Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by D'Cruz, O. J.
Right arrow Articles by Uckun, F. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by D'Cruz, O. J.
Right arrow Articles by Uckun, F. M.
Agricola
Right arrow Articles by D'Cruz, O. J.
Right arrow Articles by Uckun, F. M.
Biology of Reproduction 62, 939-949 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Studies in Humans on the Mechanism of Potent Spermicidal and Apoptosis-Inducing Activities of Vanadocene Complexes

Osmond J. D'Cruz1,b, Alexei Vassilevc, and Fatih M. Uckuna

a Drug Discovery Program, b Departments of Reproductive Biology and c Biochemistry, Parker Hughes Institute, St. Paul, Minnesota 55113


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously demonstrated that bis-cyclopentadienyl (Cp) complexes of vanadium(IV) (vanadocenes) are potent spermicidal and apoptosis-inducing agents. To gain further insight into the structure-function relationships controlling these two properties of vanadocenes, we have synthesized analogues in which the bis-Cp rings were substituted with one or five electron-donating methyl groups. The three complexes included vanadocene dichloride (VDC), bis(methylcyclopentadienyl) vanadium dichloride (VMDC), and bis(pentamethylcyclopentadienyl) vanadium dichloride (VPMDC). The concentration-dependent effect of these vanadocenes on sperm-immobilizing activity (SIA), mitochondrial membrane potential ({Delta}{Psi}m), axonemal dynein ATPase activity, and tyrosine phosphorylation of global and axoneme-specific sperm proteins was assessed by computer-assisted sperm analysis, flow cytometry, colorimetry, and immunoblotting, respectively. Apoptosis-inducing ability was quantitated by the two-color flow cytometric terminal dideoxynucleotidyl transferase-based assay that labels 3'-hydroxyl ends of fragmented DNA. All three vanadocenes induced rapid sperm immobilization (T1/2 < 15 sec). Substitution of the bis-Cp rings by five methyl groups augmented the SIA of VDC by 10-fold. The EC50 values (50% inhibitory concentration) for VDC, VMDC, and VPMDC were 7.5 µM, 4.3 µM, and 0.7 µM, respectively. Whereas SIA of vanadocenes was apparent at low micromolar concentrations, the apoptosis-inducing property was evident only at higher micromolar concentrations. The concentrations of VDC, VMDC, and VPMDC required for 50% apoptosis were 49 µM, 67 µM, and 153 µM, and for 50% reduction in sperm {Delta}{Psi}m were 435 µM, 173 µM, and 124 µM, respectively. Spermicidal activity of vanadocenes was not dependent on the inhibition of ATPase or tyrosine phosphorylation of global and sperm axonemal proteins. Due to the ability of these vanadocene complexes to rapidly generate hydroxyl radicals in the presence of oxidant, our findings provide unprecedented evidence for a novel mechanism of action for spermicidal vanadocenes. The differential concentration-dependent spermicidal and apoptosis-inducing properties of vanadocenes gives them particular utility as a new class of vaginal contraceptives.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Organometallic complexes of vanadium(IV) with bis(cyclopentadienyl) (Cp2) rings or vanadocenes are a new class of potent spermicides [1–3]. The disubstituted vanadocenes are "bent-sandwich" complexes where the bis-Cp2 rings in a tetrahedral symmetry are positioned in a bent conformation with respect to the central vanadium(IV) atom [4, 5]. Our recent structure-activity relationship studies of 12 monodentate and 7 bidentate Cp2-vanadium(IV) complexes demonstrated that these complexes have potent sperm-immobilizing activity (SIA) as well as apoptosis-inducing property against human sperm [2, 3]. The SIA of vanadocenes was dependent on vanadium(IV) as the central metal ion within the Cp2-metal complex, since its replacement with other oxidation state IV transition metal ions such as hafnium, molybdenum, titanium, or zirconium had minimal or no effect on sperm motility [2]. Unlike inorganic vanadium salts such as vanadyl sulfate (IV), sodium metavanadate (V), or orthovanadate (V), which had no effect on human sperm motility, vanadocene dihalides and diacido derivatives immobilized sperm within 15 sec without affecting the sperm plasma and acrosomal membrane integrity [2]. The efficacy factor varied from nanomolar to micromolar concentrations depending on the nature of diacido groups and bidentate ligands present at the ancillary positions of the respective vanadocenes [2, 3].

We hypothesized that the unique susceptibility of sperm to vanadocene-mediated rapid loss of motility without disruption of the membrane integrity may be due to their exquisite sensitivity to oxidative stress. The cationic form of vanadium complexes with oxidation state IV function as modulators of cellular redox potential, regulate enzymatic phosphorylation, and exert pleiotropic effects in multiple biological systems by catalyzing the generation of reactive oxygen species (ROS) [6–12]. Although inorganic salts of vanadium(IV) have the potential to generate ROS in the presence of oxidants, the lack of a demonstrable effect of inorganic salts of vanadium(IV) on human sperm motion parameters even at millimolar concentrations indicated the unique properties of vanadocenes to interact with sperm membranes to bring about this effect. Using zwitterionic and negative unilamellar liposomes, the membrane interactions of spermicidal vanadocenes were found to be due to their unique configurational preferences that alter membranes by intercalation and Fe2+-initiated lipid peroxidation [13]. These features of spermicidal vanadocenes fundamentally differ from those of currently used membrane-active surfactant spermicides such a nonoxynol-9, octoxynol-9, sodium docusate, chlorhexidine, menfegol, and benzalkonium chlorides that induce membrane permeability and membrane fusion [14–16]. Unlike the detergent spermicides, vanadocenes do not contain long hydrocarbon chains and are much shorter than the 30–40 Å required to span the bilayer and form channels. Because of the exquisite susceptibility of human sperm to oxidative stress and the ability of spermicidal vanadocenes to catalyze the generation of ROS through Fe2+-initiated lipid peroxidation without cytotoxic effect on female reproductive tract epithelial cells, they have unique clinical potential as a new class of contraceptive agents.

To develop a better understanding of how vanadocenes induce sperm motility loss, we have explored another set of vanadium(IV) compounds. Additionally, their apoptosis-inducing properties were compared to address whether SIA and apoptosis induction occur concurrently or proceed through different mechanisms. Three vanadocenes, vanadocene dichloride, bis(methylcyclopentadienyl) vanadium dichloride, and bis(pentamethylcyclopentadienyl) vanadium dichloride, were synthesized and tested for spermicidal and apoptosis-inducing activities to observe any difference in efficacy with and without one or five electron-donating methyl substitutions in the Cp2 rings. All three vanadocenes induced complete sperm immobilization within seconds. However, introduction into the bis-Cp rings of five methyl groups (VPMDC) increased the spermicidal potency of VDC by 10-fold. Unlike their SIA, the apoptosis-inducing ability of vanadocenes was apparent only at high micromolar concentrations. Vanadocene-induced SIA was not associated with inhibition of dynein ATPase activity or tyrosine phosphorylation of global and axonemal-specific sperm proteins. These findings provide unprecedented evidence for a novel mechanism of action for spermicidal vanadocenes. The differential concentration-dependent spermicidal and apoptosis-inducing properties of vanadocenes give them particular utility as a new class of vaginal contraceptives.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Vanadocene Complexes

The chemical structures of three vanadocene dihalides—vanadocene dichloride (VDC), bis(methylcyclopentadienyl) vanadium dichloride (VMDC), and bis(pentamethylcyclopentadienyl) vanadium dichloride (VPMDC)—analyzed in this study are depicted in Figure 1. VDC, VMDC, and VPMDC were prepared according to the published procedures [17–19]. VDC was purified from Soxhlet extraction with CH2Cl2 under argon, followed by recrystallization with hexane/CH2Cl2 saturated with HCl. All three complexes were characterized by UV-visible, fourier transform-infra red, electron pararesonance (EPR), and nuclear magnetic resonance spectroscopy [18]. Other reagents used were of reagent grade quality, and all solvents were used as received (Sure Seal bottle, < 0.005% water) from Aldrich Chemical Co. (Milwaukee, WI). Elemental analyses were performed by Atlantic Microlab, Inc. (Norcross, GA), and the chemical purity of the vanadocenes was >99%.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 1. Chemical composition of vanadocenes: VDC, VMDC, and VPMDC. The vanadocenes described here are tetrahedral in geometry, with two Cp rings positioned in a bent sandwich conformation with respect to the central vanadium(IV) ion

Sperm Immobilization Assay

Spermicidal effects of VDC, VMDC, and VPMDC were examined using highly motile fraction of pooled donor sperm obtained from fresh donor semen (n = 8) by discontinuous (90–45%) centrifugation using either Percoll or Enhance-S-Plus cell isolation media (Conception Technologies, San Diego, CA) followed by the "swim-up" method as described previously [20, 21]. All donor specimens were obtained after informed consent and were 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 Whittingam'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 (250–0.24 µM) in 0.25% dimethyl sulfoxide (DMSO). For each experiment, fresh stock solutions (100 mM) of vanadocenes were prepared in DMSO. A corresponding volume of DMSO (0.25%) was added to control sperm suspension. After 60 min of incubation at 37°C, the percentage of motile sperm was evaluated by computer-assisted sperm motion analysis (CASA) as described previously [2, 21, 22]. Three separate experiments were performed to assess the effect of vanadocenes on sperm motility.

To test the effect of duration of incubation on SIA in the presence of three vanadocenes, immediately after addition of 100 µM each of the three complexes or 0.1% DMSO alone to sperm suspension (107/ml) in 1 ml of BWW-0.3% BSA medium at 37°C, aliquots (4 µl) were transferred to 20-µm Microcell (Conception Technologies) chambers, and sperm motility was assessed by CASA.

Sperm Kinematic Parameters

For CASA, 4 µl each of 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 version 10 instrument (Hamilton Thorne Research, Danvers, 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 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), 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.

MTT Assay

We used MTT (3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide)-based colorimetric assays (Boehringer Mannheim, Indianapolis, IN) for evaluation of the cytotoxicity of VDC, VMDC, and VPMDC against normal human genital tract epithelial cells [23, 24]. Briefly, exponentially growing normal human vaginal, ectocervical, and endocervical epithelial cells (Clonetics Corporation, San Diego, CA) were seeded into 96-well tissue culture plates at a density of 2 x 105 cells per well in triplicate. After 24-h incubation, the culture medium was replaced with 100 µl of fresh medium containing serial 2-fold dilutions of VDC, VMDC, and VPMDC to yield final concentration ranging from 7.8 µM to 1 mM. Control wells contained medium plus 0.25% DMSO. Culture plates were then incubated for 3 h before addition of 10 µl of MTT solution (5 mg/ml). Wells containing only medium and MTT were used as controls for each plate. The formazan crystals formed were solubilized, and the optical densities (OD) at 540 nm were measured using a 96-well multiscan autoreader. The OD540 values were translated into the number of live cells in each well from the cell number curves generated for the vaginal, ectocervical, and endocervical epithelial cells as described previously [23, 24]. The IC50 values (drug concentration inhibiting cell growth by 50%) were calculated by nonlinear regression analysis. Three separate experiments were performed to assess the cytotoxicity potential of vanadocenes against reproductive tract epithelial cells.

Flow Cytometric Assay for Mitochondrial Transmembrane Potential ({Delta}{Psi}m) Using JC-1 Dye

The loss of {Delta}{Psi}m was quantitated by flow cytometry using the lipophilic cationic dye, 5,5',6,6'-tetrachloro 1,1',3,3'-tetraethylbenzimidazolecarbocyanine iodide (JC-1) [25]. This dye accumulates in the mitochondrial matrix under the influence of the {Delta}{Psi}m [26]. The molecule is able to selectively enter into mitochondria, the monomeric form emitting green fluorescence, and assumes a dimeric configuration emitting red fluorescence in a reaction driven by {Delta}{Psi}m [27]. The color of the dye changes reversibly from green to greenish orange as {Delta}{Psi}m is polarized. To quantify changes in sperm {Delta}{Psi}m following vanadocene treatment, 1-ml aliquots of motile fraction of sperm (107/ml) were incubated at 37°C for 3 h in BWW-0.3% medium in the presence and absence of increasing concentrations (0.24–500 µM) of VDC, VMDC, and VPMDC. After incubation, 5 µg/ml JC-1 (Molecular Probes, Eugene, OR) was added from a stock solution in DMSO (1 mg/ml) to the sperm suspension and 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, 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 vanadocene-treated sperm and vehicle (0.5% DMSO)-treated control sperm. The percentages of sperm positive for green and red/orange were determined using the cutoff signals for JC-1-labeled motile sperm. Three separate experiments were performed to assess JC-1 incorporation following exposure of sperm to vanadocenes.

Flow Cytometric Quantitation of DNA Fragmentation Using In Situ DNA Nick-End Labeling

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) [28]. The comparative effect of VDC, VMDC, and VPMDC to induce apoptosis was tested by incubating 1-ml aliquots of motile fraction of sperm (107/ml) in BWW-0.3% BSA medium at 37°C for 24 h in the presence and absence (0.5% DMSO alone) of increasing concentrations of (0.24–500 µM in 0.5% DMSO) each of the three vanadocenes. After treatment, sperm were washed in PBS-1% BSA and then 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 fluorescein (FITC)-conjugated dUTP according to the manufacturer's recommendations (Boehringer-Mannheim). Sperm aliquots incubated without TdT enzyme served as negative controls. Non-apoptotic sperm do not incorporate significant amounts of dUTP due to lack of exposed 3'-OH ends, and consequently have much less fluorescence than do apoptotic cells, which have an abundance of 3'-OH ends. Vanadocene-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. Three separate experiments were performed to assess dUTP incorporation following exposure of sperm to vanadocenes.

Anti-Phosphotyrosine Western Blot Analysis

Highly motile fraction of sperm (20 x 106/ml) in BWW-0.3% BSA medium was incubated in the presence and absence of increasing concentrations (0.001, 0.1, 1, 10, and 100 µM) of VDC, VMDC, and VPMDC for 60 min. Treated and control sperm were pelleted by centrifugation 1900 x g for 5 min, washed twice in cold PBS, and resuspended in extraction buffer containing 1% Triton X-100, 0.1 M NaCl, 4 mM MgSO4, 1 mM CaCl2, 0.1 mM EDTA, 0.1 mM ATP, 7 mM 2-mercaptoethanol, 5 mM imidazole buffer, pH 7.0. Samples of sperm lysates were resolved by SDS-PAGE on 10% polyacrylamide slab gels. Molecular weight standards of 14–97 kDa were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) at 120 mA for 60 min in a Hoefer (San Francisco, CA) TE 70 series semidry transfer unit. Membranes were then blocked for 1 h with 3% BSA in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Tween 20 (TBST). The membranes were incubated with anti-phosphotyrosine primary antibodies labeled with horseradish peroxidase (Transduction Laboratories, Lexington, KY; 1:2000 dilution in TBST) and developed using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech) [29].

Preparation of Dynein-Containing Axonemes from Sperm

Human sperm axonemes were prepared following the modification of the protocol developed for axoneme preparation of sea urchin sperm [30]. Highly motile fraction of sperm (20 x 106/ml) in BWW-0.3% BSA medium was incubated in the presence and absence of increasing concentrations (0.001, 0.1, 1, 10, and 100 µM) of VDC, VMDC, and VPMDC for 60 min. Treated and control sperm were pelleted by centrifugation at 1900 x g for 5 min, washed twice in cold PBS, and resuspended in extraction buffer containing 1% Triton X-100, 0.1 M NaCl, 4 mM MgSO4, 1 mM CaCl2, 0.1 mM EDTA, 0.1 mM ATP, 7 mM 2-mercaptoethanol, 5 mM imidazole buffer, pH 7.0, and disrupted with eight strokes of a Dounce homogenizer. The homogenized suspension was centrifuged at 1500 x g for 5 min to remove sperm heads, and the supernatant was recentrifuged at 12 000 x g to recover the broken axonemes.

The axoneme pellet was resuspended in extraction buffer and centrifuged at 1500 x g for 5 min to remove residual sperm heads, and then centrifuged at 12 000 x g to harvest the axonemes. The pelleted axonemes were washed three times with the same buffer but without Triton X-100. The pellet was used for the phosphate assay using the EnzCheck phosphate assay kit (Molecular Probes).

Phosphate Detection Assay

The generation of inorganic phosphate (Pi) catalyzed by sperm ATPases and GTPases was quantitated by the spectrophotometric method using the EnzChek phosphate assay (Molecular Probes) according to the manufacturer's instructions. In the presence of Pi, the substrate, 2-amino-6-mercapto-7-methylpurine riboside (MESG), is converted enzymatically by purine nucleoside phosphorylase to ribose-1-phosphate and 2-amino-6-mercapto-7-methylpurine [31]. Enzymatic conversion of MESG results in a shift in absorbance from 330 nm for the substrate to 360 nm for the product. Sensitivity of the assay is in the range of 2–150 nM Pi in a 1-ml volume. The axonemal preparations from 5 x 106 sperm were added to the reaction mixture containing MESG and purine nucleoside phosphorylase. After the reaction mixture was incubated for 30 min at 22°C, the absorbance was read at 360 min. Positive and negative controls included samples with and without ATP in the reaction mixture. Four separate experiments were performed to assay axonemal ATPase following exposure of sperm to vanadocenes.

Statistical Analysis

Results are presented as the mean or mean ± SD values from independent experiments. Nonlinear regression analysis using the GraphPad PRISM version 2.0 software (San Diego, CA) was used to find the 50% concentration of vanadocenes required to inhibit sperm motility (EC50) and endocervical epithelial cell viability (IC50) and to induce 50% of the sperm to undergo loss of {Delta}{Psi}m or become apoptotic. One-way ANOVA followed by Dunnett's test was used to obtain statistical significance between mean control and test results. A P value of < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Methyl Substitution on Cyclopentadienyl Rings Enhanced the Spermicidal Potency of Vanadocenes

Vanadocenes are potent spermicidal agents [1–3]. Although the SIA is dependent on vanadium(IV) as the central metal ion within the bis-cyclopentadienyl-metal complex, the variation of diacido groups, and/or replacement with bidentate ligands coordinated to the Cp2-vanadium(IV) moiety, significantly modulated the SIA [1–3]. To assess whether substitution in the Cp rings can also affect the spermicidal potency of vanadocenes, we synthesized a mono methyl (VMDC) and penta methyl-substituted (VPMDC) vanadium dichloride and compared their SIA with that of unsubstituted VDC using CASA. The SIA of these vanadocenes were tested side by side and at 11 different concentrations ranging from 0.24 µM to 250 µM. All three vanadocenes coordinated as ligands to the central vanadium(IV) ion induced concentration-dependent inhibition of sperm motility (Fig. 2). However, marked differences were noted in their potency. The order of potency was VPMDC > VMDC > VDC. Vanadocenes substituted with 5 electron-donating methyl groups on each Cp ring had superior SIA in comparison to unsubstituted or mono methyl-substituted vanadocene dichlorides. The mean EC50 values calculated from the concentration-response curves for VDC, VMDC, and VPMDC were 7.5 µM, 4.3 µM, and 0.7 µM, respectively (Table 1). This 10-fold increase in potency of the SIA elicited by VPMDC suggests that spermicidal potency of vanadocenes can be modulated by substitution with electron-donating methyl groups on the Cp2 rings.



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 2. Concentration-dependent inhibition of human sperm motility by VDC, VMDC, and VPMDC. Motile fraction of sperm was incubated for 60 min with increasing 2-fold concentrations (0.24–250 µM) of VDC, VMDC, VPMDC, or 0.25% DMSO alone in the assay medium, and the percentages of motile sperm were evaluated by CASA. Results are expressed as the mean ± SD for 3 separate experiments


View this table:
[in this window]
[in a new window]
  		TABLE 1. Comparison of the effect of VDC, VMDC, and VPMDC on spermicidal and apoptosis-inducing properties

The concentration-dependent SIA by vanadocenes was associated with significant changes (P < 0.001) in the movement characteristics of the surviving sperm with respect to track speed (VCL), path velocity (VAP), and straight-line velocity (VSL). All three vanadocenes irrespective of their potency showed identical concentration-dependent decline in progressive motility that was accompanied by a parallel decline in VCL, VAP, and VSL (Fig. 3). Complete loss of sperm motility was evident with VDC, VMDC, and VPMDC at concentrations of 31 µM, 15.6 µM, and 3.9 µM, respectively. By contrast, sperm motility and kinematic parameters of control sperm showed no significant changes during the 60-min monitoring period (data not shown).



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 3. Concentration-dependent effect of VDC (A), VMDC (B), and VPMDC (C) on sperm motion parameters. Motile fraction of sperm was incubated with increasing concentrations of vanadocenes or 0.25% DMSO in the assay medium, and the effects on VCL, VAP, and VSL were evaluated by CASA. Results are expressed as the mean ± SD for 3 separate experiments

The kinetics of sperm immobilization by the three vanadocenes was very rapid. The time required for complete sperm motility loss of progressively motile sperm exposed to these complexes at 100 µM was <15 sec. By comparison, sperm motility in vehicle control samples was unaffected (>95% motile) during a 60-min monitoring period (data not shown).

Selective Spermicidal Activity of VDC, VMDC, and VPMDC

The MTT cell viability assay was used to test the potential in vitro cytotoxicity of VDC, VMDC, and VPMDC against confluent monolayers of normal human vaginal, ectocervical, and endocervical epithelial cells. Cells were exposed to these vanadocenes at 8 concentrations ranging from 7.8 µM to 1 mM for 3 h. The mean IC50 values calculated from the concentration-dependent cell survival curves for VDC, VMDC, and VPMDC were >400 µM (Table 2). All three vanadocenes showed high selectivity indices against these cells (SI: 59 to 965 for vaginal cells, >133 to >1428 for ectocervical cells, and 88 to 1180 for endocervical cells, respectively). Thus, these spermicidal vanadocenes were significantly less cytotoxic against normal reproductive tract epithelial cells.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Comparison of the cytotoxic profiles of VDC, VMDC, and VPMDC against normal human vaginal, ectocervical, and endocervical epithelial cells as quantitated by MTT assay

Spermicidal Activity of Vanadocenes Was Not Concomitantly Accompanied by Reduction in Sperm {Delta}{Psi}m

We hypothesized that the rapid SIA of vanadocenes may be due to their ability to affect sperm mitochondrial function, particularly {Delta}{Psi}m. Therefore, concentration-dependent changes in sperm {Delta}{Psi}m were monitored by flow cytometry using the lipophilic cationic dye JC-1 [25]. This fluorescent dye accumulates in the mitochondrial matrix under the influence of the {Delta}{Psi}m [26]. The molecule is able to selectively enter into mitochondria, the monomeric form emitting green fluorescence, and assumes a dimeric configuration emitting red fluorescence in a reaction driven by {Delta}{Psi}m. After treatment of highly motile fraction of sperm with VDC, VMDC, and VPMDC at increasing concentrations ranging from 0.24 µM to 500 µM for 3 h, we observed a progressive reduction in sperm {Delta}{Psi}m as shown by the reduction in JC-1 red/orange fluorescence with concomitant increase in JC-1 green fluorescence. Figure 4 shows the fluorescence contour plots of sperm mitochondria probed with JC-1 in situ. Both control sperm and sperm treated with VPMDC well above (44-fold) its spermicidal concentration (31.2 µM) showed a high degree of red/orange fluorescence, indicating lack of effect of VPMDC on sperm {Delta}{Psi}m at spermicidal concentrations. Marked reduction in red/orange fluorescence was evident when sperm were treated with VPMDC only at high (250 µM and above) micromolar concentrations, indicating that the mitochondria were unable to maintain their {Delta}{Psi}m. All three vanadocenes induced marked perturbations in {Delta}{Psi}m only at high micromolar (>125 µM) concentrations (Fig. 5). The mean fraction of JC-1 green fluorescence-positive sperm increased from <5% in vehicle-treated control cells to 45%, 72%, and 85% in cells treated with 250 µM of VDC, VMDC, and VPMDC, respectively. Although the potency order of vanadocenes was similar to their spermicidal potency, the mean EC50({Delta}{Psi}m) for VDC, VMDC, and VPMDC-induced depolarization of mitochondria, as measured by decreased JC-1 red/orange fluorescence, were 435 ± 231 µM, 173 ± 58 µM, and 124 ± 19 µM, respectively (Table 1). Despite complete SIA of these vanadocenes at the low micromolar range (~10 µM), even a 3-h treatment with >100 µM concentrations of VDC, VMDC, and VPMDC resulted in only partial (<25%) increase in JC-1 monomer-positive sperm (green). Thus, although vanadocenes induced extinction of the red fluorescence due to alteration in {Delta}{Psi}m, this effect was independent of SIA. These results collectively demonstrate that the potent SIA of vanadocenes was not due to reduction in sperm {Delta}{Psi}m.



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 4. Flow cytometric analysis of VPMDC-induced depolarization of sperm {Delta}{Psi}m. Motile fraction of sperm was incubated without (A) and with low (31.2 µM; B) and high (250 µM; C) micromolar concentrations of VPMDC for 3 h, stained with JC-1 fluorescent probe to assess the {Delta}{Psi}m, and then analyzed by flow cytometry. VPMDC caused a reduction in {Delta}{Psi}m only at high micromolar concentration. Representative analysis from one of 3 experiments with identical results



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5. Concentration-dependent changes in sperm {Delta}{Psi}m quantitated by flow cytometry using JC-1 staining of sperm exposed to VDC, VMDC, and VPMDC. Motile sperm were incubated for 3 h in either control medium (0.5% DMSO) or medium supplemented with increasing 2-fold concentrations (0.24–250 µM) of VDC, VMDC, or VPMDC in the assay medium; stained with the {Delta}{Psi}m-sensitive dye JC-1; and analyzed by flow cytometry as described in Materials and Methods. The increase in JC-1 monomer (green)-positive sperm was indicative of loss of sperm {Delta}{Psi}m. Results are expressed as mean ± SD for 3 separate experiments.

Methyl Substitution on Cyclopentadienyl Rings Diminished the Apoptosis-Inducing Property of Vanadocenes

We next used the in situ TdT-mediated labeling of 3'-OH termini with digoxigenin-conjugated UTP assay method to demonstrate whether the enhanced spermicidal potency of methyl-substituted vanadocenes correlates with their ability to induce apoptosis in sperm. At 24 h after treatment of highly motile fraction of sperm with VDC, VMDC, and VPMDC at concentrations ranging from 0.24 µM to 500 µM, sperm were examined for digoxigenin-dUTP incorporation using FITC-conjugated anti-digoxigenin (green fluorescence) and PI counterstaining (red fluorescence). Figure 6 depicts the two-color flow cytometric contour plots of sperm nuclei of control and test sperm treated with low (31.2 µM) and high (250 µM) micromolar concentrations of VPMDC after staining with dUTP followed by immunodetection of the incorporated dUTP with anti-digoxigenin FITC monoclonal antibody and counterstaining with PI. Sperm treated with high micromolar concentration of VPMDC showed dual fluorescence, consistent with DNA fragmentation during apoptosis. The percentages of sperm nuclei positive for dUTP incorporation quantitated by the flow cytometric TUNEL assay for the three vanadocenes are shown in Figure 7. The percentage of apoptotic sperm increased in a concentration-dependent fashion with a mean EC50 of 49 ± 4 µM, 67 ± 10 µM, and 153 ± 32 µM for VDC, VMDC, and VPMDC, respectively (Table 1). Thus, the order of potency was VDC > VMDC > VPMDC. In contrast to their spermicidal potency, maximum dUTP incorporation was obtained when sperm were treated with vanadocenes only at concentrations >100 µM for 24 h. With VDC, >95% of sperm were apoptotic at the concentration of 125 µM and above. With VMDC, >90% of apoptotic sperm were observed at 250 µM. However, with VPMDC, only 50% of sperm were apoptotic even at 250 µM—a concentration that is 357-fold higher than its EC50 value for SIA. The differential concentration-dependent effect of vanadocenes on SIA and apoptosis indicate that SIA and apoptosis induction are mediated by two different mechanisms.



View larger version (20K):
[in this window]
[in a new window]
  		FIG. 6. Flow cytometric analysis of VPMDC-induced dUTP incorporation. Motile fraction of sperm was incubated without (A) and with low (31.2 µM; B) and high (250 µM; C) micromolar concentrations of VPMDC for 24 h, fixed, permeabilized, and visualized for DNA fragmentation using FITC-dUTP in a TUNEL assay as described in Materials and Methods. Sperm nuclei were counterstained with PI. VPMDC caused an increase in the percentage of sperm with dUTP incorporation only at high micromolar concentration. Representative analysis from one of 3 experiments with identical results



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 7. Concentration-dependent changes in sperm dUTP incorporation quantitated by the flow cytometric in situ DNA nick-end labeling assay of sperm exposed to VDC, VMDC, and VPMDC. Motile sperm were incubated for 24 h in medium with increasing 2-fold concentrations (0.24–250 µM) of VDC, VMDC, VPMDC, or 0.25% DMSO alone in the assay medium; they were then fixed, permeabilized, and visualized for DNA fragmentation using FITC-dUTP in a TUNEL assay. Sperm nuclei were counterstained with PI. Results are expressed as mean ± SD for 3 separate experiments

Lack of Inhibition of Axonemal Dynein ATPase Activity by Spermicidal Vanadocenes

Vanadate is an inhibitor of dynein ATPase [32, 33]. Dynein ATPase is essential for microtubule-dependent sperm motility. To test whether the rapid and concentration-dependent SIA of vanadocenes is due to their ability to inhibit sperm dynein ATPase activity, we treated highly motile fraction of sperm with VDC, VMDC, and VPMDC at 10-fold concentrations ranging from 0.01 µM to 100 µM for 60 min. After vanadocene treatment, sperm axonemes were isolated by sonication and differential centrifugation and assayed for ATPase activity by the spectrophotometric method using the EnzChek phosphate assay. The sensitivity of the assay is in the range of 2–150 nM of Pi in a 1-ml volume. In four separate experiments, none of the vanadocenes significantly (P > 0.05) affected the activity of dynein ATPase even at 100 µM—a concentration 13- to 130-fold higher than their spermicidal EC50 values (Fig. 8). The ATPase activity of control sperm was similar to that of vanadocene-treated sperm (n = 72). Specific activity of control ATPase was in the range of 10–15 nM of Pi per axonemes obtained from 5 x 106 sperm. The amount of free Pi present in preparations of sperm axonemes or ATP that was used as substrate was <0.2 nM. These results suggest that the observed SIA of vanadocenes observed was not due to inhibition of axonemal dynein ATPase activity.



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 8. Effect of exposure of sperm to VDC, VMDC, and VPMDC on sperm axonemal ATPase activity. Highly motile fraction of sperm was incubated for 60 min with increasing 10-fold concentrations (0.01–100 µM) of VDC, VMDC, VPMDC, or 0.1% DMSO alone in the medium and washed in PBS; axonemal proteins from Triton X-100 sperm lysates were isolated by ultracentrifugation and assayed for the generation of Pi by the spectrophotometric method using the EnzCheck phosphate assay as described in Materials and Methods. Results are expressed as mean ± SD for 4 separate experiments

Lack of Increased Tyrosine Phosphorylation of Sperm Global and Axoneme-Specific Proteins at Spermicidal Concentration

Vanadate is an inhibitor of protein tyrosine phosphatases [34]. We treated highly motile fraction of sperm for 60 min with and without increasing concentrations of each of the three vanadocenes. Axonemal-specific proteins of VDC, VMDC, and VPMDC-treated sperm were isolated by detergent extraction of homogenized sperm pellets followed by differential centrifugation and were subjected to SDS-polyacrylamide gel and Western blotting analysis with an anti-phosphotyrosine antibody. Figure 9 illustrates the representative pattern of sperm pellet phosphoproteins obtained after treatment of sperm with and without increasing concentrations of VDC or VPMDC. At the spermicidal concentrations, sperm treated with VDC or VPMDC showed no marked increase in phosphotyrosine content of several proteins using the whole sperm lysate. At the spermicidal concentration of 1–10 µM, identical phosphotyrosine signals were observed in comparison with those in vehicle controls. Figure 10 illustrates the pattern of sperm axonemal phosphoproteins obtained following treatment of sperm with and without increasing concentrations of VDC, VMDC, and VPMDC, respectively. Two major phosphoprotein bands with molecular masses of 105 kDa and 85 kDa were apparent. The intensity of these protein bands was essentially unaltered at the spermicidal concentration (<10 µM). An increase in protein phosphorylation of sperm axonemal proteins was evident only at concentrations above 10 µM (data not shown). These data demonstrate that SIA of vanadocenes was not dependent on inhibition of sperm phosphatases with concomitant increase in phosphotyrosine content of global or axonemal-specific sperm proteins.



View larger version (63K):
[in this window]
[in a new window]
  		FIG. 9. Concentration-dependent effect of exposure of sperm to VDC and VPMDC on total phosphotyrosine content of sperm proteins. Motile sperm were incubated for 60 min in BWW-0.3% BSA medium with increasing 10-fold concentrations (0.01–10 µM) of each of the 2 vanadocenes or 0.01% DMSO alone in the assay medium. Sperm proteins were separated by SDS electrophoresis, blotted onto PVDF membrane, probed with anti-phosphotyrosine antibody, and visualized by chemiluminescence as described in Materials and Methods. A) Immunoblot analysis of VDC-treated sperm. B) Immunoblot analysis of VPMDC-treated sperm. The positions of molecular weight marker proteins (x10-3) are indicated on the left. Representative Western blots from one of 4 experiments with identical results



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 10. Concentration-dependent effect of exposure of sperm to VDC, VMDC, and VPMDC on the phosphotyrosine content of sperm axoneme-specific proteins. Highly motile fraction of sperm was treated with or without increasing 10-fold concentrations (0.01–10 µM) of VDC, VMDC, or VPMDC for 60 min. Axonemal proteins from Triton X-100 sperm lysates were isolated by differential centrifugation and separated by SDS electrophoresis, blotted onto PVDF membrane, probed with anti-phosphotyrosine antibody, and visualized by chemiluminescence as described in Materials and Methods. A) Immunoblot analysis of VDC-treated sperm. B) Immunoblot analysis of VMDC-treated sperm. C) Immunoblot analysis of VPMDC-treated sperm. The positions of molecular weight marker proteins (x10-3) are indicated on the left. Representative Western blots from one of 4 experiments with identical results


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the marked increase in spermicidal potency observed with methyl substitutions on the Cp rings of vanadocene dihalide provided the opportunity to gain insight into the structure-function relationships controlling the potent spermicidal activity and apoptosis-inducing properties of vanadocenes. Although substitution of Cp rings with electron-donating methyl groups enhanced the SIA of vanadocene dihalide, VDC, by 10-fold, their apoptosis-inducing properties, evident only at high concentrations, were diminished. Whereas the spermicidal order of efficacy was VPMDC > VMDC > VDC, the reverse was true for induction of apoptosis as quantitated by the extent of DNA fragmentation. Furthermore, all three vanadocenes affected sperm {Delta}{Psi}m and protein tyrosine phosphorylation of total and axonemal-specific sperm proteins variably only at higher micromolar concentrations. These findings suggest that the concentration-dependent spermicidal and apoptosis-inducing properties of vanadocenes are independent of each other.

It has been previously reported that inorganic vanadium compounds inhibit flagellar motility of demembranated sea urchin sperm by inhibition of dynein ATPase activity [33]. Dyneins are microtubule motors that possesses ATPase activity. The movements of flagellar axonemes are generated by an ATP-dependent sliding of adjacent microtubule doublets produced by the intermittent interaction of the dynein arms on the A-subfiber with the adjacent B-subfiber triggered by the binding and hydrolysis of ATP. Therefore, we investigated whether the rapid and potent SIA of vanadocenes observed was due to inhibition of dynein ATPase activity. Interestingly, exposure of motile sperm even at 13- to 142-fold excess of their spermicidal concentration had no significant effect on sperm axonemal dynein ATPase activity. Furthermore, inorganic vanadium(IV/V) salts had no effect on the human sperm motion parameters [2]. Taken together, these results clearly demonstrate that the rapid SIA of vanadocenes observed with membrane-intact sperm was not due to inhibition of dynein ATPase activity. Whether vanadocenes can indirectly affect ATPase function of dynein, or interfere with the energy transport from the mitochondria to the flagellar axonemes or target some other enzymes that in turn can affect microtubule-dependent motility function, remains to be demonstrated.

Vanadate is an inhibitor of protein phospho tyrosine phosphatases [34]. Because a relationship between protein tyrosine phosphorylation and sperm motility has been proposed for bull, fowl, trout, and human sperm [35–39], we evaluated whether vanadocene-mediated SIA was a result of localized effect on certain phosphatases essential for sperm motility. However, at spermicidal concentrations, none of the vanadocenes significantly enhanced the tyrosine phosphorylation of global or axoneme-specific sperm proteins despite complete loss of sperm motility. Increased tyrosine phosphorylation of global or axonemal-specific sperm proteins was observed when membrane-intact sperm were treated with vanadocenes at 100 µM. This may indicate that vanadocenes at higher concentrations are acting on other components involved in tyrosine phosphorylation of proteins. The absence of a detectable increase in phosphotyrosine signal at spermicidal concentrations clearly demonstrates that inhibition of sperm motility is not due to inhibition of tyrosine phosphatases with a concomitant increase in phosphotyrosine signal of global and axonemal proteins. It is possible that vanadocenes may preferentially associate with the axoneme or basal bodies, thus creating local high concentration that affects tyrosine kinases in this cell motility compartment. Polymethylation on Cp2 rings probably allows more rapid penetration of vanadocenes through the sperm membranes in comparison with unsubstituted vanadocenes thereby, requiring a much lower concentration to affect sperm motility. Since spermicidal activity of all three vanadocenes is essentially complete in 15 sec, any biochemical changes in the sperm flagella that are critical for sperm motility loss must occur within seconds of vanadocene exposure.

Whereas the SIA was evident within seconds, induction of sperm apoptosis detected by DNA fragmentation required prolonged incubation. We used two independent methods that quantitatively assess apoptotic changes in the mitochondria and nuclear compartment. Mitochondria are the primary targets for apoptosis, and alterations in mitochondrial structure and function are early events of apoptotic cell death [27]. Our studies demonstrated that the SIA of vanadocenes could not be due to their ability to depolarize sperm mitochondria, since despite complete sperm immobilization, dramatic changes in {Delta}{Psi}m were apparent only at 100-fold excess of their EC50 values. Furthermore, even after 24-h exposure to these vanadocenes, variable increase in dUTP incorporation in the nuclei of treated sperm was evident only at 100-fold excess of their EC50 values. Since vanadium(IV) compounds by themselves do not cleave DNA [40], the dUTP incorporation by vanadocene-treated sperm is most likely due to their reaction with H2O2 forming ·OH radicals in a Fenton-like reaction [18, 41]. EPR spin trapping studies clearly demonstrated that VDC, VMDC, and VPMDC generate ·OH radicals in the presence of spin trap and H2O2, resulting in oxidation of vanadium(IV) [18]. These findings suggest that ROS generated by the vanadocenes may contribute to the SIA that culminates in apoptosis.

Recently, the membrane interactions of spermicidal vanadocenes have been studied using zwitterionic and negative unilamellar liposomes [13]. None of the spermicidal vanadocenes tested caused appreciable liposome aggregation, fusion, or changes in packing order of the liposomes as observed from UV/visible spectroscopy, fluorescence energy resonance transfer, and fluorescence polarization studies [13]. It was inferred that vanadocenes have unique configurational preferences that alter the membranes by intercalation, creating "leaky patches" in the liposomal membranes. The metal ions were found to alter the membrane by creating rigid clusters where the hydrophobic chains are close together, thus increasing the susceptibility of the lipids to peroxidation. The high concentration of n-3 polyunsaturated fatty acids in sperm plasma membranes makes them particularly vulnerable to peroxidative changes, since these fatty acids containing two or more double bonds are readily attacked by oxygen radicals [42, 43]. Thus the ability of vanadocenes to rapidly immobilize sperm via membrane intercalation and lipid peroxidation fundamentally differs from the mechanism of currently used detergent-type spermicides that induce nonspecific membrane permeability and membrane fusion [14–16].

In our ongoing preclinical animal toxicity studies, unlike the intravaginally applied N-9, repetitive intravaginal application of spermicidal vanadocenes via a gel-microemulsion nearly 1000-fold their EC50 values showed no vaginal irritation, mucosal toxicity, or systemic absorption of vanadium in the rabbit vaginal tolerance test. At spermicidal doses, vanadocenes were noncytotoxic to normal human reproductive tract epithelial cells. Therefore, unlike N-9, vanadocenes may provide a safe and effective contraceptive practice without mucosal erosion and local inflammation. Spermicidal vanadocenes were nongenotoxic when tested by the yeast DEL recombination assay and transcriptional activation of genotoxic stress-specific promoters in human hepatoma cells using the CAT-Tox(L) assay [44]. Results of our ongoing preclinical studies demonstrated that vanadocenes, because of their potent spermicidal activity and lack of inflammatory and toxic effects, may be useful as safe and effective vaginal contraceptives.

In previous studies, through a progressive series of analogues we demonstrated that the oxidation state of vanadium, the geometry of the complex, and the nature of ancillary ligands attached to the Cp2 rings are essential for the potent SIA of vanadocenes [1–3, 45]. In this study, we have demonstrated that methyl substitution on the Cp2 enhances their spermicidal potency. Due to their marked ability to rapidly generate ·OH radicals, the observed SIA and apoptosis-inducing properties of these vanadocenes may be mediated, in part, through lipid peroxidation and DNA modification by ROS, respectively [13, 18]. The rapid and potent SIA of vanadocenes without the concomitant induction of apoptosis as demonstrated in this study is of particular advantage for the development of vanadocenes as a new class of effective vaginal contraceptive agents.


   FOOTNOTES
 
First decision: 27 October 1999.

1 Correspondence: Osmond J. D'Cruz, Parker Hughes Institute, 2665 Long Lake Road, Suite 330, St. Paul, MN 55113. FAX: 651 697 0645; odcruz{at}ih.org Back

Accepted: November 16, 1999.

Received: September 23, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. D'Cruz OJ, Uckun FM. Vanadocenes as a new class of effective spermicides. Adv Reprod 1998; 1:102–123.
  2. D'Cruz OJ, Ghosh P, Uckun FM. Spermicidal activity of metallocene complexes containing vanadium(IV) in humans. Biol Reprod 1998; 58:1515–1526.[Abstract/Free Full Text]
  3. D'Cruz OJ, Ghosh P, Uckun FM. Spermicidal activity of chelated complexes of bis(cyclopentadienyl)vanadium(IV). Mol Hum Reprod 1998; 4:683–693.[Abstract/Free Full Text]
  4. Wilkinson G, Birmingham JM. Bis-cyclopentadienyl compounds of Ti, Zr, V, Nb, and Ta. J Am Chem Soc 1954; 76:4281–4284.[CrossRef]
  5. Wilkinson G. Comprehensive Organometallic Chemistry. New York: Pergamon; 1982: 554–646.
  6. Byczkowski JZ, Wan JZ, Kulkarni AP. Vanadium-mediated lipid peroxidation in microsomes from human term placenta. Bull Environ Contam Toxicol 1988; 41:696–703.[CrossRef][Medline]
  7. Ozawa T, Hanaki A. ESR evidence for the formation of hydroxyl radicals during the reaction of vanadyl ions with hydrogen peroxide. Chem Pharm Bull (Tokyo) 1989; 37:1407–1409.
  8. Carmichael AJ. Vanadyl-induced Fenton like reaction in RNA: an ESR and spin trapping study. FEBS Lett 1990; 261:165–170.[CrossRef][Medline]
  9. Keller RJ, Sharma RP, Grover TA, Piette LH. Vanadium and lipid peroxidation: evidence for involvement of vanadyl and hydroxyl radical. Arch Biochem Biophys 1988; 265:524–533.[CrossRef][Medline]
  10. Younes M, Strubelt O. Vanadate-induced toxicity towards isolated perfused rat livers: The role of lipid peroxidation. Toxicology 1991; 66:63–74.[CrossRef][Medline]
  11. Sakurai H, Tamura H, Okatani K. Mechanism for a new antitumor vanadium complex hydroxyl radical-dependent DNA-cleavage by 1,10-phenanthroline-vanadyl complex in the presence of hydrogen peroxide. Biochem Biophys Res Commun 1995; 206:133–137.[CrossRef][Medline]
  12. Shi X, Wang P, Jiang H, Mao Y, Ahmed N, Dalal N. Vanadium(IV) causes 2'-deoxyguanosine hydroxylation and deoxyribonucleic acid damage via free radical reactions. Ann Clin Lab Sci 1996; 26:39–49.[Abstract]
  13. Kotchevar AT, Ghosh P, Uckun FM. Interactions of vanadocene(IV)-chelated complexes with artificial membranes. J Phys Chem B 1998; 102:10925–10930.[CrossRef]
  14. Schill WB, Wolff HH. Ultrastructure of human spermatozoa in the presence of the spermicide nonoxynol-9 and a vaginal contraceptive containing nonoxynol-9. Andrologia 1981; 13:42–49.[Medline]
  15. Wilborn WHH, Hahn DW, McGuire JJ. Scanning electron microscopy of human spermatozoa after incubation with the spermicide nonoxynol-9. Fertil Steril 1983; 39:717–719.[Medline]
  16. Furuse K, Ishizeki C, Twahara S. Studies on spermicidal activity of surfactants. I. Correlation between spermicidal effect and physicochemical properties of p-menthanylphenyl polyoxethylene (8.8) ether and related surfactants. J Pharmacobio-Dyn 1983; 6:359–372.[Medline]
  17. Ghosh P, Ghosh S, D'Cruz OJ, Uckun FM. Structural and biological characterization of a novel spermicidal vanadium(IV) complex: bis({pi}-cyclopentadienyl)-,N,N-diethyl dithiocarbamato vanadium(IV) tetrafluoro borate, [VCp2(DeDtc)(BF4)]. J Inorg Biochem 1998; 72:89–98.[CrossRef][Medline]
  18. Ghosh P, D'Cruz OJ, DuMez DD, Peitersen J, Uckun FM. Structural and functional characterization of seven spermicidal vanadium(IV) complexes: potentiation of activity by methyl substitution on the cyclopentadienyl rings. J Inorg Biochem 1998; 75:135–143.[CrossRef]
  19. Cardin DJ, Lappart MF, Raston CL, Riley PI. Comprehensive organometallic chemistry 1982; 3:554–646.
  20. D'Cruz OJ, Toth CA, Haas GG Jr. Recombinant soluble human complement receptor type 1 inhibits antisperm antibody-and neutrophil-mediated injury to human sperm. Biol Reprod 1996; 54:1217–1228.[Abstract]
  21. D'Cruz OJ, Uckun FM. Novel derivatives of phenethyl-5-bromopyridylthiourea (PBT) and dihydroalkoxybenzyloxopyrimidine (DABO) are dual-function spermicides with potent anti-HIV activity. Biol Reprod 1999; 60:1419–1428.[Abstract/Free Full Text]
  22. D'Cruz OJ, Venkatachalam TK, Zhu Z, Shih M, Uckun FM. Bromo-methoxy and aryl phosphate derivatives of azidothymidine are dual function spermicides with potent anti-HIV activity. Biol Reprod 1998; 59:503–515.[Abstract/Free Full Text]
  23. D'Cruz OJ, Shih M-J, Yiv SH, Chen CL, Uckun FM. Synthesis of a novel phenyl phosphate derivative of bromo-methoxy zidovudine (compound WHI-07) with potent anti-HIV and spermicidal activities. Mol Hum Reprod 1998; 5:421–432.[Abstract/Free Full Text]
  24. D'Cruz OJ, Zhu Z, Yiv SH, Chen CL, Waurzyniak B, Uckun FM. WHI-05, a novel bromo-methoxy substituted phenyl phosphate derivative of zidovudine is a dual-action spermicide with potent anti-HIV activity. Contraception 1999; 59:319–331.[CrossRef][Medline]
  25. Cossarizza A, Contri-Baccarani M, Kalashnikova G, Francesschi C. A new method for the cytofluorometric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cations 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 1993; 197:40–45.[CrossRef][Medline]
  26. Zamzani N, Marchetti P, Castedo M, Zanin C, Vayssiere J-L, Petit PX, Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 1995; 181:1661–1672.[Abstract/Free Full Text]
  27. Smiley ST, Reers M, Mottola-Harshorn C, Lin M, Chen A, Smith TW, Steele GD Jr, Chen LB. Intracellular heterogeniety in mitochondrial membrane potentials revealed by J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci USA 1991; 88:3671–3675.[Abstract/Free Full Text]
  28. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119:493–501.[Abstract/Free Full Text]
  29. Vassilev A, Ozer Z, Navara C, Mahajan S, Uckun FM. Bruton's tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex. J Biol Chem 1999; 274:1646–1656.[Abstract/Free Full Text]
  30. Gibbons IR, Lee-Eiford A, Mocz G, Phillipson CA, Tang WJ, Gibbons BH. Photosensitized cleavage of dynein heavy chains. Cleavage of the "VI site" by irradiation at 365 nm in the presence of ATP and vanadate. J Biol Chem 1987; 262:2780–2786.[Abstract/Free Full Text]
  31. Webb MR. A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci USA 1992; 89:4884–4887.[Abstract/Free Full Text]
  32. Shimizu T. Inhibitors of dynein ATPase and ciliary and flagellar motility. Methods Cell Biol 1995; 47:497–501.[Medline]
  33. Gibbons IR, Cosson MP, Evans JA, Gibbons BH, Houck B, Martinson KH, Sale WS, Tang W-JY. Potent inhibition of dynein adenosine triphosphatase and of the motility of cilia and sperm flagella by vanadate. Proc Natl Acad Sci USA 1978; 75:2220–2224.[Abstract/Free Full Text]
  34. Gordon JA. Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods Enzymol 1991; 201:477–482.[Medline]
  35. Vijayaraghavan S, Trautman KD, Goueli SA, Car DW. A tyrosine-phosphorylated 55-kilodalton motility-associated bovine sperm protein is regulated by cyclic adenosine 3',5'-monophosphate and calcium. Biol Reprod 1997; 56:1450–1457.[Abstract]
  36. Ashizawa K, Higashio M, Tsuzuki Y. Effects of tyrosine kinase inhibitor on the motility and ATP concentrations of fowl spermatozoa. Mol Reprod Dev 1998; 49:196–202.[CrossRef][Medline]
  37. Hayashi H, Yamamoto K, Yonekawa H, Morisawa M. Involvement of tyrosine protein kinase in the initiation of flagellar movement in rainbow trout spermatozoa. J Biol Chem 1987; 262:16692–16698.[Abstract/Free Full Text]
  38. Leclerc P, de Lamirande E, Gagnon C. Cyclic adenosine 3',5' monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. Biol Reprod 1996; 55:684–692.[Abstract]
  39. Turner RM, Ericksson RL, Gerton GL, Moss SB. Relationship between sperm motility and the processing and tyrosine phosphorylation of two human sperm fibrous sheath proteins, pro-hAKAP82 and hAKAP82. Mol Hum Reprod 1999; 5:816–824.[Abstract/Free Full Text]
  40. Kuo YL, Liu AH, Marks TJ. Metallocene interactions with DNA and DNA-processing enzymes. In: Sigel H (ed.), Metal Ions in Biological Systems. New York: Marcel Dekker; 1996: 53–85.
  41. Sakurai H, Nakai M, Miki T, Tsuchiya K, Takada J, Matsushita R. DNA cleavage by hydroxyl radicals generated in a vanadyl ion hydrogen peroxide system. Biochem Biophys Res Commun 1992; 189:1090–1095.[CrossRef][Medline]
  42. Kim JG, Parthasarathy S. Oxidation and the spermatozoa. Semin Reprod Endocrinol 1998; 16:235–239.[Medline]
  43. Chen CS, Chao HT, Pan RL, Wei YH. Hydroxyl radical-induced decline in motility and increase in lipid peroxidation and DNA modification in human sperm. Biochem Mol Biol Int 1997; 43:291–303.[Medline]
  44. Aubrecht J, Narla RK, Ghosh P, Stanek J, Uckun FM. Molecular genotoxicity profiles of apoptosis-inducing vanadocene complexes. Toxicol Appl Pharmacol 1999; 154:228–235.[CrossRef][Medline]
  45. D'Cruz OJ, Dong Y, Uckun FM. Spermicidal activity of oxovanadium(IV) complexes of 1,1'-phenanthroline, 2,2'-bipyridyl, 5'-bromo-2'-hydroxyacetophenone and derivatives in humans. Biol Reprod 1999; 60:435–444.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Toxicol PatholHome page
O. J. D'Cruz and F. M. Uckun
Preclinical Evaluation of a Dual-Acting Microbicidal Prodrug WHI-07 in Combination with Vanadocene Dithiocarbamate in the Female Reproductive Tract of Rabbit, Pig, and Cat
Toxicol Pathol, December 1, 2007; 35(7): 910 - 927.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
O. J. D'Cruz and F. M. Uckun
Discovery of 2,5-dimethoxy-substituted 5-bromopyridyl thiourea (PHI-236) as a potent broad-spectrum anti-human immunodeficiency virus microbicide
Mol. Hum. Reprod., October 1, 2005; 11(10): 767 - 777.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
B. Sotolongo, T. T. F. Huang, E. Isenberger, and W. S. Ward
An Endogenous Nuclease in Hamster, Mouse, and Human Spermatozoa Cleaves DNA into Loop-Sized Fragments
J Androl, March 1, 2005; 26(2): 272 - 280.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
O. J. D'Cruz, P. Samuel, and F. M. Uckun
PHI-443: A Novel Noncontraceptive Broad-Spectrum Anti-Human Immunodeficiency Virus Microbicide
Biol Reprod, December 1, 2004; 71(6): 2037 - 2047.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow