133 The activity of metabolic enzymes in bovine oocytes derived from ovaries with heterogenous physiological conditions

One of the key factors for an oocyte’s quality and consequently for embryo quality is the microenvironment where the gamete develops. Metabolic homeostasis between the variable sources of energy substrates plays an important role in oocyte maturation. Therefore, we aimed to determine the abundance and activity of metabolic enzymes; AMPK, PKM2, PDH, LDHA, and LDHC in oocytes derived from ovaries in different physiological phases. Slaughterhouse ovaries were sorted according to the following categories: ovaries with visible cysts (Cystic ovaries), ovaries with an active corpus luteum (CL ovaries), ovaries with a regressing CL and fewer than eight follicles (CL-8 ovaries), and ovaries without CL and with multiple follicles normally selected for IVF (control). A total of three replicates of cumulus-oocyte complexes (COCs; 20 COCs/replicate) were collected for each group. Hyaluronidase was used to remove the cumulus cells. Oocytes were washed twice with PBS-(1%) PVP and lysed in 10 µL of radioimmunpreciptation assay (RIPA) buffer. Samples were subjected to capillary western blotting using the Jess system (Protein Simple). Antibodies against both the total (t) and phosphorylated (p) proteins of the metabolic enzymes were utilised. Total protein abundance in each sample was quantified, and the abundance of every target metabolic protein was normalised against the total protein abundance in the sample. The ratio between t and p was used to calculate enzyme activity. Data were analysed using one-way ANOVA followed by Fisher’s l.s.d. multiple comparisons test. Oocytes of control and cystic ovaries tended to have increased abundance of tAMPK compared to oocytes from CL (P = 0.09) and CL-8 (P = 0.07) ovaries. Similarly, abundance of pAMPK in oocytes from cystic ovaries was significantly (P < 0.05) higher than that of oocytes from both control and CL-8 ovaries and tended (P = 0.08) to be increased compared to oocytes from CL ovaries. Thus, activity of AMPK tended to be lower in control oocytes compared to the other ovarian categories. Abundance of both tPKM2 and pPKM2 tend to be lower in oocytes from control ovaries, while oocytes from CL-8 ovaries have significantly higher (P < 0.05) phosphorylation compared to control. Activity of PKM2 is significantly higher (P < 0.01) in oocytes from CL-8 ovaries compared to the other ovarian categories. This suggests that an alternative pathway, namely aerobic glycolysis or the Warburg effect, is more pronounced in oocytes from cystic and CL-8 ovaries compared to control and CL ovaries There were no differences in the abundance or activity of PDH among oocytes from all ovarian groups. Abundance of LDHC tends to be higher in oocytes from cystic and CL ovaries compared to control (P = 0.19) and CL-8 ovaries (P = 0.16). However, abundance of LDHA was undetectable in all ovarian categories. Oocytes from control ovaries had lower variability in abundance and activity of these metabolic enzymes compared to the other ovarian groups. In conclusion, these results suggest that ovarian physiological status affects metabolic enzyme quantity and activity in bovine oocytes. Differential oocyte metabolism as a result of ovarian environment could impact oocyte quality and may be correlated with subsequent IVF outcome.