Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: RESULTS(7)
Examination of FITC-lectin-, TOTO-3-, and Nile red-stained sperm by confocal microscopy revealed an intense acrosomal staining with FITC-lectin (green), nuclear staining with TOTO-3 (blue), and membrane staining (red) with Nile red, respectively (Fig. 5, upper panels). In acrosome-intact sperm, more than half of the sperm head (the acrosomal region) exhibited a uniform, bright green fluorescence in sperm exposed to vehicle (i.e., 1% DMSO) alone (Fig. 5A), 100 |xM WHI-05 (Fig. 5B), and 100 |xM WHI-07 (Fig. 5C) for 3 h. By comparison, in sperm exposed to 100 ^M of N-9 for 3 h under identical conditions, green fluorescence was absent due to disruption of membrane integrity and acrosomal loss (Fig. 5D), consistent with previous SEM observations. Thus, the spermicidal activity of the dual-function AZT derivatives was not accompanied by a loss of membrane integrity. canadian health&care mall
Topographical imaging of drug-treated sperm heads by HR-LVSEM revealed intact acrosomes (Fig. 5, middle panels) with smooth contiguous surfaces in sperm exposed to vehicle (Fig. 5A’) or 100 ^M WHI-05 (Fig. 5B’), whereas sperm treated with WHI-07 (Fig. 5C’) revealed signs of a mild acrosomal membrane ruffling. By comparison, sperm exposed to 10 ^M CaI A23187, as a positive control, revealed characteristic blebbing or vesiculation, fenestration, and loss of plasma and acrosomal membranes (Fig. 5D’).
FIG. 5. Upper panels) Laser scanning confocal fluorescence images of sperm. Triple labeling of sperm with FITC-Pisum sativum lectin for acro-some (green), TOTO-3 iodide for DNA (blue), and Nile red for membrane lipid (red) incubated in the absence (A) and presence of WHI-05 (B), WHI-07 (C), and N-9 (D) for 3 h. An intense acrosomal staining with FITC-lectin (green), nuclear staining with TOTO-3 (blue), and plasma membrane staining (red) of the sperm tail region with Nile red are apparent. In acrosome-intact sperm, the acrosomal region of the sperm heads exhibited a uniform, bright green fluorescence. In acrosome-reacted sperm, green fluorescence was either absent or restricted to the equatorial segment of the sperm heads. Sperm exposed to 1% DMSO alone (A), WHI-05 (B), and WHI-07 (C) did not reveal increased acrosome reaction at 3 h of incubation. Sperm exposed to 100 ^M of N-9 under identical conditions revealed only acrosome-reacted sperm (D) (original magnification X1000). Middle panels) High-resolution low-voltage scanning electron micrographs of sperm incubated in the absence (A’) and presence of 100 ^M each of WHI-05 (B’), WHI-07 (C’), and 10 ^M CaI (D’) for 3 h (X18 000 magnification). Postfixation with OsO4 and ruthenium red yielded very smooth plasma membrane over the acrosome-intact sperm head. The smooth acrosomal surface is delimited from the postacrosomal region by a equatorial band (A’). Sperm exposed for 3 h to WHI-05 and WHI-07, respectively, reveal intact acrosome with various degrees of ruffling of the plasma membrane. CaI-treated sperm reveal vesiculation, blebbing, and loss of the plasma and acrosomal membranes and well-preserved postacrosomal membrane. Bottom panels) Transmission electron micrographs of human sperm incubated in the absence (A”) and presence of 100 each of WHI-05 (B”), WHI-07 (C”), or 10 ^M CaI (D”) for 3 h (X18 000 magnification). The plasma membrane is present over the sperm head. Both acrosomal and postacrosomal membranes are clearly visible after 3 h of incubation with WHI-05 and WHI-07. Note the complete loss of acrosome in CaI-treated sperm (D”). Figure reproduced at 84% of original.