Molecular mechanisms contributing to the age-related decline in oocyte quality

Maternal ageing is accompanied by a precipitous decline in oocyte quality, culminating in increased rates of congenital disabilities, miscarriage, and ultimately, declining fertility. Indeed, the incidence of chromosome separation errors is significantly increased in the oocytes of women after the age of 35. The aetiology behind this age-related decline in oocyte quality is undoubtedly complex and multifactorial. Thus, while the consequences accompanying oocyte ageing are well defined, the molecular mechanisms underlying the loss of oocyte quality are still far from being resolved. Accordingly, the studies described in this thesis were formulated to explore mechanisms that could contribute to the demise of functionality of the aged oocyte. A primary focus for these studies was investigation of the impact of oxidative stress on protein homeostasis in the aged oocyte. Accordingly, we first explored the extent to which reactive lipid aldehydes, generated as a by-product of oxidative stress, are able to adduct and modify key elements of the oocyte proteome. Through the course of these studies, we confirmed that oocyte ageing is accompanied by a significant elevation in the dominant lipid-derived aldehyde, 4-hydroxynonenal (4-HNE). Moreover, we were able to correlate 4-HNE accumulation with impaired oocyte quality, both in the context of naturally aged oocytes and those subjected to an acute 4-HNE exposure model. Illustrative of this damage, oocytes challenged with 4-HNE were characterised by a significant increase in 4-HNE adduction of tubulin proteins, chromosomal misalignments, meiotic spindle abnormalities, and increased rates of aneuploidy. Compounding this situation, we also showed that the fidelity of the mechanisms that an oocyte can employ to mitigate the impact of 4-HNE damage becomes severely compromised in aged oocytes. Thus, the oocyte’s proteasomal machinery is itself vulnerable to oxidative damage, with several components of the core proteasome being identified as targets for 4-HNE modification. This situation leads to a loss of proteasome activity, which retards the clearance of alternate 4-HNE adducted proteins (including tubulin) and enables them to accumulate to detrimental levels within the oocyte. In addition to compromising enzymatic activity, oxidative damage to the oocyte’s DNA and stored RNA also has the potential to influence protein homeostasis via dysregulation of gene expression. In view of this potential, we also elected to explore the expression profile of small non-protein-coding RNAs (sncRNA), which regulate mRNA stability and transcription efficiency, within young and aged oocytes. These studies build on an established body of evidence that altered profiles of gene expression within aged oocytes is associated with the induction of aneuploidy. Here, we demonstrated that aged oocytes are characterised by altered expression profiles of several classes of sncRNA, including the endogenous-siRNAs (endo-siRNAs) and microRNAs (miRNAs). Notably, kinesin family members C1, (Kifc1) and C5B (Kifc5b), which have been implicated in meiotic division, were identified as putative targets of multiple dysregulated endo-siRNA. Accordingly, these kinesin family members were, in turn, downregulated at both an mRNA transcript and protein level in aged oocytes. Importantly, endo-siRNA-mediated knock down of Kifc1 and Kifc5b, as well as pharmacological inhibition of these proteins, increased the age-associated phenotype of elevated aneuploidy. Collectively, the findings reported in this thesis enhance our understanding of the mechanisms contributing to the age-related decrease in oocyte quality. This body of work also serves to emphasise the multifactorial and complex nature of oocyte ageing, information that may aid future goals of developing therapeutic interventions to counter the deterioration of the aged oocyte.