The primordial follicle pool is the main source of developing follicles in the ovary. The length of reproductive life and the onset of menopause are governed by the amount of primordial follicles in the ovary. The genetic factors and molecular mechanisms that maintain the primordial follicles in a dormant and surviving state for the whole of reproductive life are not well understood. The phosphatidylinositol 3 kinase (PI3K) signaling pathways in the oocyte that control oocyte growth and early follicular development are largely unknown. The major aim of this thesis was to investigate the functional role of the intra-oocyte PI3K pathway in the regulation of primordial follicle activation and survival.  Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a major negative regulator of PI3K. The conditional deletion of Pten in the oocytes of primordial follicles led to the overgrowth of oocytes and activation of the entire pool of primordial follicles. There were higher numbers of activated primordial follicles at postnatal day 8 (PD8) in ovaries lacking PTEN in oocytes; by PD35 all the primordial follicles were activated and all the follicles were depleted by 12 weeks, causing premature ovarian failure (POF). In addition, the rate of follicular death that occurs during sexual maturity is reduced in ovaries that lack PTEN in oocytes. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling and upregulation of both expression and activation of ribosomal protein S6 (rpS6). The overactivation of primordial follicles in ovaries that lack PTEN in oocytes is believed to be due to elevated expression and activation of rpS6. PTEN in oocytes is indispensable for the maintenance of primordial follicles in dormancy.  To study the role of the intra-oocyte PI3K signaling pathway in controlling the survival and maintenance of primordial follicles, 3-phosphoinositide-dependent protein kinase-1 (PDK1) was deleted in oocytes of primordial follicle. The loss of Pdk1 in oocytes led to the depletion of most primordial follicles around the onset of sexual maturity, causing POF during early adulthood. Furthermore, the activation of Akt, p70 S6 kinase 1 (S6K1), and rpS6 was impaired in oocytes that lacked PDK1. The suppressed PDK1–Akt–S6K1–rpS6 signaling in oocytes appears to be responsible for the loss of primordial follicles. The excessive activation of primordial follicles seen in the absence of Pten in oocytes could be reversed by concurrent deletion of Pdk1. In addition, the elevated activation of Akt and S6K1 in the absence of PTEN in oocytes was not observed in PTEN and PDK1 double mutant mice. Similarly, the hyperphosphorylation of rpS6 in oocytes that lack PTEN was prevented in double mutant mice, which was most likely due to downregulation of S6K1 activation. Thus, inactivation of rpS6 in double mutant mice might be the reason for the prevention of excessive primordial follicular activation and survival.  PTEN and PDK1 in oocytes are essential for the maintenance of quiescence and survival of primordial follicles. The molecular network involving PI3K/PTEN–PDK1 signaling in oocyte controls the survival, loss, and activation of primordial follicles, which together govern reproductive aging and determine the length of reproductive life in females. The results of the above studies indicate that the mammalian oocyte serves as the seat of programming of follicular activation and survival.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 2 uppsatser. Även tryckt utgåva.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Os objetivos deste estudo foram investigar o efeito de diferentes concentraÃÃes de jacalina e da interaÃÃo de jacalina e FSH sobre a sobrevivÃncia, ativaÃÃo e expressÃo gÃnica em folÃculos primordiais caprinos cultivados in vitro. Para isto, os fragmentos do cÃrtex ovariano foram cultivados em Meio Essencial MÃnimo (MEM) suplementado com diferentes concentraÃÃes de jacalina (0, 10, 25, 50 e 100 μg/mL - experimento I), por um e seis dias. ApÃs o tÃrmino do perÃodo de cultivo, os fragmentos de cÃrtex ovariano foram fixados para histologia clÃssica. Em seguida, avaliou-se a percentagem de folÃculos primordiais ou em desenvolvimento no controle nÃo cultivado e no tecido ovariano cultivado nos diferentes tratamentos. ApÃs a determinaÃÃo da concentraÃÃo de jacalina mais eficiente (50 μg/mL), realizou-se o cultivo de fragmentos de cÃrtex ovariano em MEM suplementado com a jacalina (50 μg/mL), FSH (50 ng/mL) ou ambos (experimento II). ApÃs 6 dias de cultivo, os fragmentos ovarianos foram fixados para histologia clÃssica. AlÃm disso, para cada tratamento, foram coletadas amostras de tecido para avaliar o perfil de expressÃo de RNAs mensageiros para BMP-15, KL, c-kit, GDF-9 e PCNA em folÃculos ovarianos caprinos cultivado in vitro por 6 dias. Os resultados demonstraram que apÃs seis dias de cultivo, a presenÃa de 50 μg/mL de jacalina no meio de cultivo promoveu um aumento de folÃculos morfologicamente normais, bem como uma reduÃÃo da percentagem de folÃculos primordiais e aumento de folÃculos em desenvolvimento, quando comparado ao meio controle. No experimento II, demonstrou-se que jacalina ou FSH estimulam a ativaÃÃo dos folÃculos e contribuem para a manutenÃÃo da viabilidade, mas nÃo foi observada uma interaÃÃo positiva entres essas duas substÃncias. A expressÃo de RNAm para a BMP-15 e KL apÃs o cultivo in vitro de fragmentos de ovÃrio nos diferentes tratamentos por 6 dias nÃo foi alterada. No entanto, a presenÃa de FSH aumentou os nÃveis de RNAm para o c-kit e para o PCNA, enquanto que o GDF-9 teve sua expressÃo reduzida em meio suplementado com jacalina. Em conclusÃo, jacalina e FSH foram capazes de promover a sobrevivÃncia e ativaÃÃo de folÃculos primordiais caprinos apÃs 6 dias de cultivo. A presenÃa de FSH aumentou a expressÃo do RNAm para PCNA e c-kit, enquanto que a presenÃa da jacalina reduziu a expressÃo do GDF-9. / The aims of this study were to investigate the effect of different concentrations of jacalin and the interaction of jacalin and FSH on survival, activation and gene expression of goat primordial follicles cultured in vitro. For this, fragments of ovarian cortex were cultured in Minimum Essential Medium (MEM) supplemented with different concentrations of jacalin (0, 10, 25, 50 and 100 mg/mL - experiment I) for one and six days. After the end of cultured period, the fragments of ovarian cortex were fixed for histology. Then, the percentage of primordial follicles in uncultured or cultured ovarian tissue in different treatments were evaluated. After determining the most effective concentration of jacalin (50 μg/mL), ovarian cortex fragments were cultured in MEM supplemented with jacalin (50 μg/ml), FSH (50 ng/ml) or both (experiment II). After 6 days of culture, the ovarian fragments were fixed for histology. Furthermore, for each treatment, tissue samples were collected to evaluate the expression profile of mRNA for c-kit, KL, GDF-9, BMP-15 and PCNA in goats ovarian follicles cultured in vitro for 6 days. The results showed that after six days of culture, the presence of 50 μg/ml jacalin in culture medium increased the percentage of normal follicles, and promoted a reduction in the percentage of primordial follicles and increase of developing follicles, when compared to control medium. In experiment II, jacalin or FSH stimulated primordial follicle activation and contributed to maintain follicle viability, but there was no positive interaction between these two substances. The levels of mRNA for BMP-15 and KL after in vitro culture of ovarian fragments in different treatments was not altered. However, the presence of FSH increased levels of mRNA for c-kit and PCNA, while the GDF-9 expression was reduced in medium supplemented with jacalin. In conclusion, jacalin and FSH were able to promote the survival and activation of goat primordial follicles after 6 days of culture. The presence of FSH increased expression mRNA of PCNA and c-kit, while the presence of jacalin reduced the expression of GDF-9.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O objetivo deste estudo foi investigar a expressÃo do sistema interleucina 1 (proteÃnas e RNAm de ligantes e receptores) e sua distribuiÃÃo nos ovÃrios de vacas cÃclicas, bem como avaliar os efeitos da IL-1β na sobrevivÃncia e ativaÃÃo de folÃculos primordiais in vitro. Os ovÃrios foram processados para a localizaÃÃo do sistema interleucina 1 em folÃculos prÃ-antrais e antrais utilizando as tÃcnicas de imunohistoquÃmica, qPCR e anÃlise de Western blot. Para os estudos in vitro, fragmentos ovarianos foram cultivados em α-MEM+ suplementado com IL-1β (0, 1, 10, 50 ou 100 ng/mL), e apÃs 6 dias foram processados para anÃlise histolÃgica. Os resultados de imunohistoquÃmica mostraram que a proteÃna para os integrantes do sistema interleucina I (IL-1β, IL-1RA, IL-1RI e IL-1RII) foram detectados em diferentes compartimentos foliculares. Infelizmente, o anticorpo testado para localizaÃÃo de IL-1α nÃo reagiu em ovÃrios bovinos. Todas as proteÃnas testadas foram observados no citoplasma dos oÃcitos e nas cÃlulas da granulosa de todas as categorias foliculares, e nas cÃlulas da teca de folÃculos antrais, com a exceÃÃo da IL-1α, que nÃo foi encontrada em nenhuma das cÃlula analisados. Foram observados nÃveis variÃveis de RNAm para o sistema interleucina 1 nas diferentes categorias foliculares analisadas. ApÃs 6 dias de cultivo, a presenÃa de IL-1β (10 ou 50 ng/mL) foi capaz de manter a percentagem de folÃculos normais e de promover a ativaÃÃo dos folÃculos primordiais. Em conclusÃo, os componentes do sistema de interleucina 1 sÃo expressos diferencialmente em cÃlulas ovarianas de acordo com a fase do desenvolvimento folicular. AlÃm disso, a IL-1β promove o desenvolvimento de folÃculos primordiais in vitro. Estes resultados sugerem um importante papel do sistema interleucina 1 na regulaÃÃo da foliculogÃnese em bovinos. / This study aims to investigate the expression of interleukin 1 system (proteins and mRNA of ligands and receptors) and its distribution in ovaries of cyclic cows, as well as to evaluate the effects of IL-1β on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of interleukin 1 system in preantral and antral follicles by immunohistochemical, qPCR and western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM+ supplemented with IL-1β (0, 1, 10, 50 or 100 ng/mL), and aftes 6 days the tissues cultured were processed for histological analysis. Immunohistochemical results showed that the proteins for interleukin 1 system (IL-1β, IL-1RA, IL-1RI and IL-1RII) were detected in the various follicular compartments. Unfortunately, IL-1α antibodies tested did not react in bovine ovaries. All the proteins tested were observed in the cytoplasm of oocytes and granulosa cells from all follicular categories, and theca cells of antral follicles, with the exception that IL-1α has not been found in any analyzed cell. Variable levels of mRNA for the interleukin 1 system in the follicular size and classes analysed. After 6 days of culture, the presence of IL-1β (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, interleukin 1 system is differentially expressed in the ovarian cells according to the stage of follicular development. Moreover, IL-1β promotes the development of primordial follicles. These results suggest an important role of the interleukin 1 system in the regulation of folliculogenesis in bovine species.