Intracellular communication between the cumulus cell complex and the oocyte is essential for numerous processes during oocyte maturation. The aim of this study was to determine the interaction between oocyte-secreted factors and the metabolic activity of bovine cumulus cell complexes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were aspirated from ovaries derived from an abattoir and divided into four treatment groups: (i) intact COCs, (ii) oocytectomized complexes (OOX), in which the ooplasm was microsurgically removed, (iii) OOX co-cultured with denuded oocytes (OOX + DO) and (iv) DO. The complexes were cultured individually in IVM media. After 0-4, 10-14 and 20-24 h of culture, the utilization of oxygen, glucose, pyruvate and L-lactate by the complexes was measured. The metabolic activity of the DO was undetectable. There were no significant differences in metabolic measurement among any of the treatment groups, indicating that the metabolism of the cumulus complex is not affected by the presence of the oocyte. When metabolic activity for the complexes was analysed relative to time in culture, there was an approximate twofold increase in the consumption of oxygen, glucose and pyruvate over the 24 h period (P < 0.05), although production of L-lactate remained constant. The relationship between total glucose uptake and L-lactate production indicated that the majority of glucose consumed at the start of culture was being utilized via glycolysis, but by the cessation of the maturation period, there was significant utilization of glucose elsewhere, possibly for the formation of cumulus extracellular matrix. These results indicate that metabolism of COC does not reflect biochemical activity of the oocyte. Nevertheless, the metabolic requirements of the COC increase throughout maturation.

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.

In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.

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.