Consumption of Xenoestrogen-Contaminated Fish during Lactation Alters Adult Male Reproductive Function

Jayaprakash Aravindakshan*, Mary Gregory*, David J. Marcogliese{dagger}, Michel Fournier* and Daniel G. Cyr*,1

* INRS-Institut Armand Frappier, Université du Québec, Montreal, Quebec, Canada H9R 1G6; and {dagger} St. Lawrence Centre, Environment Canada, Montreal, Quebec, Canada

Received March 13, 2004; accepted May 11, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
Little information exists on the transfer of endocrine-disrupting effects through the food chain. The transfer of chemicals, particularly from the aquatic ecosystem, that can cause such effects on fish-eating predators must be established. Fish from the St. Lawrence River are exposed to xenoestrogens causing male reproductive dysfunction. The objective of this study was to determine if lactational exposure to contaminated fish could alter the development of the male reproductive system in rats. Three experimental groups were used: rats (dams) gavaged with (a) distilled water (control), or (b) homogenized fish from a reference site (Îles de la Paix) or (c) homogenized fish from a xenoestrogen-contaminated site (Îlet Vert). Pups were exposed via lactation and sampled on either day 21 (weaning) or day 91 (adults). There was no effect on the body weights or in the male reproductive organ weights between groups except for adult epididymal weight, which was significantly decreased in the xenoestrogen group. Adult sperm concentrations and sperm motility parameters were all significantly decreased in the xeonestrogen group as compared to the reference and control groups. Furthermore, the distribution of stages of spermatogenesis was altered in the xenoestrogen group, indicating an effect on the kinetics of spermatogenesis. Immunoreactivity of connexin43 (Cx43), a gap-junctional protein, was markedly decreased in the seminiferous epithelium of the xenoestrogen group, suggesting that the intercellular coordination of testicular function may be affected. These data indicate that contaminants from xenoestrogen environments may pass through the food chain and exert effects on male reproductive functions.

Key Words: xenoestrogen; sperm motility; spermatogenesis; gap junctions.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
There is substantial evidence that prenatal or early postnatal exposure to endocrine-disrupting actions of various environmental contaminants, such as polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT), can adversely affect wildlife populations (de Solla et al., 1998Go; Giesy et al., 1994Go; Guillette et al., 1994Go, 1996Go). Studies have shown that bald eagles nesting along the shores of the Great Lakes and feeding primarily on contaminated Great Lakes fish have lower reproductive success (Bowerman et al., 1995Go; Colborn 1991Go). The most direct evidence for adverse human health effects from environmental pollution is found in a series of studies linking PCB exposure to consumption of contaminated fish (Fein et al., 1984Go; Jacobson et al., 1984Go; Jacobson and Jacobson 1988Go). These studies were able to associate the maternal consumption of contaminated fish with adverse health effects in their children who were exposed to the PCBs via lactation.

Contaminants such as 17-{alpha}-ethinyl estradiol, bisphenol A, and alkyl phenols that are present in treated sewage effluent can act as estrogen-mimics and induce a wide range of effects on fish. These effects include feminization and hermaphroditism in males (Arukwe 2001Go; Jobling and Tyler, 2003Go; Rothcell and Ostrander, 2003Go). In fact, fish can serve as "barometers" of the effects of xenoestrogen contamination in the aquatic ecosystem. One index of exposure is the presence of vitellogenin (Vtg), an egg yolk protein, in male fish. Normally, females synthesize Vtg in the liver in response to estradiol. Several reports have shown an upregulation of Vtg in male and immature fish collected near sewage effluents (Aravindakshan et al., 2004Go; Jobling and Sumpter, 1993Go; Tyler et al., 1996Go) and other industrial effluents.

The effluent generated by the municipal sewage treatment facility of the City of Montreal, Quebec, Canada, is discharged into the St. Lawrence River at a single site near the eastern tip of the Island of Montreal. We have shown that spottail shiners exposed to this effluent exhibit induced Vtg levels, delayed spermatogenesis, reduced spermatozoal production, decreased sperm motility, and a high incidence of intersexuality (Aravindakshan et al., 2004Go).

We noted (while sampling fish in the St. Lawrence River to study the presence and effects of endocrine-disrupting chemicals) the presence of a sport fishery in this area, suggesting that people eat fish from sites where we have reported estrogenic effects on fish (Aravindakshan et al., 2004Go). Kosatsky et al. (1999a)Go reported that in the Montreal area, sport fishers eat their catch as often as three times weekly and can consume in excess of 18 kg of St. Lawrence River–caught fish annually. Heavy consumers of these fish have higher hair mercury levels and higher circulating levels of PCBs and dichlorodiphenyldichloroethylene (DDE) than do infrequent consumers. Studies also suggest that Montrealers of Asian origin consume more fish from the St. Lawrence River than other sport fishers do and, in turn, that they have higher levels of contaminants than median levels found in other sport fishers (Kosatsky et al., 1999bGo). The fact that fish in the St. Lawrence River are exposed to estrogenic compounds suggests that people, as well as fish-eating mammals consuming these fish, may be exposed to the same endocrine-disrupting compounds, assuming that these chemicals can bioaccumulate.

Exposure to environmental contaminants during critical periods of development may represent a far greater risk to animals and humans than exposure as adults. Endocrine-disrupting chemicals are particularly problematic to developing animals, because the timing of endocrine-mediated events may be deregulated, resulting in permanent physiological defects to the immune, nervous, or reproductive system (Arukwe 2001Go; Jobling and Tyler, 2003Go; Rothcell and Ostrander, 2003Go). The reproductive systems of mammals undergo substantial development both in utero and after birth, prior to puberty. In rats, the male reproductive tract undergoes substantial development in the first three weeks of life. During this period, cells of the testis and epididymis differentiate into cells resembling those of the adult (Pelletier, 2001Go; Rodriguez et al., 2002Go). The blood–testis and blood–epididymal barriers are formed, and the first wave of spermatogenesis is initiated (Cyr, 2001Go; Cyr et al., 2002Go; Pelletier, 2001Go). Thus the period of lactation, when the mother can pass along contaminants to her offspring, represents a critical period of reproductive development for the male pups. As such, this critical period is among the most vulnerable to xenobiotics, including endocrine disruptors.

Organochlorinated compounds are known to pass up the food chain and become biomagnified in top predators, yet we know relatively little about whether endocrine-disrupting compounds can be passed up the food chain and cause endocrine disruption to higher vertebrates. This possibility is particularly relevant, given the increasing number of aquatic ecosystems in which endocrine-disrupting chemicals reportedly affect aquatic organisms. The transfer of these chemicals and their effects to fish-eating predators and humans must therefore be established, in order to evaluate the potential risk of endocrine disruptors in aquatic ecosystems to riverine mammalian species. The objective of the present study was to determine whether the maternal consumption of fish from a xenoestrogen-contaminated environment results in adverse reproductive consequences to weaning male pups, and if so, whether such effects become apparent only when the pup reaches adulthood. This information will assist us in developing a better understanding of the exposure and risks associated with eating fish from environments contaminated with estrogenic compounds.


    Materials and Methods
 TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
Collection of fish. Previous studies from our laboratory established the presence of xenoestrogens in the St. Lawrence River in proximity to the Island of Montreal by monitoring Vtg mRNA levels in spottail shiners (Notropis hudsonius; Aravindakshan et al., 2004Go). Based on these results, a reference site (Îles de la Paix) and a site with a high level of xenoestrogen-contamination (Îlet Vert) were identified. For the present study, immature spottail shiners of the same age and size were captured at each of these sites using a beach seine. Fish were returned to the laboratory and euthanized. The livers of a subset of immature fish were dissected, immediately frozen in liquid nitrogen, and stored at –80°C. The rest of the fish were subsequently stored at –80°C.

Vtg mRNA levels. Total RNA was extracted from livers of immature spottail shiners captured at both the reference site and the xenoestrogen-contaminated sites using the guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987Go). After RNA isolation, reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine Vtg mRNA levels as described previously (Aravindakshan et al., 2004Go). The resulting Vtg amplicons were separated on an agarose gel, stained with ethidium bromide, and their relative levels determined by densitometry using a Bio-Rad Fluor Image analyzer (Bio-Rad Laboratories, Mississauga, ON). The 28S rRNA was used as an internal standard. Data were expressed as the intensity of the Vtg amplification product relative to that of the 28S rRNA.

Experimental protocol. Timed-pregnant Sprague-Dawley rats (250 ± 5 g; 6–8 weeks old) were purchased from Charles River Canada, Ltd (St. Constant, Montreal, QC) one week prior to parturition and kept under standard controlled temperature (22°C) and lighting (12 h light, 12 h darkness). On the day of birth, 120 male pups were randomly mixed, and 10 male pups were placed with a lactating dam. All rats were fed Purina rat chow and given water ad libitum. Three experimental groups (n = 40 rats per group) were used: (1) control, (2) rats fed fish from the reference site; (3) rats fed fish from the xenoestrogen-contaminated site. Fish from either the reference site or the contaminated site were homogenized in distilled water and fed to the dams in each of the respective experimental groups by gavage. The control group received distilled water alone with no fish. The dams were gavaged three times a week at a dosage of 1% of their body weight, starting from the day of parturition to the time the pups were weaned (day 21). Therefore, an average 250 g female received 2.5 g of fish (wet weight) three times per week, or an average of 1.43 g of fish/kg/day. We based this dose on previous studies around the Island of Montreal that indicate that fishers consume as much as 18 kg of fish per year on average, but that consumption can be higher depending on ethnic background. There is virtually no ice fishing on the St. Lawrence around the Island of Montreal; therefore fishing is limited to the months from late spring until early autumn. If we assume an average person weighing 65 kg eating 18 kg of fish over a 6-month period, then the daily consumption averages approximately 0.76 g fish/kg/day. The US average fish consumption is 0.25 g/kg/day and the Canadian average is 0.17 g/kg/day for the general population, although fish consumption is greater among certain aboriginal populations (Boyer et al., 1991Go; Chan et al., 1999Go).

After weaning, the pups were subsequently maintained on a standard diet of rat chow and water ad libitum. The dams and pups were weighed three times each week. The pups were sampled on either day 21 or day 91 (adult). All animal protocols used in the present study were approved by the University Animal Care Committee.

Male rats (21 or 91 days of age) were anaesthetized with an intraperitoneal injection of ketamine/xylazine (50:10 mg/kg). The animals were weighed, bled, and euthanized. Paired testis, epididymis, seminal vesicle (empty), and ventral prostate weights were recorded. Tissues were then frozen in liquid nitrogen and stored at –80°C or fixed in Bouin's solution for further histological analyses.

Testosterone radioimmunoassays. Blood samples from adult rats (day 91; n = 25 per group) were obtained by puncturing the dorsal aorta prior to euthanasia. Blood samples were allowed to clot overnight at 4°C and were subsequently centrifuged to obtain the serum. Serum samples were frozen at –80°C until the time of assay. Serum testosterone levels were determined by means of a commercial assay kit used according to the manufacturer's protocol (ImmuChem Double Antibody Testosterone RIA kit, ICN Biomedicals, Costa Mesa, CA). Intra-assay variability was assessed to be less than 2.9%, and inter-assay variability was calculated at 6.3%.

Sperm motility parameters. Functional analysis of male reproduction was determined by measuring sperm motility of adult rats (n = 10 per group), using an IVOS semen analyzer (Hamilton-Thorne Research, Beverly, MA). The cauda epididymidis was clamped both proximally and distally, removed from the epididymis, and rinsed in Medium 199 (with Hank's salts supplemented with 0.5% w/v BSA, pH 7.4; GIBCO, Mississauga, ON) in a 35-mm plastic Petri dish at 37°C. The cauda epididymidis was punctured with a surgical scalpel blade (No. 11; Fisher Scientific, Ottawa, ON) allowing the sperm to flow out. The cauda epididymidis was removed from the media and the Petri dish was returned to the incubator kept at 37°C in a 5% CO2 atmosphere for 5 min, to allow the sperm to disperse. The sperm were diluted 1:10 in medium prior to analyses for motility parameters. Among those motility parameters measured were the following: Percent motility—the percent of motile sperm within the analysis field divided by the sum of the motile plus immotile sperm within the analysis field; Path Velocity (VAP)—the average velocity of the smoothed cell path, expressed in microns per second); Progressive Velocity (VSL)—the average velocity measured in a straight line from the beginning to the end of the track; Curvilinear Velocity (VCL)—the sum of the incremental distances moved in each frame along the sampled path divided by the time taken for the sperm to cover the track; Beat Cross Frequency (BCF)—the frequency with which the sperm track crosses the sperm path; Straightness (STR)—the departure of the cell path from a straight line; Linearity (LIN)—the departure of the cell track from a straight line.

The other cauda epididymidis was dissected and frozen (–20°C) for subsequent analysis of sperm concentration. Briefly, the cauda epididymis was thawed and homogenized in a 50 ml conical tube containing 20 ml of distilled water. The "IDENT fluorescent dye" (Hamilton-Thorne Research) was resuspended in 100 µl of distilled water in a small 1.5 ml microcentrifuge tube. A 100 µl aliquot of the homogenized sample was added to the resuspended IDENT solution and incubated at room temperature for 2 min. The solution was mixed and a 5 µl aliquot was placed on a 20 µm sperm analysis chamber (2X Cel; Hamilton-Thorne Research) slide and analyzed with the IVOS semen analyzer under ultraviolet light.

Morphological analyses. At day 21 (n = 15 per group) and at day 91 (n = 15 per group), testes and epididymides were fixed by immersion in Bouin's fixative for 24 h and subsequently transferred to 70% ethanol until processing. Fixed tissues were then dehydrated in graded ethanol, cleared in xylene, and mounted in paraffin for light microscope and immunocytochemical analysis.

To identify the stages of spermatogenesis in the testis, sections of adult testes were stained with periodic acid–Schiff (PAS) stain (Sigma-Aldrich Canada Ltd, Mississauga, ON) according to the manufacturer's instructions. Anatomical and morphological changes in the testis and epididymis were assessed at the light microscopic level. The diameters of 50 round seminiferous tubules were measured, and staging of spermatogenesis was accomplished by observing 200 tubules from each testis according to the criteria described by Leblond and Clermont (1952)Go.

Immunohistochemistry. Immunoperoxidase staining of testicular sections (n = 5 rats per group) was performed according to previously published protocols (Oko and Clermont, 1989Go). A Heat Induced Epitope Retrieval (HIER) step was included, using citrate buffer (1.8 mM citric acid, 8.2 mM sodium citrate). Sections in citrate buffer were heated in a microwave for 15 min. Immunolocalization of connexin 43 (Cx43) was performed using rabbit polyclonal antisera raised against Cx43 (100 µg/µl; Zymed Laboratories, South San Francisco, CA). The DAKO CSA System HRP kit (DAKO, Carpinteria, CA) was then used to localize Cx43 according to the manufacturer's instructions. Negative control slides, in which the primary antibody was replaced with normal goat serum, were also performed concurrently.

Statistical analysis. The data were tested for normality and homogeneity of variance using the Kolmogorov-Smirnov (K-S) test. All data were analyzed using one-way analysis of variance (ANOVA; SigmaStat, Version 2.0, Jandel Corporation, San Rafael, CA). If the effects were significant, the Student-Newman-Keuls test was used for post-ANOVA multiple comparisons (p < 0.05). All data are presented as the mean ± standard error of the mean (SEM), unless otherwise indicated.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitellogenin Induction in Spottail Shiners
Semi-quantitative RT-PCR results indicate that Vtg mRNA levels were significantly higher in fish captured at Îlet Vert (xenoestrogen-contaminated site) as compared to the reference site at Îles de la Paix (Fig. 1). These results confirmed that fish from Îlet Vert were exposed to xenoestrogens.



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FIG. 1. Hepatic Vtg mRNA levels in immature spottail shiners captured at the reference site (Îles de la Paix) and the contaminated site (Îlet Vert) along the St. Lawrence River. Total cellular RNA was extracted from fish liver and subjected to RT-PCR with specific Vtg primers. Data were standardized to the 28S rRNA. Each reaction was done within the linear range of the assay. Data are expressed as the mean ± SEM. Different superscripts indicate significant differences between groups (p < 0.05).

 
Body and Tissue Weights and Testosterone Concentrations
The body weights of the dams and the pups did not differ significantly between treatment groups throughout the experimental period. At 21 days, paired testis, epididymis, seminal vesicles, and ventral prostate weights were not altered by treatment (Table 1). Morphologically, the diameter of the seminiferous tubules of control rats and of rats whose mothers were fed fish from the reference site and the xenoestrogen-contaminated site were not statistically different. Testes from rats in all experimental groups displayed a well-defined lumen by 21 days of age.


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Table 1 Body and Organ Weights as Well as Circulating Serum Testosterone Levels in Adult Rats Whose Mothers Were Given Either Water or Fish from a Reference Site of Xenoestrogen-Contaminated Fish during Lactation

 
When the pups reached adulthood (91 days), however, there was a significant decrease in the weight of the epididymis in pups whose mothers were fed fish from the xenoestrogen-contaminated site, as compared to either the control or reference site group (Table 1). Paired seminal vesicle weights and ventral prostate weights were not altered by treatment, suggesting that biologically active androgen levels were not altered by treatment (Table 1). Further analyses of serum testosterone levels confirmed that they were not altered by treatment (Table 1).

Sperm Motility Parameters
Indicators of sperm concentrations and their motility were assessed for the three experimental groups. Cauda epididymidis spermatozoal concentrations were significantly decreased in the xenoestrogen-exposed group as compared to both the controls and the group whose mothers were fed fish from the reference site (Fig. 2). In the xenoestrogen-exposed group, the percentage of motile spermatozoa was decreased by approximately 20% (Fig. 3a) and the percentage of motile spermatozoa with progressive motility was significantly decreased as compared to the controls and the group exposed to reference site fish (Fig. 3b). The VAP, VSL, and VCL (Figs. 4a, 3b, and 3c) were all reduced in the xenoestrogen-exposed group, BCF was not different from the control group (Fig. 4d). Straightness and linearity were also lower in the xenoestrogen-exposed group than in both the controls and the group exposed to reference fish (Fig. 5a and 5b). When spermatozoa were classified according to their velocity, there was a reduction in the percentage of rapidly moving cells and an increase in the percentage of static cells in the xenoestrogen-exposed group, as compared to the controls and the reference group (Fig. 6).



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FIG. 2. Mean sperm concentration in adult rats (n = 10 per group) whose mothers were give either water or fish from a reference site of xenoestrogen-contaminated fish during lactation. Sperm concentrations were assessed using the IVOS semen analyzer, and the data are expressed as the mean ± SEM. Different superscripts indicate significant differences between groups (i.e., "a" is different from "b").

 


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FIG. 3. Sperm motility (A) and progressive motility (B) in adult rats (n = 10 per group) whose mothers were give either water or fish from a reference site of xenoestrogen-contaminated fish during lactation. Data are expressed as the mean ± SEM. Different superscripts indicate significant differences between groups (i.e., "a" is different from "b").

 


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FIG. 4. Sperm Path Velocity (VAP; A), Progressive Velocity (VSL; B), Curvilinear Velocity (VCL; C), Beat Frequency (BCF; D) in adult rats (n = 10 per group) whose mothers were give either water or fish from a reference site of xenoestrogen-contaminated fish during lactation. Analyses were determined using the IVOS semen analyzer. Data are expressed as the mean ± SEM. Different superscripts indicate significant differences between groups (i.e., "a" is different from "b").

 


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FIG. 5. Straightness (A) and linearity (B) of sperm displacement in adult rats (n = 10 per group) whose mothers were give either water or fish from a reference site of xenoestrogen-contaminated fish during lactation. Analyses were done using the IVOS semen analyzer, and the data are expressed as the mean ± SEM. Lowercase letters indicate significant differences between groups (i.e., "a" is different from "b").

 


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FIG. 6. Mean percentage of Rapid, Medium, Slow, and Static cells in adult rats whose mothers were given either water or fish from a reference site of xenoestrogen-contaminated fish during lactation. Data are expressed as the mean ± SEM. Different superscripts indicate significant differences between groups (i.e., "a" is different from "b").

 
Effects on Spermatogenesis
Because sperm concentrations and motility were reduced, we wanted to determine whether their reduction was due to an effect on spermatogenesis. Histological examination of the testes indicated that there were no significant differences in the diameter of the seminiferous tubules in any of the three experimental groups (data not shown). When tubules were staged according to the different stages of spermatogenesis, shifts in the frequencies of stages were observed in adult rats. There were no differences in the stages of spermatogenesis between the control group and the group whose mothers were fed reference site fish. In the xenoestrogen-exposed group, however, the frequency of tubules at stages I through VIII was decreased. In addition, a significant decrease in the incidence of both stages VII and XIV in the xenoestrogen-exposed group as compared to the two other experimental groups was noted (Fig. 7). Stage XIV is the stage at which the second meiotic division occurs, whereas sperm are released into the lumen of the seminiferous tubule between stages VII and VIII. These results suggest that these two critical stages of development are delayed as a result of lactational exposure to contaminants from the fish captured in the xenoestrogen-contaminated site.



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FIG. 7. Distribution of the stages of spermatogenesis in adult rats (n = 15 per group) whose mothers were give either water or fish from a reference site of xenoestrogen-contaminated fish during lactation. Testes were removed, fixed in Bouin's solution, and embedded in paraffin. Sections (5 µm) were mounted on glass slides and stained with periodic acid–Schiff (PAS) stain. Staging of spermatogenesis was done by evaluating 200 tubules from each adult rat testis. "a" indicates a significant difference between the xenoestrogen group and both the control and reference group.

 
Connexin 43 in the Testes
The process of spermatogenesis requires highly coordinated intercellular communication between developing germ cells and Sertoli cells. This is accomplished by gap junctions. In the testis, gap junctions are predominantly composed of Cx43 (Batias et al., 2000Go). We therefore wanted to determine whether Cx43 was altered in the testis of rats whose mothers were fed fish from the xenoestrogen-contaminated site. In controls and in rats whose mothers were fed fish from the reference site, Cx43 was localized at the base of the tubules between adjacent Sertoli cells. The immunoperoxidase reaction resembled discrete ribbon-like strands localized in the area of the Sertoli–Sertoli junctional complex (Fig. 8A). A sparse punctate immunoreaction was also observed higher up in the seminiferous tubule between developing germ cells and Sertoli cells. The immunoreaction in some of the tubules from stage II to VII was more intense than in tubules at stages IX–XIV. Although this finding is consistent with previously published studies (Riley et al., 1992; Tan et al., 1996Go; Lablak et al., 1998), the mechanisms that drive this stage-specific expression of Cx43 is not known.. Immunostaining was also observed outside the seminiferous tubule between Leydig cells (Fig. 8B). There were no differences in the immunoperoxidase staining pattern between the control group and the Site I group (data not shown). In testes from rats whose mothers had been fed xenoestrogen-contaminated fish, Cx43 immunostaining along the plasma membrane was less pronounced than in either the control group or the reference group (Fig. 8C and 8D). In some tubules the staining between adjacent Sertoli cells appeared to be completely absent. Likewise, the immunoreaction between adjacent Leydig cells appeared to be less intense, although the effect was not as pronounced as in Sertoli cells. There was no immunoperoxidase reaction observed in sections incubated without primary antibody, which were used as negative controls (data not shown).



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FIG. 8. Immunolocalization of Cx43 in testis. Histological sections of adult rat testes (n = 5 per group) whose mothers were give either water (A, B) or fish from a xenoestrogen-contaminated site (C, D) during lactation were immunostained with anti-Cx43 antibody and a horseradish peroxidase-linked secondary antibody. Cx43 immunostaining is indicated by arrows. The micrographs indicate that there is less Cx43 immunostaining in rats exposed to xenoestrogens. Lu = lumen, L = Leydig cells, IT = intertubular space. Magnification x400 (A, C); x640 (B, D).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 RESULTS
 DISCUSSION
 REFERENCES
 
The long-term adverse effects resulting from the consumption of fish captured in xenoestrogen-contaminated environments are an important consideration in assessing the risk of endocrine-disrupting chemicals. We have shown, both in this study and in a previous study, that xenoestrogens are present in the St. Lawrence River over a distance exceeding 75 km (Aravindakshan et al., 2004Go). Our observations indicate an active sport fishery in the St. Lawrence River, and other investigators have reported that these fishers often eat their catch and some may consume sizeable quantities of fish (Kosatsky et al., 1999aGo). In fact, heavy eaters of these fish have elevated levels of mercury in hair, and elevated circulating levels of DDE and PCBs (Kosatsky et al., 1999aGo). We can therefore infer that humans are exposed to contaminants present in fish from the St. Lawrence River and that the chemicals responsible for inducing an estrogenic response in the fish are transferred to humans. Furthermore, other predators such as fish-eating mammals and birds are likely targets for these contaminants, although, at the moment, there is no information regarding possible endocrine-disrupting effects on these species in the St. Lawrence River.

The body weights of the dams and the pups in our rat population did not differ among any of the treatment groups in this study, suggesting that fish consumption did not have an effect on the growth rates of the rats. This finding also suggests that there do not appear to be any differences in the nutritive content of fish from the reference site and those from the xenoestrogen-contaminated site, a finding in accordance with our previous studies, which showed no differences in the condition factors of the fish between these two sites (Aravindakshan et al., 2004Go).

In 21-day-old rats, there were no effects on the weights of male reproductive organs or in their histological appearance. However, when the rats reached adulthood, there was a significant decrease in the weight of the epididymis. Other tissue weights were unaffected. Sperm production occurs in the testis, but the epididymis is where sperm acquire the ability to swim and fertilize and where they are stored until the time of ejaculation (Yeung and Cooper, 2002Go). The lower epididymal weight may result from a decrease in the concentration of spermatozoa that are stored in the cauda region of the epididymis. Previous studies with estrogenic compounds have shown that the time required for sperm transport through the epididymis can be altered (Hess, 1998Go; Klinefelter and Suarez, 1997Go). It is possible that an increase in transit time of the sperm through the caput/corpus region of the epididymis may account for a decrease in epididymal weights. Epididymal weight is also dependent on circulating levels of testosterone. However, serum testosterone levels were not different between experimental groups, and since both ventral prostate and seminal vesicle weights also did not differ, this indicates that circulating androgen levels and activity were not affected in rats whose mothers were fed xenoestrogen-contaminated fish.

Sperm concentration, progressive motility, and linearity were significantly reduced in rats exposed to xenoestrogen-contaminated fish. It has been reported that in humans, there is a significant correlation between fertilization rates and linearity (Hirano et al., 2001Go). The reduction in the sperm motion (manifested as decreases in the percentage of motile sperm) and linear velocity may be significant factors in the onset of infertility. Furthermore, cells that were designated as slow and static were not included in the average calculated for our velocity parameters. The high incidences of slow and static cells, the decreased velocity parameters, and the decreased linearity seen in the rats exposed to xenoestrogens all suggest that the sperm in rats from this group may have reduced fertilizing potential. Goyal et al. (2003)Go reported that the administration of the potent estrogen, diethylstilbestrol (DES), to rats at 10 µg given on alternate days for the first 12 days neonatally resulted in a decrease in sperm motility and linearity. However, in contrast to the present study, they also observed hypertrophy of both the seminiferous tubule and intertubular cells. The fact that we observed effects on sperm motility parameters without extensive testicular tissue damage suggests that estrogenic chemicals may exert more subtle effects at environmentally relevant doses.

Spermatogenesis is a linear process in which adjacent cellular associations (14 stages in the rat; Leblond and Clermont, 1952Go) advance from stage to stage in wavelike formation (Parvinen and Vanha Perttula, 1973). There is limited information regarding the 14 stages of rat spermatogenesis after treatment with environmentally relevant doses of xenoestrogens. In the present study, the frequency of the stages of spermatogenesis was altered, an indication of a disturbance in the kinetics of spermato- genesis. These observations raise the questions of whether this alteration is due to certain stages maturing more rapidly or whether certain cell types experience germ cell arrest. Alternatively, the primary effect may have been on the germ cells that are outside the blood–testis barrier during the early postnatal period. Environmental contaminants such as diethylcarbamyl methyl-2–4-dinitropyrrole (Patanelli and Nelson, 1964Go), 2,5-hexanedione, (Chapin et al.; 1983Go), and ethylene glycol monomethyl ether (Chapin et al., 1984Go; Creasy et al., 1985Go) have all been shown to alter the stage frequencies of spermatogenesis. The changes in stage kinetics and the absence of gross histopathological effect on germ cells observed in this study suggest that the xenoestrogens could be causing more subtle effects on male reproductive function.

Spermatogenesis is a synchronized and spatially patterned process of cell proliferation and differentiation in which gap-junctional intercellular coupling plays an important role in coordinating these functions. The relatively large number of different connexins and their localization in the testis is suggestive of the importance of intercellular gap-junctional communication in testicular functions (Batias et al., 2000Go; Risley et al., 1992Go; Tan et al., 1996Go). In rodents, both Sertoli–Sertoli cell interaction and germ cell (spermatogonia and spermatocytes)–Sertoli cell communication are mediated by gap junctions containing Cx43 (Batias et al., 2000Go). Testicular intercellular communication mediated by Cx43 gap junctions is believed to represent an essential process for spermatogenesis, because spermatogenesis is arrested in testes that lack Cx43 (Roscoe et al., 2001Go). Immunocytochemical localization of Cx43 indicated that in each experimental group, some tubules displayed a more intense Cx43 immunoreaction than others. Previous studies have shown that Cx43 immunostaining is dependent on the stage of the tubule (Batias et al., 2000Go; Risley et al., 1992Go; St-Pierre et al., 2003Go; Tan et al., 1996Go). Our results indicate that the expression of testicular Cx43 is reduced in rats that were exposed to xenoestrogenic compounds during lactation. The reduced expression of Cx43 may be responsible, in part, for alterations observed in the kinetics of spermatogenesis. Recent studies have shown that lindane can alter the expression and targeting of Cx43 in Sertoli cells (Mograbi et al., 2003Go). Furthermore, Defamie et al. (2003)Go reported alterations in the expression of Cx43 in patients with azoospermia and undifferentiated Sertoli cells, a finding that suggests pathological consequences associated with altered gap-junctional communication.

Although the nature of the chemicals responsible for causing these effects is at present unknown, chemical analyses of sediments around the Island of Montreal suggest that contaminant levels are generally low, with the exception of zinc in sewage effluent and alkyl phenols that are present downstream from the Montreal sewage discharge point into the St. Lawrence River (Gagnon and Saulnier 2003Go; Sabik et al., 2003Go). Whether these chemicals or other, unidentified chemicals are responsible for the reproductive toxicity effects observed in this study remains to be established. It is clear, however, either that certain chemicals at the sampling site can be transferred to developing offspring and exert permanent effects on male reproductive development, or that the effects of such chemicals on the mothers can be transferred and cause latent pathological effects in the male offspring.

In summary, the present findings show that exposure of immature rats to fish from a xenoestrogen-contaminated environment during the early postnatal period exerts long-lasting effects on epididymal sperm concentration and motility. This effect appears to result in part from alterations in spermatogenesis that are associated with a decrease in the gap- junctional protein Cx43. Together, these results suggest that the consumption of fish from xenoestrogen-contaminated ecosystems may alter the postnatal development of the male reproductive tract, resulting in permanent effects on male reproductive parameters. These results may be particularly important for both fish-eating humans and other riverine mammalian species and they raise serious concerns regarding the transmission of endocrine-disrupting effects through the food chain.


    ACKNOWLEDGMENTS
 
The authors thank Ms A. Gendron for her assistance with the field sampling. This study was supported by the Canadian Network of Toxicology Centres (CNTC), the Fonds D'Action Québécois Pour le Développement Durable (FAQDD), and Environment Canada.


    NOTES
 

1 To whom correspondence should be addressed at INRS-Institut Armand Frappier, Université du Québec, 245 Hymus Boulevard, Pointe-Claire, Québec, H9R 1G6. Fax: (514) 630-8850. E-mail: daniel.cyr{at}INRS-IAF.uquebec.ca.


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