Abstract
Reproductive success declines with maternal age, largely due to the accumulation of errors in oocyte development. As women age, repeated cycles of folliculogenesis lead to diminished negative feedback on follicle-stimulating hormone (FSH), promoting the recruitment of lower-quality follicles. Superovulation, which artificially increases FSH levels, has been linked to reduced mitochondrial activity in oocytes, suggesting a potential similarity between aged and superovulated oocytes. To explore this relationship, this study quantified mitochondrial activity in aged oocytes.
During my honours research, inconsistencies were observed in how the fluorescent dyes Tetramethylrhodamine Methyl Ester (TMRM) and MitoTracker Green (MG) bound to oocyte mitochondria. Therefore, this project first examined the binding properties of these dyes to ensure reliable mitochondrial activity measurements. Three experiments were conducted to investigate this; (1) sequential addition of TMRM followed by MG and vice versa to test labelling interference, (2) FCCP a mitochondrial membrane depolariser was used to test if TMRM and MG fluorescence was dependent on active oxidative phosphorylation and (3) transmission electron microscopy to test if dye localisation was consistent with mitochondrial location. Results confirmed that TMRM accurately indicated mitochondrial activity, binding to regions where mitochondria were present. However, MG does not bind in all locations where mitochondria were confirmed to be, which challenges the understanding of MG binding characteristics.
Using TMRM as a marker, mitochondrial activity was tested in oocytes from aged mice. The experiment examined 58 oocytes from mice aged 10 months, and 47 oocytes from mice aged between 2-3 months. Mice were monitored through their oestrous cycles, and naturally ovulated oocytes were collected. Oocytes were stained with MitoTracker Green, TMRM, and Hoechst Blue and fluorescence was quantified using ImageJ revealing significantly higher mitochondrial activity in aged oocytes compared to younger counterparts. This finding contradicts the initial hypothesis that aged and superovulated oocytes would exhibit similar mitochondrial activity, suggesting that FSH elevation may have distinct effects depending on oocyte age. The increased mitochondrial activity in aged oocytes may reflect compensatory mechanisms addressing accumulated cellular damage.