Fluorescent Staining of Fertilized Oocytes

The same staining protocol was used following both IVF experiments mentioned above. After three washings in medium (PBS supplemented with 0.5% Triton-X and 2% fetal bovine serum), non-specific binding sites were blocked in saturation medium (PBS supplemented with 20% fetal calf serum and 0.5% Triton-X) for 1 h at 38.5 °C. Samples were then incubated with Hoechst staining solution (1 µg/mL) for 10 min, at room temperature, before observation under a microscope fitted with epifluorescence (Olympus BX 41). Percentages of fertilized oocytes (presence of 2 pronuclei) and development to the 2-cell stage was recorded at 17 h post-insemination. Spot Basic 5.1 software (Diagnostics Instruments) was used to capture images of oocytes and embryos. As previously reported, higher proportions of 2-cell stage at the 17 hr time point indicated a faster first cell cycle because of functional sperm centrosomes [4 (link),25 (link)].