No fluorescence appeared on the epididymal spermatozoa after they had been immunoreacted successively with the P12 antiserum and the fluorescein-conjugated anti-rabbit IgG, manifesting the lack of P12 on the cell surface. When the cells were preincubated with P12 before cyto-chemical staining, a crescent fluorescence zone on the anterior region of the sperm acrosome was visible (Fig. 6), indicating P12-binding sites on the acrosomal region. antibiotic levaquin
The proportion of epididymal spermatozoa having intact acro-somes was compared to the proportion of epididymal sperm with the P12-binding zone after they had been incubated in a modified Tyrode’s solution according to the previously mentioned study (Table 1). Around 86% and 85% of the fresh cells from caudal epididymis had an intact acro-some and P12-binding zone, respectively. The population of cells with an intact acrosome decreased progressively as long as the incubation proceeded. This was parallel to the decrease in the cell population with the P12-binding zone. At any incubation time, the population of cells with an intact acrosome was nearly equal to that with the P12-bind-ing zone, and the cell morphology showed no visible change in the presence of P12 in the cell incubation, indicating P12-binding sites on intact acrosome.
FIG. 6. Immunolocalization of P12-binding zone on the spermatozoa. Fresh cells were dried on glass slides. Slides were incubated in PBS in the presence of P12 for 30 min. The P12-binding zone on the cell was im-munolocalized by the indirect fluorescence method with P12 antiserum and fluorescein-conjugated anti-rabbit IgG. The slides were observed by a light microscope (A) or a fluorescence microscope (B). Bar = 10 ^m.
TABLE 1. Correlation of intact acrosome and the P12-binding zone on the sperm head treated under several conditions.3
|Incubation time (min)||Additional incubation in P12b (min)||Percentage of cells with intact acrosome||Percentage of cells with P12-binding zone|
|0||—||86 ± 4||3±58|
|60||—||77 ± 8||76 ± 7|
|70||—||74 ± 7||75 ± 6|
|120||—||68 ± 6||70 ± 5|
|60||10||72 ± 6||75 ± 7|
|60||60||67 ± 5||69 ± 6|
a Fresh spermatozoa from caudal epididymis were incubated in modified Tyrode’s solution at 37 °C for cell capacitation (see text for details). b The protein was added to the cell suspension at a final concentration of 1.5 ^M.