127 Temporal regulation of molecular pathways, metabolic enzymes and growth factor receptors during bovine oocyte maturation in vitro

Abstract

Although great efforts have been made to improve in vitro oocyte maturation (IVM) medium, we have yet to achieve competence equivalent to in vivo-matured oocytes. The failure in development of culture conditions for IVM yielding high quality eggs is attributed to an incomplete understanding of molecular pathways regulating oocyte and cumulus cell metabolism. The objective of the present study was to characterise the expression and functional activity of cell signalling pathways (mTOR, AKT, 4EBP1, ERK1/2), metabolic enzymes (PKM2, PDH, LDHA, AMPK), and growth factor receptors (IGF1R, IGFIIR, EGFR, FGFR1) in bovine oocytes and cumulus cells before and after in vitro maturation. In vitro-derived cumulus-oocyte complexes were collected at germinal vesicle (GV) and metaphase II (MII) stages (20 cumulus-oocyte complexes per stage; n = 3 replicates) and subjected separately to Western blot analysis using antibodies against both phosphorylated (p) and total (t) protein abundance; the ratio p:t was used to determine the activity of each pathway. Results demonstrate increased (P < 0.05) mTOR and ERK1/2 signalling, with no change in AKT and 4EBP1 activity, in oocytes during IVM. We observed increased (P < 0.05) abundance of oocyte t-ERK from the GV to MII stage, but total expression of AKT, mTOR and 4EBP1 did not change. In cumulus cells, there was a significant (P < 0.05) reduction in mTOR and 4EBP1, an increase in AKT, and no significant change in ERK activity. Analysis of metabolic enzymes in oocytes demonstrated increased (P < 0.05) PDH, reduced AMPK, and unchanged PKM2 and LDHA phosphorylation during IVM. However, increased expression of t-PKM2 abundance was observed from the GV to MII stage. In cumulus cells, tAMPK abundance was reduced (P > 0.05), but no significant change was observed in the activity of other metabolic enzymes analysed during IVM. Finally, we observed abundant expression of IGF2R in the oocyte compared with other growth factor receptors analysed, although IGF2R was significantly (P < 0.05) reduced from GV to MII oocytes. In cumulus cells, both IGF1R and IGF2R were highly abundant compared with EGFR and FGFR but did not change during IVM. Data were analysed using one-way ANOVA. Results suggest that regulatory mechanisms including AKT/mTOR/4EBP1 and ERK are entirely different in oocytes and cumulus cells during maturation. An increase in the inhibitory phosphorylation of oocyte PDH ^(S293) toward the end of maturation suggests low metabolism of pyruvate via the Krebs cycle at that time. Similarly, dephosphorylation of AMPK ^(T172) suggests reduced AMPK activity and reduced fatty acid oxidation in mature oocytes. In addition, temporal regulation of IGF1R in the oocyte and EGFR in cumulus cells suggests an important role for these growth factor receptors during maturation and that these growth factors could be used to improve IVM medium in the bovine. Collectively, these results increase our understanding of the molecular pathways regulating oocyte metabolism during maturation and provide a strategy to improve the IVM environment for assisted reproductive technology.