* INRS-Institut Armand Frappier, Université du Québec, Montreal, Quebec, Canada H9R 1G6; and St. Lawrence Centre, Environment Canada, Montreal, Quebec, Canada
Received March 13, 2004; accepted May 11, 2004
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ABSTRACT |
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Key Words: xenoestrogen; sperm motility; spermatogenesis; gap junctions.
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INTRODUCTION |
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Contaminants such as 17--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 2001
; Jobling and Tyler, 2003
; Rothcell and Ostrander, 2003
). 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., 2004
; Jobling and Sumpter, 1993
; Tyler et al., 1996
) 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., 2004).
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., 2004). Kosatsky et al. (1999a)
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 Rivercaught 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., 1999b
). 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 2001; Jobling and Tyler, 2003
; Rothcell and Ostrander, 2003
). 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, 2001
; Rodriguez et al., 2002
). The bloodtestis and bloodepididymal barriers are formed, and the first wave of spermatogenesis is initiated (Cyr, 2001
; Cyr et al., 2002
; Pelletier, 2001
). 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.
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Materials and Methods |
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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, 1987). After RNA isolation, reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine Vtg mRNA levels as described previously (Aravindakshan et al., 2004
). 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; 68 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., 1991; Chan et al., 1999
).
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 motilitythe 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 acidSchiff (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).
Immunohistochemistry. Immunoperoxidase staining of testicular sections (n = 5 rats per group) was performed according to previously published protocols (Oko and Clermont, 1989). 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.
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RESULTS |
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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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DISCUSSION |
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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., 2004).
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, 2002). 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, 1998
; Klinefelter and Suarez, 1997
). 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., 2001). 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)
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, 1952) 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 bloodtestis barrier during the early postnatal period. Environmental contaminants such as diethylcarbamyl methyl-24-dinitropyrrole (Patanelli and Nelson, 1964
), 2,5-hexanedione, (Chapin et al.; 1983
), and ethylene glycol monomethyl ether (Chapin et al., 1984
; Creasy et al., 1985
) 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., 2000; Risley et al., 1992
; Tan et al., 1996
). In rodents, both SertoliSertoli cell interaction and germ cell (spermatogonia and spermatocytes)Sertoli cell communication are mediated by gap junctions containing Cx43 (Batias et al., 2000
). 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., 2001
). 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., 2000
; Risley et al., 1992
; St-Pierre et al., 2003
; Tan et al., 1996
). 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., 2003
). Furthermore, Defamie et al. (2003)
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 2003; Sabik et al., 2003
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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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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