Exposure to Trichloroethylene and its Metabolites Causes Impairment of Sperm Fertilizing Ability in Mice

Hongbin Xu*, Nongnuj Tanphaichitr*,{dagger}, Poh-Gek Forkert{ddagger}, Araya Anupriwan*,2, Wattana Weerachatyanukul*,3, Renaud Vincent§, Arthur Leader{dagger} and Michael G. Wade§,1

* Ottawa Health Research Institute, Hormones, Growth, Development, and Department of Biochemistry/Microbiology/Immunology, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9; {dagger} The Ottawa Hospital, and Department of Obstetrics and Gynecology, Division of Reproductive Medicine, University of Ottawa, Ottawa, Ontario, Canada K1Y 1J8; {ddagger} Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6; and § Environmental Health Research Division, Health Canada, Tunney's Pasture, Ottawa, Canada K1A 0L2

Received May 21, 2004; accepted August 26, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichloroethylene (TCE) is a prevalent occupational and environmental contaminant that has been reported to cause a variety of toxic effects. Here, we have undertaken studies to test the hypothesis that TCE exposure adversely affects sperm function and fertilization. Sperm retrieved from mice exposed to TCE (1000 ppm) by inhalation for 1 to 6 weeks were incubated in vitro with eggs isolated from superovulated female mice. The number of sperm bound per egg was significantly decreased when mice were exposed to TCE for 2 and 6 weeks but not at exposures of 1 and 4 weeks. In vivo fertilization was also determined in superovulated female mice mated with males exposed to TCE for 2 to 6 weeks. The percentages of eggs fertilized, as assessed by the presence of two pronuclei, were significantly decreased after 2 and 6 weeks of TCE exposure. A slight but insignificant decrease was observed after 4 weeks of TCE exposure. The direct effects of TCE and its metabolites, chloral hydrate (CH) and trichloroethanol (TCOH), on in vitro sperm–egg binding were also investigated. Sperm–egg binding was significantly decreased when sperm were pretreated with CH (0.1–10 µg/mL). Significantly lower levels of sperm–egg binding were also detected with TCOH (0.1–10 µg/mL), although the decreases were not as pronounced as those for CH. These results showed that TCE exposure leads to impairment of sperm fertilizing ability, which may be attributed to TCE metabolites, CH, and TCOH.

Key Words: trichloroethylene; chloral hydrate; fertilization; sperm–egg binding; trichloroethanol.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichloroethylene (TCE) is used in a variety of industrial and commercial applications. Annual production rates are estimated at 320 million pounds in the United States alone (ATSDR, 1997Go). It is used principally for vapor degreasing and cold cleaning of metal parts, and hence occupational exposures are common especially in the metal, aviation, and automotive industries. It has been estimated that 3.5 million workers are exposed to TCE (NTP, 1988Go, 1990Go). Environmental release of TCE in the United States reported for the years 1988 to 1997 exceeded 345 million pounds (Toxics Release Inventory, 2004Go), leading to TCE becoming a major contaminant in soil and groundwater (ATSDR, 1997Go).

Data from previous studies indicated that TCE exposure is associated with impairment of male fertility. In a study that evaluated 55 occupational categories for occupation-related infertility, men employed as mechanics and involved in degreasing of engine parts appeared to have the highest risk of idiopathic infertility (Rachootin and Olsen, 1983Go). In other studies, TCE exposure was associated with reduced sperm density in a cohort of men exposed occupationally to TCE (Chia et al., 1996Go). Multigenerational studies in mice and rats exposed orally to TCE identified reduced fertility and increased incidence of abnormal sperm in some but not all generations (NTP, 1985Go, 1986Go). Inhalation exposure to TCE has also been shown to cause male infertility, reduced testis size, reduced epididymal sperm number and motility, reduced serum testosterone levels (Kumar et al., 2000Go, 2001Go), and a significant increase in the proportion of misshapen sperm nuclei (Land et al., 1981Go). More recent studies reported a decrease in the percentage of zona-free eggs fertilized by sperm from rats exposed to TCE in drinking water (DuTeaux et al., 2004Go).

The metabolism of TCE is mediated through two major pathways: conjugation with glutathione and oxidation via cytochrome P450 (Fig. 1). Conjugation with glutathione produces S-(1, 2-dichlorovinyl) glutathione and subsequently S-(1, 2-dichlorovinyl)-L-cysteine (Bruckner et al., 1989Go; Lash et al., 2000aGo). Oxidative metabolism of TCE occurs in the liver and produces the primary transient metabolite TCE oxide in one pathway, which is then converted to dichloroacetyl chloride. Chloral is the major metabolite in another pathway where it is rapidly converted to its hydrate (chloral hydrate, CH), which then undergoes reduction and oxidation to form trichloroethanol (TCOH) and trichloroacetic acid (TCA), respectively (Kimmerle and Eben, 1973Go). TCOH and TCA are major circulating and urinary TCE metabolites (Lash et al., 2000aGo). Dichloroacetyl chloride metabolism generates dichloroacetic acid, which can also be formed via dechlorination of TCA. The toxicity of TCE is ascribed to its bioactivation, although the specific metabolites implicated in particular outcomes have not been fully resolved (Bull, 2000Go; Lash et al., 2000aGo; Odum et al., 1992Go). However, it has been proposed that CH, dichloroacetic acid, and TCA are TCE metabolites associated with liver and lung toxicity, whereas S-(1, 2-dichlorovinyl)-L-cysteine is implicated in kidney toxicity (Lash et al., 2000bGo).



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FIG. 1. Proposed scheme of the oxidative pathway of trichloroethylene (TCE) metabolism. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase.

 
Several P450 enzymes including CYP1A1/2, CYP2B1/2, 2C11, and CYP2E1 have been reported to be involved in hepatic metabolism of TCE (Guengerich et al., 1991Go; Nakajima et al., 1992aGo, bGo). However, the available evidence indicated that CYP2E1 is the predominant P450 enzyme responsible for the bioactivation of TCE in both humans and rodents (Lipscomb et al., 1998Go; Lash et al., 2000aGo). The CYP2E1 enzyme has been identified in the testis and epididymis of rodents (Forkert et al., 2002Go; Healy et al., 1999Go; Jiang et al., 1998Go; DuTeaux et al., 2003Go) and non-human primates and humans (Forkert et al., 2003Go). Furthermore, the TCE metabolite chloral was generated in incubations of epididymal and testicular microsomes in mice (Forkert et al., 2002Go). The amount of chloral produced by epididymal microsomes was about fourfold higher than that produced by testicular microsomes, and it coincided with damage to the epididymal epithelium. Chloral, as well as TCA and TCOH, was also identified in seminal plasma of men exposed to TCE in the workplace (Forkert et al., 2003Go). These findings suggested that TCE is bioactivated in the male reproductive system, resulting in production of metabolites deleterious to tissues including the epididymal epithelium.

In this study, we have investigated the potential effects of TCE exposure on sperm function. We have examined the impact of TCE inhalation on the capacity of mouse spermatozoa to bind to mature zona-intact eggs in vitro and to fertilize eggs in vivo. In addition, we have determined the toxic effects of TCE and its metabolites on sperm–egg binding. Our results showed that sperm–egg binding and fertilization are decreased in male mice exposed to TCE, and this decrease is likely due to the direct effects of CH and TCOH on sperm.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials. Trichloroethylene, spectrophotometric grade (>99.5% purity) was purchased from Aldrich Chemical (Milwaukee,WI). Trichloroethanol, pregnant mare's serum gonadotropin (PMSG), and human chorionic gonadotropin (hCG) were purchased from Sigma Chemical Co. (St. Louis, MO). Chloral hydrate was purchased from BDH (Toronto, ON). Hoechst 33258 was obtained from Molecular Probes (Eugene, OR), and paraformaldehyde from Polysciences Inc. (Warrington, PA). All other chemicals were of reagent grade and were obtained from standard chemical suppliers.

Animals. Male CD-1 mice (80–90 days of age at the start of exposure) were purchased from Charles River Canada (St. Constant, Quebec), and were individually housed in polycarbonate cages with free access to Purina rodent chow 5010 (Ralston-Purina, St. Louis, MO) and water under constant photoperiod (12:12 h light/dark). Sexually mature female CF-1 mice were purchased from Charles River Canada and were used for mating studies and for egg retrieval in the sperm–egg binding studies. All procedures relating to animal treatment adhered to Canadian Council on Animal Care guidelines and were approved by Animal Care Ethics Committees at Health Canada and the Ottawa Health Research Institute.

Inhalation exposure. After acclimation to the facility for 1 week, mice were transferred to 2.5 m3 inhalation chambers and housed individually in suspended stainless-steel wire-mesh cages with an integral food dish and a water spigot. The chambers were operated at an air flow of 500 liters/min of HEPA and activated charcoal filtered air (ca. 10 chamber volumes/h), with an internal chamber temperature of 19°–24°C, and relative humidity of 30–50%. Mice were housed in these cages until the completion of all exposures.

Animals were exposed by inhalation to atmospheres containing a TCE concentration of 1000 ppm (5.37 mg/l) for 1 to 6 weeks (6 h/day, 5 days/week). The atmospheres were generated by evaporating TCE through a glass evaporative system, with the resulting vapor being carried by an air stream into the chamber inlet and mixed with the incoming air. Concentrations of TCE in air from both chambers (i.e., TCE and control) and from the surrounding room were monitored every 6 min throughout the exposure period by gas chromatography (X-Tra Process gas chromatography, Amscor, U.S.) connected to the chambers through a multi-valve system (Douglas et al., 1999Go). At the end of the exposure period, the flow of TCE was terminated without changing the flow rate of the incoming air stream. Control animals were used for each experiment and were treated identically in an adjacent chamber, except that the TCE evaporating system was not connected to the air intake. Food containers were removed from both TCE and control chambers for the duration of the exposure to prevent oral exposure to TCE by consumption of TCE-laden food.

Assessment of body weight, testis and epididymis weight, and sperm number and motility. After each TCE exposure regimen, body weights of both control and TCE-exposed mice were recorded. Mice were sacrificed by cervical dislocation, and right testis and epididymis were dissected, freed of fat pads, blotted on tissue paper, and weighed. The left cauda epididymides were longitudinally scored once with a surgical blade and the sperm-containing epididymal fluid was squeezed out from this incision into HEPES-buffered Krebs Ringers bicarbonate (KRB-HEPES) medium supplemented with 0.3% bovine serum albumin (BSA) (KRB-HEPES: 119.4 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM Mg2SO4, 21 mM HEPES, 5 mM NaHCO3, 25 mM sodium lactate, 1 mM sodium pyruvate, 5.6 mM glucose, 2.8 µM phenol red, pH 7.4). Sperm were also squeezed from the vas deferens and combined with the caudal epididymal sperm suspension. An appropriate dilution of the sperm suspension was placed into a hemocytometer, and the number of total sperm and motile sperm were immediately counted under a Nikon inverted microscope (DIAPHOT-TMD, Nikon Canada, Mississauga, ON) at 200x magnification.

Assessment of sperm spontaneous acrosome reaction. Cauda epididymal and vas deferens sperm, prepared as described above, were washed once with KRB supplemented with 0.3% BSA (KRB-BSA): (KRB: same ingredients as KRB-HEPES except that 21 mM HEPES + 5 mM NaHCO3 buffering system was substituted with 25 mM NaHCO3) by centrifugation (350 g, 10 min, 27°C) resuspended in KRB-BSA at 10 million/mL and incubated for 30 min at 37°C under 5% CO2. These conditions have previously been shown to allow sperm capacitation (Tanphaichitr et al., 1993Go). Spontaneous acrosome reaction was determined by evaluating the sperm acrosomal status following a previously described method (Bleil and Wassarman, 1990Go). Briefly, an aliquot of the capacitated sperm suspension (~1 million sperm) from each sample was fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Sperm were washed twice with 100 mM ammonium acetate (pH 9.0) and resuspended in the same buffer, after which an aliquot of the sperm suspension was placed onto a microscope glass slide coated with gelatin and air dried. Sperm on the slides were incubated in freshly made Coomassie blue dye (0.22% Coomassie blue G-250 (Bio-Rad, Hercules, CA), 50% methanol, 10% glacial acetic acid, 40% water) for 2 min. Slides were washed thoroughly with distilled water to remove unbound dye. Slides were air-dried, topped with Permount mounting medium (Fisher Scientific, Nepean, ON), and covered with coverslips. Stained sperm were examined under a brightfield microscope (Zeiss Canada, Toronto, ON) at 400x or 630x. Specifically, aldehyde-fixed acrosome-intact sperm are stained with Coomassie blue at their head convex ridge (the site of the acrosome), whereas acrosome-reacted sperm show negative staining.

In vitro sperm–egg binding. Sperm–egg binding was determined using procedures described previously (Tanphaichitr et al., 1993Go). Cumulus masses containing mature eggs were collected from superovulated female mice into KRB-HEPES-BSA using established procedures (Hogan et al., 1994Go). Zona pellucida (ZP)–intact eggs were freed from cumulus cells by 0.1% hyaluronidase digestion and washed in KRB-BSA, pre-warmed at 37°C under 5% CO2. Capacitated sperm from mice exposed to air (controls) and TCE were prepared as described above. Eggs (n = 20–30) were then incubated with 60,000 motile capacitated sperm from control or TCE-treated mice, in a 60 µl droplet of KRB-BSA for 30 min at 37°C under 5% CO2. Subsequently, sperm–egg complexes were gently washed 4 times in KRB-BSA through a drawn Pasteur pipette (inner diameter 200 µm) to remove loosely attached sperm, placed into a well of a sera culture slide, and overlaid with mineral oil. The number of sperm bound to the ZP per egg was counted under an inverted microscope. Because numerous sperm bound to the ZP, only those in the focal plane of the zona diameter were counted. The nonspecific binding level of sperm to the egg ZP was determined by incubating sperm with fertilized eggs (Hogan et al., 1994Go). This nonspecific binding level was 5–10% of the positive control value (i.e., when mature eggs were incubated with capacitated sperm) (Tanphaichitr et al., 1993Go). The average number of sperm bound per egg from each control or TCE-treated mouse was then calculated, and the data from all control or TCE-treated mice were expressed as mean ± S.D., except for the data from the two control mice at week 4 and week 6 time points, which were simply expressed as an average of the two animals.

In alternate experiments where the direct effects of TCE and its metabolites were investigated, capacitated sperm were first incubated with various concentrations of TCE, CH, or TCOH for 30 min. Control sperm were those incubated in KRB-BSA, containing vehicle at the same concentration as that used for TCE solubilization (0.5% ethanol in the case of TCE and TCOH), or those incubated simply in KRB-BSA (in the case of CH). In each experiment, sperm from the same animal were used for control and treatments with TCE, CH, and TCOH. Both control and pretreated sperm were washed by centrifugation with KRB-BSA prior to incubation with mature eggs and the numbers of sperm bound per egg were determined as described above. Data of the number of sperm bound per egg were expressed as mean ± S.D. of means obtained from the three replicate experiments performed on different days.

In Vivo fertilization. Immediately after the TCE exposure, control and TCE-exposed mice were individually caged with a single female CF-1 mouse that had been superovulated by sequential i.p. injections of PMSG (5 IU) and hCG (10 IU), with a 48 h interval (Hogan et al., 1994Go). Caging of each female with a male was done immediately after the hCG injection. The females were examined for the presence of copulation plugs 14 h after the initiation of cohabitation. Females showing the copulation plugs were sacrificed, and cumulus masses containing mature eggs were collected from the oviducts into KRB-HEPES-BSA. Eggs were freed from cumulus cells by hyaluronidase digestion (Hogan et al., 1994Go), washed in KRB-HEPES containing 0.1% polyvinylpyrrolidone, and fixed in 4% paraformaldehyde in PBS. Fixed eggs were stained with 5 µg/mL Hoechst 33258 and examined for fertilization, based on the presence of 2 pronuclei, under a Zeiss IM35 epifluorescence microscope. The number of eggs retrieved from each mated female that had two pronuclei was recorded, and the percentage of eggs fertilized was calculated setting total eggs retrieved as 100%. Data were then expressed as mean ± S.D. of percent eggs fertilized from all females mated one-to-one with control or TCE-treated males.

Statistical analysis. Data of percent eggs fertilized per male from fertilization studies were analyzed by 2-way factorial analysis of variance (ANOVA) using treatment (control vs. TCE) and weeks of exposure as tested factors. Significant differences indicated by ANOVA were further evaluated by Dunnett's method. Similar analyses were performed for mean sperm–egg binding data per male for sperm collected from animals exposed for 1 or 2 weeks, where the number of control and TCE-treated mice was 3 or more. However, sperm–egg binding data were available for only 2 control males each for the 4- and 6-week exposures, precluding ANOVA for these time points. Therefore, sperm–egg binding data for these time points were analyzed separately using Student's t-test. Data from the studies on the effects of sperm pretreatment with TCE or its metabolites (TCOH, CH) on in vitro sperm–egg binding were also subjected to 2-way ANOVA with pretreatment dose and experimental repeat number being the two tested factors. Significant effects were evaluated using Dunnett's method. The analyses were performed using SigmaStat (SPSS, Chicago, IL).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhalation exposure to TCE did not result in significant changes in body weight, or in the testis and epididymis weights of the mice. Nor did it alter the number and percent motility of epididymal + vas deferens sperm of the TCE-treated mice, relative to sperm from the control mice, at all exposure time points (Table 1). More than 95% of sperm retrieved from the cauda epididymis and vas deferens of both TCE-exposed and control mice showed normal morphology. This indicated that there was no abnormality in production of sperm with normal morphology and motility, and that the TCE-inhaling animals were generally in good health.


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TABLE 1 Lack of Effect of TCE Exposure on Body, Testis, and Epididymis Weights and on Epididymal Sperm Reserves

 
We first examined the ability of sperm from TCE-exposed males to bind to mature eggs in vitro. The number of sperm bound to the ZP per egg ranged from 13 to 22 for 12 control males at all treatment time points. In contrast, the average number of sperm bound to the ZP per egg was significantly decreased in sperm retrieved from 11 animals treated with TCE for 2 weeks, as compared to sperm collected from 5 corresponding control males (9 ± 5 vs. 18 ± 2; p <0.001; Fig. 2B). A similar result was observed in sperm retrieved from 3 mice inhaling TCE for 6 weeks, with the average number of sperm bound to the ZP per egg being 12 ± 2 versus 20 (19, 21) observed for sperm from the two corresponding controls (Fig. 2D). Student's t-test indicated that this effect was significant either when the average value per male was used as the experimental variable (p = 0.018) or when the values for all eggs were used (p ≤ 0.001). This represented a reduction of 47% and 38% in ZP binding ability of sperm from mice exposed to TCE for 2 and 6 weeks, respectively (Fig. 2B and 2D). In contrast, the average number of sperm bound per egg for sperm from the 4 males treated with TCE for 1 week was very similar to that for sperm from the 3 control mice (Fig. 2A). The average number of sperm bound per egg from 4 males treated with TCE for 4 weeks was also similar to that observed in sperm from the two corresponding controls (14 ± 6 vs. 17 and 13 for control). However, these values from individual TCE-treated males at that time point appeared to be variable, ranging from 6 to 19 (Fig. 2C). Notably, sperm retrieved from mice exposed to TCE at all time points possessed similar motility (ranging from 60% to 80% with an average of 61% of TCE versus 67% of control) and similar percentages of the acrosome-intact population (85% to 90%). Therefore, the results described above likely reflected direct adverse effects of TCE and/or its metabolites on sperm ability to bind to the egg ZP.



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FIG. 2. Decreases in in vitro egg binding ability of sperm retrieved from mice exposed to TCE. Mice were exposed to TCE for 1 (A), 2 (B), 4 (C) or 6 (D) weeks. Data of the number of sperm bound per egg (expressed as mean ± S.D., with the number of males (n) being a variable) were shown for both control and TCE-treated mice, except those for control mice at week 4 and week 6, which were an average value obtained from the two male mice. The figure in parentheses denotes the total number of eggs assessed. Student's t-test was used to analyze a significant difference of the number of sperm bound per egg between control and TCE-treated animals for each time point. For week 1 and week 2, regardless of whether the number of males or the number of total eggs was used as a variable, similar results were obtained. However, because the number of control males for week 4 and week 6 was only 2, only the number of total eggs could be used as a variable for Student's t-test. ANOVA was also performed for data from week 1 and week 2. An asterisk denotes a significant difference (p < 0.001).

 
In in vivo fertilization experiments, the proportion of retrieved eggs possessing two pronuclei was recorded as an index of fertility. Percentages of eggs fertilized ranged from 67% to 100% for each mated female among 18 control males used for mating (Fig. 3). Male mice exposed to TCE produced sperm that fertilized significantly fewer eggs. An average fertilization rate of 44 ± 38% and 34 ± 29% was seen in 15 and 5 females mated one-to-one with male mice exposed to TCE for 2 and 6 weeks, respectively (Fig. 3A and 3C). This accounted for a 50% and 60% reduction in fertilization when compared to the corresponding percent eggs fertilized in 9 and 3 females mated by control males for week 2 and week 6, respectively (i.e., 89 ± 11% and 84 ± 8%). However, a slight and insignificant decrease in in vivo fertilizing capacity of 4 males exposed to TCE for 4 weeks was observed, i.e., 79 ± 15%, as compared to 88 ± 8% observed in 6 females mated by control males (Fig. 3B). All females that were caged with males, both control mice and TCE-exposed mice, were found to have vaginal plugs, indicating that TCE exposure did not impair mating efficiency.



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FIG. 3. Decreases in the percentages of eggs fertilized in vivo by sperm from TCE-exposed mice. Mice were exposed to TCE for 2 (A), 4 (B), or 6 (C) weeks. Each female was mated with a single male mouse. Data of the percentage of eggs fertilized (expressed as mean ± S.D. of n males) were shown for both control and TCE-exposed mice. The figure in parentheses alongside the number of males denotes the total number of eggs assessed. An asterisk denotes a significant difference (p < 0.05).

 
To determine if the observed decreases in sperm ability to bind to and to fertilize eggs were due to direct effects of TCE and/or its metabolites on sperm–egg binding, sperm were treated with TCE (1, 10, or 100 µg/mL), CH (0.1, 1.0, or 10 µg/mL), or TCOH (0.1, 1.0, or 10 µg/mL) prior to co-incubation with mature eggs in vitro. In all experiments, an average of 25 ± 3 control sperm bound to each egg. In the three replicate experiments performed on different days, treatment of sperm with TCE at concentrations from 1 to 100 µg/mL had no significant effect on sperm–ZP binding, with 24 ± 3, 23 ± 3, and 22 ± 3 sperm bound per egg, respectively, compared to 26 ± 3 of the control level (Fig. 4A). Treatment of sperm with TCOH caused a slight but significant inhibition of sperm–egg binding at all concentrations tested (p < 0.001), with the number of sperm bound per egg being 20 ± 4, 22 ± 4, and 19 ± 2, respectively, compared to 23 ± 3 of control mouse sperm (Fig. 4B). A decrease of 16% was detected at the highest concentration (10 µg/mL) of TCOH. Treatment with CH led to significant decreases (38–71%) in sperm binding to eggs at the three concentrations tested, 0.1, 1.0, and 10 µg/mL, resulting in 15 ± 3, 9 ± 4, and 7 ± 3 sperm bound per egg, respectively, compared to 25 ± 4 for control mouse sperm (p < 0.001; Fig. 4C).



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FIG. 4. Changes of ZP binding ability of sperm pretreated in vitro with TCE and its metabolites. Treatments: TCE (A), trichloroethanol (TCOH; B), or chloral hydrate (CH; C). Data of the number of sperm bound per egg were expressed as mean ± S.D. of means obtained from the three replicate experiments performed on different days. The figure in parentheses above the bar denotes the total number of eggs assessed. The percentage indicated above the bar represents the percent value of controls. A single asterisk and a double asterisk denote a significant difference with p < 0.05 and p < 0.001, respectively.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have shown that inhalation exposure of mice to TCE, using concentrations and durations similar to those in this study, elicits epididymal toxicity that manifested as necrosis and sloughing of the epithelium (Forkert et al., 2002Go). Because the epididymis plays a central role in sperm maturation, it is likely that epididymal sperm are also a target of TCE-induced toxicity. In light of these assumptions, we have postulated that TCE exposure will lead to decreased fertility due to impaired ability of defective sperm to bind to and to fertilize eggs. In this study, sperm from mice that were exposed to TCE by inhalation were incubated in vitro with mature eggs retrieved from superovulated female mice. Our results showed that the number of sperm bound to the ZP per egg was significantly reduced when sperm were collected from mice exposed to TCE for 2 and 6 weeks (Fig. 2). However, a significant reduction in sperm–egg binding was not obvious among the four TCE-treated mice after 4 weeks of exposure, although the high S.D. of the data reflected a great variation of the responses to TCE treatment among these four animals (Fig. 2C). Although the diminished number of sperm bound to eggs suggested that fertilization was affected, this parameter did not by itself constitute direct evidence to demonstrate impairment in in vivo fertilization. In order to assess this possibility, male mice that had been exposed to TCE were mated with superovulated females. Eggs were then retrieved from the females on the following day and evaluated for the fertilized population, as assessed by the presence of two pronuclei. Results in Figure 3 reveal that the levels of in vivo fertilization were significantly decreased when male mice exposed to TCE for 2 or 6 weeks were used for the mating. However, a decline in the percentage of eggs fertilized was not detected on exposure to TCE for 4 weeks. These findings suggested that the extent of sperm–egg binding correlated well with the level of in vivo fertilization (Figs. 2 and 3). The underlying basis for the lack of apparent adverse effects of TCE inhalation on sperm–egg binding and in vivo fertilization after 4 weeks of exposure is not clear. The possibility that a defensive mechanism transiently developed in these mice requires further investigation with a higher number of mice. Because sperm motility and the level of spontaneous acrosome reaction were not affected in the TCE-exposed mice, the decreases in sperm–egg binding observed in mice exposed for 2 or 6 weeks likely reflected the direct effect of TCE metabolites on the function of ZP receptors on the sperm head surface. Our results demonstrating the adverse effects of TCE inhalation on mouse sperm fertilizing ability corroborate those of DuTeaux et al. (2004)Go, which revealed reduced levels of zona-free eggs fertilized by sperm from rats consuming TCE-containing water. The question of whether sperm from TCE-exposed mice have impaired ability to bind to and penetrate the oolemma, in addition to impaired zona binding capacity, is being addressed in our laboratory.

It is well established that bioactivation of TCE is a prerequisite for evoking toxicity (Lash et al., 2000aGo). Although several P450 enzymes have been implicated in TCE metabolism, CYP2E1 appears to be the major P450 enzyme catalyzing the oxidation of TCE with the highest efficiency (Lash et al., 2000aGo; Lipscomb et al., 1998Go). In the male reproductive system of mice, CYP2E1 is localized to the epididymal epithelium and testicular Leydig cells (Forkert et al., 2002Go), and to the efferent ductules (DuTeaux et al., 2003Go). CYP2E1-dependent catalytic activity, as assessed by p-nitrophenol hydroxylation, was twofold higher in the epididymis than in the testis. CYP2E1 has also been identified as a P450 enzyme involved in TCE metabolism in the male reproductive tract (Forkert et al., 2002Go). Incubations of microsomes from the epididymis and testis produced concentration-dependent formation of chloral that was considerably higher in the microsomes than in the epididymis and testis. These findings indicated that CYP2E1 is responsible, in part, for TCE bioactivation in the male reproductive tract. Hence, it was of interest to investigate the direct effects of the TCE metabolites CH and TCOH on the sperm ability to bind to the zona-intact eggs. Although previous results revealed a 40% decrease in in vitro fertilization in mouse gametes incubated in medium containing TCOH at a concentration 75-fold higher than that used in the current study, it was not clear whether the sperm, the eggs, or both were affected (Cosby and Dukelow, 1992Go). Our results showed that both of these TCE metabolites caused significant decreases in the ability of washed epididymal sperm to bind to the egg ZP in vitro, with CH exerting a far more potent effect than TCOH (Fig. 4). It should be noted that the concentrations of TCE, TCOH, and CH used in our in vitro studies are comparable to their described levels in the epididymis homogenate of mice exposed to TCE by inhalation for 2 weeks or more (Forkert et al., 2003Go). Our observed reactivity of CH on mouse sperm corroborates previous findings describing abnormalities of spermatogenic cells in mice treated with CH (Allen et al., 1994Go; Nutley et al., 1996Go; Russo et al., 1984Go). In addition, treatment of rats with dichloroacetic acid produced abnormal sperm morphology and decreased motility (Linder et al., 1997Go). These findings, in addition to the lack of an effect by the parental compound, TCE (Fig. 4), support the concept that bioactivation of TCE to its metabolites is responsible for the adverse effects of the compound on the sperm maturation process in the epididymis. This may be one of the causes of the observed decreases in sperm–egg binding and fertilization observed in TCE-inhaling mice. The mechanisms of how CH and TCOH affect the sperm plasma membrane and metabolism are under investigation in our laboratory. We are also examining whether the defensive mechanism, exerted by the animals at a certain time period after TCE exposure, is via secretion of inhibitors of the TCE metabolites' action into the epididymal fluid.

Substantial data have accrued revealing the adverse effects of occupational exposures to chemicals including TCE on male reproductive health. In a study of occupational exposure, sperm density was decreased and abnormal sperm increased in subjects exposed to TCE (Chia et al., 1996Go). A more recent study reported that exposure to chlorinated solvents, including TCE, decreased semen concentration and sperm motility and increased the number of abnormal sperm (Tielemans et al., 1999Go). These findings indicated that TCE exposure has adverse effects on sperm production and quality. Other studies have revealed that paternal exposure to solvents increases the time for achieving conception (Sallmén et al., 1998Go), decreases the rates of successful implantation after in vitro fertilization (Tielemans et al., 2000Go), and increases the relative risk and incidence of spontaneous abortions (Lindbohm et al., 1991Go; Taskinen et al., 1989Go). Furthermore, a case control study examining the link between occupation and infertility revealed that men employed as auto mechanics were about 9 times more likely to have idiopathic infertility (Rachootin and Olsen, 1983Go). Our current results, as well as our previous findings describing the presence of TCE metabolites (chloral, TCOH, TCA, and DCA) in seminal plasma of mechanics exposed to TCE in the workplace, as well as in the epididymis of mice exposed to TCE (Forkert et al., 2003Go), strongly suggest that the observed adverse effects of TCE inhalation on male reproductive health are caused by the reactivity of TCE metabolites.


    ACKNOWLEDGMENTS
 
We thank D. J. McIntyre for the operation of the inhalation facility. We also acknowledge the technical assistance provided by Alice Lee, Stephen Bjarnason, and Avril McMahon. This study was supported by grant funding from Health Canada and Environment Canada (Toxic Substances Research Initiative). A. A. and W. W. each received a Ph.D. studentship, from the Thailand Research Fund and the National Science and Technology Development Agency of Thailand, respectively.


    NOTES
 
2 Present address: Department of Anatomy, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand. Back

3 Present address: Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. Back

1 To whom correspondence should be addressed at PL 0803D, Tunney's Pasture, Ottawa, K1A 0L2, Canada. Fax: 613-957-8800. E-mail: Mike_Wade{at}hc-sc.gc.ca.


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