The intra-oocyte signaling pathways that control oocyte growth and early follicular development are largely unknown. The aim of this thesis was to investigate the regulation and functions of phosphatidylinositol 3 kinase (PI3K) pathway in the oocyte, focusing in the roles of Foxo3a, p27, and Pten (phosphatase and tensin homolog deleted on chromosome ten). The physiological significance of Foxo3a in oocytes had been investigated by generating a transgenic mouse, whereby constitutively active Foxo3a is maintained in oocytes using the oocyte-specific ZP3 (Zona pellucida) promoter. The expression of the constantly active “negative” molecule Foxo3a in mouse oocytes was found to cause retardation of oocyte growth, resulting in a significant reduction in oocyte volume in secondary follicles. The transgenic mice also showed arrested follicular development and were infertile. In addition, when Foxo3a was overexpressed in oocytes of primary follicles, oocyte growth and follicular development were retarded. One of the causes of this phenotype may be the retained expression of the cyclin-dependent kinase (Cdk) inhibitor 1B (Cdkn1b), commonly known as p27kip1 or p27, in the nuclei of oocytes. The role and related mechanisms of p27 in controlling early follicular development and oocyte growth were then investigated using wild-type and p27-deficient (p27-/-) mice, and we demonstrated that (i) p27 suppresses follicle endowment/formation and activation, (ii) p27 induces follicle atresia that occurs prior to sexual maturity, and (iii) the overactivated follicles in p27-/- ovaries are depleted in early adulthood, causing premature ovarian failure (POF). In this thesis, we also provide genetic evidence that in mice with conditional deletion of Pten a major negative regulator of PI3K in oocytes, the entire pool of primordial follicles becomes activated, and subsequently all activated follicles are depleted in young adulthood, causing POF. Further mechanistic studies revealed that loss of Pten in oocytes resulted in elevated Akt signaling, which led to upregulation of both expression and activation of ribosomal protein S6 (rpS6) in oocytes. The results thus show that the mammalian oocyte serves as the headquarters of programming of the occurrence of follicle activation, and that the PI3K pathway of the oocyte governs follicle activation through control of initiation of oocyte growth.

The migration of T lymphocytes is a vital component of the immune system, with roles in immunosurveillance and inflammation. The role of Phosphoinositide 3-kinase within T lymphocyte migration is unclear, with some evidence that it may be a disposable signal. Here, using Staphylococcal Enterotoxin B activated peripheral blood mononuclear cells and the T cell line CEM cells, the role of Phosphoinositide 3-kinase and its downstream kinases was investigated. CCL22 mediated CEM cell migration and CXCL12 mediated peripheral blood mononuclear cell migration were shown to be independent of Phosphoinositide 3-kinase using several different broad-spectrum Phosphoinositide 3-kinase inhibitors. However, these cells were Akt-dependent, as demonstrated by incubation with the Akt inhibitor Akti-1/2. Differences in the effect of the inhibitors on Akt activity were discovered, indicating that either Akt can be activated in the absence of Phosphoinositide 3-kinase, or differences exist regarding the relative abundance of each protein within the cell. Th17 cells are a subtype of the T helper cell family and have been shown to be involved in inflammation and immune diseases. Mouse splenocytes were polarised to a Th17 phenotype and analysed for the surface expression of chemokine receptors. CCR2, CCR6 and CCR9 were shown to be expressed on Th17 cells and upregulated under Th17 polarising conditions. However, only CCR2 and CCR6 induced migration of Th17 cells. This migration was sensitive to Phosphoinositide 3-kinase and Akt inhibitors. This data reveals a model for the migration of Th17 cells to areas of inflammation, and sheds light on the role of Phosphoinositide 3-kinase during this process.

The phosphoinositide 3-kinase (PI3K) enzymes are well known for their regulation of pro-survival signalling cascades that result in increased cell survival and proliferation. However, most of what we understand is based on Class I PI3K enzymes and much less is understood about the Class II enzymes. Loss of PI3K C2α in mice results in embryonic lethality, or severe glomerular injury with increased morbidity. In contrast, PI3K C2β deficient mice display no apparent phenotype and are healthy and viable. Previous work in our laboratory revealed that administration of a sub-nephritogenic dose of nephrotoxic serum led to an augmented immune response resulting in glomerular damage and impaired renal function, which was associated with T-cell infiltration. Elucidating the immunological basis of this sensitivity was the basis of my project. In response to a subcutaneous injection of sheep IgG in complete Freund’s adjuvant, the spleens of PI3K C2β-/- mice showed prominent germinal centre associated cell proliferation that was absent in the controls. Analysis of splenocyte populations revealed that PI3K C2β-/- mice had an increased population of CD4+ T-cells and when cultured in vitro within a total splenocyte population, the increased CD4+ T-cell population was maintained. However, this effect was lost when T-cells were purified and maintained ex vivo. These data suggest that the increased PI3K C2β-/- CD4+ proliferation may be due to additional factors within the total splenocyte population. B-cell populations from the spleens of PI3K C2β-/- mice had higher CD19 expression compared to B-cells from control mice. Elevated levels of CD19 are associated with a reduced activation threshold. In response to stimulation with a sub-optimal dose of LPS and IL-4 PI3K C2β-/- B-cells underwent increased class switch recombination, displayed increased metabolic activity and remained viable for longer than B-cells from control mice. B-cell lysates from PI3K C2β-/- mice also revealed increased levels of phosphorylated MEK1/2. These data indicate that PI3K C2β may serve as a negative regulator of B-cell function and that loss of this PI3K enzyme isoform activity produces a heightened immune response which may lead to a predisposition to associated pathologies.

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

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