Little is known about the metabolic profile of cumulus-oocyte complexes (COCs) during maturation. The aim of this study was to determine the differential participation of enzymatic activity in cumulus cells and the oocyte during in vitro maturation of bovine oocytes, by measuring the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) and lipolysis (lipase). COCs were matured in medium 199 plus 10% (v/v) steer serum for 22-24 h at 39 degrees C in 5% CO(2):95% humidified air. Phosphofructokinase, glucose-6-phosphate dehydrogenase and lipase activities were measured in immature and in vitro matured COCs, denuded oocytes and cumulus cells, respectively. Phosphofructokinase and glucose-6-phosphate dehydrogenase activities (enzymatic units) remained constant during in vitro maturation of COCs, but there was a significant decrease in lipase activity (units) (P < 0.05), as activity in cumulus cells decreased significantly (P < 0.05). For the three enzymes studied, enzyme activity (units) remained unchanged in the oocyte during in vitro maturation. Specific activity increased in the oocyte (P < 0.05) and decreased in cumulus cells as a result of maturation (P < 0.05). In cumulus cells, phosphofructokinase was the most abundant of the three enzymes followed by glucose-6-phosphate dehydrogenase and then lipase (P < 0.05), whereas in the denuded oocyte this order was reversed (P < 0.05). Thus, the metabolism of cumulus cells is adapted to control the flow of metabolites toward the oocyte, which maintains its enzymatic activity even when dissociated from cumulus cells during maturation. The high activity of phosphofructokinase in cumulus cells indicates that glucose is metabolized mainly via the glycolytic pathway in these cells. The greater relative activity of glucose-6-phosphate dehydrogenase recorded in the oocyte indicates that glucose uptake could be directed mainly toward the pentose phosphate pathway. The marked lipolytic activity concentrated in the oocyte indicates an active participation in lipid catabolism during maturation.
Reactive oxygen species (ROS) production is a normal process of cell metabolism. In vitro environments usually increase cell production of ROS, which has been implicated as a main cause of cell damage. Nevertheless, the role of ROS in oocyte in vitro maturation (IVM) is controversial. In most cells, enzymatic antioxidant systems can attenuate the effect of oxidative stress by scavenging ROS. The aim of this work was to determine whether: (1) standard conditions of bovine oocyte IVM are responsible for oxidative stress; (2) cumulus cells participate in protection against oxidative stress of the oocyte; and (3) enzymatic antioxidant activity is present in oocytes and cumulus cells. Cumulus-oocyte complexes (COCs) were matured in TCM-199 + 10% steer serum for 24 h at 39 degrees C in 5% CO2:95% humidified air. Oxidative stress was determined by the 2',7'-dichlorofluorescein diacetate assay. Superoxide dismutase (SOD), glutathione peroxidase, and catalase activities were measured spectrophotometrically. Under standard conditions of in vitro maturation, there was no increase in ROS production per COC (P > 0.05), but ROS level per cumulus cell diminished. There was no modification in ROS levels in oocytes matured in the presence versus the absence of their surrounding cumulus cells ( P > 0.05). To the best of our knowledge, the presence of SOD, glutathione peroxidase and catalase activities were detected in oocytes and cumulus cells for the first time. Enzymatic units were lower in denuded oocytes with respect to cumulus (P < 0.05), accounting for 37% for SOD, 25% for glutathione peroxidase, and 11% for catalase of the total COC units. Specific enzyme activity diminished in cumulus cells (P > 0.05) and increased in oocytes due to maturation (P > 0.05). The presence of activity of an enzymatic antioxidant system in the bovine oocyte would regulate in part ROS levels during IVM. Oocytes could be capable of controlling the increase in ROS because of the presence of their own enzymatic antioxidant system, SOD having the highest specific activity with respect to cumulus cells.
During cumulus-oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-L-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.
Few studies demonstrate at a biochemical level the metabolic profile of both cumulus cells and the oocyte during maturation. The aim of the present study was to investigate the differential participation of enzymatic activity in cumulus cells and in the oocyte during in vitro maturation (IVM) by studying the activity of enzymes involved in the control of amino acid metabolism, alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and the tricarboxylic acid (TCA) cycle, isocitrate dehydrogenase (IDH) and malate dehydrogenase (MDH). No NAD-dependent isocitrate dehydrogenase (NAD-IDH) activity was recorded in cumulus-oocyte complexes (COCs). ALT, AST, NADP-dependent isocitrate dehydrogenase (NADP-IDH) and MDH enzymatic units remained constant in cumulus cells and oocytes during IVM. Specific activities increased in oocytes and decreased in cumuluscells as a result of IVM (P < 0.05). Similar activity of both transaminases was detected in cumulus cells, unlike in the oocyte, in which activity of AST was 4.4 times greater than that of ALT (P < 0.05). High NADP-IDH and MDH activity was detected in the oocyte. Addition of alanine, aspartate, isocitrate + NADP, oxaloacetate or malate + NAD to maturation media increased the percentage of denuded oocytes reaching maturation (P < 0.05), in contrast to COCs in which differences were not observed by addition of these substrates and co-enzymes. The activity of studied enzymes and the use of oxidative substrates denotes a major participation of transaminations and the TCA cycle in the process of gamete maturation. The oocyte thus seems versatile in the use of several oxidative substrates depending on the redox state.
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