Implications of Glycolytic and Pentose Phosphate Pathways on the oxidative status and active mitochondria of the porcine oocyte during in vitro maturation Theriogenology Online

22 23 The gGlycolysis and the pentose phosphate pathway (PPP) were modulated in porcine 24 cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of 25 inhibitors and stimulators of key enzymes of the pathways, with the aim of to analizing 26 analyze their participation influence on the oxidative status, mitochondrial activity and 27 maturation of the oocyte. The influence of Glycolysis and PPP in COCs were evaluated by 28 lactate production-glucose uptake and by the Brilliant Cresyl Blue test, respectively. 29 Oocyte oxidative and mitochondrial activities were evaluated by Redox Sensor Red CC-1 30 and Mito Tracker Green FM, respectively. pharmacological and physiological inhibitors of 31 glycolysis (NaF and ATP) and PPP (6-AN and NADPH) we validating by assessing 32 glucose and lactate turnover and brilliant cresyl blue staining in oocytes. Modulators of 33 glycolysis and PPP activity significantly perturbed nuclear maturation, oxidative 34 metabolism (Redox Sensor Red CC-1) and mitochondrial mass (Mitotracker Green FM) 35 within oocytes (P < 0.05). In comparison,Oocyte nuclear maturation rate and oxidative and 36 mitochondrial activities decreased in the presence of the pharmacological (NaF) or the 37 physiological (ATP) inhibitors of glycolysis (P<0.05).The pharmacological (6-AN) and the 38 physiological (NADPH) inhibitors of PPP induced a decrease in the oocyte nuclear 39 maturation rate and oxidative and mitochondrial activities (P<0.05). The physiological 40 stimulators of glycolysis (AMP) and PPP (NADP) caused nodid not effect on any of 41 evaluated parameter. In the absence of modulators, We found fluctuations in the oocyte 42 oxidative activity and mitochondrial activities mass were observed during porcine IVM. 43 Comment [MM1]: Mitotracker Green measures mitochondria mass (number), independent of membrane potential (MMP), so it actually doesn’t indicate activity. Rosamine based mitotracker dyes (i.e. Mitotracker Red CMX Ros) do measure MMP. Comment [MM2]: Is this right???