The role of hypoxia inducible factors in regulating ovarian function.
Date
2010
Authors
Tam, Kimberley Kai Yen
Editors
Advisors
Thompson, Jeremy Gilbert E.
Kind, Karen Lee
Russell, Darryl Lyndon
Kind, Karen Lee
Russell, Darryl Lyndon
Journal Title
Journal ISSN
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Thesis
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Abstract
Hypoxia inducible factors (HIFs) are heterodimeric bHLH-PAS domain transcriptional factors that mediate physiological responses to low oxygen. There are 3 known isoforms. In normoxic conditions, HIFs are rapidly degraded via the ubiquitin-proteasome pathway. In hypoxic conditions, HIFs are stabilised and translocate to the nucleus, where they activate the Hypoxia Response Elements (HRE) upstream of numerous target genes involved in angiogenesis and glycolysis. Hypoxia has been established as the major inducer of HIFs, but other factors, such as transition metals and hormones, can also induce HIF target gene expression by increasing HIF protein stability and/or expression.
In the ovary, oocytes grow in follicle structures surrounded by a basement membrane that excludes the vasculature. Follicular development occurs under the influence of signals from both the oocyte and the systemic environment mediated by the ovarian stroma. During follicular development, the follicle develops an antrum. In the antral follicle, an oxygen gradient is thought to exist across the follicle, potentially limiting the oocyte’s ability for oxidative phosphorylation. In contrast, following the ovulatory signal, blood vessels cross the basement membrane between theca and granulosa layers and continue a rapid growth to sustain corpus luteum development and function.
This study hypothesised that these transitional events in follicular growth, especially the formation of an antrum and the formation of the corpus luteum involve HIF-signalling.
To assist with unravelling the role of HIFs in the ovary, this study utilised a transgenic C57BL/6-Tg(HRE(4)-SV40-EGFP) HIF reporter mouse line, designed to produce EGFP following HIF binding to the HRE. Examination of ovaries collected from cycling and pregnant mice revealed that EGFP was absent from all primordial, primary and preantral follicles. EGFP was, however, variably detected within the theca of antral follicles of all sizes. Furthermore, FSH was unable to induce HIF 1α protein or increased expression of HIF activated genes in cultured mouse granulosa cells, despite readily doing so in the presence of hypoxic mimetics (e.g. CoCl₂ ). In contrast, I observed a stimulatory effect of hCG on HIF 1α protein levels within granulosa cells. Temporal analyses following eCG/hCG treatment in vivo revealed a time-dependent increase in EGFP localisation within granulosa cells post hCG, in synchrony with immunoreactive HIF 1α protein levels in granulosa cells in vivo, with maximal levels around the time of ovulation. Corpora lutea (CL) also expressed EGFP, suggesting that HIFs play a role during follicle differentiation and luteinisation as a response to ovulatory signals.
As there was little evidence of HIF activity in developing follicles in vivo, I assessed whether cumulus cells displayed oxygen sensitive-gene expression. Previous studies in our laboratory used microarrays to examine cumulus cell gene expression following in vitro maturation (IVM) at varying oxygen concentrations (2, 5, 10 & 20%) and demonstrated up-regulation of known HIF-regulated genes. I confirmed using quantitative Real Time RT-PCR that expression of these known HIF-regulated genes in cumulus cells was regulated by oxygen concentration in vitro. Surprisingly, I was unable to stabilise HIF 1α protein in cumulus cells, despite investigating a variety of incubation conditions. This contrast with results for mural granulosa cells, where HIF 1α is readily detectable and levels can be altered by oxygen concentration, hypoxia mimetics, proteasome inhibition and the oocyte-specific TGFβ family member, GDF-9. These results demonstrate a clear distinction between HIF activity within cumulus cells and mural granulosa cells and may lead to a new understanding of how follicular development, especially antrum formation and growth, can occur within an avascular environment.
School/Discipline
School of Paediatrics and Reproductive Health : Obstetrics and Gynaecology
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2010