Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(11)
Frozen human eggs (n = 46) were thawed and rinsed three times in BWW-0.3% BSA, and then three times in BWW-3.5% BSA, before addition of sperm suspension. The process of egg recovery, freezing, thawing, and manipulation in vitro invariably resulted in cumulus-free, non-viable eggs. To investigate the effect of WHI-05 and WHI-07 on the binding of human sperm to human zona, the motile fraction of capacitated sperm was divided into two aliquots of 107/ml and labeled with two cell-permeant DNA-specific dyes (SYBR-14 and SYTO-17). The first aliquot was incubated with 2.5 ^M SYBR-14 (Molecular Probes), and the second was incubated with 5 ^M SYTO-17. After 30 min of incubation, sperm motility was assessed by CASA. Motile sperm with intensely stained green or blue nuclei were washed thrice in BWW, and one million motile sperm labeled with SYBR-14 (green; control) and SYTO-17 (blue; control or test) were added to a dish containing human zona (n = 2) under mineral oil and coincubated for 4 h. ventolin inhalers
After extensive washing of eggs to dislodge any loosely adherent sperm, the eggs were mounted on glass slides and examined under a Bio-Rad MRC-1024 Laser Scanning Confocal Microscope as described above. Using fluorescence imaging, the fluorescence emission of SYBR-14 and SYTO-17 localized on the sperm head was simultaneously recorded using 598/40 nm and 680 DF32 emission/filter, respectively. Confocal images of Z-sections were obtained using a Nikon X20 (NA 1.4) objective and Kalman collection filter. Digitized images were processed as described above. The number of tightly bound green-and blue-colored sperm nuclei adherent to each human egg was counted from the Z-sections of fluorescence images of each egg on all focal planes and was expressed as a binding ratio.