Inaug.-Diss.--Rostock.

Mammalian females form their germ cells (oocytes) before or shortly after birth. The oocytes interact with somatic cells to form primordial follicles, creating the quiescent population from which oocytes will be recruited to grow throughout life. A female’s fertility life span is therefore, dependant on the size of this pool and the rate at which primordial follicle are activated to grow. However, there is still much we do not know about the quiescent follicle population and the mechanisms that control their recruitment into the growing follicle population are still unclear. There is evidence that the phosphoinositide-3-kinase (PI3K) pathway is key to activation of follicle growth. The role of the PI3K pathway has been primarily explored in the rodent model and has highlighted this pathway’s importance both in the activation of quiescent follicle growth and maintaining dormancy of the quiescent follicle population. This thesis aimed to explore if the PI3K pathway played a similar role in a large mono-ovulatory species as it does in the small polyovulatory rodent species. Bovine is a mono-ovulate species, which has similar attributes in its reproduction and folliculogenesis to the human in vivo; therefore using an in vitro bovine model might be a valuable indication of the role of the PI3K pathway in the human. Initial experiments tested if the bovine was a good model for human primordial follicle activation in an in vitro environment. It was observed that the bovine and human had comparative levels of activation and subsequent increases in both the primary and secondary follicle populations within an in vitro culture system. These similarities indicate that the bovine is a relevant model for the human in vitro. It is not possible to culture the entire bovine ovary. Therefore knowing the location of the primordial follicles is important to establishing what region(s) of the ovary to use. The overall concentration of ovarian follicles was higher in the cortex and gradually declined through the consecutive inner layers of the ovary. The distribution of the ovarian follicle populations were different in each distinctive region of the ovary with the quiescent follicles representing a much larger proportion of the ovarian follicle population in the cortex compared to the inner regions of the ovary. The location an ovarian follicle in the ovary was seen to influence its health in both the quiescent and growing follicle populations, with reduced health seen in the IV inner layers of the ovary compared to the cortex. This resulted in very few healthy quiescent follicles outside of the cortex region making it the more favourable region to culture in functional studies. The role of the PI3K pathway was therefore explored using an in vitro bovine model using the pharmacological compounds bpV (HOpic) and 740Y-P, both of which caused an up-regulation of the PI3K-pathway. It was observed that up-regulation of the PI3K pathway caused an increase in the activation of the quiescent follicle population, and the resulting primary follicles were larger in size. However, there was reduced health in both the growing and quiescent follicle populations. The ill health appears to be due to a disruption in the co-ordination of growth between oocyte and granulosa cells in the ovarian follicles, leading to enlarged oocytes in both the primary follicles and quiescent follicles. Although the PI3K pathway caused an increase in quiescent follicle activation and larger primary follicles there was no increase in the number of viable large secondary follicles obtained. The growth of the secondary follicles was unaltered by the initial activation of the quiescent follicles via the PI3K pathway. These experiments show that the PI3K pathway plays a role in primordial follicle activation in large mono-ovulate species. However, up-regulating the PI3K pathway results in a decrease in health of the quiescent and primary follicle populations, thus limiting its immediate value as a therapeutic target. This study has improved our understanding of the role of the PI3K pathway in primordial follicle activation in a large mono-ovulate species. It has highlighted that the up-regulation of the PI3K pathway using both bpV (HOpic) and 740Y-P increases the activation of the bovine ovarian follicles in vitro. However, up-regulating the PI3K pathway disrupts the development of the ovarian follicles resulting in retarded growth and thereby a decrease in the survival of both the quiescent and growing follicle populations. The similarities in activation, growth and development between the bovine and the human in vitro indicate that the results observed in the bovine are a good indication of what would occur in the human. This study has also improved our understanding of the location, distribution and viability of the ovarian follicle population within the ovary.

Lodgepole pine (Pinus contorta var. latifolia) is the most wide-ranging pine in North America. Populations in British Columbia vary widely in phenotypic and genotypic characteristics. The effect of climate on variation in reproductive characteristics has never been examined, yet is vital to the production of seed necessary for reforestation. This study aims to determine the relationship between the climate in B.C and variation in female cone and seed characteristics. The study makes use of the Illingworth provenance trial, sixty common garden plots that are distributed throughout British Columbia. Female cones from six provenances were collected at 21 sites during the summer of 2012. The number of scales was counted and maximum length was measured for each cone. Seed was extracted and counted. Variables were pooled for each tree. The climate at each site was described using data from ClimateWNA. Principal components analysis was used to reduce the highly correlated data set to the first two principal components (PC1 and PC2), which together described 76.7% of the variation in the data. PC1 was most closely aligned with variables related to temperature, the number of frost-free days, and degree-days above 5°C or below 0°C. PC2 was most closely aligned with precipitation and moisture variables. The reproductive variables were moderately positively correlated with one another. Analysis of variance indicated that average cone length and the average number of seeds per cone were significantly affected by both site and provenance, however the average number of seeds per cone was not. Average values of each reproductive trait for each site were modelled against the first two principal components using multiple analysis of variance and univariate linear modelling. The best-fit model for the average number of scales per cone included PC1 and PC2, however the model only described 4.9% of the variation in the data. The best-fit model for the number of seeds included only PC1, and iv the model only explained 4.1% of the variation in the data. The model for average cone length had the strongest results, with a model that included PC2 and explained 18.7% of the data. The results of the study indicate that climate is not the most important factor in predicting reproductive characteristics such as cone length, and the number of scales and seed per cone. The significant effect of moisture on average cone length was the strongest relationship identified in the study. The reproductive traits were best described by their stability across the climates of the test sites. High genetic variation in lodgepole pine populations may be contributing to the stability of reproductive traits. Lodgepole pine female cone and seed traits were stable for mature trees over a wide range of provenances and climate regions. / Graduate / 0309 / 0478 / anne.berland@gmail.com

Orientador: Sebastião Roberto Taboga / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T18:00:13Z (GMT). No. of bitstreams: 1 Corradi_LaraSilvia_D.pdf: 6064748 bytes, checksum: 6595f8861a86f33fa6761af9a8f6b0a8 (MD5) Previous issue date: 2008 / Resumo: Os andrógenos têm papel central na biologia da próstata, mas os estrógenos também podem afetar o crescimento e a diferenciação desta glândula. Em tecidos específicos do corpo, a proporção entre andrógenos e estrógenos pode diferir significativamente daquela encontrada no plasma sanguíneo. As concentrações intracelulares desses esteróides nos tecidos alvos são reguladas pelo metabolismo local de hormônios, altamente dependente de enzimas específicas metabolizadoras de esteróides, como a 5a-redutase (5a-r) e a Aromatase (aro). Na próstata, a ação androgênica é acentuada devido à conversão de testosterona em dihidrotestosterona por ação da enzima 5a-r, enquanto que a aro é responsável pela aromatização da testosterona em estrógenos. Assim, a síntese local destes esteróides assume grande importância em doenças que acometem tecidos glandulares, como o câncer de próstata, onde níveis anormais de hormônios promovem o desenvolvimento e o aumento de ocorrência de malignidades. Desse modo, os papéis dos andrógenos e estrógenos ativados podem ainda ser melhor compreendidos na manutenção das interações homeostáticas epitélio-estromais prostáticas. Estudos com inibidores específicos destas enzimas vêm sendo desenvolvidos a fim de se tentar esclarecer os reais papéis enzimáticos na manutenção da fisiologia prostática, assim como no surgimento e desenvolvimento de doenças. De modo geral, após 30 dias consecutivos de inibição, simultânea ou não, das enzimas 5a-r e aro, respectivamente por ação da Finasterida e do Letrozol, a próstata de gerbilos jovens, adultos e velhos mostrou-se com o compartimento epitelial e o estromal alterados e reorganizados na tentativa de adaptar-se à nova condição hormonal induzida. Imediatamente após o término de administração das drogas, na fase inicial do período de pós-tratamentos, as concentrações de testosterona e estrógenos modificaram-se, as células epiteliais tiveram o padrão de atividade secretora alterada e, no estroma, a matriz extracelular ficou totalmente remodelada, além das células musculares lisas e dos fibroblastos fenotipicamente alterados. Na fase final do período de pós-tratamento, ficou evidente a tentativa da próstata em se normalizar morfologica e fisiologicamente, porém, em nenhuma das idades isso aconteceu de modo efetivo. O bloqueio da metabolização de hormônios esteróides na próstata pode ser uma ferramenta importante para o estudo da interação epitélio-estroma tanto na fisiologia normal, quanto nas doenças da próstata. Sugere-se portanto, que o desbalanço entre as concentrações de andrógenos e estrógenos provocados pela Finasterida e de Letrozol, juntos ou separados, alterou significativamente a interação epitélio-estroma. Todos os resultados obtidos parecem ser indicativos de importantes sinais do novo cenário hormonal intraprostático. A recuperação do equilíbrio entre as concentrações hormonais da próstata fica comprometida após o bloqueio enzimático, dando, portanto, às enzimas em estudo, status de crucial importância para o desenvolvimento e manutenção da próstata. O estabelecimento de modelos experimentais para o estudo das relações entre epitélio e estroma e o conhecimento dos componentes celulares e macromoleculares da próstata tornam-se instrumentos muito importantes para o entendimento do desenvolvimento, da estrutura e da fisiologia desta glândula / Abstract: Androgens have substantial role in the biology of the prostate, but estrogens also can affect the growth and differentiation of this gland. In specific tissues of the body, the ratio between androgens and estrogens can differ significantly from that found into plasma. The intracellular concentrations of these steroids in target tissues are mediated by a local hormone metabolism, by specifcs steroid-metabolizing enzymes, as the 5a-redutase (5a -r) and the aromatase (aro). In the prostate, the androgenic action is accented due to the conversion of testosterone in dihydrotestosterone by the activity of the 5a -r, while aro enzyme is an alternative pathway for the aromatization of testosterone into estrogens. These locally syntheses of steroids hormones assume thus, a great importance to the prostate cancer, where abnormal hormone levels can promote development and proliferation of malignancy to this gland. The activated functions of androgens and estrogens may be better understood focusing the maintenance of homeostatic prostatic epithelial-stromal interactions. Studies conducted with inhibitors of these specific enzymes have been carried out in an effort to clarify the real role of steroid-metabolizing enzymes in the maintenance of prostatic physiology, as well as into malignant progression prostatic cancer. After 30 consecutive days of inhibition, simultaneous or not, of the enzymes 5a -r and aro, respectively by Finasterida and Letrozol, the prostate of young, adult and old gerbils revealed that epithelial and estromal compartments were totally modified and reorganized in the attempt to adapt it to the new induced hormonal condition. Immediately after the end of drugs administration, in the early phase of the post-treatments period, the concentrations of testosterone and estrogens had been altered, the epithelial cells had their secretore activity pattern modified and, in the stromal compartment, the extracellular matrix was totally remodeled. The smooth muscle cells and fibroblasts became fenotipically modified. In the late phase of the post-treatments period, it was evident the attempt of the prostate gland in became morphologically and physiologically as a normal gland, however, this was not achieved in any analyzed gerbil. These steroids hormone metabolization blockade seems to be a good tool for the study of the epithelium-stroma interaction both in normal and abnormal prostate gland. Based on this data, it is suggested that the unbalance between androgens and estrogens intraprostatic concentrations provoked by Finasteride and Letrozol, together or not, interfered into the prostatic compartments interactions significantly. The results seem to be indicative of important signals of the new locally hormonal scene within the prostate gland. The recovery of balance hormonal concentrations of the prostate is damaged after the enzymatic blockade what gives, therefore, to this to 5a -r and aro enzymes, a status of crucial key for the normal and abnormal prostate gland. The establishment of experimental models for this kind of study and the knowledge of the cellular and macromolecular components of the prostate are also fundamental for the agreement of development, structure and physiology of prostate / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Made available in DSpace on 2017-07-10T14:17:04Z (GMT). No. of bitstreams: 1 Leonardo.pdf: 2065969 bytes, checksum: ecaa0d3dc812ef2017f1f16fc1f49d2a (MD5) Previous issue date: 2013-07-01 / The induction of obesity in experimental animals with cafeteria diet simulates the high-calorie food that humans eat, nowadays, thus enabling to study obesity and comorbidities resulting from this condition. The male reproductive system is directly affected by obesity with increased lipid peroxidation, increase of apoptotic bodies, changes in parameters espermatobioscópicos and reduced fertility. The aim of the study was to evaluate whether there are changes caused by obesity in epithelia of efferent ducts and epididymis , both in relation to morphology and expression of aquaporins 1 and 9 ( AQPs 1 and 9 ) in adult Wistar rats . After weaning , the animals were randomly divided into two groups : control group ( CON ) received standard diet and water ad libitum ( n = 10 ) and group cafeteria ( CAF ) , which received cafeteria diet soda and degassed ad libitum ( n = 10). The treatment group CAF began after the 12th week of life, totaling 40 weeks of consumption of cafeteria diet, being all dead animals with 52 weeks of age. The efferent ducts , along with initial segments ( Si ) , head ( Cç ) , body ( Co ) and tail ( Cd ) epididymis were submitted to routine histological and immunohistochemical staining for AQP- 1 and AQP- 9 . In the present study we evaluated the data: body mass, length nasoanal, waist circumference, Lee index and retroperitoneal fat mass and perigonadais; also evaluated dimensions, using morphometry and morphology of the epithelia of the efferent ducts and segments Si, Cç, Co and Cd epididymis and analyzed the expression of AQP- 9 and AQP- 1 in the efferent ducts and epididymal segments above. Changes in expression of AQPs in the efferent ducts and the epididymal epithelium were observed with increased expression of AQP- 1 and AQP- 9 reduction in efferent ducts and epididymal cauda, respectively, in animals treated with cafeteria diet. Also, there were morphological changes in the epididymal epithelium, such as increasing the number and size of cells and clear halo; increased endocytic organelles, and heterogeneous distribution of stereocilia, in animals with diet-induced obesity cafeteria. Given the results, it is believed that these changes should cause a profound impact on the luminal environment and interfere in the transport process, maturation, maintenance, protection and storage of sperm, being responsible for obesity alter the expression of AQP- 1 in efferent ducts, AQP- 9 in the epididymal cauda, the relative distribution of cells and clear halo and morphological characteristics of these parts via the sperm. However, more studies are needed to elucidate the specific mechanisms by which the cafeteria diet affects sperm morphology via specifically efferent ducts and epididymal ducts / A indução à obesidade, no modelo experimental animal com dieta de cafeteria, simula a alimentação com alto teor calórico que os seres humanos ingerem, na atualidade, possibilitando, assim, estudar a obesidade bem como as comorbidades advindas desta condição. O sistema genital masculino é afetado diretamente pela obesidade com aumento de peroxidação lipídica, aumento de corpos apoptóticos, mudanças nos parâmetros espermatobioscópicos e redução de fertilidade. O objetivo do estudo foi avaliar se há alterações causadas pela obesidade nos epitélios dos ductos eferentes e epididimário, tanto em relação à morfologia quanto à expressão de aquaporinas 1 e 9 (AQPs 1 e 9) em ratos Wistar adultos. Após o desmame, os animais foram divididos aleatoriamente em dois grupos: grupo controle (CON), que recebeu dieta padrão e água ad libitum (n=10) e grupo cafeteria (CAF), que recebeu dieta de cafeteria e refrigerante desgaseificado ad libitum (n=10). O tratamento do grupo CAF teve início depois da 12º semana de vida, totalizando 40 semanas de consumo da dieta de cafeteria, sendo todos os animais mortos com 52º semanas de idade. Os ductos eferentes, juntamente com segmentos inicial (Si), cabeça (Cç), corpo (Co) e cauda (Cd) do epidídimo, foram submetidos à rotina histológica e imunohistoquímica para AQP-1 e AQP-9. No presente estudo foram avaliados os dados: massa corporal, comprimento nasoanal, circunferência abdominal, índice de Lee e massa das gorduras retroperitoniais e perigonadais; também, avaliou-se dimensões, através de morfometria, e características morfológicas dos epitélios dos ductos eferentes e segmentos Si, Cç, Co e Cd do epidídimo, bem como foram analisadas as expressões de AQP-9 e AQP-1 nos ductos eferentes e segmentos epididimários supracitados. Alterações na expressão das AQPs, nos ductos eferentes e parte do epitélio epididimário, foram observadas com aumento na expressão de AQP-1 e redução de AQP-9 nos ductos eferentes e cauda epididimária, respectivamente, dos animais tratados com dieta de cafeteria. Também, ocorreram alterações morfológicas no epitélio epididimário, como aumento do número e tamanho de células halo e claras; aumento de organelas endocíticas; e distribuição heterogênea de estereocílios, nos animais com obesidade induzida por dieta de cafeteria. Em vista dos resultados, acredita-se que essas alterações devem causar um profundo impacto sobre o ambiente luminal e interferir no processo de transporte, maturação, manutenção, proteção e armazenamento dos espermatozoides, sendo a obesidade responsável por alterar a expressão de AQP-1 nos ductos eferentes, da AQP-9 na cauda epididimária, a distribuição relativa de células claras e halo e características morfológicas destas partes da via espermática. No entanto, mais estudos são necessários para esclarecer os mecanismos específicos pelos quais a dieta de cafeteria afeta a morfologia da via espermática, especificamente dos ductos eferentes e ductos epididimários

En aquest treball de tesi doctoral es descriu l'estructura gonadal, gametogènesi i cicle anual de Scorpaena porcus, S. scrofa i S. elongata per tal d'aprofundir en el coneixement dels diferents graus d'especialització dins l'oviparisme en peixos. Es pretén fer èmfasi en aquells trets inusuals que difereixen del què s'ha descrit fins ara per la majoria de les espècies ovípares de teleostis, i relacionar-los amb l'estratègia reproductiva de les espècies que els presentenEl mostreig s'ha dut a terme de forma intensiva entre l'any 2000 i 2002, tot i que s'ha continuat recol·lectant mostres fins a l'agost de 2004, amb més o menys freqüència segons la necessitat. En total s'han obtingut 258 exemplars de S. porcus, 119 de S. scrofa i 46 de S. elongata.S'ha pogut veure com S. porcus, S. scrofa i S. elongata són espècies ovípares ovulípares especialitzades, ja que la seva estructura ovàrica presenta tot una sèrie de trets molt particulars i poc comuns a la resta de peixos ovípars estudiats fins ara: posició central del raquis musculo-connectiu que es troba envoltat completament pel lumen i la paret ovàrica; presència de peduncles en oòcits en fase de creixement secundari; escassetat i mida petita dels alvèols corticals; poc gruix de la zona radiata; i secreció d'una massa gelatinosa que engloba els ous en el moment de la posta. Totes aquestes característiques també s'han observat en Scorpaena notata, suggerint que l'especialització de l'oviparisme ovulípar es dóna en tot el gènere Scorpaena.L'estructura testicular i l'espermatogènesi d'aquestes espècies també és força particular. Per una banda els testicles són del tipus lobular no-restringit, tot i que presenten algunes característiques com la ordenació parcial dels cists segons l'estadi de maduració de les cèl·lules germinals que contenen i l'absència de lumen central, que actualment només s'ha observat en testicles del tipus lobular restringit de les espècies de la sèrie Aterinomorpha. Per l'altre banda, l'espermatogènesi és del tipus semicística, és a dir, els cists s'obren abans d'acabar tot el procés i les cèl·lules germinals s'acaben de desenvolupar a la llum del lòbul fins a espermatozoides.La posta d'aquestes espècies és múltipla i constisteix en una massa gelatinosa d'ous pelàgica, que en el cas de S. porcus s'alliberen entre els mesos de juny i agost, i en S. scrofa, a partir del mes de juliol. S'ha relacionat la presència d'aquesta massa amb un aparellament mascle-femella, de manera que el mascle alliberaria l'esperma directament sobre la massa d'ous, assegurant així la fartilització total dels mateixos. / The present work describes gonadal structure, gametogenesis and annual cycle of Scorpaena porcus, S. scrofa and S. elongate, to go more deeply into the knowledge of different degree of oviparity specialization in fishes. We pretend to emphasize unusual features that diverge from those that have been described until now for oviparous teleosts.Sampling was performed intensively between years 2000 and 2002. However, collection of specimens was continued until August 2004 sporadically according to experimental needs. We obtained a total of 258 specimens of S. porcus, 119 of S. scrofa and 46 of S. elongata.S. porcus, S. scrofa and S. elongata are specialized ovuliparous species. The ovary showed many characteristics that are not typical for oviparous fishes: central ovarian stroma, pedunculate oocytes, scarcity and small size of cortical alveoli, thinness of the zona radiata, and a gelatinous matrix secreted during the maturation. All these features also were observed in Scorpaena notata suggesting that specialized ovuliparity probably occurs in the entire genus Scorpaena.Testicular structure and spermatogenesis are unusual too. On one hand, these three species have an unrestricted lobular testis. However, they present a partial arrangement of cists according to the stage of development of germinal cells, and they don't have central lumen, two features that currently have only been described for the restricted lobular testes of aterinomorph fishes. On the other hand, spermatogenesis is semicystic: cysts open before the end of spermatogenesis, which is then completed in the lobule lumen.These species are multiple spawning fishes, and they release a pelagic gelatinous mass of eggs. Spawning in S. porcus occurs between June and August, and in S. scrofa begins in July. The presence of the gelatinous mass of eggs has been related to a mating behaviour between male and female that guarantees the fertilization of the whole pond.