Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(10)
Transmission Electron Microscopy (TEM)
For TEM, treated sperm were fixed in 3% glutaraldehyde in 5% sucrose in 0.2 M cacodylate buffer for 3 h at room temperature. The samples were postfixed in 1% OsO4, 5% sucrose, and 0.1% ruthenium red for 2 h at 4°C. The samples were dehydrated in ascending ethanol series and embedded in Spurr’s epoxy. Thin sections (90 nm) were prepared on a Reichert-Jung (Nossloch, Germany) Ultracut E and stained with uranyl acetate and lead citrate. The grids were examined using a JEOL-1200 EX II electron microscope (JEOL, Paris, France) at a accelerating voltage of 60 kV. Sperm were observed under low (X25 000) and high (X100 000) magnification, and representative sperm were photographed. In each specimen evaluated, 100-200 sperm were evaluated for the intactness of sperm head membranes. flovent inhaler
Homologous Sperm-Zona Binding Assay
Specifically, human eggs that failed to fertilize in vitro were separately inseminated with a mixture of equal numbers of SYBR 14 (green)- and SYTO 17 (blue)-labeled sperm, the latter pretreated with three concentrations (25, 50, and 100 ^M) of the tested compounds. After coincubation, the number of tightly bound green (SYBR-14) and blue (SYTO-17) sperm were assessed by confocal laser scanning microscopy.