Glucose metabolism via the PPP was assessed by measuring the 14CO2 produced from [1-14C]glucose (55 mCi/ mmol; Amersham). [1-14C]Glucose (55 ^Ci/ml; 1 mM) was added to sperm suspensions containing no unlabeled glucose. In addition, some experiments were performed with [6-14C]glucose (55 mCi/mmol; Amersham) to evaluate the oxidation of labeled glucose by the Krebs cycle. buy flovent inhaler
Measurements were performed in a closed incubation system, essentially as described by Rieger et al.. Spermatozoa were incubated with radiolabeled glucose in 3-^l droplets placed on the inner side of the lids of Eppendorf (Hamburg, Germany) tubes previously filled with 1.5 ml of 25 mM NaHCO3, whose function was to trap 3H2O and 14CO2 released from [5-3H]glucose and [1-14C]glucose, respectively. Both M16 medium and NaHCO3 were equilibrated with 5% CO2 in air at 37°C before measurements. As soon as the 3-^l droplets were deposited on the lids, the Eppendorf tubes were closed and incubated at 37°C. Sham preparations, containing no spermatozoa but 3-^l droplets of medium supplemented with radiolabeled glucose, were included in each experiment and for each medium tested.