227 A transcriptomic quest to untangle equine oocyte maturation

Changes in the nucleus, cytoplasm, and cumulus during oocyte maturation are directly associated with the developmental competence of this highly specialised cell. Cumulus cells (CC) play a critical role as they communicate with the oocyte, orchestrating these events and providing metabolic support. In vivo maturation (IVV) yields oocytes with higher developmental competence when compared with in vitro maturation (IVM). Additionally, the cumulus expansion observed in IVV is greater than in IVM. Therefore, we aimed to characterise the dynamics of gene expression patterns in equine cumulus cells during IVV and IVM to determine biological pathways that might explain these differences. CC were obtained from cumulus-oocyte complexes (COCs) of immature oocytes (GV; n = 6), in vitro-matured oocytes (IVM; n = 12), and in vivo-matured oocytes (IVV; n = 12). Extracted total RNA from CC was sequenced and an average of 24 million reads/sample was generated. The raw reads were trimmed (Trimmgalore) and mapped (STAR) to the current equine reference genome. Mapped reads were quantified (Featurecount) and compared between groups using the DESEqn 2 package (R software, FDR cutoff 0.1; Min fold change 1.5). We found an average of 15,000 expressed genes. The three groups of samples clustered separated from each other, with IVV samples showing a similar gene expression pattern among the replicates as observed in the principal component analysis. By comparing IVV to GV samples, we found 4,345 differentially expressed genes (DEG) with 1,894 upregulated and 2,451 downregulated in IVV. Associated pathways to downregulated genes were regulation of cell cycle, nuclear division, and cellular response to growth factor stimulus, while upregulated genes were related to metabolic pathways, glycolysis, gluconeogenesis, fructose, and mannose metabolism. Additionally, as opposed to previous findings on IVM, the follicle-stimulating hormone receptor gene (FSHR) showed reduced expression during IVV. When comparing IVM to IVV samples, we found 6,677 DEG (2,187 upregulated and 4,490 downregulated in IVM). Upregulated genes were involved in pathways of chromosome segregation, DNA repair, sister chromatid segregation, DNA replication, mitotic cell cycle process, and nuclear division, while downregulated genes were associated with transmembrane signalling receptors and chemical stimulus detection. Additionally, we found a reduced expression of genes involved in cumulus expansion (HAS2) and different expressions of gonadotrophin receptors (FSHR, LHCGR) in the IVM group. Lastly, to investigate the specific effect of different IVM media, we compared the gene expression patterns of CC from three different maturation media and determined that all CC showed a dissimilar gene expression profile compared with IVV samples. These data provide a fundamental basis to understand the equine oocyte maturation process, which will guide us to optimise IVM conditions by identifying the pathways altered during IVM.