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a Drug Discovery Program,
b Departments of Reproductive Biology and
c Chemistry, Hughes Institute, St. Paul, Minnesota 55113
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human sperm are exquisitely sensitive to oxidative stress. This is due to the high content of polyunsaturated fatty acids in their cell membranes, the low levels of cytoplasmic enzymes for scavenging the ROS, which initiate lipid peroxidation, and the reduced activity of repair enzymes to recover from oxidative damage [14–17]. ROS such as hydrogen peroxide (H2O2) and hydroxyl radicals (OH;tb) affect sperm motility by peroxidation of membrane lipids and proteins [14, 15, 18, 19]. Oxidative damage to sperm proteins, carbohydrates, and DNA is an important pathophysiological mechanism in the onset of male infertility [18, 20, 21]. Superoxide radicals generated by the action of xanthine oxidase exert a direct, suppressive effect on sperm function, leading to loss of motility, impaired capacitation, and poor sperm-egg interaction [22].
Because of the ability of vanadium(IV)-containing complexes to catalyze the generation of ROS, we synthesized diverse organovanadium and oxovanadium complexes with different ligands linked to the central vanadium(IV) atom by carbon, nitrogen, or oxygen atoms. Our recent studies on 12 monodentate diacido- and 7 bidentate-coordinated complexes of bis(cyclopentadienyl)vanadium(IV) demonstrated that these vanadocenes have potent spermicidal and apoptosis-inducing properties against human sperm [23–26]. In fact, very short (< 1 min) exposure to vanadocenes at nanomolar to micromolar concentrations was sufficient to induce complete sperm motility loss, whereas prolonged exposure of sperm to millimolar concentrations of inorganic vanadium (oxidation state IV and V) salts had no effect on sperm motility [24]. Furthermore, none of the other metallocene dichloro complexes of oxidation state IV containing titanium, zirconium, molybdenum, or hafnium exhibited spermicidal activity [24].
Since the redox potential and the stability of metal complexes are greatly affected by the ancillary groups, different ligands were selected to test their effects on spermicidal activity and stability. The stability of organometallic complexes with monodentate ligands in aqueous solutions was found to be improved by chelating effects of certain bidentate ligands, particularly dithiocarbamate and acetylacetonate [25, 26]. Because of the pharmacological and biochemical importance of vanadium compounds, and our novel finding of vanadocenes as a new class of effective spermicidal agents [23–26], we have synthesized 11 stable oxovanadium(IV) complexes. These included oxovanadium complexes with a square pyramidal geometry with the oxoligand in the axial position. The coordination complexes were stabilized with 5-membered mono- and bis-1,10-phenanthroline (phen): [VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2-phen)2, VO(Cl-phen), and VO(Cl-phen)2], mono- and bis-2,2'-bipyridyl (bipy): [VO(bipy), VO(bipy)2, VO(Me2-bipy), and VO(Me2-bipy)2], and bis-5'-bromo-2'-hydroxyacetophenone (acph): [VO(Br,OH-acph)2], as ancillary ligands linked via nitrogen or oxygen atoms. The phenanthroline-peroxovanadate complex potentiates the generation of ROS in cells [27]. Since sperm motility and function have been shown to be exquisitely susceptible to ROS, we set out to examine these compounds for spermicidal activity using computer-assisted sperm analysis (CASA). Our results presented herein provide unprecedented evidence that vanadium IV-bound to phenanthroline, bipyridyl and acetophenone as ancillary ligands and their derivatives are potent spermicidal agents and also induce apoptosis in human sperm.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The names of the 11 oxovanadium complexes synthesized and tested in this study are listed in Table 1. The chemical structures of 10 cationic complexes with either mono- or bis-1,10-phenanthroline and 2,2'-bipyridyl, and one neutral complex, bis-5'-bromo-2'-hydroxyacetophenone, are depicted in Figure 1. Nine novel oxovanadium(IV) complexes were synthesized on the basis of the previously published chemistry of VO(phen) and VO(phen)2 complexes [27]. Briefly, these complexes were synthesized by reacting an aqueous solution of vanadyl sulfate with an ethanol solution or a chloroform solution of the ligands. The complexes purified from chloroform, ether, and/or water were characterized by Fourier transform infrared spectroscopy (FT-Nicolet model Protege 460; Nicolet Instrument Corp., Madison, WI), UV-visible spectroscopy (DU 7500 spectrophotometer; Beckman Instruments, Fullerton, CA), mass spectrometry, and elemental analysis (Atlantic Microlab, Inc., Norcross, GA). The choice of these three ancillary ligands (phenanthroline, bipyridyl, and acetophenone) was based on the report that the cationic oxovanadium(IV) complex of phenanthroline is superior to cisplatin (cis-diaminedichloroplatinum([II]) with respect to antitumor activity [27] and on the structural similarity of bipyridyl ring to phenanthroline, as well as on the neutral nature of acetophenone complex of oxovanadium(IV). Structural variations of the ligands included addition of bromo, chloro, or methyl groups on the phenanthroline, bipyridyl or acetophenone rings.
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Sperm Immobilization Assay (SIA)
To evaluate the spermicidal effects of inorganic complexes of oxovanadium(IV)—VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2-phen)2, VO(Cl-phen), VO(Cl-phen)2, VO(bipy), VO(bipy)2, VO(Me2-bipy), VO(Me2-bipy)2, and VO(Br,OH-acph)2—a highly motile fraction of pooled donor sperm (n = 5) was prepared by discontinuous (90–45%) Percoll gradient (Conception Technologies, San Diego, CA) centrifugation and the swim-up method as described previously [23, 24, 28]. All donor specimens were obtained after informed consent and in compliance with the guidelines of the Hughes Institutional Review Board. Motile sperm (
10 x 106/ml) were suspended in 1 ml of Biggers, Whitten, and Whittingham's medium (BWW) containing 0.3% BSA (fraction V; Sigma Chemical Co., St. Louis, MO) in the presence and absence of serial 2-fold dilutions of test substance (250–1.9 µM) in 0.25% dimethyl sulfoxide (DMSO). For each experiment, fresh stock solutions (100 mM) of vanadium compounds were prepared in DMSO. A corresponding volume of DMSO (0.25%) was added to the control sperm suspensions. After 3 h of incubation at 37°C, the percentage of motile sperm was evaluated by CASA as described previously [24, 25]. The percentages of motilities were compared with those of sham-treated control suspensions of motile sperm. The spermicidal activity of test compounds was expressed as the EC50 values (the final concentration of the compound in medium that decreased the proportion of motile sperm by 50%).
To test the effect of duration of incubation on SIA in the presence of the 11 oxovanadium(IV) complexes, a motile fraction of sperm (107/ml) was incubated at 37°C in 1 ml of BWW-0.3% BSA in the presence of 200 µM each of the 11 complexes or 0.2% DMSO alone. At timed intervals of 2, 5, or 10 min, aliquots (4 µl) were transferred to two 20-µm Microcell (Conception Technologies) chambers, and sperm motility was assessed by CASA.
Sperm Kinematic Parameters
For CASA, 4 µl of each sperm suspension was loaded into two 20-µm Microcell chambers placed onto a counting chamber at 37°C. At least 5–8 fields per chamber were scanned for analysis using a Hamilton Thorne Integrated Visual Optical System (IVOS) version 10 instrument (Hamilton Thorne Research Inc., Beverly, MA). Each field was recorded for 30 sec. The Hamilton Thorne computer calibrations were set at 30 frames at a frame rate of 30 images/sec. Other settings were as follows: minimum contrast 8; minimum size 6; low-size gate, 1.0; high-size gate, 2.9; low-intensity gate, 0.6; high-intensity gate, 1.4; phase-contrast illumination; low path velocity at 10 µm/sec and threshold straightness at 80%; magnification factor, 1.95. The performance of the analyzer was periodically checked using the playback function.
The attributes of sperm kinematic parameters evaluated included numbers of motile (MOT) and progressively (PRG) motile sperm; curvilinear velocity (VCL; a measure of the total distance traveled by a given sperm during the acquisition divided by the time elapsed); average path velocity (VAP; the spatially averaged path that eliminates the wobble of the sperm head), straight-line velocity (VSL; the straight-line distance from beginning to end of track divided by time taken), beat-cross frequency (BCF; frequency of lateral head displacement), amplitude of lateral sperm head displacement (ALH; the mean width of sperm head oscillation), and the derivatives straightness (STR = VSL divided by VAP x 100) and linearity (LIN = VSL divided by VCL x 100, departure of sperm track from a straight line). Data from each individual cell track were recorded and analyzed. At least 200 sperm were analyzed for each aliquot sampled.
Flow Cytometric Quantitation of SpermAcrosome Reaction
In experiments designed to assess the comparative effects of 11 oxovanadium(IV) complexes and a commercial detergent-based spermicide, N-9, on sperm acrosome reaction, motile fractions of sperm (107/ml) prepared from a single donor were incubated in 1 ml of BWW-0.3% BSA in the presence of 100 µM each of the 11 vanadocene complexes—VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2-phen)2, VO(Cl-phen), VO(Cl-phen)2, VO(bipy), VO(bipy)2, VO(Me2-bipy), VO(Me2-bipy)2, and VO(Br,OH-acph)2—in 0.1% DMSO, N-9, or DMSO (0.1%) alone at 37°C. After 3 h, 5 µg/ml of purified, phycoerythrin (PE)-conjugated murine anti-CD46 monoclonal antibody (mAb; clone 122–2; Research Diagnostics, Flanders, NJ) was added, and the sperm suspensions were incubated for an additional 30 min. The suspensions were washed in Tyrode's salt solution (Sigma) containing 1% BSA (1% TBSA), and the percentages of CD46-positive sperm were analyzed by flow cytometry using a FACS Vantage flow cytometer (Becton Dickinson, Mountain View, CA), as described previously [29, 30]. Two separate experiments were performed to determine acrosomal loss following exposure of sperm to oxovanadium(IV) complexes.
Flow Cytometric Assays for Oxovanadium(IV) Complex-Induced Apoptosis
We used three independent flow cytometric apoptotic assays to determine oxovanadium(IV)-mediated quantitative changes at the mitochondrial, surface membrane, and sperm nuclear compartments.
Mitochondrial Transmembrane Potential (
m) UsingJC-1 Dye
The loss of m, an early marker for apoptosis, was quantitated by flow cytometry using the lipophilic cationic dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolecarbocyanine iodide (JC-1) [31]. This dye accumulates in the mitochondrial matrix under the influence of the m [32]. The molecule is able to selectively enter into mitochondria, the monomeric form emitting at 527 nm after excitation at 490 nm. However, depending on the membrane potential, JC-1 is able to form J-aggregates that are associated with a large shift in emission (590 nm). The color of the dye changes reversibly from green to greenish orange as m becomes more polarized [33]. To quantitate changes in sperm m following oxovanadium(IV) complex exposure, highly motile fractions of sperm (107/ml) in duplicate aliquots were incubated at 37°C for 3 h in BWW-0.3% BSA medium in the presence and absence of 100 µM each of the 11 oxovanadium complexes. After incubation, 10 µg/ml JC-1 (Molecular Probes, Eugene, OR) was added from a stock solution in DMSO (1 mg/ml) to the sperm suspension, which was then incubated for an additional 10 min. At the end of the incubation period, sperm were washed in Tyrode's salt solution (Sigma), resuspended in 200 µl of Tyrode's salt solution, and analyzed by flow cytometry for JC-1-specific fluorescence. The excitation was at 488 nm; the emissions for green and red/orange fluorescence were 530 nm and 575 nm respectively. JC-1 monomer and aggregated fluorescence were simultaneously measured in oxovanadium(IV) complex-exposed and control sperm. The percentages of sperm positive for green, orange, and greenish orange were determined using the cutoff signals for JC-1-labeled motile sperm. Two separate experiments were performed to assess JC-1 incorporation following exposure of sperm to oxovanadium(IV) complexes.
Sperm Membrane Changes Using Fluorescein Isothiocyanate (FITC) Annexin V
In order to examine the expression of phosphatidyl serine on the sperm surface after oxovanadium(IV) complex exposure, we used flow cytometry to evaluate surface binding of FITC-Annexin V [34]. One-milliliter aliquots of highly motile sperm (107) in triplicate were incubated in BWW-0.3% BSA at 37°C for 12 h with and without 100 µM of each of the 11 oxovanadium(IV) complexes in 0.1% DMSO. After exposure to these complexes, sperm were washed with 1% TBSA, and the pellets were resuspended in the same medium. The sperm suspension was reacted for 30 min at room temperature with 6 µg/ml of FITC-conjugated recombinant human Annexin V (Caltag Laboratories, San Francisco, CA). After two washes in Tyrode's salt solution, sperm were resuspended in 1% TBSA containing 1 µg/ml propidium iodide (PI) and analyzed for surface-bound Annexin V and PI permeability by quantitative flow cytometry using an argon laser for excitation of fluorescence. Annexin V and PI binding were simultaneously measured in oxovanadium(IV) complex-exposed and control sperm as described previously [25]. The percentages of sperm positive for Annexin V and PI were determined using the cutoff signals for membrane-intact motile sperm. Two separate experiments were performed to assess the surface expression of phosphatidyl serine following exposure of sperm to oxovanadium(IV) complexes.
DNA Fragmentation Using In Situ DNA Nick-End Labeling by the TUNEL Method
A flow cytometric two-color terminal deoxynucleotidyl transferase (TdT) assay was employed to detect apoptotic sperm nuclei by TdT-mediated digoxigenin-uridine triphosphate (dUTP) nick-end labeling (TUNEL [35]). The abilities of 11 oxovanadium(IV) complexes to induce apoptosis were compared by incubating 1-ml duplicate aliquots of motile sperm (107/ml) in BWW-0.3% BSA at 37°C for 24 h with and without each of the test compounds at a 100-µM concentration. Sperm were washed in PBS-1% BSA and fixed in 4% paraformaldehyde in PBS for 15 min. After two washes in PBS, they were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice, and washed twice with PBS. Labeling of exposed 3'-hydroxyl (3'-OH) ends of fragmented sperm nuclear DNA was performed using TdT and detected by FITC-conjugated dUTP according to the manufacturer's recommendations (Boehringer-Mannheim, Indianapolis, IN). Sperm aliquots incubated without TdT enzyme served as negative controls. Non-apoptotic sperm do not incorporate significant amounts of dUTP because of lack of exposed 3'-OH ends, and consequently have much less fluorescence compared to apoptotic cells, which have an abundance of 3'-OH ends. Oxovanadium(IV)-induced apoptosis of sperm was shown by an increase in the number of cells staining with FITC-dUTP (M2 gates). The M1 and M2 gates were used to identify non-apoptotic and apoptotic PI-counterstained sperm populations, respectively. Two separate experiments were performed to assess dUTP incorporation following exposure of sperm to oxovanadium(IV) complexes.
Confocal Laser Scanning Microscopy
Confocal microscopy of TUNEL-positive and control sperm was performed using a Bio-Rad MRC 1024 Laser Scanning Confocal Microscope (Bio-Rad Labs., Richmond, CA) equipped with an argon-ion laser (excitation at 488 nm and emission at 540 nm) and mounted on a Nikon Eclipse E800 series upright microscope (Nikon Instruments, Garden City, NY) with high numerical aperture objectives. Confocal images were obtained using a Nikon x100 (NA 1.4) numerical aperture objective and Kalman collection filter as described previously [24, 25]. Digital data were processed using Lasersharp (Bio-Rad), and digitized images were saved on a Jaz disk (Iomega Corp., Roy, UT) and processed with Adobe Photoshop software (Adobe Systems, Mountain View, CA). Final images were printed using a Fuji Pictography 3000 (Fuji Photo Film Co., Tokyo, Japan) color printer.
Statistical Analysis
Sperm functional parameters are presented as mean ± SD values. Nonlinear regression analysis was used to find the EC50 values (i.e., concentrations of compound that result in 50% sperm motility loss) from the concentration-effect curves using GraphPad PRISM Version 2.0 software (San Diego, CA). One-way ANOVA followed by Dunnett's test was used to obtain statistical significance between control and test results. Linear regression analysis was performed to furnish the correlation coefficient, r2. A p value of < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of the reported ability of an oxovanadium(IV) complex of 1,10-phenanthroline to induce hydroxyl radical-mediated cell damage [27, 36], we synthesized a series of 11 oxovanadium(IV) complexes including six phenanthroline (phen)-linked [VO(phen), VO(phen)2, VO(Me2-phen), VO(Me2-phen)2, VO(Cl-phen), and VO(Cl-phen)2] and four bipyridyl (bipy)-linked [VO(bipy), VO(bipy)2, VO(Me2-bipy), and VO(Me2-bipy)2] via nitrogen atoms, and one acetophenone (acph)-linked [VO(Br,OH-acph)2] via oxygen atoms; and tested them for spermicidal activity using CASA. These complexes were tested side-by-side and at 8 different concentrations ranging from 1.9 µM to 250 µM.
All 11 oxovanadium complexes induced concentration-dependent inhibition of sperm motility assessed after a 3-h incubation in BWW-0.3% BSA medium. However, marked differences were noted in their potency. Table 2 shows the EC50 values calculated from the concentration-response curves. Among the 6 phenanthroline-linked cationic complexes, the bis-1,10-phenanthroline complex, VO(phen)2, and its 5-chloro derivative, VO(Cl-phen)2, were the most potent, with EC50 values of 6.5 µM and 5.5 µM, respectively. Among the 4 bipyridyl-linked cationic complexes, the bis-2,2'-bipyridyl complex, VO(bipy)2, and its 4,7-dimethyl derivative, VO(Me2-bipy)2, were the most active, with EC50 values of 35 µM and 73 µM, respectively. The mono-2,2'-bipyridal complex, VO(bipy), was the least active (EC50 = 118 µM). The 5-bromo derivative of bis-2'-hydroxyacetophenone, a neutral complex, was also potent, with an EC50 value of 13.4 µM. These marked differences (21-fold) in potency of the spermicidal activity elicited by the three ancillary heteroligands and their derivatives suggest that the spermicidal potency of oxovanadium(IV)-complexes is modulated by the 5-membered bidentate ligands. The spermicidal activity of the most potent oxovanadium(IV) complex, VO(Cl-phen)2, was 14-fold more potent than that of the commercial detergent-based spermicide, N-9 (79 µM), when tested under identical experimental conditions.
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Figure 2 shows the concentration-response curves of spermicidal effects of 7 representative oxovanadium(IV) complexes: VO(phen), VO(phen)2, VO(Cl-phen)2, VO(Me2-phen), VO(bipy)2, VO(Me2-bipy), and VO(Me2-bipy)2. The spermicidal activity of the oxovanadium(IV) complexes was strongly dependent on the type of coordinated heteroligands. The oxovanadium(IV) complexes stabilized with 5-membered bis-chelated ligands of phenanthroline, bipyridyl, and acetophenone, with a vanadium(IV) atom conferring a "butterfly structure," had superior spermicidal activity when compared with diaqua monochelated complexes (Fig. 1).
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Also, in comparison to N-9, the spermicidal activity of oxovanadium(IV) complexes was not associated with a concomitant loss of acrosomal membrane or membrane damage as quantitated by the flow cytometric anti-CD46 mAb binding assay using unfixed sperm suspensions [29,30]. Despite complete sperm motility loss quantitated after a 3-h incubation period, 76–97% of the treated sperm remained anti-CD46-negative (acrosome-intact) (Table 2). The most potent oxovanadium(IV) complexes, VO(Cl-phen)2 and VO(Br,OH-acph)2, after a 3-h incubation period induced a 4- to 5-fold increase (21.5% ± 0.5% and 24.3% ± 0.6%, respectively, p < 0.05) in acrosome reactions over control (5.2% ± 0.7%). However, complete sperm motility loss with these complexes was achieved within 2 and 10 min of exposure. Thus, the spermicidal activity of oxovanadium(IV) complexes was not concomitantly associated with disruption of sperm membranes.
Kinetics of Sperm Immobilization by Oxovanadium(IV) Complexes Was Variable
Interestingly, the kinetics of sperm immobilization by the 11 oxovanadium(IV) complexes was variable. The corresponding times required for 50% motility loss of progressively motile sperm exposed to these complexes ranged from < 1 min to > 60 min. Sperm immobilization by the neutral complex, VO(Br,OH-acph)2, was the fastest, followed by VO(Cl-phen)2, with T1/2 values of 38 sec and 7.3 min, respectively. The other cationic oxovanadium(IV) complexes showed a lag period of 30–60 min to bring about > 50% sperm motility loss. By comparison, sperm motility in control samples remained stable during the 3-h monitoring period.
Oxovanadium(IV) Complexes Affected Sperm Kinematics
The observed concentration- and time-dependent decreases in sperm motility after exposure to the 11 oxovanadium(IV) complexes were associated with significant changes in the centroid-derived movement characteristics of the surviving sperm, particularly with respect to the track speed (VCL), straight-line velocity (VSL), and path velocity (VAP). The representative sperm kinematic parameters observed for VO(Cl-phen)2 versus concentration and time are shown in Figure 3, A and B, respectively. The decreases in VCL, VSL, and VAP were similar in magnitude with increasing concentrations of VO(Cl-phen)2 or exposure time. However, the linearity (LIN) of the sperm tracks and the straightness (STR) of the swimming pattern were affected only with increasing concentration of the drug. The beat-cross frequency (BCF) and the amplitude of lateral sperm head displacement (ALH) were relatively uniform as the proportion of motile sperm declined with increasing concentration (0–15.6 µM) or exposure time (0–10 min). By contrast, the sperm motion parameters of control sperm showed insignificant changes during the 3-h exposure.
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Oxovanadium(IV) Complexes of 1,10-Phenanthroline, 2,2'-Bipyridyl, and 5'-Bromo-2'-Hydroxyacetophenone and Derivatives Induced Apoptosis in Human Sperm
Because the phenanthroline complex of vanadium(IV) has been shown to induce hydroxyl-mediated DNA strand breaks [27, 36], we tested the effects of the 11 oxovanadium(IV) complexes with phenanthroline, bipyridyl, and acetophenone as ancillary ligands to induce apoptosis in human sperm. We used three independent apoptosis assays to quantitatively assess changes at the mitochondrial, surface membrane, and nuclear level. Analysis by flow cytometry of the mitochondrial membrane potential changes occurring during apoptosis were analyzed with a m indicator, JC-1, a carbocyanine cationic dye, by following fluorescence associated with the uptake of JC-1 to evaluate m modifications [33]. 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 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). 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, m modifications, evaluated by the uptake of cationic lipophilic dye, were detected early in the process of apoptosis induced by oxovanadium(IV) complexes.
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Changes in the plasma membrane of the cell surface also appear early in cells undergoing apoptosis [37, 38]. In apoptotic cells, the membrane phospholipid phosphatidyl serine is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing phosphatidyl serine to the external cellular environment [38]. Annexin V binds to phosphatidyl serine residues that are exposed on the surface of cells undergoing apoptosis. The apoptosis-dependent surface binding of FITC-labeled recombinant human Annexin V with 10 of the 11 oxovanadium(IV) complex-treated sperm showed a dramatic increase in binding of Annexin V to sperm membrane (Table 3). After 12 h of incubation, 26–99% (p < 0.05) of the treated sperm were apoptotic. Control sperm exhibited minimal fluorescence (Fig. 4C). By contrast, 99% of VO(Cl-phen)2-treated sperm were positive for FITC-Annexin V (Fig. 4D), indicating that surface membrane alteration occurred after prolonged exposure to oxovanadium(IV) complexes. Control sperm treated with 0.1% DMSO alone showed only 7 ± 3% Annexin V positivity at 12 h. The most potent spermicidal complexes, VO(Cl-phen)2 and VO(phen)2, also induced maximum Annexin V positivity.
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 oxovanadium(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 µM 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). 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 µM 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The kinetics of sperm immobilization by the oxovanadium(IV) complexes was dependent on their net charge. Structure-activity relationship analyses of 19 vanadocenes [23–25] and 11 oxovanadium(IV) complexes clearly demonstrated that the spermicidal properties of these complexes were determined by the oxidation state of the vanadium(IV) atom. The various ancillary ligands linked by either carbon, nitrogen, or oxygen atoms to the central vanadium(IV) atom significantly contributed either to fine tuning of the spermicidal potency or enhancing the stability of these complexes in aqueous solution. In addition, similar to our earlier findings with neutral complexes of vanadocenes [24], the neutral complex of oxovanadium(IV), VO(Br,OH-acph)2, rapidly inactivated sperm in seconds in comparison to the cationic chelated oxovanadium(IV) complexes or cationic chelated vanadocenes, which required a lag period of several minutes [25]. Therefore, it appears from our study that despite the tetrahedral geometry of the "bent-sandwich" structures of vanadocenes or the square pyramidal geometry/"butterfly" structures of oxovanadium(IV) complexes, the rapidity of vanadium(IV)-mediated spermicidal activity was dependent on the neutrality of these complexes. Because the neutral complex of oxovanadium(IV), VO(Br,OH-acph)2, was a rapid spermicidal agent, it is likely that this complex of oxovanadium(IV) is rapidly transported across the sperm cell membranes. Because of its rapidity and potency, VO(Br,OH-acph)2 may be useful as a contraceptive agent.
The mechanism of sperm motility loss induced by oxovanadium(IV) complexes is yet to be determined. Both the vanadocenes(IV) and oxovanadium(IV) complexes also have antitumor activity [11, 12, 27]. The antitumor effects of vanadium(IV) complexes are thought to be due to their reaction with the vanadyl-H2O2 system, which results in the generation of hydroxyl radicals in a Fenton-like reaction [7, 9, 27]. In particular, the oxovanadium(IV) complex, after dissociation of phenanthroline rings, results in the formation of peroxocompounds that in the presence of H2O2 generate hydroxyl radicals. In support of this hypothesis is the observation that the vanadyl-phen complex induces hydroxyl radical-dependent DNA cleavage in the presence of H2O2 [36, 39]. The vanadyl complex [VO(phen)(H2O)2]2+ has high antitumor activity toward human nasopharyngeal carcinoma [27]. Hydrogen peroxide is formed in cells by dismutation of superoxide anions, which are generated in various systems such as xanthine-oxidase, NADPH oxidase, and NADH-dependent cytochrome P450 and neutrophils [40]. Thus, H2O2 is thought to react with oxovanadium(IV) bound to DNA to generate ROS, resulting in cleavage of DNA. It is likely that oxovanadium(IV)-induced sperm motility loss and apoptosis are mediated primarily by the ability of these complexes to induce ROS-mediated damage to sperm. In sperm, an NADPH-dependent superoxide-generating system has been demonstrated [41]. In addition, the ability of H2O2 generating Lactobacillus acidophilus, which is present in the vaginas of most normal women, can further potentiate the spermicidal activity of intravaginally applied oxovanadium(IV) complex. This is in contrast to the commercial vaginal detergent spermicide, N-9, which is selectively toxic to Lactobacilli [42, 43]. Furthermore, unlike N-9, the spermicidal activity of the oxovanadium(IV) complexes was not concomitantly associated with membrane disruption.
We ascribe the irreversible nature of the sperm-immobilizing activity of oxovanadium(IV) complexes to their ability to induce apoptosis. We used three independent methods that quantitatively assess apoptotic changes in the mitochondria, surface membrane, and nuclear compartment. Mitochondria are the primary targets for apoptosis, and alterations in mitochondrial structure and function are early events of apoptotic cell death [32]. Our studies demonstrated that spermicidal oxovanadium(IV) complexes induced depolarization of sperm mitochondria, an early marker for apoptotic cell death. Prolonged exposure of sperm to these spermicidal complexes also resulted in increased FITC-Annexin V binding to sperm surface due to membrane changes during apoptosis, as well as increased dUTP incorporation in the nuclei of treated sperm. Since vanadium(IV) compounds by themselves do not cleave DNA [44], the dramatic uptake of dUTP observed in our study appears to be due to the cleavage of the DNA polymer as a result of cytotoxicity induced by ROS-mediated effects of oxovanadium(IV) complexes. The fact that human sperm are exquisitely sensitive to oxidative stress, and the ability of oxovanadium(IV) complexes to potentiate these effects establishes these oxovanadium(IV) complexes as a new class of gentle contraceptive agents. In addition, our unpublished observations indicate that some of these oxovanadium(IV) complexes selectively inhibit the growth of human testicular cancer cells via the induction of apoptosis. This activity profile indicates that oxovanadium(IV) complexes could be useful as male antifertility agents as well as anticancer agents against testicular germ cell tumors.
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FOOTNOTES |
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Accepted: September 22, 1998.
Received: July 15, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cyclopentadienyl)-,N,N-diethyl dithiocarbamato vanadium(IV) tetrafluoro borate, [VCp2(DeDtc)(BF4)]. J Inorg Biochem 1998; (in press).This article has been cited by other articles:
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  G. Martin, O. Sabido, P. Durand, and R. Levy Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction Hum. Reprod., December 1, 2005; 20(12): 3459 - 3468. [Abstract] [Full Text] [PDF]
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 S.L. Taylor, S.L. Weng, P. Fox, E.H. Duran, M.S. Morshedi, S. Oehninger, and S.J. Beebe Somatic cell apoptosis markers and pathways in human ejaculated sperm: potential utility as indicators of sperm quality Mol. Hum. Reprod., November 1, 2004; 10(11): 825 - 834. [Abstract] [Full Text] [PDF]
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 O. J. D'Cruz, B. Waurzyniak, and F. M. Uckun Antiretroviral Spermicide WHI-07 Prevents Vaginal and Rectal Transmission of Feline Immunodeficiency Virus in Domestic Cats Antimicrob. Agents Chemother., April 1, 2004; 48(4): 1082 - 1088. [Abstract] [Full Text] [PDF]
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S.-L. Weng, S. L. Taylor, M. Morshedi, A. Schuffner, E.H. Duran, S. Beebe, and S. Oehninger Caspase activity and apoptotic markers in ejaculated human sperm Mol. Hum. Reprod., November 1, 2002; 8(11): 984 - 991. [Abstract] [Full Text] [PDF]
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 O. J. D'Cruz, T. K. Venkatachalam, and F. M. Uckun Novel Thiourea Compounds as Dual-Function Microbicides Biol Reprod, July 1, 2000; 63(1): 196 - 205. [Abstract] [Full Text]
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G. Barroso, M. Morshedi, and S. Oehninger Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa Hum. Reprod., June 1, 2000; 15(6): 1338 - 1344. [Abstract] [Full Text] [PDF]
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O. J. D'Cruz, A. Vassilev, and F. M. Uckun Studies in Humans on the Mechanism of Potent Spermicidal and Apoptosis-Inducing Activities of Vanadocene Complexes Biol Reprod, April 1, 2000; 62(4): 939 - 949. [Abstract] [Full Text]
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 R. K. Narla, Y. Dong, O. J. D’Cruz, C. Navara, and F. M. Uckun Bis(4,7-dimethyl-1,10-phenanthroline) Sulfatooxovanadium(IV) as a Novel Apoptosis-inducing Anticancer Agent Clin. Cancer Res., April 1, 2000; 6(4): 1546 - 1556. [Abstract] [Full Text]
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O. J. D'Cruz, T. K. Venkatachalam, and F. M. Uckun Structural Requirements for Potent Human Spermicidal Activity of Dual-Function Aryl Phosphate Derivative of Bromo-Methoxy Zidovudine (Compound WHI-07) Biol Reprod, January 1, 2000; 62(1): 37 - 44. [Abstract] [Full Text]
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