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Type: Thesis
Title: The Role of Haemoglobin in Healthy Oocyte Development
Author: Lim, Megan
Issue Date: 2020
School/Discipline: Adelaide Medical School
Abstract: Haemoglobin is present in a diverse group of non-erythroid tissues, with proposed roles in oxygen regulation and sequestering reactive oxygen species (ROS) or nitric oxide (NO). Previous studies discovered in vivo matured cumulus oocyte-complexes (COCs) of mouse and human origin express haemoglobin in high amounts, compared to drastically low levels following maturation in vitro. In vitro maturation (IVM) is known to produce poorer quality COCs, likely due to changes in gene expression and irregular ROS/NO levels. In this thesis, I aim to investigate the role of haemoglobin in oxygen regulation and sequestering ROS and NO, which are vital for maintaining healthy oocyte development. Haemoglobin interacts with partner molecule 2,3-bisphosphoglycerate (2,3-BPG), synthesised by bisphosphoglycerate mutase (Bpgm), to facilitate the release of oxygen. The maintenance of a low oxygen environment is crucial to ensuring oocyte developmental success. This thesis provides new insights on the characterisation of Bpgm in the in vivo matured and IVM COC, demonstrating a contrasting expression pattern between Bpgm and haemoglobin. Interestingly, Bpgm is highly expressed in IVM COCs relative to in vivo, and modifications to IVM were made to uncover how it is regulated. A CRISPR-Cas9 knockdown of Bpgm was carried out, with exciting initial results suggesting Bpgm might be involved in the upregulation of oxygen-regulated genes. During COC development, its metabolic needs rely heavily on oxidative phosphorylation, which can result in undesirably high levels of ROS. I investigated the antioxidant potential of haemoglobin to nullify ROS by testing different in vitro models of oxidative stress. Menadione induced ROS production in granulosa cells and COCs, while compromised culture conditions, simulating a poor IVM environment, increased ROS levels in denuded oocytes (DOs). Although exogenous haemoglobin quenched ROS in granulosa cells, this was not observed in DOs. This result brings to light the vulnerability of DOs in poor IVM conditions and warrants further investigation to combat oxidative insult induced by the culture environment. Nitric oxide (NO) may play a role in oocyte meiotic arrest due to its ability to increase levels of cyclic GMP, a known phosphodiesterase inhibitor, thus maintaining high cyclic AMP levels within the oocyte and meiotic arrest. As haemoglobin is known to sequester NO, I assessed the expression of NO synthase (NOS) during in vivo maturation and whether this was dysregulated during IVM. Interestingly, gene expression patterns were remarkably similar between in vivo and IVM. Also, Nos2 expression correlated with time points of meiotic progression. The addition of haemoglobin was unable to change Nos2 levels in COCs. This however could be due to the chosen model, and the utilisation of alternative models may determine whether haemoglobin has a biological effect on NOS gene expression. This thesis broadly expands on earlier findings of the presence of haemoglobin in COCs, demonstrating the association of haemoglobin and Bpgm with low oxygen, and informing future experimental designs to test the role of haemoglobin in ROS and NO regulation. By advancing haemoglobin research, this work adds to the understanding of methods to improve IVM conditions and enhance in vitro oocyte developmental competence.
Advisor: Thompson, Jeremy
Dunning, Kylie
Brown, Hannah
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020
Keywords: haemoglobin
reactive oxygen species
nitric oxide
oocyte maturation
Provenance: This thesis is currently under Embargo and not available.
Appears in Collections:Research Theses

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