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BOR - Papers in Press, published online ahead of print January 21, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.022210
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BIOLOGY OF REPRODUCTION 70, 1518–1526 (2004)
DOI: 10.1095/biolreprod.103.022210
© 2004 by the Society for the Study of Reproduction, Inc.

Toxicology

Male Reproductive Toxicity of Trichloroethylene: Sperm Protein Oxidation and Decreased Fertilizing Ability1

Shelley Brown DuTeaux3, Trish Berger4, Rex A. Hess5, Becky L. Sartini4, and Marion G. Miller2,3

Department of Environmental Toxicology3 and Department of Animal Science,4 University of California, Davis, California 95616 Department of Veterinary Biosciences,5 University of Illinois, Urbana, Illinois 61802


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of the present study was to characterize and investigate potential mechanisms for the male reproductive toxicity of trichloroethylene (TCE). Male rats exposed to TCE in drinking water exhibited a dose-dependent decrease in the ability to fertilize oocytes from untreated females. This reduction in fertilizing ability occurred in the absence of treatment-related changes in combined testes/epididymides weight, sperm concentration, or sperm motility. In addition, flow cytometric analysis showed that there were no treatment-related differences in sperm mitochondrial membrane potential or acrosomal stability. TCE caused slight histological changes in efferent ductule epithelium, coinciding with the previously reported ductule localization of cytochrome P450 2E1. However, no alterations were noted in the testis or in any segment of the epididymis. Because there were no treatment-related changes to sperm indices and no clear pathological lesions to explain the reduced fertilization, the present study investigated TCE-mediated sperm oxidative damage. Oxidized proteins were detected by immunochemical techniques following the derivatization of sperm protein carbonyls with dinitrophenyl hydrazine. Immunochemical staining of whole, intact sperm showed the presence of halos of oxidized proteins around the head and midpiece of sperm from TCE-treated animals. The presence of oxidized sperm proteins was confirmed by Western blotting using in vitro-oxidized sperm as a positive control. Thiobarbituric acid reactive substances analyses showed a dose-dependent increase in the level of lipid peroxidation in sperm from treated animals, as well. Oxidative damage to sperm may explain the diminished fertilizing capacity of exposed animals and provide another mechanism by which TCE can adversely affect reproductive capabilities in the male.

epididymis, in vitro fertilization, male reproductive tract, sperm, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichloroethylene (TCE; ClHC=CCl2) is a small molecular weight halogenated solvent that is considered a potential health hazard to humans [1]. Because of its widespread industrial use and historic disposal practices, TCE is one of the most common water contaminants in the United States [2]. It is estimated that over 3 million individuals are exposed to TCE through occupational and environmental exposures annually [3].

Recent epidemiological studies have shown associations between exposure to chlorinated solvents (including TCE) and decreased semen concentration, sperm motility, and increased percentages of abnormal sperm [4, 5]. Paternal TCE and solvent exposure have also been associated with an increased risk of spontaneous abortion, decreased implantation rates, decreased fertility, and delayed conception [6–9]. Studies in mice and rats support an association between TCE exposure and altered sperm parameters. Mice exposed to TCE via inhalation (~150 ppm) had increased percentages of abnormal sperm [10]. TCE inhalation (376 ppm) also decreased epididymal sperm count and motility in rats [11]. An increased percentage of abnormal sperm was observed in rats following drinking-water exposure to TCE (9.5 ppm) in combination with other drinking-water contaminants [12].

One mechanism to explain the toxic effects of TCE is the in situ metabolism of TCE by enzymes located in the tissues where toxicity is observed [13]. The level of TCE metabolism is highest in the liver and the kidney [14, 15]. Following TCE exposure, toxicity is found in the centrilobular regions of the liver and kidney proximal tubules, where cytochrome P450 enzymes are localized [14, 15]. Recent experiments show that the rat excurrent ducts can oxidatively metabolize TCE [16]. Such findings support the hypothesis that male reproductive toxicity may arise from the metabolism of TCE within the reproductive tract.

TCE is metabolized primarily by cytochrome P450 2E1 [17]. Oxidative metabolism of TCE generates a variety of reactive intermediates, including the unstable epoxide 2,2,3-trichlorooxirane and TCE-oxide [14, 18], oxygen-free radicals [19], and trichlorinated carbon-centered radicals [20]. Reactive oxygen species and radicals can damage cellular components, resulting in increased protein turnover, decreased protein function, and lipid peroxidation [21].

Production of reactive oxygen species by sperm is considered a normal physiological reaction [22]. However, an imbalance between the production of oxygen radicals and the detoxification/scavenging of these radicals can alter reproductive fitness [22]. Peroxidation of sperm membrane lipids and oxidation of sperm proteins are associated with decreased motility and fertilizing ability [23, 24]. The purpose of the present study is threefold: to determine if TCE exposure results in reproductive tract or sperm pathology; to determine if TCE exposure alters the ability of sperm to fertilize oocytes in vitro; and to determine if TCE exposure results in oxidative damage to sperm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Adult male rats were obtained from two sources: Charles River Sprague-Dawley rats (CR rats) from Charles River Laboratories (Hollister, CA) and from the University of California, Davis breeding colony of Simonson albino rats (UCD rats). Female Sprague-Dawley rats (28–45 days; Charles River) were purchased for oocyte retrieval. All animals were kept under a 12L:12D cycle in a temperature (22 ± 2°C) and humidity (40– 70%) controlled facility. Rats were maintained on Formulab Purina 5008 rat chow (Purina, St. Louis, MO) ad libitum. The University of California, Davis Animal Use and Care Administrative Advisory Committee approved all animal use.

Chemicals and Antibodies

Analytical-grade TCE, 25% glutaraldehyde (w/v), hormones, rabbit anti-dinitrophenyl antibody, bovine serum albumin, fraction V (BSA), and acid Tyrode were purchased from Sigma (St. Louis, MO). Ethoxylated castor oil (Alkumuls 719) was a gift from Rhodia (Cranbury, NJ). The dual emission fluorophore 5,5,'6,6'-tetrachloro-1,1,'3,3'-tetraethylbenzimidazolycarbocyanine iodide (JC-1), AlexaFluor488 goat anti-rabbit fluorescent conjugated IgG, SYTO-17, propidium iodide, and fluorescein isothiocyanate (FITC)-labeled peanut agglutinin were purchased from Molecular Probes (Eugene, OR). Goat anti-rabbit alkaline phosphatase-conjugate IgG was purchased from Promega (Madison, WI). The Bioxytech MDA-586 kit was purchased from OXIS Health Products Inc. (Portland, OR).

Chemical Treatment

Drinking water was distilled and deionized, treated by reverse osmosis, and filtered through granular activated carbon to minimize any chemical contamination. Male rats were exposed to 0, 0.2%, or 0.4% TCE (v/v) in a solution of 3% ethoxylated castor oil (v/v) in drinking water for 14 days. Glass water bottles, fit with Teflon septa and rubber/stainless steel stoppers, were filled every 24 h with fresh solutions to minimize headspace and TCE volatilization.

Gamete Retrieval

TCE-treated and control male rats were killed by CO2 inhalation in a warm (30–32°C) room. Weights were recorded for the whole animal and for the testis and attached epididymis. The cauda epididymidis and vas deferens were removed, rinsed in warmed fertilization medium (a modified Tyrode medium containing 50 µg/ml gentamycin sulfate, 4 mg BSA/ml, and 20 mM Hepes), and placed in 3–4 ml of warmed medium in a 35 x 10 mm culture dish. Sperm were collected by retrograde flush of the vas deferens and cauda epididymis. Briefly, several small slits were made in the distal cauda and a known volume of warm in vitro fertilization (IVF) buffer was injected into the cauda from the vas to flush out the sperm. The flush was complete when the fluid flowing from the distal cauda became clear. This method of sperm collection insures a representative sperm population and one that is not biased toward collecting only motile sperm [25]. Sperm were diluted to 0.5 x 106 sperm/ml and incubated (3 h, 37°C, 5% CO2:95% air) for sperm capacitation [26].

Female rats were induced to ovulate by i.p. injection of equine chorionic gonadotropin (eCG; 15 IU) followed 48 h later by 15 IU of human chorionic gonadotropin (hCG) [26]. Female rats were killed by CO2 inhalation followed by cervical dislocation 16–18 h after hCG injection. Oviducts and attached ovaries were dissected. Oviductal cumulus masses were teased out into warm saline-BSA (0.9% NaCl [w/v], 1 mg BSA/ml) and the cumulus matrix was dispersed with 1 mg hyaluronidase/ml saline- BSA. The zona pellucida was removed by brief transfer to acid Tyrode (pH 2.5). Oocytes were diluted with 1 ml saline-BSA, removed from the dish, and rinsed with three drops of fertilization medium [26].

In Vitro Fertilization

Oocytes were added to 100-µl drops of preequilibrated medium under oil and mixed with 10 µl of diluted sperm from each treatment (final volume 120 µl). After incubating for 20 h at 37°C under 5% CO2:95% air, oocytes were rinsed with three drops of fertilization medium to remove the loosely attached spermatozoa. Oocytes were incubated for 10 min with 0.08 mg/ml Hoechst 33342, rinsed, and placed on slides. Decondensed sperm heads and attached spermatozoa were counted using a fluorescent microscope (400x magnification) [26].

Assessment of Sperm Parameters

Sperm concentration was determined on 20-µl aliquots of diluted sperm using a hemacytometer. Sperm motility was assessed visually using phase-contrast optics. This was followed by a more detailed computer- assisted semen analysis (CASA) using a Hobson Sperm Tracker sperm motility analyzer (Hobson Tracking System Ltd., Sheffield, UK). The motility parameters measured included average path velocity (VAP), straight- line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and linearity (LIN). Parameter settings were based on manufacturer recommendations and empirical alterations were used to optimize the tracking [27]. The frame rate was 30 frames/sec (i.e., continuous analysis of the sperm head motion for 30 points/sec). A minimum of 100 tracks were analyzed for each male in each treatment group.

Flow Cytometry

Acrosomal status and viability were evaluated with SYTO 17/propidium iodide (PI) and FITC-labeled peanut agglutinin (PNA). SYTO 17 labels live sperm a dull red, PI labels dead sperm a bright red, and FITC PNA labels the acrosomal membrane when it is accessible [28]. In nonpermeabilized cells, FITC PNA labeling suggests the cell is undergoing an acrosome reaction [29]. FITC PNA (150 µl of 0.1 g/L) and SYTO 17 (1 µl of a 10% [v/v] stock solution) were added to 500 µl rat sperm (25 x 106/ml). After a 10-min incubation, 3 µl PI (2.99 mM) was added. A 50-µl aliquot was immediately diluted with 450 µl fertilization medium and fluorescence was analyzed on a FACScan equipped with a 488 argon laser, FL1 530-nm band-pass filters, and FL3 620-nm long-pass filters (Becton-Dickinson, San Jose, CA). Samples were evaluated after 0, 3, and 6 h of in vitro capacitation.

The dual emission fluorophore JC-1 was used to assess the mitochondrial membrane potential in sperm from treated and control animals [30]. Green fluorescence is emitted at low mitochondria membrane potential, and orange fluorescence is emitted at high mitochondrial membrane potential. Three microliters of a prepared solution (1.53 mM JC-1 in dimethyl sulfoxide) was added to a 500-µl sample of sperm (25 x 106/ml) and incubated for 10 min at 37°C. Labeled sperm were diluted 1:10 in Holloway medium and immediately assessed for low and high membrane potential using flow cytometry on a FACScan [30]. Fluorescent labeling patterns of a minimum of 10 000 spermatozoa per animal per treatment were evaluated. Samples were evaluated after 0, 3, and 6 h of in vitro capacitation.

Histology

Perfusion fixation was modified from a previously published method [31]. Briefly, treated and control rats (n = 3 per group) were anesthetized with a mixture of 80 mg/kg ketamine and 20 mg/kg xylazine. An incision was made in the abdomen and 0.4 ml heparin was injected into the vena cava. The animal was perfused through the aorta with Ringer bicarbonate solution (pH 7.4) and perfusion fixed for 12–15 min with 4% glutaraldehyde (v/v). The testis and attached epididymis (fat pad intact) was dissected and stored in 4% glutaraldehyde. Prior to embedding, the efferent ductules were dissected away from the testis and epididymis. The caput, corpus, and cauda epididymides were sectioned laterally. The tissues were embedded in glycol methacrylate resin, thin-sectioned (2.5 µm), and stained with 1% toluidine blue (v/v) or periodic acid-Schiff (PAS) with hematoxylin counterstaining. Histology was observed using light microscopy.

Immunochemical Detection of Oxidized Proteins on Spermatozoa

Caudal sperm from treated and control rats (n = 3 per group) were washed with sodium phosphate buffer (pH 7.4) containing 50 µg/ml gentamycin sulfate and pelleted by gentle centrifugation (500 x g, 5 min). Sperm were resuspended and smeared onto poly-L lysine-coated slides. Remaining sperm were retained for Western blotting and analysis of lipid peroxidation (below). After drying, smears were rehydrated in sodium phosphate buffer (pH 7.4). Protein carbonyls were derivatized by incubating the smears in a solution of 5 mM 2,4-dinitrophenylhydrazine in sodium phosphate buffer (pH 6.3) for 45 min at 22°C. Following derivatization, smears were rinsed twice in phosphate buffered saline (PBS), blocked with 5% normal goat serum (v/v) in 1% BSA (w/v)-PBS, and incubated with rabbit anti-dinitrophenyl antibody (1:250). Following extensive washing, smears were incubated with AlexaFluor488-conjugate goat anti-rabbit IgG antibody (1:1000), rinsed, and cover slipped with VectaShield (Vector Laboratories, Burlingame, CA). Background fluorescence was tested by incubating duplicate sections with normal rabbit serum (1:250) in place of the primary antibody. Sperm were visualized on an Olympus AX70 Provis Microscope (Olympus America, Inc., Melville, NY) and images were captured digitally by a Zeiss AxioCam CCD camera (Carl Zeiss, Thornwood, NY).

Oxidized proteins were also detected using Western blotting. Briefly, washed whole sperm (20 x 106/ml) were derivatized in vitro with a solution of 5 mM 2,4-dinitrophenyl hydrazine in sodium phosphate buffer (pH 6.3) for 45 min at 22°C (1 ml total volume). Sperm were rinsed twice by adding 3 ml sodium phosphate buffer (pH 7.4), gently mixing, and centrifuging at 500 x g for 5 min. Excess dinitrophenyl hydrazine was removed, and sperm were resuspended in sodium phosphate buffer (pH 7.4) and homogenized with a glass and Teflon mortar and pestle. For a positive control, caudal sperm collected from untreated rats were oxidized in vitro using a mixture of 1.8 mM ADP, 0.23 or 2.3 mM H2O2, and 2.7 mM FeSO4 [32]. These sperm were derivatized, washed, and homogenized as above. Sperm homogenates (100 µg total protein) were solubilized by heating with 10% SDS (w/v), separated on 10% SDS-PAGE, and electroblotted to 0.45 m nitrocellulose using a wet transfer apparatus (Bio- Rad, Hercules CA). Membranes were blocked with 5% milk (w/v) in Tris- buffered saline (50 mM Tris-HCl, 150 mM NaCl; pH 7.5) with 0.05% Tween-20 (v/v; TTBS). Following blocking, membranes were incubated with rabbit anti-dinitrophenyl antibody (1:1000), washed, and incubated with goat anti-rabbit alkaline phosphatase-conjugate IgG antibody (1: 20 000). Immunoreactive bands were visualized using Western Blue alkaline phosphatase substrate (Promega, Madison, WI).

Lipid Peroxidation (Thiobarbituric Acid Reactive Substances Assay)

The level of lipid peroxidation in sperm from treated and control animals (n = 3 per group) was assayed using the Bioxytech MDA-586 kit for spectrophotometric analysis of malondialdehyde (MDA). Sperm were gently resuspended in sodium phosphate buffer (pH 7.4) containing 50 µg/ml gentamycin sulfate. Sperm were diluted to a final concentration of 20 x 106/ml and homogenized by hand in a glass and Teflon mortar and pestle with butylated hydroxytoluene added as an antioxidant. Sperm homogenate (200 µl) was placed in a microcentrifuge tube, to which was added 10 µl probucol and 640 µl N-methyl-2-phenylindole in a 75% acetonitrile:25% methanol (v/v) mixture according to kit directions. Samples were acidified with concentrated hydrochloric acid (150 µl), vortexed thoroughly, and incubated for 60 min in a 45°C water bath. Following the incubation, samples were centrifuged (10 000 rpm, 10 min) and the absorbance of the cleared supernatant was measured at {lambda} = 586 nm in triplicate samples. Duplicate six-point standard curves (0, 0.2, 0.4, 0.8, 1.6, 2.4 µmol MDA) were produced by the acid-incubation of the MDA precursor tetramethoxypropane according to kit directions. Standards were analyzed in duplicate at the beginning and end of each set of samples and a standard curve was generated from the average absorption at each point. Sample mean absorbances were converted to µmol MDA using linear regression from the standard curve, and results were expressed as µmol MDA/106 sperm.

Data Analysis

In vitro fertilization data, sperm motion parameters, and flow cytometry data were subjected to analysis of variance (ANOVA) using SAS (SAS Statistical Programs, Cary, NC). For the in vitro fertilization data, ANOVA was treatment (fixed factor) x male (random factor). Percentage of oocytes fertilized was analyzed before and after transformation to the arc-sin square root, a common transformation used to improve normality for percentage data [33]. For motion parameters, percentiles derived from individual track data were subjected to ANOVA [34]. Data for each sperm motion parameter for each sample were sorted from a file of the individual track data and values at the 50th percentile (median) were determined. Percentile values for PNA labeling of acrosomal status and JC-1 labeling from flow cytometric analysis were similarly analyzed. In addition, acrosomal status and mitochondrial membrane potential data were subjected to quadrant analysis (separation into live vs. dead and acrosome-reacting vs. nonreacting or high vs. low membrane potential). Differences in animal and organ weight parameters were calculated as mean ± SD (n = 3 per treatment group) and lipid peroxidation data are presented as mean ± SEM (triplicate incubations from each animal in each treatment group). Significance for both was measured using the Student t-test with P < 0.05. To avoid repetition of similar experimental results, data reported are for CR rats unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TCE Adversely Affects In Vitro Fertilization

In the present study, male rats were exposed to drinking water with 0%, 0.2% (2.73 mg/L), or 0.4% (5.46 mg/L) TCE emulsified in 3% ethoxylated castor oil. This was the only source of fluid for male rats for the duration of the experiment. Based on animal weight and average daily water consumption of 28 ml per animal, the calculated oral doses of TCE ranged from 1.6–2.0 mg kg–1day–1 for the low-dose group and 3.4–3.7 mg kg–1day–1 for the high- dose group. The lethal dose, LD50 (oral, rat), for TCE is 5650 mg/kg [3]. The calculated doses in the present study are approximately 1/100 and 1/200 of the LD50 for the high- and low-dose groups, respectively.

TCE caused a dose-dependent decrease in the ability of sperm to fertilize zona-free oocytes from untreated females (Fig. 1). Qualitatively, the effect was the same in the two trials. However, the magnitude of the effect was different between the CR rats and the UCD rats. For the CR rats, the percentage of oocytes fertilized was 33% and 46% for the high-dose and low-dose groups, respectively. The effect was significant when compared with percentage fertilized in the control group (P < 0.005 for 0.4% TCE treatment; P < 0.05 for 0.2% TCE treatment). For UCD rats, percentage of oocytes fertilized was 4% for the high-dose group and 18% for the low-dose groups. The effect of both treatments was significant when compared with the percentage fertilized in the control group (P < 0.05). The percentage of oocytes fertilized by sperm collected from control animals varied from 34% (UCD rats) to 80% (CR rats), although the treatment protocol was replicated in the two trials. Some of the variance in control values may be due to biological variability between groups of animals, as previously found in this laboratory [27]. A difference in proficiency of IVF technicians may also account for some of the differences in control values between trials.



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  		FIG. 1. Decreased fertilizibility of oocytes from untreated female rats when fertilized in vitro with sperm recovered from males dosed with 0, 0.2%, or 0.4% trichloroethylene (TCE) (v/v) in ethoxylated castor oil vehicle (3% v/v) for 14 days. Two separate IVF trials were conducted, one with Charles River Sprague-Dawley (CR) rats and one with University of California, Davis breeding colony (UCD) rats. Both groups demonstrated a dose-dependent decrease in percentage of fertilized oocytes. When compared with UCD controls, ANOVA results indicated a significant decrease in the percentage of oocytes fertilized with sperm from 0.2% TCE- treated UCD rats (P < 0.05, n = 3) and 0.4% TCE-treated UCD rats (P < 0.05, n = 3). When compared with CR controls, ANOVA results indicated a significant decrease in percentage of oocytes fertilized with sperm from 0.2% TCE-treated CR rats (P < 0.05; n = 2) and 0.4% TCE- treated CR rats (P < 0.005; n = 3). In these trials, the UCD controls averaged 34% fertilization and the CR controls averaged 80% fertilization

No Treatment-Related Effects on Whole Animal or Sperm Parameters

There were no treatment-related effects on final animal weights, testis/epididymis weights, or organ:body weight ratios in any of the treatment groups (Table 1). There was, however, a decrease in weight gain over the course of the experiment in both TCE treatment groups. No differences were noted in either sperm concentration or percent motility between treated and control males (Table 1). Results from computer-assisted semen analysis indicated that there were no treatment-related changes in VAP, VCL, ALH, BCF, VSL, or LIN (Table 2). There were no TCE treatment-related effects on acrosomal stability or mitochondrial membrane potential at any of the time points (Table 2).


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  		TABLE 1. Weight and reproductive parameters in male rats after exposure to trichloroethylene (TCE) in drinking water.*


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  		TABLE 2. Motility, acrosomal integrity, and mitochondrial membrane potential in sperm from male rats exposed to trichloroethylene (TCE) in drinking water.*

TCE Induced Treatment-Related Effects in the Efferent Ductules

Neither the testis nor the caput, corpus, or cauda showed histological changes following TCE treatment. However, exposure to 0.2% and 0.4% TCE resulted in slight changes within the efferent ductule epithelium (Fig. 2). In general, there appeared to be effects in the apical cytoplasm, the region between the nucleus and microvilli, in the nonciliated cells of the proximal efferent ductules (Fig. 2, C and E). There also appeared to be a nonuniform increase in cell height in the conus efferent ductules at the highest dose level (Fig. 2F), although this was not quantified. There may be increased endocytosis, as shown by increased staining of endosomes and PAS-positive lysosomes in the apical cytoplasm, particularly in animals treated at the highest dose. This may represent increased reabsorption and concentration of sperm entering the epididymis; however, epididymal sperm counts were not different from controls (Table 1). The common duct that opens into the initial segment of the epididymis showed no consistent effect (data not shown). Effects observed were consistent in control and treated animals from two separate dosing experiments.



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  		FIG. 2. The efferent ductule epithelium following trichloroethylene treatment. Control regions showing proximal and conus efferent ductules (A, B) with columnar epithelium (double arrow), nonciliated (N) and ciliated cells (Ci). The apical cytoplasm contains endosomes and lysosomes (arrow) and a prominent brush border. PAS-positive granules are generally more prominent in the conus region of the efferent ductules. In 0.2% TCE-treated animals, the proximal ductule epithelium is taller than controls (double arrow) and the endocytotic apparatus appears more extensive in nonciliated cells (arrow) (C, D). No change was seen in the ciliated cells. Endosomes and lysosomes in the conus region show reduced PAS staining (arrow) when compared with controls. In 0.4% TCE-treated animals, the proximal ductule epithelium is taller than controls (double arrow) and the endocytotic apparatus appears more extensive in nonciliated cells (arrow) (E, F). No change was seen in the ciliated cells. In the conus region, endosomes and lysosomes show more extensive PAS staining (arrow) than controls. TCE, Trichloroethylene; Prox, proximal efferent ductules. Bar = 50 µm. Micrographs are representative of 11 animals

Oxidized Proteins Detected on Spermatozoa from Treated Animals

The formation of carbonyl groups, although not a specific indicator, can be used as an index of oxidative modification of proteins [35]. The reaction of protein carbonyls with 2,4-dinitrophenyl hydrazine forms stable protein hydrazones that can be detected immunochemically [36, 37]. In the present study, oxidized proteins (in the form of dinitrophenyl hydrazine-derivatized protein carbonyls) were detected on the surface of spermatozoa from TCE-treated rats using an antibody to the dinitrophenyl moiety (Fig. 3). Very little protein oxidation was detected in spermatozoa collected from vehicle control animals (Fig. 3A) when compared with spermatozoa from 0.2% TCE- (Fig. 3B) and 0.4% TCE- (Fig. 3C) treated animals. No fluorescence was detected in slides incubated with normal serum in place of the primary antibody (data not shown). Under higher magnification, it appears that the fluorescence is localized to the sperm plasma membrane and the lateral acrosome of sperm from 0.2% TCE-treated animals (Fig. 3D). Along with the fluorescence on the plasma membrane, a distinct halo of oxidized proteins was visible around individual spermatozoa from 0.4% TCE-treated animals (Fig. 3E). There was a consistent increase in the intensity of plasma membrane staining and appearance of oxidized protein halos from low to high dose.



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  		FIG. 3. Localization of oxidized proteins on surface on spermatozoa from vehicle control animals and 0.2% and 0.4% trichloroethylene-treated animals (A, C). Higher magnification images detail protein oxidation present in plasma membrane around the acrosome, head, and midpiece from 0.2% (D) and 0.4% (E) trichloroethylene-treated animals. Micrographs are representative of replicates from three animals per treatment group. Bar = 25 µm (A– C), 10 µm (D–E)

The presence of oxidized sperm proteins was confirmed using Western blotting with the same anti-dinitrophenyl antibody (Fig. 4). Immunoreactive proteins are visible in sperm from vehicle control rats (Fig. 4, lane 1). Compared with sperm from vehicle control rats, oxidized sperm proteins were more abundant after treatment with either 0.2% TCE (Fig. 4, lane 2) or 0.4% TCE (Fig. 4, lane 3). The prevalence of oxidized proteins increased from low to high dose. Spermatozoa oxidized in vitro with 0.23 mM H2O2 (Fig. 4, lane 4) and 2.3 mM H2O2 (Fig. 4, lane 5) served as positive controls, and exhibited a similar increase in immunoreactivity. Several oxidized proteins visible in the H2O2-treated sperm were consistent in molecular mass to proteins in the TCE-exposed spermatozoa. One band of oxidized protein (~40 kDa) appeared to be unique to spermatozoa from 0.4% TCE-treated animals.



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  		FIG. 4. Western blot analysis of oxidized proteins in sperm collected from vehicle control, 0.2% trichloroethylene-, and 0.4% trichloroethylene-treated animals (lanes 1–3). Oxidation is present in sperm from untreated animals treated in vitro with 0.23 mM H2O2 (lane 4) and 2.3 mM H2O2 (lane 5). Approximately 100 µg total protein of whole sperm homogenate per lane. Numbers represent relative molecular masses of protein standards. Blot representative of three replicates

Sperm from TCE-Treated Animals Exhibited Increased Lipid Peroxidation

Lipid peroxidation is initiated by free radicals in biological systems [38]. Lipid peroxides derived from polyunsaturated fatty acids decompose to form a series of more stable products, the most common of which is MDA [39]. The quantification of MDA is a convenient proxy for assessment of lipid peroxidation in biological systems [39]. To evaluate the relationship between in vivo TCE exposure and lipid peroxidation, sperm from treated animals were examined for the degree of lipid peroxidation using an assay to detect total MDA (free and protein-bound Schiff base conjugates). The level of lipid peroxidation in control sperm (0.050 ± 0.002 µmol MDA/106 sperm) was considered a baseline level [23]. There was a dose-dependent increase in the level of lipid peroxidation in sperm after TCE exposure (Fig. 5). The level of MDA was 0.071 ± 0.002 µmol MDA/106 sperm for the low-dose group and 0.084 ± 0.009 µmol MDA/106 sperm for the high-dose group. These levels were significantly increased from the level of lipid peroxidation in control sperm (P < 0.005). In comparison, sperm collected from untreated animals and oxidized in vitro with 2.3 mM H2O2 produced 0.92 ± 0.21 µmol MDA/106 sperm.



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  		FIG. 5. Increased lipid peroxidation in spermatozoa from trichloroethylene-treated animals using a thiobarbituric acid-reactive substances assay. The error bars represent the SEM of triplicate incubations from each animal (n = 3 per treatment group). The level of lipid peroxidation was significantly higher in both the 0.2% and 0.4% trichloroethylene-treated groups when compared with the vehicle controls (P < 0.005). MDA, Malondialdehyde; TCE, trichloroethylene


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study reports for the first time that in vivo exposure of male rats to TCE results in a decline in the ability of sperm to fertilize oocytes in vitro. The decrease in the percentage of fertilized oocytes occurred without any observed effects on sperm concentration, motility, energy (mitochondrial) status, or acrosome integrity. Although sperm morphology was not specifically evaluated, there is no strong evidence that TCE has an effect on morphologic parameters [40, 41].

Drinking water administration is an environmentally relevant route of exposure. By administering TCE in drinking water, the water bottle acts as a point source for both oral exposure and inhalation of small volumes of volatilized TCE. This mimics the combined oral and inhalation routes previously modeled for human TCE exposure from contaminated water [42]. Effects observed in the present study occurred at approximately 1/100 and 1/200 of the LD50 for the high- and low-dose groups, respectively [3]. The total effective dose may be slightly higher based on the (noncalculated) inhalation of TCE by each animal at the water bottle nozzle. In previous studies, changes in sperm morphology and motility were noted in mice after exposure to 1/43 of the LC50, in rats at 1/33 the LC50, and in rabbits at 1/120 the LD50 [10–12]. In general, sperm abnormalities occurred at higher in vivo TCE doses than the dose necessary to elicit a change in fertilizing ability of sperm in the present study.

The major enzyme responsible for the oxidative metabolism of TCE is cytochrome P450 2E1 (Fig. 6) [14, 17]. Cytochrome P450 2E1 has been localized to the rat efferent ductules and the mouse epididymis, and these tissues are capable of oxidatively metabolizing TCE [16, 43]. Several TCE metabolites, including chloral hydrate, trichloroacetic acid, and dichloroacetic acid cause male reproductive toxicity when administered to animals [44–46]. Observed effects include decreased epididymal sperm count and motility, formation of distorted sperm heads and acrosomes, and reduced fertility [47, 48]. TCE metabolites have been detected in the epididymis following in vivo TCE dosing [41]. In addition, when trichloroethanol, trichloroacetic acid, or dichloroacetic acid was added to IVF medium, there was a decrease in the number of oocytes fertilized with sperm from untreated mice [49]. Although specific metabolites were not measured in the present study, the presence of cytochrome P450 2E1 in the efferent ductules and alterations in ductule epithelium suggest that adverse reproductive effects could be due to TCE metabolites formed in situ.



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  		FIG. 6. Proposed scheme for the oxidative metabolism of trichloroethylene (TCE).[14]

TCE treatment in the present study resulted in alterations to the epithelium of the rat efferent ductules, which contain active forms of cytochrome P450 2E1 [16]. The primary functions of the efferent ductules are sperm transport and reabsorption of water, ions, and proteins released with the sperm from the rete testis [50]. The nonciliated cells, which contain a brush border, apical vesicles, and a variety of vacuoles, are specialized for fluid reabsorption [50]. Certain toxicants alter fluid reabsorption, resulting in increased sperm concentrations, compacted luminal contents, and dilated efferent ductules [51]. In severe cases, ductule occlusions can produce a fluid build-up and testicular atrophy as well as decreased cauda sperm concentrations [51]. Observations in the present study indicate that TCE may increase the cytoplasmic volume and epithelial cell height of the efferent ductules and the staining pattern of the endosomes and lysosomes in the efferent ductules. The histological changes observed in the present study, however, were not sufficient to alter sperm concentrations, cause ductule occlusions, or result in testicular atrophy, as previously observed with other toxicants [51].

Bioactivation of TCE by cytochrome P450 2E1 forms reactive epoxides [14] and carbon and oxygen-centered radicals [19, 20]. These radical species can react rapidly with oxygen, transition metals, and organic molecules to generate secondary free radicals and reactive oxygen species [38]. These cascades may alter the reducing milieu of the epididymis and result in an environment where sperm may undergo oxidative damage. Alternatively, it is possible that TCE and/or its metabolites are not directly involved in oxidative damage of sperm. Another possible explanation for the observed protein and lipid oxidation of sperm is biochemical cascades and oxidative processes that occur because of epithelial cell damage and loss of function.

The fertilizing ability of spermatozoa is dependent in part on the integrity and fluidity of the sperm plasma membrane, which is in turn dependent on the content of polyunsaturated fatty acids [52]. The sperm plasma membrane contains a characteristically high level of polyunsaturated fatty acids, which make spermatozoa particularly susceptible to oxidative damage [30]. Spermatozoa are also highly dependent on their surrounding environment to protect them from reactive oxygen species during epididymal transit and maturation [22, 53]. Oxidatively damaged spermatozoa are characterized by the accumulation of lipid hydroperoxides in the plasma membrane [52]. Studies indicate that lipid peroxidation can profoundly affect sperm quality, including percent motility and specific motility parameters [52]. Results from the present study indicate that there is a dose-dependent increase in the level of lipid peroxidation in sperm following in vivo TCE exposure. However, the effects of TCE were not severe enough to detect a treatment-related difference in motility parameters using CASA.

Lipids are not the only possible target for oxidative damage by TCE. Proteins are also vulnerable to oxidative damage [21]. The ability of capacitated sperm to fuse to oocytes depends on the presence of functional sperm plasma membrane proteins [26]. If these proteins are oxidatively damaged, one might expect that sperm-oocyte interactions would also be adversely affected. One indicator of oxidative modification of proteins is the increase in carbonyl content (i.e., aldehyde and ketones) [35, 36]. An increase in carbonyl content and protein oxidation may occur as the consequence of attack by free radicals [35]. Carbonyl groups may be introduced into proteins by primary reactions such as metal-catalyzed oxidations, radiation-mediated oxidation, and oxidation by ozone or nitrogen oxides or in secondary reactions where proteins are oxidized by reactive species generated by the oxidation of other molecules [35].

In the present study, oxidized proteins in the form of protein carbonyls were detected on sperm from treated animals. The presence of oxidized proteins increased in a dose-dependent manner. The proteins that became oxidized may have existed on spermatozoa before spermiation or may have been added or modified during epididymal transit. Because the animals were dosed with TCE for 14 days, sperm sampled from the cauda would have been exposed to TCE during the final stages of development (late spermatid), spermiation, and epididymal transit [54]. However, without sampling directly from the testis, caput, corpus, and cauda, it is not possible to implicate a specific site within the male reproductive tract. It is probable that, along with other proteins, a number of sperm surface proteins associated with egg binding and fusion may have been oxidatively damaged following in vivo dosing. Together, these findings suggest that TCE and/or its reactive metabolites may create an environment where sperm plasma membrane proteins are oxidatively damaged. The protein oxidation observed did not correlate with any sperm parameter change, with the exception of decreased fertilizibility. That is, as protein oxidation increased, percent fertilization decreased.

The levels of oxidized protein appears similar in sperm collected from animals treated with 0.4% TCE in vivo and sperm exposed to 0.23 mM H2O2 in vitro. A likely question is, therefore, whether H2O2 affects sperm motility and fertility in a manner similar to TCE. A study by Aitken and colleagues [55] showed that sperm-oocyte fusion declined when the sperm were incubated in vitro with 50–200 µM H2O2. The authors also showed that motility was generally not affected except at the highest in vitro concentrations (200 µM H2O2). In the present study, sperm collected from 0.4% TCE-treated animals exhibited a dramatic decrease in IVF but showed little or no change in motility. This suggests that TCE and H2O2 may alter the fertilizibility of sperm in similar ways, possibly by oxidatively damaging important sperm-oocyte binding proteins.

Another important question is whether TCE can directly target sperm. Referring to the work by Cosby and Dukelow [49], the authors found no change in percentage of fertilized oocytes when TCE (1000 ppm) was added to IVF culture medium. Unpublished work from this lab indicates that TCE (1300 ppm) added to sperm in vitro did not affect motility as assessed by CASA. Not until in vitro TCE concentrations reached 6500 ppm (50 mM) did changes in motility (ALH) occur. Therefore, TCE may not be directly responsible for the changes in reproductive parameters measured in this and other studies. It may be that TCE needs to be bioactivated to elicit the effects observed. This hypothesis is supported by data from previous studies showing the presence of cytochrome P450 2E1 in the epididymis and efferent ducts and the ability of those tissues to metabolize TCE [16, 43]. In addition, TCE metabolites have been measured in the seminal fluid of male automotive workers, although effects on fertility were not studied [56]. To date, there are no published reports of animal or human spermatozoa containing cytochrome P450 2E1.

Findings from the present study suggest that TCE may establish an environment in the excurrent ducts in which oxidative damage can occur. The results clearly indicate an effect of TCE treatment on IVF. It is possible that other pro-oxidant chemicals or environmental exposures known to cause oxidative damage will alter fertility without drastic changes to sperm indices. The present study suggests that chemicals could reduce fertility in subtle ways not readily detected by conventional histological techniques or sperm parameter analysis.


   ACKNOWLEDGMENTS
 
The authors would like to acknowledge Dr. Bruce Winder, who initially investigated protein carbonyls in rat sperm. The authors would also like to thank Ms. Kay Carnes for her assistance with the histological assessments.


   FOOTNOTES
 
1 Partially supported by the U.S. Environmental Protection Agency (U.S. EPA) grant/cooperative agreement R825325 to T.B. and M.G.M. This research has not been subjected to U.S. EPA's required peer review and does not necessarily reflect the views of the U.S. EPA. No official endorsement should be inferred. Back

2 Correspondence: Marion G. Miller, Department of Environmental Toxicology, University of California, One Shields Avenue, Davis, California, 95616. FAX: 530 752 3394; mgmiller{at}ucdavis.edu Back

Received: 11 August 2003.

First decision: 4 September 2003.

Accepted: 19 January 2004.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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