Ovaries were placed in 1 ml М2 medium in a 30-mm Petri dish (Falcon, Becton & Dickinson, Oxford, UK) under a stereomicroscope (X30), and their antral follicles were punctured with a thin and sharp pulled sterile Pasteur pipette until oocytes were released into the medium. Those oocytes that still showed the presence of follicle cells surrounding the zona pellucida were gently pipetted through a narrow Pasteur pipette. Only oocytes free of follicle cells and with a diameter > 70 |xm were used in further experiments. Oocytes showing signs of degeneration were discarded. flovent inhaler
Oocyte Classification, Culture, and Fertilization
Cumulus-free oocytes were individually transferred into a 7.5-jjl1 drop of М2 medium containing 0.05 (xg/ml Hoechst 33342, overlaid with mineral oil, and incubated for 15 min at 37°C in 5% C02 in air. After staining, oocytes were classified into NSN or SN groups under an inverted fluorescence microscope (Leitz, Diavert; X300 or X500; UG1 Schott-beamsplitter excitation filter and A Leitz emission block), where they were exposed to fluorescence irradiation for no more than 3 sec. Oocytes were washed 34 times in М2 medium, then washed for 10 min in 200 ml a-MEM prewarmed at 37°C, and subsequently transferred, in groups of 20, into culture drops of a-MEM. Fifteen h (eCG-hCG experiments) or 24 h (eCG-only experiments) later, all the oocytes were prewashed in Wt medium and transferred into Wt medium fertilization drops containing capacitated sperm. After isolation, a control sample of 1015 oocytes for each experiment was neither stained nor classified but instead transferred into either Wt medium or a-MEM, cultured, and subsequently fertilized in parallel to Hoechst-treated oocytes.