Spermicidal Activity of Oxovanadium: RESULTS(8)
Next, TdT-mediated labeling of exposed 3′-OH termini of nuclear DNA with FITC-conjugated dUTP by the in situ TUNEL method was employed to demonstrate that oxo-vanadium(IV) complexes induced apoptosis in the sperm nuclear compartment. Figure 4, E and F, depicts the two-color flow cytometric contour plots of sperm nuclei of control sperm (E) treated with 0.1% DMSO and test sperm (F), respectively, treated with 100 ^M of VO(Cl-phen)2 in 0.1% DMSO after staining with FITC-dUTP and counterstaining with PI. More than 97% of VO(Cl-phen)2-treated sperm became apoptotic (TUNEL-positive) after 24 h of incubation (Fig. 4F). A 24-h exposure of sperm with any one of the 11 oxovanadium(IV) complexes evaluated resulted in a marked increase of TUNEL-positive cells, observed as a 43-98% (p < 0.05) increase in FITC-dUTP fluorescence (Table 3). buy ventolin inhalers
By contrast, < 10% of control sperm treated with 0.1% DMSO alone showed apoptotic nuclei after 24 h of incubation. The percentages of apoptotic sperm quantitated by the flow cytometric TUNEL assay correlated well with the potency (EC50 values) of these oxovanadium(IV) complexes in sperm immobilization assays (r2 = 0.557; p < 0.05). Figure 5 depicts confocal microscopy images of sperm nuclei treated with 100 ^M VO(Cl-phen)2 in 0.1% DMSO after incubation with TdT and FITC-dUTP with (A and C) and without (B) PI counterstaining. Confocal images of TUNEL-positive sperm clearly indicated that the fluorescence was localized to the sperm nuclear region. Nuclei of VO(Cl-phen)2-treated sperm showed dual fluorescence (C) consistent with apoptosis.
FIG. 5. Confocal laser scanning microscopy images of sperm nuclei undergoing oxovanadium-induced apoptosis. Motile sperm were incubated for 24 h in medium with 100 ^M VO(Cl-phen)2, fixed, permeabilized, and visualized for DNA degradation in aTUNEL assay. A) Sperm nuclei counterstained with PI (red). B) Sperm nuclei visualized for FITC-dUTP incorporation (green). C) Nuclei of VO(Cl-phen)2-treated sperm show dual fluorescence. Apoptotic nuclei appear yellow because of superimposed labels. Original magnification X1000 (reproduced at 89%).