P-172 Mitochondria are dysregulated in poor quality oocytes and can be targeted in vitro to alter oocyte quality

Abstract Study question Does mitochondrial gene expression differ between good and poor quality oocytes and can mitochondria be targeted in vitro to alter oocyte quality? Summary answer Mitochondrial-related genes are dysregulated in poor quality oocytes and mitochondrial inhibitors can be used during IVM to alter oocyte quality. What is known already Good oocyte quality is essential for successful pregnancy yet we still have a poor understanding of the optimal composition of a good egg. Mitochondrial number and activity are important for oocyte quality and during maturation the ultrastructure of the mitochondria changes to a hooded or ring form. We have observed differences in the timing of this morphological change between good and poor quality oocytes in a sheep model. Oligomycin treatment of oocytes decreases the proportion of hooded mitochondria and increases mitochondrial membrane potential (MMP) indicating that hooded mitochondria may form during maturation to reduce mitochondrial activity prior to fertilisation. Study design, size, duration Gene expression differences were compared between poorer quality peripubertal lamb oocytes and adult oocytes harvested during estrus from pools of ∼80 abattoir-derived ovaries on three separate dates for each age. Denuded oocytes (GV stage) were frozen in three pools of ∼20 per age and total RNA isolated. Sheep oocytes (∼30 per group) were treated with oligomycin, FCCP or vehicle control in final 2 hours of IVM on five separate dates, fertilised and cultured. Participants/materials, setting, methods RNA sequencing cleaned reads were mapped using HISAT2 on sheep genome version ARS-UIRambᵥ2. Count matrix were generated using featureCount and differential gene expression was performed using DESeq2. After normalisation, Wald test was used to define genes differentially expressed. Genes were considered significantly altered with a FDR 5% after Benjamini-Hochberg correction. Mean cleavage rate was compared to vehicle control in five replicate experiments using one-way ANOVA with Benjamini, Krieger and Yekutieli FDR 5%. Main results and the role of chance Oligomycin (ATP synthase inhibitor) decreased cleavage rate compared to control (P 0. 001) while FCCP (electron transport chain uncoupler) had no effect. There were 163 genes up-regulated and 194 genes down-regulated in lamb GV oocytes compared to adult GV stage (FDR 5%). Metascape pathway analysis of the up-regulated genes in lamb oocytes showed the most enriched pathway was Thermogenesis which included these genes: ACOT9, AJUBA, ATP5ME, ATP5MF, CALML4, CREB3L3, CRPPA, DNAJC15, DPM3, FMC1, LDHA, NDUFA12, NDUFAF2, NDUFS5, NT5C, PHGDH, POLR2G, PPARG, PRPS1L1, SDHAF3, SLIRP, TIMM8B and UBB. Six of these up-regulated genes were in the top 20 most highly expressed genes. Thermogenesis is the process by which the mitochondrial electron transport chain becomes uncoupled to dissipate heat and may also control production of free radicals by mitochondria and their response to oxidants. These genes and other mitochondrial related genes that were up-regulated in lamb GV oocytes (CKS2, DCN, PCP4L1, SLC51B) are involved in formation of the electron transport chain complexes, oxidative phosphorylation, ATP synthesis, mitochondrial organization and regulation of membrane potential. Together these results indicate that mitochondrial function is dysregulated in poorer quality oocytes and that treatment with mitochondrial inhibitors during IVM can alter oocyte quality. Limitations, reasons for caution This study was carried out in sheep oocytes and results would need to be confirmed in human oocytes if possible. Further research is needed to determine if decreasing MMP can improve oocyte quality and embryo development. Wider implications of the findings This animal model study indicates that mitochondria may be targeted during IVM to alter oocyte quality and this knowledge could be applied to develop ways of increasing human IVM and ART success rates. Trial registration number not applicable