Activity of key enzymes involved in the energetic metabolism of porcine oocytes during in vitro maturation

ELIZABETH BREININGER; GABRIEL MARTIN ALVAREZ; CYNTHIA GUTNISKY; PABLO DANIEL CETICA

Bologna

Congreso; ESDAR Conference 2013; 2013

ESDAR

Little is known about the metabolic profile of cumulus?oocyte complexes (COCs). Our purpose was to determine the activity of key enzymes of the glycolytic pathway (phosphofructokinase, PFK) and the tricarboxylic acid cycle (isocitrate dehydrogenase, IDH and malate dehydrogenase, MDH) in immature or in vitro matured COCs. COCs were sonicated (4 min, 50%) and centrifuged (17 0009 g, 20 min), being the activity determined in the supernatant. An enzymatic unit (U) was defined as the amount of enzyme necessary to form 1 lmol of fructose 1,6?bisphosphate/min measured as the oxidation of 2 lmoles of NADH/min for PFK or reduction of 1 lmol of NAD/min for IDH and MDH. Enzymatic activity was expressed as mean SEM in U (U/COC) and specific activity (U/mg protein) and compared by Student?s t test. The U in PFK was (2.41 0.36) 10?5 and (2.53 0.33) 10?5, in IDH was (3.56 0.56) 10?6 and (2.67 0.36) 10?6, and in MDH was (4.02 0.69) 10?5 and (3.25 0.19) 10?5 for immature and matured COCs, respectively. The specific activity in PFK was (3.70 0.60) 10?2 and (0.94 0.12) 10?2, in IDH was (6.05 1.28) 10?4 and (1.51 0.20) 10?4, and in MDH was (3.33 1.64) 10?2 and (0.35 0.02) 10?2 for immature and matured COCs, respectively. Since U remained unchanged during in vitro maturation, a significant decrease (p < 0.05) in specific activity was observed, probably due to the increase of protein amount during maturation. These results indicate that the key enzymes involved in the energetic metabolism would be already present in immature oocytes.