Chemicals and Media
The basic culture medium used in all experiments was M16 containing 23.3 mM lactate, 0.33 mM pyruvate, and 5.56 mM glucose, and supplemented with 15 mg/ml type V BSA. Cytochalasin B, phloretin, and iodoacetate were dissolved as stock solutions (1000 X concentrated in dimethyl sulfoxide) and stored at -20°C. Dilutions in culture medium were made before use. All chemicals were obtained from Sigma Pharmaceuticals (Buchs, Switzerland). The antibodies anti-GLUT3 and anti-GLUT1 were kindly provided by Dr. G.W. Gould (University of Glasgow, Scotland) and Dr. B. Thorens (University of Lausanne, Switzerland), respectively. buy cipro
Oocytes were obtained from 3- to 4-wk-old female B6D2F1 mice (IFFA-CREDO, L’Arbresle, France), which received injections of 5 IU eCG (Folligon; Veterinaria, Zurich, Switzerland) followed 48 h later by 5 IU hCG (Chol-uron; Veterinaria). Fourteen to 16 h after administration of hCG, oocytes surrounded by their cumulus cells were collected. After cumulus digestion by 0.2 mg/ml hyaluronidase (Sigma), zonae pellucidae were mechanically removed as we have previously described.