Cytoplasmic and oxidative activity consequences of glycolysis stimulation in bovine cumulus-oocyte complexes during in vitro maturation

CYNTHIA GUTNISKY; SERGIO MORADO; GABRIEL DALVIT; JEREMY THOMPSON; PABLO CETICA

Lima

Workshop; V International Campus course on Diagnostic, Therapeutic and Preservation Techniques in Reproduction; 2012

Glucose is necessary for cumulus-oocyte complex (COC) in vitro maturation (IVM); its catabolism by glycolysis within cumulus cells produces ATP and substrates which can be subsequently oxidized by the oocyte. In somatic cells, the glycolytic pathway is positively regulated by AMP by stimulation of phosphofructokinase 1. The intermediary metabolism of glucose also produces NADH which, besides being used for oxidative phosphorilation and reductive synthesis, could also regulate redox state. This work studied the effect of the stimulation of glycolysis during IVM on glucose uptake and lactate production of COCs and on the nuclear and cytoplasmic maturation of the oocyte. A further aim was to determine the effect of stimulation of COC glycolysis on oocyte oxidative and mitochondrial activities. Glucose uptake and lactate production from spent media was measured by spectrophotometric assays. COCs were matured individually in medium 199 + 5% FBS with or without gonadotropic supplementation and AMP (0, 1, 10, 20 and 40 mM). After 22h of maturation, COCs were removed from culture to evaluate the rate of meiotic completion to the metaphase II chromosome configuration. To asses cytoplasmic maturation, IVM was carried out as described above with gonadotropins (positive control), without supplementation of gonadotropins (negative control) and only supplemented with AMP 10 mM. After 21h of maturation, in vitro fertilization was performed. The cytoplasmic maturation was evaluated by the ratio of cleaved embryos. In order to study the oxidative and mitochondrial activities of the oocytes, groups of 50 COCs were cultured with 10 mM AMP. The oxidative and mitochondrial activities of the oocytes were measured at 4 different time points (0, 9, 15, 22h). Oocytes were stained with the fluorescent probes Redox Sensor Red CC-1 and MitoTracker Green FM and observed by laser scanning confocal microscopy. The images were analysed using Photoshop 9.0 software. In the presence of FSH+LH, COCs matured with AMP had no effect on glucose uptake and lactate production. However, in the absence of gonadotropins a dose dependent stimulatory effect was observed (p