Gamete Fusion Assay
Zona-free oocytes were incubated in insemination drops for 20 min to allow sperm binding to the oolemma. They were then transferred into 25-^l droplets of M16 for 45 min to allow bound sperm to enter the oocytes and to de-condense. The oocytes were then washed as described by Wolf and Hamada to remove loosely attached sperm. For assessment of sperm binding to the oolemma, and sperm entry and decondensation, the oocytes were fixed in formaldehyde and stained with Hoechst 33342 (Sigma) as we have previously described. buy yasmin online
To inhibit glucose uptake or glycolysis in sperm, cyto-chalasin B (50 ^M), phloretin (0.5 mM), and iodoacetate (10 ^M) were added to the insemination drops 60 min before addition of the oocytes. Cytochalasin B was also added 30 and 0 min before gamete mixing. When NADPH was used instead of glucose, it was added to sperm suspensions containing no glucose 1 h or 15 min before addition of the oocytes. These different compounds were present in the culture medium until the end of the experiment.
Glucose metabolism was measured in capacitated sperm essentially as described for cattle embryos by Rieger et al.. Measurements were made in M16, a bicarbonate-buffered medium containing pyruvate and lactate that supports both sperm capacitation and fertilization.
Glucose metabolism via glycolysis was measured by collecting 3H2O released from [5-3H]glucose (13.6 Ci/mmol; Amersham Rahn, Zurich, Switzerland). Tracer quantities of [5-3H]glucose (250 ^Ci/ml; about 20 ^M) were added to sperm suspensions supplemented with 1 mM of unlabeled glucose.