17 Feb

Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: RESULTS(9)

The mean sperm-zona binding ratio for control eggs coincubated with SYBR 14- and SYTO 17-labeled control sperm was 1.07. In contrast, the mean sperm-zona binding ratios for eggs coincubated with control (SYBR 14) and 25, 50, and 100 ^M WHI-07-treated sperm (SYTO 17) were 0.088, 0.039, and 0.009, respectively. The corresponding mean ratios for sperm treated with 25, 50, and 100 ^M of WHI-05 were 0.18, 0.07, and 0.10, respectively. Figure 6 shows the representative sperm-zona binding patterns of green and blue sperm to a control egg (Fig. 6A) and the test eggs in which the blue sperm were pretreated with either 100 ^M of WHI-05 (Fig. 6B) or 25 ^M of WHI-07 (Fig. 6C) prior to coincubation with green-labeled sperm. Despite the variable number of sperm binding to individual zona, a clear reduction in the number of blue-colored sperm bound to test eggs is apparent in comparison to control eggs. flovent inhaler

Inhibitory Effects of WHI-05 and WHI-07 on Heterologous Sperm-Zona-Free Hamster Egg Binding and Penetration Assay

The heterologous zona-free hamster egg penetration assay appears to correlate with the in vivo fertilizing capacity of human sperm as well as the ability of human sperm to fertilize intact human eggs in vitro. Therefore, the inhibitory effects of WHI-05 and WHI-07 on sperm-egg interaction were further confirmed using the zona-free hamster egg penetration assay. As expected, preincubation of capacitated sperm with either compound resulted in significant inhibition (p < 0.001) of sperm binding to zona-free eggs (Table 2). Similarly, the penetration rate of human sperm to zona-free hamster eggs following exposure to increasing concentrations of WHI-05 or WHI-07 showed marked inhibition (41-100% inhibition when compared with control). Thus, pretreatment of human sperm with spermicidal aryl phosphate derivatives of bromo-methoxy-AZT resulted in a concentration-dependent loss of the ability to bind and penetrate zona-free hamster eggs as well as inhibition of binding to human eggs.
Fig6Aryl Phosphate Derivatives
FIG. 6. Laser scanning confocal fluorescence images of two-color human sperm-zona interaction. Motile sperm were labeled separately with cell-permeant DNA-specific dyes, SYBR 14 (green) or SYTO 17 (blue), combined in equal proportion and coincubated with zona-intact human eggs with or without prior treatment of SYTO 1 7-labeled sperm with 25, 50, and 100 ^M of WHI-05 or WHI-07. A) Binding of two-colored untreated control sperm (SYBR 14 + SYTO 17) to human zona of a control egg. The zona pellucida is not visible with the fluorescent optics. B) Binding of SYBR 14-labeled untreated and SYTO 17-labeled WHI-05-treated (100 ^M) sperm to human zona of a test egg. C) Binding of SYBR 14-labeled untreated and SYTO 17-labeled WHI-07-treated (25 ^M) sperm to human zona of a test egg. Note the binding of both green and blue sperm to control egg and the predominance of green sperm binding to test eggs. The number of sperm bound to individual zona is variable (original magnification X200).

TABLE 2. Inhibitory effects of WHI-05 and WHI-07 on human sperm binding and penetration of zona-free hamster eggs.

Treatment Zona-free hamster eggs
Binding” Penetrationbc (%)
None 15.0 ± 14.8 68/121
25 ^M 5.6 ± 7.0d 7/28
100 0.1 ± 0.4d 0/15
25 ^M 4.1 ± 7.3d 11/88
100 ^M 5.8 ± 7.0d 10/59

a Expressed as the average number (mean ± SD) of tightly bound sperm per egg.
b A hamster egg was considered penetrated if there were one or more decondensed sperm heads in egg cytoplasm.
c The fraction represents the number of eggs penetrated over the total number of eggs inseminated. dp < 0.001.

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