The role of the hexosamine biosynthesis pathway and β-O-linked glycosylation in determining oocyte developmental competence.

Abstract

Maternal diabetes and conditions such as obesity in which blood glucose levels are elevated are associated with reduced fertility and poor pregnancy outcomes. Many studies have examined the effects of hyperglycaemia on the early embryo and fetus; however, it is becoming increasingly evident that the peri-conceptual environment surrounding the oocyte has a significant impact on developmental competence and the long-term health of offspring. In this thesis, I aimed to investigate the role of the hexosamine biosynthesis pathway (HBP) in oocyte developmental competence. The HBP is a glucose-metabolising pathway which can also be upregulated by glucosamine, a potent hyperglycaemic mimetic which enters the HBP downstream of the rate-limiting enzyme. The HBP produces uridine diphosphate-N acetylglucosamine, which can be used for the β-O-linked glycosylation (O-GlcNAcylation) of proteins, regulating their function in a similar manner to phosphorylation. Firstly I established the effect of hyper- and hypo-glycaemic conditions during in vitro maturation (IVM) of mouse cumulus-oocyte complexes (COCs) on a range of measures associated with oocyte developmental competence, including cumulus expansion, meiotic maturation, cleavage and blastocyst development rates. A low (1 mM) glucose concentration achieved optimal oocyte competence, and glucose supplementation during only the first hour of IVM was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine was able to substitute for glucose during this first hour. In the absence of glucose throughout IVM, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on these outcomes. These experiments underscored the importance of the other glucose metabolic pathways, during COC maturation, and supported the concept that excess flux through the HBP has detrimental consequences. Using Western blots and immunohistochemistry, it was shown that both glucosamine and high glucose levels induced an increase in total O-GlcNAcylation in COCs, which was reduced in the presence of an inhibitor of the β-O-linked glycosyltransferase enzyme. Several specific proteins were identified using mass spectrometry as potential targets of O-GlcNAcylation in COCs, including heat-shock protein 90 (HSP90, both α and β isoforms). While glucosamine treatment of COCs significantly decreased blastocyst development rate, inhibiting HSP90 with 17-allylamino-17-demethoxygeldanamycin during IVM in the presence of glucosamine recovered blastocyst rates to control levels. This effect was not due to an increase in overall HSP90 levels, since inhibiting HSP90 in control COCs did not affect blastocyst rate. These results suggest O-GlcNacylated HSP90 has an aberrant function in the COC. This study is the first to examine in detail O-GlcNAcylation levels in the COC, and their correlation to oocyte developmental competence. HSP90 was identified as a potential target of O-GlcNAcylation in the COC, and subsequently shown to mediate oocyte developmental competence. This research is significant because of the increasing numbers of women wishing to become pregnant who have high blood glucose levels due to diabetes, obesity or poor diet. I have generated critically needed knowledge towards understanding how these lifestyle factors affect fertility and identifying possible avenues for new therapies.