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BOR - Papers in Press, published online ahead of print May 4, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.037374
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BIOLOGY OF REPRODUCTION 73, 414–426 (2005)
DOI: 10.1095/biolreprod.104.037374
© 2005 by the Society for the Study of Reproduction, Inc.

In Utero and Lactational Exposure to an Environmentally Relevant Organochlorine Mixture Disrupts Reproductive Development and Function in Male Rats1

Mohamed-Kheir Idris Anas 3, Christine Guillemette 3, Pierre Ayotte 4,5 , Daria Pereg 4, Francine Giguère 3, and Janice L. Bailey 2, 3

Centre de Recherche en Biologie de la Reproduction, Département de Sciences Animales,3 Unité de Recherche en Santé Publique (CHUL-CHUQ),4 Département de Médecine Sociale et Préventive,5 Université Laval, Québec, Canada G1K 7P4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We hypothesized that in utero and lactational exposure of male rats to a mixture of more than 15 organochlorines, resembling that found in blubber from northern Quebec seals, alters reproductive development and function. Female rats were gavaged with either corn oil (controls) or the organochlorine mixture in increasing doses (low, medium, and high) for 5 wk before mating and through gestation. Developmental effects were monitored in the male offspring from Postnatal Day (PND) 2 until PND 90. The high-dose mixture reduced the number of pups per litter, percentage of live offspring, and pup weights (P < 0.05). Because only three rats from the high-dose treatment survived, data from this group beyond PND 2 were not included in the statistical analyses. As assessed by the time of preputial separation, puberty was delayed in the pups from treated dams (P < 0.05). Testes weights in the medium-dose group were greater than those in controls on PND 21 (P < 0.05). Ventral prostate weights were lower for the medium-dose group on PND 60 (P < 0.05). On PND 90, weights of the epididymis, ventral prostate, and seminal vesicle of the medium-dose rats were reduced compared to those of controls (P < 0.05). On PND 90, sperm motility parameters assessed by computer-assisted sperm analysis were altered in the low- and medium-dose groups (P < 0.05). Testicular and epididymal morphology was severely affected in rats exposed to the high dose of the mixture. Serum testosterone, LH, FSH, prolactin, and total thyroxine levels did not differ because of organochlorine treatment. Therefore, in utero and lactational exposure to an environmentally relevant organochlorine mixture adversely affects the reproductive system of male rats, perhaps via antiandrogenic effects during testis development, suggesting a possible reproductive health hazard for humans and other species.

environment, male reproductive tract, puberty, sperm, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Certain environmental pollutants interfere with normal hormonal function in animals and humans and, thus, are termed endocrine disruptors [1]. It is assumed that endocrine-disrupting chemicals exert their effects by mimicking or antagonizing endogenous hormones, modifying hormone-receptor levels, or altering the synthesis and metabolism of endogenous hormones [2]. Because hormones play a central role in regulating reproductive development and functions, the impact of endocrine disruptors on reproductive health is of interest. To date, reproductive abnormalities, including feminization of males, abnormal sexual behavior, birth defects, altered sex ratios, decreased sperm production, reduced testicular size, infertility, and thyroid dysfunction, have been reported in laboratory animals and wildlife exposed to endocrine-disrupting chemicals [1, 3– 7]. In humans, impaired fertility, altered birth-sex ratio, declining sperm count and quality, and undescended testes also have been attributed to exposure to environmental contaminants [8–11]. The causal association between altered reproductive health and exposure to environmental pollutants, however, remains a subject of controversy.

One group of environmental pollutants includes chlorinated pesticides and polychlorinated biphenyl (PCBs), collectively termed organochlorines. Because of their resistance to degradation, high lipophilicity, and long-range atmospheric transport, organochlorines are present in the environment and contaminate food chains throughout the world despite being banned in many developed countries. Various organochlorines have been detected in human blood, milk, and tissues in different parts of the world [12– 19]. Furthermore, the presence of organochlorine compounds in the follicular fluid and semen of humans and farm species has been reported [20–24]. Given their lipophilic character, organochlorines are found at higher concentrations in fatty tissues. Human exposure to organochlorines often is related to the frequent consumption of contaminated fish and game, and it occurs both more frequently and at higher levels in populations that rely on hunting and fishing products for sustenance. Native populations in the Arctic, such as the Inuit, consume culturally important diets that contain large amounts of fatty tissues from sea mammals. Consequently, the Inuit are exposed to high doses of several organochlorines relative to the general populations in North America and Western Europe [25, 26]. Although preliminary data indicate no differences in sperm count among fertile Inuit men from four regions of Greenland, the poorest sperm motility occurred in the Inuit from the east coast, where organochlorine exposure is higher [27].

Several organochlorines have been identified as hormonally active agents [28–30]. When examined individually, various organochlorines have been shown to adversely affect the reproductive performance of laboratory animals [31–36]. Organochlorines are ubiquitously present as complex mixtures in the environment. Thus, the reproductive hazard of exposure to combinations of these chemicals is of concern, because these pollutants might act in additive, synergistic, or antagonistic ways. As a result of their particular sensitivity to hormonal fluctuations, embryonic/fetal developmental responses to endocrine disruptors are different from the responses of adults. Therefore, embryonic/fetal exposure to low levels of exogenous hormones or toxicants may cause permanent physiological changes that would not manifest in adults exposed to similar or even greater levels [37].

The present study was carried out to test the hypothesis that in utero and lactational exposure of male rats to an environmentally relevant mixture of organochlorines modulates their reproductive development and function. The mixture used comprised more than 15 organochlorines and was shown previously to interrupt gamete competence and subsequent embryo development in vitro in the porcine model [38, 39].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Organochlorine Mixture

The organochlorine mixture was designed to resemble that found in Ringed Seal blubber from northern Quebec [40], which is part of the traditional Inuit diet. The major component of the mixture is a custom PCB neat mix (AccuStandard, New Haven, CT) containing the following components: 2,4,4'-trichlorobiphenyl (320 mg, 8.46% of total PCBs), 2,2',4,4'-tetrachlorobiphenyl (256 mg, 6.77% of total PCBs), 3,3,4,4'-tetrachlorobiphenyl (1.4 mg, 0.04% of total PCBs), 3,3',4,4',5-pentachlorobiphenyl (6.7 mg, 0.18% of total PCBs), Aroclor 1254 (12.8 g, 33.82% of total PCBs), and Aroclor 1260 (19.2 g, 50.73% of total PCBs). 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (p,p'-DDT), technical toxaphene, {alpha}-hexachlorocyclohexane (HCH), aldrin, dieldrin, 1,2,4,5-tetrachlorobenzene, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (p,p'-DDD), ß-HCH, hexachlorobenzene, mirex, {gamma}-HCH, and pentachlorobenzene were from Aldrich Chemical Co. (Milwaukee, WI). Technical chlordane was from Ceriliant (Austin, TX). Chemicals were weighed and dissolved in corn oil to yield a stock solution containing 23.3 mg PCB/ml corn oil plus the other organochlorines at the relative proportions listed in Table 1. The stock solution was kept shielded from light at room temperature and was diluted with corn oil before treatment to obtain the appropriate dosing solutions (1:20 for the high dose, 1:200 for the medium dose, and 1:2000 for the low dose).


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  		TABLE 1. Composition of the organochlorine mixture used in this study

Animals and Treatments

Figure 1 summarizes the experiment conducted. Five-week-old female Sprague-Dawley rats were housed two per cage in a room with a 12L: 12D photoperiod (lights-on, 0645–1845 h), a temperature range of 22 ± 1°C, and a humidity range of 46% ± 10%. All rats were identified using the Aramis Laboratory Animal Microtattoo System (Ketchum Manufacturing, Inc., Ottawa, ON, Canada). Animals were given access to food and chlorinated water ad libitum. Animal care and handling were in accordance with the guidelines of the university committees for animal care, chemical safety, and ethics.



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  		FIG. 1. Schematic diagram of the experimental plan

After 10 days of acclimatization, the rats were weighed and randomly assigned to four groups (n = 10 per group) and administered either corn oil (control group) or the organochlorine mixture thrice weekly by gavage (1 ml/100 g body weight). Each day, the low-dose group received 50 µg/ kg, the medium-dose group 500 µg/kg, and the high-dose group 5000 µg/ kg of PCBs. Corresponding doses of other organochlorines included in the mixture are listed in Table 1. After 5 wk of treatment, each pair of female rats was housed with an unexposed male rat for 10 days, and mating was confirmed by the presence of a vaginal plug. Organochlorine treatment continued through mating and gestation until terminated at parturition. The day of parturition was considered to be Postnatal Day (PND) 1. On PND 2, number of litters, litter size, sex ratio, percentage of live offspring, pup body weights, and pup anogenital distance were determined. Litters with more than 10 pups were then culled to 10 (male and female), and those with less than seven pups were excluded. Because only three male rats from the high-dose group, all from the same dam, survived to the end of the experiment, data from this group beyond PND 2 were excluded from the statistical analyses. Pups remained with their dams until weaning (PND 21), at which time blood samples were collected from the dams and some of the pups (samples were pooled per sex per litter, n = 4) by cardiac puncture after being anesthetized with ketamine and xylazine. Serum and plasma were separated by centrifugation (900 x g, 30 min) and held at –20°C until the time of hormone and organochlorine analyses. The anesthetized dams and male pups were then killed by cervical dislocation, and livers, spleens, uteri, ovaries, testes and epididymides, ventral prostates, and seminal vesicles were collected, cleared from the surrounding fat, and weighed; organ weights were assessed relative to animal body weight. Preputial separation in the remaining male rats (n = 20 males/treatment) was monitored daily from PND 35. Ten of these pups were killed on PND 60, and the remaining 10 were killed on PND 90. Again, blood samples were collected for hormone and organochlorine analyses, and livers, spleens, and reproductive organs were removed and weighed as described above. The right caudae epididymides were used for examining sperm motility and morphology.

Evaluation of Epididymal Sperm Motility

On PNDs 60 and 90, the removed right cauda epididymis was placed in a 35-mm plastic Petri dish containing 2 ml of TCM-199 (Gibco Laboratories, Grand Island, NY) supplemented with 0.5% fatty acid-free BSA (Fraction V; Sigma, St. Louis, MO) on a 37°C heating stage. They were then nicked with a scalpel several times and incubated for 15 min in a humidified 5% CO2 incubator to allow sperm to diffuse. The sperm were then analyzed for motility, progressive motility, average path velocity, straight-line velocity, curvilinear velocity, amplitude of lateral head displacement, beat cross frequency, straightness, and linearity with a Hamilton-Thorne CEROS analyzer (version 12; Beverly, MA). Analysis was performed after 15, 30, and 60 min of incubation. The analyzer setting was as follows: frame rate, 60 Hz; frames acquired, 30; minimum contrast, 80; and minimum cell size, seven.

Sperm Morphology

Fifty microliters of the sperm suspension used for motility analysis were diluted further with 0.5 µl of TCM-199 containing 10 µl of concentrated (37%) formalin. A 10-µl aliquot of the formalized sperm sample was placed on a glass slide, cover-slipped, and examined for morphology with a phase-contrast microscope (400x total magnification). Two-hundred sperm from each animal were evaluated for head and tail abnormalities [41].

Tissue Preparation for Histology

Some of the collected testes and epididymides (n = 3) were fixed in Bouin fixative (Sigma) for 2 wk and then transferred to 70% and then 50% ethanol. Next, organs were embedded in paraffin, sectioned (thickness, 5 µm), mounted on slides, stained with hematoxylin-eosin (Sigma), and cover-slipped. Slides were examined by light microscopy for any marked histological changes. One-hundred seminiferous tubules from each rat were assessed to determine the tubule differentiation index, expressed as the percentage of seminiferous tubules undergoing spermatogenesis beyond formation of B spermatogonia [42].

Organochlorine Assays

The PCB congeners (International Union for Pure and Applied Chemistry nos. 28, 52, 99, 101, 105, 118, 128, 138, 153, 156, 170, 180, 183, and 187) and 18 chlorinated pesticides and metabolites (aldrin, {alpha}-chlordane, {gamma}-chlordane, p,p'-DDD, p,p'-DDE, p,p'-DDT, dieldrin, {alpha}-HCH, ß-HCH, {gamma}-HCH, heptachlor, heptachlor epoxide, hexachlorobenzene, mirex, cis-nonachlor, trans-nonachlor, oxychlordane, and pentachlorobenzene) were determined by high-resolution gas chromatography with electron-capture detection (HRGC-ECD). Plasma samples (2 ml) were extracted with an ammonium sulfate:ethanol:hexane (1:1:3) solution, cleaned on Florisil columns (J.T.Baker, Phillipsburg, PA), and analyzed on an HP-5890 series II gas chromatograph equipped with dual-capillary columns (Ultra-1 and Ultra-2; Hewlett-Packard, Palo Alto, CA) and dual Ni-63 electron-capture detectors. Peaks were identified by their relative retention times obtained on the two columns using a computer program developed in-house. Quantification was performed mainly on the Ultra-1 column. Detection limits were 0.2 µg/L for PCB congeners, 0.3 µg/L for p,p'-DDT and ß-HCH, 0.8 µg/L for dieldrin and heptachlor epoxide, and 0.2 µg/L for the other pesticides and metabolites. Plasma samples also were analyzed for five toxaphene congeners (Parlar nos. 26, 32, 50, 62, and 69) by HRGC with mass-spectrometry detection, but these compounds were not detected in any samples (detection limits: 0.08 µg/L for Parlars 62 and 68, and 0.2 µg/L for Parlars 26, 32, and 50). Quality-control procedures of the HRGC-ECD method as well as the accuracy and precision data were as previously reported [43].

Hormone Analyses

Serum samples collected on PND 21 were pooled per sex per litter for hormone analyses. Solid-phase radioimmunoassays were used to determine concentrations of testosterone, total thyroxine (T4; Coat-A-Count; Diagnostic Products Corporation, Los Angeles, CA), FSH (Biocode S.A, Liege, Belgium), as well as LH and prolactin (MP Biomedicals, Asse-Relegem, Belgium) according to the manufacturer's protocols. Each hormone assay was completed using a single kit and in one run for each hormone, with intra-assay coefficients of variation of 6.2%, 6.6%, 3.8%, 11.7%, and 4.8% for testosterone, T4, FSH, LH, and prolactin, respectively. Minimum detectable concentrations were 4 ng/dl, 0.22 µg/dl, 0.2 ng/ ml, 0.14 ng/ml, and 0.5 ng/ml for testosterone, T4, FSH, LH, and prolactin, respectively.

Statistical Analyses

Data are presented as the mean ± SEM. Percentages were arcsine transformed before statistical analyses. Analysis of variance was performed using the general linear models procedure (SAS Institute, Inc., Cary, NC). Differences were considered to be statistically significant at P < 0.05. When the main effect of treatment was significant, treatments were compared using the Student-Newman-Keuls multiple-comparison test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fertility and Developmental Data of Dams

The effect of organochlorine exposure on dam fertility is presented in Table 2. No treatment differences in the mating, fertility, and pregnancy indices were observed. The high dose reduced the number of pups per litter and the percentage of live offspring relative to all other groups (P < 0.05). No differences in the number of implantation sites, sex ratio, and duration of gestation were observed among treatments. None of the dams died before the end of the experiment, and no apparent signs of general toxicity, such as behavioral changes, ruffled coats, or increased excitability, were observed. No differences were seen in the body weight gain of dams after 5 wk of treatment (Fig. 2). Dams from the high-dose group had reduced weight gain at the time of parturition compared to all other groups (P < 0.05). The body weight gain also was reduced at the time of weaning in the high-dose group relative to that in the control group (P < 0.05). Dams treated with the high dose of the mixture had lower spleen weights (relative to body weight) compared to all other treatments (Fig. 3). Relative uteri weights in dams from the high-dose group were higher than those in the low-dose group (P < 0.05) but did not differ from controls. Relative ovary weights were not affected by treatment.


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  		TABLE 2. Fertility data of female rats treated with the organochlorine mixture or corn oil (vehicle)



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  		FIG. 2. Body weight gain of dams treated orally with the organochlorine mixture or corn oil (vehicle) for 5 wk before mating and during gestation. Each bar represents the mean ± SEM for 10 animals. Different letters indicate significant differences within each sampling time



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  		FIG. 3. Organ weights (g/kg body weight) in dams treated orally with the organochlorine mixture or corn oil (vehicle) for 5 wk before mating and during pregnancy. Dams were killed at pup weaning (PND 21). Each bar represents the mean ± SEM for 10 animals. Different letters indicate significant differences. Uterus and ovary weights were not affected by treatment

Developmental Data of Male Pups

No overt signs of general toxicity were observed in the pups at any stage of the protocol. Table 3 presents data regarding growth and parameters indicative of in utero sex-hormone disruption for F1 males according to treatment. Anogenital distance was shorter in pups from the high-dose group compared to all other groups (P < 0.05), but not when expressed relative to body weight. Nipple retention also was unaffected; however, a dose-related increase in age at preputial separation was observed in pups exposed to the organochlorine mixture (P < 0.05). Mean body weight of pups from the high-dose group on PND 2 was lower compared to the other groups (P < 0.05). The body weights of pups from the low- and medium-dose groups did not differ from controls at all stages. F1 rats in the medium-dose group had higher testes weights (relative to body weight) on PND 21 (P < 0.05) but not on PND 60 or PND 90 (Fig. 4). On PND 60, relative ventral prostate weights were lower in rats from the medium-dose group compared to the other groups (P < 0.05). Furthermore, the organochlorine treatment caused a dose-related decrease in the relative weights of epididymides, seminal vesicles, and ventral prostate on PND 90 (P < 0.05). As for the nonreproductive organs, relative liver weights were slightly greater in rats from the medium-dose group on PND 21 relative to the control and low-dose groups (P < 0.05), and spleen weights never differed. Data from the high-dose group were not included in the statistical analyses, because only three rats from this group survived beyond PND 2. Nevertheless, compared to the other treatments, rats from the high-dose group appeared to have markedly lower relative weights of testes, epididymides, seminal vesicles, and ventral prostates, whereas relative liver and spleen weights were slightly higher.


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  		TABLE 3. Developmental data (mean ± SEM) of male rats exposed in utero and during lactation to the organochlorine mixture or corn oil (vehicle)



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  		FIG. 4. Organ weights (g/kg body weight) in male rats exposed in utero and through lactation to an organochlorine mixture or corn oil (vehicle) on PNDs 21, 60, and 90. Each bar represents the mean ± SEM for 10–12 animals. Different letters indicate significant differences within each sampling time. Spleen weight was not affected by treatment. *Data from the high-dose group were not included in the statistical analysis, because only three male rats from this group survived beyond PND 2

Sperm Motility and Morphology

The effect of organochlorine treatment on the motility of the caudal epididymal sperm is presented in Figure 5. On PND 60, the percentages of motile and progressively motile sperm were not affected by treatments; however, on PND 90, the percentages of motile and progressively motile sperm decreased in a dose-dependent manner (P < 0.05). For the low-dose group, the decrease was significant after 60 min of incubation, whereas decreases were noted at all tested time periods for rats in the medium-dose group. Amplitude of lateral head displacement and beat cross frequency were higher for rats from the medium-dose group compared to controls (P < 0.05) (Table 4), but the other motility parameters were not affected. Sperm morphology was not altered in the low- and medium-dose groups (Table 4). We were able to collect only a few sperm (17–32 sperm/ rat), all immotile, from the three surviving rats of the high-dose group. Moreover, none of the sperm examined from this group was morphologically normal. Severe defects were observed, including two-headed sperm, missing flagella, and/or separated heads and tails.



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  		FIG. 5. Percentages of motile and progressively motile sperm from rats on PND 60 and PND 90 that had been exposed in utero and through lactation to the organochlorine mixture or corn oil (vehicle). Caudal epididymal sperm were incubated in TCM-199 at 37°C in CO2 for 15, 30, or 60 min, at which time motility was assessed. Each bar represents the mean ± SEM for 10 animals. Different letters indicate significant differences within each incubation time. All sperm were immotile from the three rats in the high-dose group that survived beyond PND 21


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  		TABLE 4. PND90 sperm motion parameters and sperm morphology of rats exposed in utero and during lactation to the organochlorine mixture or corn oil (vehicle).a

Hormone Levels

Maternal organochlorine treatment did not appear to affect serum hormone concentrations in F1 male rats (Fig. 6). Serum testosterone, LH, and FSH levels on PNDs 60 and 90, as well as the T4 and prolactin levels at all tested time points, did not differ among treatments.



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  		FIG. 6. Serum testosterone, FSH, LH, T4, and prolactin in rats exposed in utero and through lactation to the organochlorine mixture or corn oil (vehicle) on PNDs 21, 60, and 90. Each bar represents the mean ± SEM for five to seven animals for PND 60 and PND 90 and four samples (pooled per litter) for PND 21. No significant differences were observed as a result of the organochlorine treatment. *Because only three rats from the high-dose group survived beyond PND 2, data from the high-dose group were excluded from the analysis

Histology

Testes from animals in the low- and medium-dose groups were morphologically similar to those of controls and displayed a 100% tubule differentiation index (Fig. 7). Based on visual estimation, however, some of the tubules from rats in the medium-dose group appeared to contain fewer spermatozoa in the lumen compared to the controls. In contrast, severe histological changes were observed in the testes of rats from the high-dose group. Testicular tubules from these animals contained several large pyknotic nuclei, with no indication of sperm release, whereas the lumens were devoid of sperm and presented cellular debris and fibrins.



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  		FIG. 7. Sections of testes and epididymides from rats exposed in utero and through lactation to the organochlorine mixture or corn oil (vehicle). Staining was performed with hematoxylin-eosin. Seminiferous tubules from animals in the control group (A) and the medium-dose group (B) are normal, as indicated by the presence of germ cells and the release of sperm. Seminiferous tubules from rats in the high-dose group are occupied by several large pyknotic nuclei, with no indication of sperm release (C). Seminiferous tubule lumens from animals in the medium-dose group (E) had relatively fewer sperm than the control (D). Seminiferous tubule lumens from rats in the high-dose group (F) are deprived of sperm and contain cellular debris and fibrins. Epididymal lumens from animals in the high-dose group (I) are smaller in size than those of the control group (G) and the medium-dose group (H) and are without sperm. Epididymal lumens from animals in the medium-dose group (K) had fewer sperm than the control group (J). Epididymal lumens from rats in the high-dose group are devoid of sperm and are filled with cellular debris (L). Original magnification x400

The morphology of the epididymides also appeared to be normal in rats from the low- and medium-dose groups, with the possible exception of fewer sperm in epididymal lumens from rats in the medium-dose group (Fig. 7). Epididymal tubules from the surviving rats in the high-dose group contained no sperm at all, were filled with cellular debris, and had a smaller diameter compared to those of the control and medium-dose groups.

Plasma Organochlorine Concentrations

Plasma concentrations of various organochlorines and their metabolites in dams and their male offspring are presented in Tables 5 and 6. Aldrin, {alpha}-chlordane, {gamma}-HCH, heptachlor, cis-nonachlor, heptachlor epoxide, pentachlorobenzene, toxaphene congeners, and PCB congeners 28 and 52 were not detected in any sample. In addition, {gamma}-chlordane, dieldrin, heptachlor, {alpha}-HCH, and trans-nonachlor were detected in plasma samples from dams of the high-dose group but not in samples from their male offspring. Plasma organochlorine concentrations in dams from the medium-dose group were two- to fivefold higher than those in dams from the low-dose group, whereas concentrations in dams from the high-dose group were 18- to 80-fold higher than those in dams from the medium-dose group. Plasma levels of all detected organochlorines in the male pups on PND 21 were similar to or slightly higher than those measured in their dams. Plasma concentrations in F1 males on PND 90 were markedly lower (at least 10-fold) than those on PND 21 for both the low- and medium-dose groups.


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  		TABLE 5. Organochlorine concentrations (µg/L) in plasma of dam rats treated orally with the organochlorine mixture or corn oil (vehicle).a


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  		TABLE 6. Organochlorine concentrations (µg/L) in plasma of male rats exposed in utero and through lactation to the organochlorine mixture or corn oil (vehicle).a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These data indicate that in utero and lactational exposure to a mixture containing several organochlorine compounds, similar to that found in blubber from northern Quebec seals, alters male rat reproductive function. The changes include delayed puberty, retarded development of reproductive organs, disrupted spermatogenesis, and reduced sperm motility, suggesting a teratogenic effect of the organochlorine mixture on the male rat reproductive system. Effects of exposure to organochlorine compounds and their metabolites on male rat reproductive development and function have been described in several reports [34, 44–49], but these studies typically focused on a single chemical, using only one or a few maternal doses at a specific stage of pregnancy/lactation or administering the compound to adult animals. Because aboriginal populations living in the Arctic are exposed to numerous organochlorines through their traditional diet, we investigated the impact of maternal exposure to a relevant complex organochlorine mixture on the male reproductive system in the rat model.

Direct Effects of the Mixture on Dams and Possible Toxicity of the High Dose

Apart from reduced litter sizes and percentages of live offspring in rats from the high-dose group, other fertility parameters were unaffected by treatment. The markedly reduced litter size in rats from the high-dose group might have resulted from dams eating their stillborn pups before they were counted, because the number of implantation sites in these dams was not different from the number in controls. Hence, the ability of the dams in the high-dose group to produce fertile oocytes and support early embryonic development and placentation was not different from that of dams in the other groups, although subsequent fetal/ pup loss occurred.

No dam from the high-dose group died before the end of the experiment, but they did show some signs of systemic toxicity: reduced spleen weight and decreased body weight gain relative to controls at the time of parturition and weaning. In contrast, high mortality and profound effects on reproductive function and development were observed in F1 male rats from the high-dose group. Only three male rats, all from one dam, survived to the end of the present study. The high mortality and altered reproductive/ developmental parameters in pups from this group may have been caused by direct teratogenic effects of the mixture in addition to adverse effects secondary to maternal toxicity. Moreover, because exposure continued in the mother's milk, delayed effects could be related to increasing body burden and direct toxicity on the high-dose pups. The lack of apparent toxicity to the low- and medium-dose dams suggests that the adverse effects on their pups likely are via teratogenic or epigenetic effects in utero. Therefore, subsequent discussion with regard to data obtained after PND 2 will emphasize the low- and medium-dose groups.

Altered Development of Pups Because of Maternal Organochlorine Exposure

Maternal exposure to the low or medium dose of the mixture did not influence the weights of male pups. The effects of prenatal exposure to organochlorines on body growth of the offspring are conflicting: Some reports have indicated a negative impact [49, 50], whereas others have shown either no consequence [34, 51] or increased growth rate until weaning, followed by a decrease thereafter [44]. The present study indicates that the effect of maternal exposure to organochlorines on pup body weight is evident only at a high dose that induces maternal toxicity (i.e., reduced gestational weight gain and reduced spleen weight).

Anogenital distance and nipple retention reflect the androgen status in male rats. Exposure of male rodents to antiandrogens decreases anogenital distance [52], whereas disappearance of the rudimentary nipples requires androgens [53]. In the present study, anogenital distance in rat offspring (relative to body weight) was unaffected, suggesting no neonatal feminization of the males. Likewise, nipple retention was observed only in rats from the high-dose group. Onset of puberty, as indicated by the age of preputial separation, is another androgen-sensitive event [54]. Here, a dose-dependent delay in preputial separation was clearly observed in males maternally exposed to the organochlorine mixture, indicating a differential response between these androgen-dependent processes, with preputial separation being the most sensitive endpoint. In contrast, administration of a single 10-µg/kg dose of a dioxin-like PCB congener (PCB 126) to female rats on Gestational Day 15 reduced anogenital distance in the male offspring without affecting the age of preputial separation [44]. Moreover, reduced anogenital distance and increased nipple retention were reported in male rats from dams treated with a high dose of p,p'-DDE (100 mg/kg) from Gestational Days 14 to 18 [33, 47]. Although this high dose of p,p'-DDE generated average pup plasma levels of 87 µg/L on PND 21, which is a concentration similar to that of pups from the medium-dose group in the present study (72.3 µg/ L), it was not sufficient to delay preputial separation. Hence, in these two studies, anogenital distance and nipple retention, but not onset of puberty, were affected by treatment [33, 47]. In contrast to the present findings, subchronic exposure of mature male rats to a mixture of organochlorines and metals had little impact on the reproductive function [55]. These differences in male reproductive system development can be caused by our mixture, which comprises estrogenic, antiandrogenic, and dioxin-like compounds, as well as the extended duration of exposure.

Noteworthy effects on reproductive organ development were observed in rats from the medium-dose group. Mean weight of testes from these rats was higher than in controls on PND 21 but not at a later age. The reason for this transient augmentation in testicular weight is unknown. Increased testes weight in response to PCB treatment has been attributed to depressed fetal thyroid hormone levels, which stimulate Sertoli cell proliferation [56]. In the present study, no treatment-induced reduction in T4 was observed at any stage of development. Reduced epididymal and seminal vesicle weights were evident only on PND 90, whereas ventral prostate weight was lower at both PND 60 and PND 90, indicating that the ventral prostate is particularly sensitive to the hormonal effects of the mixture.

Development and maintenance of the male accessory sex organs also are androgen-dependant and, thus, are vulnerable to antiandrogens. Any putative antiandrogenic effect of this organochlorine mixture, however, was not accompanied by modified circulating testosterone levels in the F1 males at any tested age. Similarly, in utero and lactational exposure to DDE and tetrachlorodibenzodioxin induced antiandrogenic effects without decreasing serum testosterone concentration in the male pups [34, 49, 57, 58]. On the other hand, the effects of antiandrogens on development of the male reproductive system are similar in many ways to those of estrogens [59], and prenatal/neonatal exposures to estrogenic compounds can cause reproductive disorders in male rodents [60–62]. Therefore, it could be argued that the adverse effects of the mixture as observed in the present study might be caused by the antiandrogenic and/or estrogenic compounds present. Moreover, direct toxic effects of the mixture on the male reproductive tract during fetal or pubertal development cannot be ruled out.

Our results show that despite the well-known hypothyroid effects of PCBs [63, 64], the organochlorine mixture in the present study did not create any observable reduction in serum T4 levels throughout development of the male rat (except for the few individuals in the high-dose group). In addition, serum concentrations of FSH, LH, and prolactin were unaltered by treatment, providing further evidence that the altered reproduction of the male pups likely was not via the inhibition of hormones that regulate reproduction.

Sperm motility and morphology are other parameters that can be used to elucidate the toxicity of a substance to the male reproductive tract [65]. In the present study, we did not observe any treatment-related decline in sperm motility at late puberty/early adulthood (PND 60). In contrast, mature adult (PND 90) sperm motility and progressive motility were reduced by the organochlorine treatment. Perhaps testicular dysgenesis accompanied by subtle spermatogenesis problems is at the root of the poor sperm motility. This is supported by our previous observations that sperm motility is reduced in men presenting very high levels of DDE in rural Mexico [66]. Some evidence indicates that high exposure to PCBs may affect sperm motility [67] and sperm counts [68] in humans. Interestingly, in the present study, sperm morphology was affected only in animals of the high-dose group, suggesting a lower sensitivity of this parameter compared to motility.

The histological findings indicate that testicular and epididymal morphology in rat offspring was not altered by treatment with the low organochlorine dose. Rats from the medium-dose group, however, had relatively fewer sperm in testicular and epididymal lumens than controls. This could explain, in part, the reduced epididymides weights observed in rats from this group on PND 90. In contrast, a pronounced alteration of spermatogenesis was observed in testes from rats exposed to the high dose of the mixture: Germ cells in seminiferous tubules from these testes were either degenerated or difficult to recognize at any stage, and tubules as well as epididymal lumens were almost completely deprived of sperm. This could explain the low sperm numbers in these epididymides for motility analysis and their deteriorated morphology. The negative effect of the mixture on spermatogenesis was not accompanied by abnormal testosterone levels in rats from the medium-dose group. This finding might indicate that the mixture acted directly on the testis during its formation [69] in addition to its possible indirect effect through the endocrine regulation of the testis.

Environmentally Relevant Organochlorine Doses

Organochlorines distribute in all lipids of the body, including plasma lipids and milk fat. Our group previously published data for humans indicating that when expressed on a lipid basis, concentrations of organochlorines in maternal blood plasma lipids, breast milk, and umbilical cord-blood plasma lipids are highly intercorrelated and relatively close to each other [70]. Hence, although we did not measure concentrations of organochlorines in milk from the dams in the present study, exposure through lactation clearly occurred. Serum organochlorine levels decreased dramatically over time in response to the pups' rapidly increasing body weight and body fat mass, where the compounds are mainly stored in the body (increased volume of distribution). Between PND 21 and PND 90, body weight increased approximately sevenfold (Table 6).

The lowest dose of the mixture resulted in mean plasma organochlorine concentrations in the dam rats and their male offspring that were either similar to (in the case of p,p'-DDT) or substantially lower than the highest concentrations recently reported in Arctic populations [71]. Specifically, concentrations as high as 60 µg/L of PCBs (expressed as Aroclor 1260), 21 µg/L of total PCBs (sum of the same 14 congeners measured in the present study), 3 µg/L of p,p'-DDT, 34 µg/L of p,p'-DDE, 0.8 µg/L of mirex, 6 µg/L of oxychlordane, and 4.5 µg/L hexachlorobenzene were detected in maternal plasma of women giving birth in Arctic Canada. Even higher concentrations were reported in the Inuit from Greenland [72]. These concentrations are comparable to those noted in the present study for dams in the medium-dose group. However, plasma organochlorine concentrations in dams from the high-dose group were 4- to 77-fold higher than those measured in Inuit women. In addition, concentrations as high as 47 µg/ L of PCBs (expressed as Aroclor 1260) were detected in plasma samples from Montrealers of Asian origin [73]. These observations indicate that the mixture and dosing protocol used in the present study generated plasma organochlorine concentrations comparable to those observed in populations experiencing unusually high exposure to organochlorines through the food chain. Our results are therefore relevant to those populations.

An Apparently Net Antiandrogenic Effect of the Mixture on F1 Male Rats

These data do not uncover specific mechanistic pathways. However, based on previous studies in the literature, we speculate that the observed phenotype of the F1 rats largely is induced by antiandrogenic effects of the mixture. It has been proposed that "testicular dysgenesis syndrome," which includes low sperm counts and other abnormalities of the male reproductive tract, is induced by endocrine disruption during fetal development [74]. Although the organochlorine mixture contains approximately one-third PCBs, which are considered to be estrogenic, in utero exposure to low levels of estrogenic contaminants typically has not induced remarkable effects on male reproductive development in an experimental setting [75–77]. On the other hand, some PCBs, such as Aroclor 1254 (39% of total PCBs in our mixture), also have been reported to be antiandrogenic [39]. An increasing body of literature shows that antiandrogens, such as p,p'-DDE [78], which makes up 20% of this mixture, may play a more significant role by reducing fetal testosterone production, on which normal testis formation depends [79]. Transient embryonic exposure to vinclozolin leads to germ cell apoptosis and reduced sperm motility in pubertal and adult rats without a decrease in testicular weights [69], not unlike our present observations. Although we did not measure fetal testosterone levels, in utero exposure to antiandrogens do not necessarily lead to altered testosterone in adults [34, 49, 57, 58], so it seems to be plausible that the principal mechanism of the organonochlorines is antiandrogenic. Future research should test this hypothesis directly. In conclusion, our data show that in utero and lactational exposure to a mixture of organochlorines retards reproductive development and function in male rats, supporting the hypothesis that certain environmental contaminants can harm reproductive biology.


   ACKNOWLEDGMENTS
 
The technical assistance of the staff at the Université Laval's Hôtel Dieu animal facility is greatly appreciated. We also thank M. Beaulieu, J.S. Cadieux, S.C. Campagna, C. Dubé, A. Morrier, and S. Peris for help with the dissections and R. Prince for transporting the animals.


   FOOTNOTES
 
1 Supported by the Northern Contaminants Program of Indian and Northern Affairs Canada. Back

2 Correspondance: Janice L Bailey, Centre de Recherche en Biologie de la Reproduction, Départment des Sciences Animales, Pavillon Paul-Comtois, Université Laval, Sainte-Foy, PQ G1K 7P4, Canada. FAX: 418 656 3766; janice.bailey{at}crbr.ulaval.ca Back

Received: 23 November 2004.

First decision: 14 January 2005.

Accepted: 27 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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