REDOX and anti - oxidant state

52 Exogenous oocyte secreted factors such as bone morphogenetic protein 15 (BMP15), together 53 with hormones traditionally used during in vitro oocyte maturation, increase the developmental 54 competence of cumulus oocyte complexes (COCs). Separately, FSH and BMP15 induce 55 different metabolic profiles within COCs, namely FSH increases glycolysis while BMP15 56 stimulates FAD and NAD(P)H auto-fluorescence within oocytes, without changing the REDOX 57 ratio. Hence, the aim of this study was to investigate if BMP15-induced increased NAD(P)H 58 was due to NADPH production. Cattle COCs were cultured with FSH and/or recombinant 59 human BMP15 (BMP15). Following culture with BMP15, there was a significant decrease in 60 glucose 6-phosphate dehydrogenase activity (P<0.05). Treatment with an inhibitor of isocitrate 61 dehydrogenase (IDH) decreased NAD(P)H intensity 3-fold in BMP15 treated oocytes, 62 suggesting BMP15 stimulates IDH and NADPH production via the TCA cycle. As NADPH is a 63 reducing agent, reduced glutathione (GSH), H2O2 and mitochondrial activity were measured. 64 FSH alone decreased GSH levels within the oocyte, with the combination of BMP15 and FSH 65 recovering levels. Expression of genes encoding glutathione-reducing enzymes were also 66 lower in oocytes cultured in the presence of FSH. However, BMP15 supplementation 67 promoted mitochondrial localisation patterns consistent with enhanced developmental 68 competence. Metabolomics revealed there was significant consumption of glutamine and 69 production of alanine by COCs +FSH +BMP15 compared to the control (P<0.05). Hence, this 70 study demonstrates that BMP15 supplementation differentially modulates reductive metabolism 71 and mitochondrial localisation within the oocyte. In comparison, FSH-stimulation alone 72 decreases the oocyte’s ability to regulate cellular stress and therefore utilizes other 73 mechanisms to improve developmental competence. 74