Pattern of Male Reproductive System Effects After in Utero and Lactational 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Exposure in Three Differentially TCDD-Sensitive Rat Lines

Ulla Simanainen*,{dagger},1, Tapio Haavisto{ddagger}, Jouni T. Tuomisto*, Jorma Paranko{ddagger}, Jorma Toppari§, Jouko Tuomisto*,{dagger}, Richard E. Peterson||,||| and Matti Viluksela*

* National Public Health Institute, Department of Environmental Health, Kuopio, Finland; {dagger} University of Kuopio, Kuopio, Finland; {ddagger} Department of Biology, Laboratory of Animal Physiology, § Department of Physiology, and Department of Pediatrics, University of Turku, Turku, Finland; and || School of Pharmacy and ||| Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53705

Received February 6, 2004; accepted March 31, 2004

ABSTRACT

Male reproductive effects induced by in utero and lactational exposure to TCDD were analyzed in three rat lines that are differently sensitive to TCDD. Rats from lines A, B, and C were selectively bred from TCDD-resistant Han/Wistar (Kuopio, H/W) and TCDD-sensitive Long-Evans (Turku/AB, L-E) rats and exhibited very different LD50 values for TCDD: >10,000, 830, and 40 µg/kg in males, respectively. The resistance in line A rats was linked to a mutated H/W-type aryl hydrocarbon receptor (Ahrhw) and in line B rats to a H/W-type unknown allele B (Bhw). Line C rats had no resistance alleles. Influence of the resistance alleles on developmentally induced male reproductive effects of TCDD was studied by exposing pregnant females to TCDD (0.03, 0.1, 0.3, or 1 µg/kg) on gestation day (GD) 15. Male progeny were sacrificed on postnatal day (PND) 70. Next, the dams were given 1 µg/kg TCDD on GD 15 and male progeny were sacrificed on PND 14, 21, 28, 35, or 49. Serum testosterone concentration, male sex organ weights, and testicular and cauda epididymal sperm numbers were analyzed; the most sensitive end point was decreased sperm numbers. The dose of 1 µg/kg TCDD reduced daily sperm production by 9.3, 25, and 36%, and cauda epididymal sperm reserves by 18, 42, and 49% in rat lines A, B, and C when measured on PND 70, respectively. The most consistent and significant effect was decreased weight of prostate lobes. The growth of the male reproductive organs was not markedly affected by the resistance alleles Ahrhw and Bhw. In contrast, the effects on sperm parameters appeared to be slightly modified by the resistance alleles. Thus, the intraspecies genetic differences in C-terminal transactivation domain of AHR appear to modify the sensitivity to only certain dioxin-induced male reproductive effects.

Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; dioxin; in utero and lactational exposure; male reproductive tract; strain differences; rat; prostate; sperm.

The endocrine system presents a number of target sites for the induction of adverse effects by the ubiquitous environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Numerous studies have demonstrated that male reproductive and developmental processes are sensitive to perinatal low-dose TCDD exposure, resulting in reduced sperm number and reduced size of reproductive organs (reviewed by Peterson et al., 1993Go; Theobald et al., 2003Go). These low-dose effects are essential in terms of risk assessment of TCDD and similar types of compounds. Sharpe and Skakkebaek (1993)Go hypothesized that in utero exposure to environmental endocrine disrupters such as TCDD may, at least in part, be responsible for decreased human sperm counts and other male reproductive tract disorders. However, epidemiological evidence is not yet available to support this hypothesis.

Various dose-dependent effects on male offspring androgenic status and growth of androgen-dependent organs have been reported in different strains of pregnant female rats administered with TCDD (Faqi et al., 1998Go; Gray et al., 1997aGo; Haavisto et al., 2001Go; Mably et al., 1992aGo,bGo,cGo; Ohsako et al., 2001Go, 2002Go; Wilker et al., 1996Go). The range of defects and the TCDD sensitivity has varied among the studies, even within the same rat strain. Mably et al., (1992a)Go and Faqi et al. (1998)Go reported that daily sperm production was highly susceptible to perinatal TCDD exposure in Holtzman and Wistar rats. However, the effect was not reproducible in Sprague-Dawley and Long-Evans rats (Gray et al., 1997aGo; Wilker et al., 1996Go), or in another study with Holtzman rats (Ohsako et al., 2001Go). Decreased sex accessory organ weights were observed in most studies, though the magnitude of the defects varied among studies. The most sensitive, persistent, and reproducible effects were seen with sperm numbers, especially the decrease in cauda epididymal and ejaculated sperm numbers.

In our laboratory, two rat strains with a great sensitivity difference to TCDD-induced acute lethality have been used (Pohjanvirta and Tuomisto, 1994Go). In Han/Wistar (Kuopio, H/W) rats, the LD50 for TCDD is over 10,000 µg/kg, whereas for Long-Evans (Turku/AB, L-E) rats it is about 10 µg/kg. The TCDD resistance of H/W rats is due to a mutated Ahr allele (Ahrhw) and to the currently unknown Bhw allele (Pohjanvirta et al., 1998Go; Tuomisto et al., 1999Go).

The Ahrhw allele has been shown to harbor a point mutation that results in an abnormal C-terminus transactivation domain and a smaller AHR protein in H/W than in L-E rats (~98 kDa vs. 106 kDa). L-E rats have clearly higher total hepatic levels of AHR than H/W rats (Franc et al., 2001Go; Pohjanvirta et al., 1998Go, 1999Go). However, no differences between the strains were found in TCDD affinity to cytosolic AHR, in the ability of their AHRs to be transformed into the DNA-binding form by TCDD, or in the specific binding of the activated AHR to DNA (Pohjanvirta et al., 1999Go). Also, the upregulation of the AHR by TCDD was similar in H/W and L-E rats (Franc et al., 2001Go).

The identity of the B allele has not yet been determined, but it may encode a protein participating in the AHR signaling pathway. Recently, it was shown that at least the differences in relative expression of the AHR dimerization partner ARNT or the AHR repressor (AHRR) do not contribute to the dioxin sensitivity differences between the H/W and L-E rats (Korkalainen et al., 2003Go, 2004Go).

Two H/W-type, TCDD resistance genes (Pohjanvirta, 1990Go), the altered Ahrhw and another unknown allele Bhw, were segregated into two new rat lines designated A and B (Tuomisto et al., 1999Go). Line A has the resistance allele Ahrhw and line B has the resistance allele Bhw; rat line C has only wild-type alleles Ahrwt and Bwt. Lines A, B, and C exhibit very different LD50 values for TCDD: >10,000, 830, and 40 µg/kg in males, respectively.

The typical end points of dioxin toxicity can be classified based on the modification of TCDD effects by the resistance alleles Ahrhw and Bhw (Simanainen et al., 2002Go, 2003Go). The type I end points (e.g., increased EROD activity, decreased thymus weight, and tooth defect) are independent of genotype variation. However, for type II end points (e.g., weight loss, liver toxicity, and increased serum bilirubin), the efficacy (magnitude of effect) of TCDD is suppressed by the resistance alleles. The Ahrhw allele is the most important resistance factor.

In contrast to other rat strains studied (Haavisto et al., 2001Go; Mably et al., 1992cGo; Roman et al., 1995Go), maternal TCDD exposure on gestational day (GD) 13 stimulated testicular testosterone synthesis and increased circulating testosterone concentrations in H/W fetuses (GD 19.5; Haavisto et al., 2001Go). Therefore, it was hypothesized that the mutated H/W-type AHR could modify the in utero TCDD effects on male reproductive end points.

The majority of single nucleotide polymorphisms found in human Ahr are located in exon 10, which covers the major portion of the C-terminal transactivation domain (Cauchi et al., 2001Go; Harper et al., 2002Go; Kawajiri et al., 1995Go; Smart and Daly, 2000Go; Wong et al., 2001Go). Interestingly, one study showed that a combination of two or three of single nucleotide polymorphisms (SNPs) found in the C-terminal end of the human Ahr failed to induce TCDD-dependent CYP1A1 expression in vitro (Wong et al., 2001Go). Therefore, the sensitivity differences based on the mutated Ahr could have relevance for human risk assessment, although there is currently no evidence for the clinical relevance of the Ahr polymorphism in humans.

The aim of the present study was to determine whether the sensitivity difference to TCDD acute lethality among the rat lines A, B, and C correlates with the magnitude of the developmental male rat reproductive tract changes induced by perinatal TCDD exposure. Accordingly, the dose response and time course of in utero and lactational TCDD exposure effects on male reproductive organ weights, serum testosterone concentrations, cauda epididymal sperm number, and daily sperm production were determined in rat lines A, B, and C.

MATERIALS AND METHODS

Dose-response study. Line A, line B, and line C rats were bred in the SPF (specific pathogen–free) barrier unit of the National Public Health Institute (Kuopio, Finland). Adult (12–15 weeks old) female rats in estrus were mated with untreated males from 9:00 to 12:00 P.M. and vaginal smears were collected and examined for the presence of sperm. The day after copulation was considered GD 0.

Pregnant females were housed singly in plastic cages that had wire-mesh covers in a room with a 12 h light/dark cycle (lighted from 0700 to 1900 h). Aspen chips (Tapvei Co., Kaavi, Finland) were used as bedding and nesting material. The temperature in the animal room was 21 ± 1°C and the relative humidity was 50 ± 10%. Rats were maintained on standard pelleted laboratory animal feed (R36, Ewos, Södertälje, Sweden) and tap water ad libitum.

In the morning on GD 15, graded single doses of TCDD (0.03, 0.1, 0.3, or 1 µg/kg) or an equivalent volume of vehicle (corn oil, 4 ml/kg) were given by gavage to pregnant rats. For each dose, 5–8 pregnant dams were used. TCDD was purchased from the UFA-Oil Institute (Ufa, Russia) and it was over 99% pure as confirmed by gas chromatography–mass spectrometry.

The day of birth was considered postnatal day (PND) 0. One day after birth, the number of live offspring and the gender ratio were recorded. When possible, by random termination of excess offspring, litters were adjusted to three males and three females to allow uniform postnatal exposure. In the rat lines used, the average number of pups per litter is only 6 ± 3 (mean ± SD). To keep the litters as homogenous as possible and without greatly increasing the number of pregnant dams needed, only litters with less than four pups were excluded. Offspring were weaned on PND 28, terminating the lactational TCDD exposure. Dams were killed and the number of uterine implantation sites was counted after staining the uteri with ammonium sulfide solution (10%, v/v). For each litter, survival from implantation to the day after birth was calculated by the following formula:

(1)
where a is the total number of implantation sites and b is the number of pups on PND 1. After weaning, the pups were housed with the same-sex littermates in plastic cages with wire-mesh covers.

Dam and pup viabilities were monitored throughout the study. Body weight of the offspring was determined on PNDs 1, 4, 7, 14, and 28. Anogenital distance (AGD) and crown-rump length (CRL) were measured on PNDs 1 and 4 by using a vernier caliber capable of resolution to 0.1 mm.

At the age of 70 days, the pups were decapitated and trunk blood was collected. For testosterone analysis, sera were stored at –80°C. Testis, cauda of the right epididymis, ventral prostate, seminal vesicles, and thymus were dissected and weighed. Seminal vesicles were weighed without fluid and coagulating glands. The organ weights were reported as both absolute and relative weights (organ weight/body weight). Cauda epididymis and testis were frozen for the analysis of sperm counts.

Time-course experiment. Pregnant rats were dosed by gavage with a single dose of 1 µg/kg TCDD or an equivalent volume of corn oil vehicle (4 ml/kg) on GD 15. One or two male pups from each litter were terminated at PND 14, 21, 28, 35, or 49. There were 4–8 pregnant dams per group and 7 ± 2 (mean ± SD) male pups were examined at each time point.

On PND 1, the number of live offspring and gender ratio were recorded, and the pups were examined for gross malformations. When possible, litters were adjusted to four males and two females. Body weights of offspring were determined weekly from PND 1 until PND 49.

At termination, the male pups were decapitated and trunk blood was collected. Separated sera were stored at –80°C. Testis, right cauda epididymis, ventral prostate, dorsolateral prostate, anterior prostate, and seminal vesicles were dissected and weighed. Fluid was not removed from the seminal vesicles. Right cauda epididymis and testis were frozen for sperm count analysis. Also, for male pups terminated on PND 35, the lungs, heart, kidneys, liver, salivary glands, and spleen were dissected and weighed.

Serum testosterone. Serum testosterone concentrations were analyzed from all samples in the time-course experiment and from the control and highest TCDD dose (1 µg/kg) samples in the dose-response study. Testosterone was measured from diethyl ether–extracted sera by time-resolved fluoroimmunoassay DELFIA (Perkin Elmer Life and Analytical Sciences, Wallac Oy, Turku, Finland). After extraction, samples were reconstituted to 100 µl of Dilution II buffer (Perkin Elmer Life and Analytical Sciences) from which 25 µl/well were taken for analysis. For enhancing the sensitivity of the assay, commercial tracer and antisera were also diluted 5:8, giving a sensitivity of 0.04 ng/ml when the limit of detection was defined as the value of two standard deviations above the mean of the zero standard measurement value. Intra- and interassay variations were below 6 and 12%, respectively, at the testosterone concentration of 1.0 ng/ml.

Daily sperm production and cauda epididymal sperm counts. For sperm counts, frozen testis and cauda epididymis were homogenized for 2 min using Ultra Turrax homogenizer (model T25 basic, IKA-WERKE GmbH & Co., Staufen Germany) in 50 ml (testis) or 20 ml (cauda epididymis) 0.9% saline, containing 0.05% Triton X-100 and 0.01% thimerosal. Homogenates were diluted to approximately 1 x 106 sperm/ml, and counts from four hemocytometer chambers were counted and averaged (Blazak et al., 1993Go).

Statistical analysis. Statistical analysis was performed using SPSS 10.0 statistical software (SPSS Inc., Chicago, IL). Litter means were used and data were analyzed by one-way analysis of variance (ANOVA). If the test showed a significant difference, the least significant difference test was used as a post hoc test. In case of nonhomogenous variances (according to Bartlett's test, p < 0.01), the nonparametric Kruskal-Wallis ANOVA was used followed by the Mann-Whitney U-test. P values less than 0.05 were considered significant.

RESULTS

Maternal Toxicity and Pup Survival
TCDD did not produce any deaths or overt toxicity to the dams as assessed by body weight gain and visual inspection (data not shown). Also, pup survival from implantation to the day after birth was not affected by TCDD. However, survival from implantation to the day after birth was surprisingly low in control line B rats in the dose-response study (41%), resulting in a significant difference compared with the two lowest doses (0.03 and 0.1 µg/kg TCDD). The average survival was 85% (range 80–89%), 64% (41–86%), and 74% (63–85%) in control lines A, B, and C rats, respectively. Corresponding values after TCDD exposure were (without clear dose-response) 83% (70–92%), 60% (28–85%), and 77% (61–86%), respectively.

For each line, the percentage of male pups that survived between PND 1 and weaning on PND 28 was 99%. The only exceptions were line B males exposed to 0.03 µg/kg TCDD and line C males exposed to 0.03 or 1 µg/kg TCDD, where the male survival rate was on average 81% (range 81–83%).

Nonreproductive Organ Weights on PND 35
The relative weights of liver, spleen, heart, lung, and kidneys tended to be increased in lines B and C male offspring. However, the increase was significant only for relative liver weight in lines A and C males and for relative lung weight in line C males (Table 1).


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TABLE 1 The Relationship of Rat Line to Absolute Organ Weights and to the Effects of a Maternal Dose of TCDD (1 µg/kg on GD 15) on Relative Organ Weights of Male Offspring at 35 Days of Age

 
Growth of the Male Offspring
The dose-response study showed that, on PND 70, significant reduction in body weight was observed only in lines B and C rats at the highest dose of 1 µg/kg TCDD (Table 2). At younger ages in the time-course study, the same dose significantly reduced body growth in all lines and at most time points measured (Table 2). Exceptionally, in 1-day-old line A males exposed to 0.03 µg/kg TCDD, body weight was increased compared to control rats (data not shown).


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TABLE 2 The Effects of GD 15 TCDD Exposure on Body Weight and Organ Weight in Male Progeny Necropsied at Different Ages

 
Anogenital Distance
AGD was measured on PNDs 1 and 4 (Table 3). On PND 1, the highest maternal dose of 1 µg/kg TCDD significantly decreased the absolute AGD only in line C rats. On PND 4, the AGD of TCDD-exposed rats did not differ from the control rats in any group. Also, when AGD was related to the cube root of pup body weight [AGD/(body weight)1/3] (Gallavan et al., 1999Go), there was no detectable effect between the TCDD-exposed and control rats at either time point.


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TABLE 3 The Effects of GD 15 TCDD (1 µg/kg) Exposure on Body Weight and AGD in Male Progeny Necropsied at PND 1 or 4

 
Serum Testosterone
Serum testosterone levels in lines A, B, and C male rats were low, below 1 ng/ml, and were not significantly affected by TCDD (data not shown). A pubertal increase in testosterone levels of lines A and B control rats was detected on day 49. In line C male rats, serum testosterone levels in the control rats and those exposed to TCDD remained low until day 49 with an increase present on day 70 (testosterone measured only in the 1 µg/kg TCDD group).

Accessory Sex Organ Weights
Until day 49, the maternal dose of 1 µg/kg TCDD decreased both absolute and relative weights of the ventral, anterior, and dorsolateral prostate in lines A, B, and C rats at most time points measured. The change was most consistent and significant in the ventral lobe (Table 2). In the group exposed to 1 µg/kg TCDD, the average decrease in absolute weight of anterior prostate was 37% (range 30–55%), 32% (18–42%), and 34% (19–45%) in lines A, B, and C rats, respectively. Similarly the average dorsolateral prostate weight was decreased by 34% (range 29–37%), 28% (16–33%), and 39% (22–59%), in lines A, B, and C rats, respectively. The effect on ventral prostate was not permanent, as the only significant decrease in weight remaining at the age of 70 days was observed in line B rats at the maternal dose of 1 µg/kg TCDD (Table 3). TCDD did not have any consistent effects on the weight of seminal vesicles (data not shown).

Testis and Epididymis Weights
The absolute weights of testis and epididymis are shown in Table 2. Except for a significant increase on PNDs 28–49, the relative weights of testis, epididymis, and cauda epididymis remained unchanged. In some rats, a maternal dose of 1 µg/kg TCDD caused severe epididymal malformations including small caput and cauda as well as a degeneration of corpus epididymis. Malformed epididymis was observed in 6 of 44 line C male rat offspring (6 of 17 litters affected) and 3 of 47 line A male rat offspring (3 of 15 litters affected).

Daily Sperm Production and Cauda Epididymal Sperm Counts
In the dose-response study, the highest TCDD dose (1 µg/kg) reduced daily sperm production by 9, 25, and 36%, and cauda epididymal sperm reserves by 18, 42, and 49% in lines A, B, and C rats, respectively, when measured on PND 70 (Fig. 1). In the time-course study, the daily sperm production measured in 49-day-old rats had decreased after TCDD exposure by 35, 17, and 32% in lines A, B, and C rats, respectively; the decrease was significant only in line A rats (data not shown).



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FIG. 1. The effect of in utero and lactational exposure to TCDD on daily sperm production (DSP) and cauda epididymal sperm reserve (mean ± SE) at the age of 70 days in lines A, B, and C rats (n = 4–7 litters/treatment). Means significantly (p < 0.05) different from corresponding controls are shown with solid symbols.

 
DISCUSSION

Sensitivity Differences Among the Rat Lines
The time-course study indicated that, until about 49 days of age, the male reproductive system of the three rat lines was similarly affected by in utero and lactational TCDD exposure. However, the dose-response study suggested that, compared to lines B and C rats, the line A rats were somewhat more resistant to certain end points of male reproductive toxicity. At the age of 70 days, sperm parameters (daily sperm production and cauda epididymal sperm reserve) were significantly decreased only in lines B and C rats. Also, the increases in relative weights of the heart, lung, and spleen were more pronounced in line C than in line A rats. Therefore, it appears that sensitivity of the developing male accessory sex organs to in utero and lactational TCDD exposure is not modified by the resistance alleles Ahrhw and Bhw. However, the intraspecies genetic differences in C-terminal transactivation domain of AHR appear to play a role in the sensitivity to TCDD-induced decreases in sperm parameters and in the development of certain nonreproductive organs.

In a recent study, we were able to show that newborn line A rats are resistant to TCDD acute lethality and that the full resistance develops during the first weeks of postnatal life (Simanainen et al., 2004Go). Also, the nonlethal biochemical and toxic short-term effects caused by TCDD exposure in adulthood could be categorized into two types according to the modification by the resistance alleles (Simanainen et al., 2002Go, 2003Go; Tuomisto et al., 1999Go). The efficacy ratio of 0.5 between Ahrhw and Ahrwt genotypes was used as a practical classification criterion between type I and type II end points (Simanainen et al., 2003Go). In the present study, the TCDD effect on selected nonreproductive organ weights and sperm parameters appeared to fulfill these criteria of type II end points with two-fold lower efficacy in line A than in line C rats. In contrast, the reduction in prostate weight is a type I end point with no sensitivity differences among the rat lines at any age.

The deletion mutation of Ahrhw is located within the transactivation domain, leaving the domains responsible for ligand and DNA binding as well as heterodimerization intact (Pohjanvirta et al., 1998Go). Therefore, the activation of transcription seems to be the critical step where the wild-type and H/W-type receptors act differently. This may also be important for the dioxin risk assessment of humans, since most polymorphisms in the human AHR are located in the transactivation domain (Harper et al., 2002Go). In theory, a mutation in human AHR C-terminal end could result in decreased sensitivity to dioxin-induced male reproductive effects, as observed in rats with H/W-type mutated AHR.

Androgenic Status
The lack of effect of TCDD on serum testosterone levels in this study is consistent with previous studies (Cooke et al., 1998Go; Faqi et al., 1998Go; Gray et al., 1995Go; Loeffler and Peterson, 1999Go; Roman et al., 1995Go), where a tendency of TCDD to decrease or have no effect on serum testosterone levels was reported in young adult rats. In our TCDD-exposed male rats, testosterone levels were most severely, though not significantly, depressed on day 49. Similar depression was also reported by Roman and coworkers (1995)Go and is probably associated with the delayed onset of puberty. After 49 days of age, the testosterone levels of TCDD-exposed male rats approached the control levels. The AGD, which is known to be an androgen-sensitive parameter in neonatal rats (Neumann et al., 1970Go), was not affected by the TCDD exposure.

Accessory Sex Organs
In the present study, no clear effect was seen on relative seminal vesicle weight. Therefore, the seminal vesicle is not as sensitive of a target as the prostate to in utero and lactational TCDD exposure. Relative organ weights were considered a better indicator than absolute organ weights, because the highest TCDD dose (1 µg/kg) significantly decreased body weight at most time points. The most reproducible effect of in utero and lactational TCDD exposure was the decrease in prostate weight. Prostate weight was decreased at the highest dose (1 µg/kg TCDD, lower doses were analyzed only on PND 70), but the effect was not permanent. Similarly, in Long-Evans hooded (Gray et al., 1997bGo), Sprague-Dawley (Wilker et al., 1996Go), and Wistar rats (Faqi et al., 1998Go), a significant inhibitory effect on prostate development was seen after a single maternal TCDD dose of 0.8, 1, or a total dose of 0.72 µg/kg, respectively. Notably, in Holtzman rats exposed in utero and via lactation to TCDD, the weight of the ventral prostate and seminal vesicles were significantly reduced after a maternal TCDD dose as low as 0.2 µg/kg, until the offspring were 120 days of age (Mably et al., 1992cGo; Ohsako et al., 2001Go). These studies imply that, although the resistance alleles studied here did not alter the sensitivity of sex accessory organ development to the inhibitory effects of TCDD, there are other strain differences in TCDD sensitivity and persistence of prostate defects, with Holtzman rats being the most sensitive.

Sperm Parameters
The decrease in cauda epididymal sperm counts in particular is among the most sensitive end points of the male rat reproductive pathogenesis after perinatal TCDD exposure (Gray et al., 1995Go, 1997bGo; Mably et al., 1992aGo; Theobald and Peterson, 1997Go). Inhibitory effects of in utero and lactational TCDD exposure on daily sperm production are less consistent. The present study supports the finding of Ohsako and coworkers (2001)Go that maternal exposure to a relatively low dose of TCDD (less than 1 µg/kg) has a minimal or no effect on testicular development and spermatogenesis. Similarly, the epididymal sperm reserves were decreased only at the highest maternal doses of TCDD. The effect on sperm parameters was observed on PND 70, which indicates that the effects on sperm numbers are more persistent than those observed in prostate development.

Testis and Epididymis Weights
In accordance with previous studies with Wistar and Holtzman rats (Faqi et al., 1998Go; Ohsako et al., 2001Go), the relative weights of testis and epididymis were not affected by in utero and lactational TCDD exposure. There was a slight correlation between the absolute weight of epididymis and cauda epididymal sperm number and a noticeably weaker correlation or no correlation between absolute weight of testis and daily sperm production. However, these correlations were not strong enough to explain the sensitivity differences among the rat lines. Line C rats remained most susceptible to decreased cauda epididymal sperm number even when the sperm number was related to the epididymis weight.

The developmental abnormalities in the corpus epididymis that we observed were similar to those reported previously in Sprague-Dawley rats (Ohsako et al., 2002Go; Wilker et al., 1996Go). Wilker and coworkers (1996)Go reported partial to complete agenesis of the corpus epididymis in approximately a quarter of the rats after a maternal dose of 2 µg/kg TCDD on GD 15. In the present study, these malformations were observed at 1 µg/kg TCDD and mainly in the sensitive line C rats (14%). This implies that line C rats may be more sensitive to this effect compared to lines A and B rats.

In conclusion, despite the great influence of resistance alleles Ahrhw and Bhw on TCDD-induced acute lethality, the data generated in this study show that these H/W-type alleles (compared with wild-type alleles) do not modify the TCDD effect on male accessory reproductive organ development after in utero and lactational TCDD exposure. However, the alleles may modify TCDD effects on sperm parameters and on the development of some nonreproductive organs, such as thymus, spleen, heart, and lung. Differences in developmental sensitivity to TCDD apparently are less profound compared to acute lethality and some other short-term exposure end points. This study confirmed that the effects on prostate development and sperm numbers, especially on the epididymal sperm counts, are sensitive and consistent signs of in utero and lactational TCDD exposure.

ACKNOWLEDGMENTS

We are grateful to Ms. Arja Tamminen and Ms. Jaana Jääskö for their excellent technical assistance. This study was financially supported by the Academy of Finland (Grant 200980 and Project 42551 of the Finnish Research Program on Environmental Health), the European Commission (Contracts QLK4-CT-1999-01446 and QLK4-CT-2001-00269), NIH grant ES01332 (to R. E. P.), the Graduate School in Environmental Health, and the Jenny and Antti Wihuri Foundation.

NOTES

1 To whom correspondence should be addressed at P.O. Box 95, FIN-70701 Kuopio, Finland. Fax: +358 17-201-265. E-mail: ulla.simanainen{at}ktl.fi.

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