evaluations to establish the conception rate on days 25-27 after mating. Pregnancy rate was determined by ultrasonography with a ALOKA SSD500 and probe of 7.5 MHz and defined as the presence of an embryonic vesicle and embryo in itself. The oestradiol determination was carried out by means of the radioimmunoassay technique (RIA) for human. The mean of oestradiol concentration was 0.78 ± 0.39 pg/ml. The values were divided in two groups according to its oestradiol level: group A (basal oestradiol level less than 0.5 pg/ml) and group B (oestradiol level > 0,5 pg/ml). The obtained results shown that majority of samples had very low, basal or undetectable concentration of oestradiol in alpacas. Oestradiol level was not affected by the categorization in pregnant and non-pregnant animals. The females pregnant presented values of oestradiol concentration (0,85 ± 0.59 pg/ml) higher than females non-pregnant (0,68 ± 0.47 pg/ml), but this difference was not statistically significant (p > 0.05).

L’œstradiol (E2) représente une cible thérapeutique potentielle contre le déclin de la mémoire associé au vieillissement. En effet, l’E2 favorise des mécanismes de plasticité synaptique, altérés par le vieillissement, censés sous-tendre la rétention mnésique. Cependant, la littérature révèle un tableau plus complexe, qui invite à reconsidérer les relations entre E2 et mémoire. Dans ce contexte, le but de cette thèse était d’améliorer notre compréhension des effets mnésiques d’une supplémentation chronique en E2 chez la souris mâle, jeune et âgée. Chez les animaux âgés, l’E2 réduit les déficits de la mémoire à long-terme dite « déclarative » en améliorant la capacité à relier des évènements séparés dans le temps en améliorant le fonctionnement du CA1 de l’hippocampe. En revanche, cette supplémentation reste sans effet sur le déficit observé dans une tâche de mémoire à court-terme sollicitant la gestion des interférences.Chez les animaux jeunes, l’E2 peut avoir des effets délétères sur la fonction mnésique normale. En effet, un prolongement de la rétention mnésique s’avère paradoxalement dommageable sur la mémoire à court-terme en aggravant les effets d’interférence. Cet effet s’accompagne d’une altération fonctionnelle centrée sur le gyrus denté hippocampique.Nos résultats comportementaux montrent que l’E2 prolonge la rétention mnésique mais que selon l’âge du sujet et la forme de mémoire mise en jeu par la tâche, cet effet « promnésiant » ne se traduit pas forcément par une amélioration de la performance.Ce travail invite aussi à considérer davantage les conséquences d’une exposition croissante aux composés œstrogénique de l’environnement sur la santé publique. / Estradiol (E2) is a potential therapeutic target against age-related memory decline. Indeed, E2 promotes synaptic plasticity processes impaired in aging and thought to underlie mnemonic retention. However, the literature reveals a much more complex picture, leading to reconsider the relationships between E2 and memory. The aim of my PhD work was to improve our understanding of the mnemonic effects of a chronic supplementation of E2 in young and aged male mice. In aged animals, E2 supplementation reduces hippocampal-dependent memory deficits called declarative memory, by improving the capacity to link temporally distant event which depends on CA1 functionality. However, the supplementation had no effect on the severe deficits of short-term/working memory which solicits not only retention but also the organization of learned information and forgetting to avoid interference. In young animals, E2 can induce deleterious effects on regular mnemonic function. Indeed, the prolongation of mnemonic retention can paradoxically imapir working memory by aggravating interference effects. This effect was associated with a functional alteration centered on dentate gyrus. Our behavioral results show that E2 prolongs mnemonic retention but, in accordance with our hypotheses, but depending on the age of the subject and the type of memory involved in the task, this promnesic effect does not necessarily induce an improved performance.Our work confirms the therapeutic interest of E2 in the context of mnemonic aging and allows pinpointing its limits. This work also invites to carefully consider the consequences of a growing exposition to environmental estrogenic compounds on public health.

Tesis presentada a la Universidad de Chile para optar al grado académico de Doctor en Farmacología / La función ovárica es controlada por un eje endocrino y una regulación nerviosa paralela. La administración de valerato de estradiol (EV) a ratas produce una condición morfológica de ovario poliquístico (PCO) que está acompañada por un tono adrenérgico aumentado. Esta condición está precedida por el aumento del factor de crecimiento nervioso (NGFB) y su receptor, NGFR. La relación causal entre la actividad nerviosa y el desarrollo de PCO se demostró, ya que la disección del nervio ovárico superior (NOS) o la administración de anticuerpos contra NGFB producen la reversión de la condición de ovario poliquístico generada por EV en ratas adultas. Esta reversión ocurre a nivel de la dinámica folicular, ciclicidad estral y tono adrenérgico. Además de estos resultados evidencias preeliminares muestran que la condición de PCO tiende a revertir en forma espontánea en estas ratas, sin embargo ésta no ocurre en la misma magnitud en ratas tratadas en la edad neonatal con EV, manteniéndose éstas acíclicas, sugiriendo una ventana en la cual estradiol produce efectos irreversibles en el control nervioso de la función ovárica. Nuestro objetivo fue determinar la ventana de mayor sensibilidad al tratamiento con EV y los cambios tempranos en NGFB/NGFR/TrkA, que programan la condición de ovario poliquístico en la rata de forma irreversible. Administramos EV a distintas edades (1, 7, 14, 21 y 30 días postnatal) y controlamos los ciclos estrales hasta los 6 meses de edad. En ese momento evaluamos la morfología ovárica y norepinefrina (NE) por HPLC. Encontramos que las ratas tratadas a los 1, 7 y 14 días de edad se mantenían acíclicas durante todo el periodo estudiado, lo que se condecía con un pobre desarrollo folicular, anovulación y presencia de quistes foliculares ováricos. En cambio las ratas que fueron tratadas a los 21 y 30 días de edad con EV recuperaron su ciclicidad estral, la capacidad ovulatoria, mostraban un patrón de desarrollo folicular similar al control. En conclusión la ventana de sensibilidad al efecto deletéreo permanente producido por EV corresponde a la etapa neonatalinfantil en la rata. Además nos propusimos buscar el mecanismo por el cual estradiol genera estos efectos. Para esto se realizó la técnica Microarray para evidenciar los genes relacionados a neurotrofinas y receptores que cambian luego de la administración neonatal de EV. Encontramos que varios genes relacionados con proliferación celular, apoptosis, diferenciación y cambios epigenéticos inducidos por EV eran regulados a la baja. Según esto realizamos inmunohistoquímica y TUNEL para evidenciar que células están muriendo o proliferando y cuales están sujetas a cambios epigenéticos en el ovario neonatal de rata. Aparentemente, existe un desbalance en la conducta proliferativa y diferenciación de células implicadas en el ensamblaje folicular, lo que repercute en un menor número de folículos primarios en las ratas inyectadas con la hormona. Por último para verificar si el aumento de NGFB fue el responsable de los cambios permanentes producidos en el desarrollo folicular, administramos un anticuerpo policlonal contra NGFB durante los 3 primeros días de vida en ratas tratadas el día 1 de edad con EV. Al analizar el ovario a los 60 días de vida, además de una baja concentración de NA y NGFB elevado, existe un aumento de folículos antrales grandes y una acumulación de estructuras foliculares prequísticas y quísticas, sugiriendo que la presencia de niveles aumentados de NGFB durante la vida adulta facilita la conversión de estructuras preovulatorias en folículos quísticos. Además la ausencia de ovulación indica fuertemente que probablemente existen mecanismos dependientes de catecolaminas, como la ovulación, y otros mecanismos que dependen de NGFB, como el desarrollo folicular, que en forma fina y coordinada regulan la función ovárica. / Ovarian function is under dual gonadotropic and neural control driven by gonadotropins and sympathetic nerves respectively. Estradiol Valerate (EV) single exposure to neonatal or adults rats develops a polycystic ovarian condition (PCO), which is preceded by an increased adrenergic tone and an increased concentration of Nerve Growth Factor (NGFB) and its low affinity receptor NGFR. A causal relationship between the nerve activity and PCO was demonstrated, since either the Superior Ovarian Nerve resection (SONX) or the blockade of NGFB with an antibody against the peptide reversed the polycystic condition in adult EV-treated rats. Previous work of our group have shown that the reversion occurs spontaneously in rats treated at different ages with the hormone, but it does not occur in rats are exposed to EV in the early life. Thus it is suggested that exists a developmental window in which estradiol produces irreversible damages in the reproductive system. Our aim was to determine this window of sensitivity to the deleterious effects of estradiol and the mechanism underlying this effect. We administered a single EV dose (10 mg/Kg) at different ages and the estrual cyclic activity was controlled until 6 month of life. At this age we analyzed the ovarian morphology and the noradrenaline (NA) ovarian concentration. We find that EV-treated rats at 1, 7 and 14 days old were acyclic during the whole period studied, and in addition they had a poor follicular development, anovulation and follicular cyst formation. In contrast, rats that were treated at days 21 and 30 of life recovered the estrual cyclic activity, the normal follicular development and the ovulatory capacity. In conclusion, the neonatal-infant period of life is the most sensitive period to the permanent damage produced by estradiol in the rat. Looking for the mechanism by which estradiol generates these effects, we performed a microarray to quantify the changes in the expression of neurotrophins, its receptors and related genes, 24h after estradiol exposure. Different changes in the expression of genes (particularly down-regulation) related to cell proliferation/differentiation and epigenetic regulations of gene expression were observed. We choose some genes specially affected and we did inmunohistochemistry against PCNA,Hystone H3 acetylated and TUNEL. Apparently there is an unbalance in the pattern of proliferation and differentiation in cells during the follicular assembly, a fact that has repercussion in the number of primary follicles. These results strongly suggest that rapid and early changes to the neurotrophin/receptors system, participates in the changes in follicular development found when rats were exposed neonatally to estradiol. Finally, and to verify if the primary increase in NGF was responsible for the permanent changes in follicular development, we administrated a policlonal antibody against NGFB during the first three days after birth to rats treated with EV the first day of age. The analysis of follicular development demonstrated that , in addition to a 90% decrease in NE concentration in the ovary and a increase in endogenous NGFB concentration, adults rats presented a further accumulation in precysts and cysts and in the preovulatory follicles strongly suggesting that the presence of increased levels of NGF during adulthood facilitates the conversion of antral follicles to cysts and because these rats did not recovered the ovulatory performance, there are probably mechanisms dependent of the presence of catecholamines (eg. ovulation) and other mechanism such us follicular development depend of NGF, strongly suggesting that ovary function is the results of coordinated regulation in which both sympathetic nerves and NGFB participates. / CONICYT; FONDECYT; MECESUP