132 Abundance and activity of metabolic enzymes in bovine cumulus cells derived from ovarian samples under variable physiological conditions

Cumulus cells (CC) play an important role in oocyte growth, maturation, and subsequent fertilisation and can be used to evaluate oocyte quality. Knowledge of CC metabolism would inform our understanding of the interaction of these cells with the enclosed oocyte and their influence on oocyte quality. Therefore, we aimed to determine the abundance and activity of enzymes involved in key points of cellular metabolism: AMPK, PKM2, PDH, LDHA, and LDHC in CC derived from ovaries with different physiological states. Abattoir ovaries were sorted into four categories: ovaries with a cyst (Cystic ovaries), ovaries with an active corpus luteum (CL ovaries), ovaries with a regressing CL and with fewer than eight small antral follicles (CL-8 ovaries), and ovaries with more than eight antral follicles and without active CL (control). A total of three replicates of cumulus-oocyte complexes (COCs; 20 COCs/replicate) were collected. The CC were separated from the oocytes using hyaluronidase and washed twice with PBS-(1%) PVA and then lysed in 20 µL of radioimmunoprecipitation assay (RIPA) buffer. Samples were subjected to capillary western blotting using the Jess system (Protein Simple). Antibodies against both total (t) and phosphorylated (p) proteins of the metabolic enzymes were used. The activity of the enzymes was determined by calculating the ratio of p to t. PDH was used to normalise the abundance of the target proteins. Data were analysed using one-way ANOVA followed by Fisher’s l.s.d. multiple comparisons test. The CC from control ovaries had a significantly higher abundance of tPKM2 compared with CC from cystic ovaries (P < 0.05) and tended to have increased tPKM2 compared to CC from CL-8 ovaries (P = 0.06). The abundance of tAMPK in CC from CL ovaries tended to be higher (P = 0.10) compared to CC from control ovaries. No other differences were detected in the abundance of tPKM2 and pPKM2. The activity of PKM2 was lower in CC from control ovaries compared to CC from cystic, CL, and CL-8 ovaries. This suggests the utilisation of aerobic glycolysis in CC from these ovarian categories. Phosphorylation of PDH tended (P = 0.08) to be higher in CC from CL ovaries compared to CC from control ovaries. The inhibitory phosphorylation of PDH could be associated with the decline of pyruvate metabolism into the Krebs cycle. Taken together, the current results suggest that the abundance and activity of metabolic enzymes in CCs result in variable physiological ovarian environments, which could differentially affect oocyte quality and subsequent IVF outcome.