Spermicidal Activity of Oxovanadium: RESULTS(6)
Analysis by flow cytometry of the mitochondrial membrane potential changes occurring during apoptosis were analyzed with a A^m indicator, JC-1, a carbocyanine cationic dye, by following fluorescence associated with the uptake of JC-1 to evaluate A^m modifications. Motile sperm exhibited intense green and red fluorescence of JC-1 (Fig. 4A). It can be seen that a 3-h treatment with the oxovanadium(IV) complex VO(Cl-phen)2 resulted in an extinction of the red fluorescence (Fig. 4B), indicating that alteration occurred after VO(Cl-phen)2 treatment. A 3-h pretreatment of sperm with 7 of the 11 oxovanadium(IV) complexes resulted in variable decreases in A^m-related fluorescence observed as 31-73% reduction (p < 0.05) in JC-1 aggregate (orange/ green) fluorescence without concomitant reduction in JC-1 monomer (green) fluorescence (Table 3). flovent inhaler
By contrast, > 90% of control sperm were positive for orange/red fluorescence. The most potent spermicidal agents, VO(Cl-phen)2 and VO(phen)2, induced the maximum shift. Therefore, A^m modifications, evaluated by the uptake of cationic lipophilic dye, were detected early in the process of apop-tosis induced by oxovanadium(IV) complexes.
FIG. 4. Flow cytometric quantitation of oxovanadium(IV) complex-induced apoptotic sperm. Motile sperm were incubated at 37°C in either control medium (0.1% DMSO) or medium supplemented with 100 ^M of a representative oxovana-dium(IV) complex, VO(Cl-phen)2. The apo-ptosis-inducing ability of VO(Cl-phen)2 (right panels) in comparison with medium control (left panels) was tested by three flow cytometric assays that quantitatively assess changes of the mitochondrial membrane potential, based on JC-1 staining (A, B); surface plasma membrane, based on FITC-Annexin V-staining (C, D); and sperm nuclear compartment, based on FITC-dUTP nick-end labeling of fragmented DNA (E, F) after 3, 12, and 24 h, respectively. Note the marked reduction in JC-1 red fluorescence (aggregates) labeling with no reduction in green emission (monomers). In C-F, sperm nuclei were counter-stained with PI.
TABLE 3. Apoptosis-inducing property of spermicidal oxovanadium(IV) complexes containing mono- and b/s-1,10-phanthroline, 2,2‘-bipyridyl and 5′-bromo-2’-hydroxyacetophenone and derivatives.
|Treatment DMSO control||.aggposper92||C-resitm+||1ate-ive(%)ab3||AnV-psper7||neosm+||xinitive(%)ab3||TUposper9||JNsitm+||EL-ive(%)ab1|
a Motile sperm were incubated at 37°C for 3, 12, and 24 h in either control medium or in medium supplemented with 100 ^M each of the 11 oxovanadium(IV) complexes; stained respectively with JC-1, FITC-An-nexin V, or FITC-dUTP; and analyzed by flow cytometry. b Mean ± SD for two separate experiments using sperm from different donors.