* Environmental Health Sciences Division, National Institute for Environmental Studies, 162 Onogawa, Tsukuba, Ibaraki 305-0053, Japan;
CREST-JST, Kawaguchi, Saitama 332-0012, Japan;
Department of Hygienic Chemistry, Science University of Tokyo, 12 Ichigaya-Funagawara-machi, Shinjuku-ku, Tokyo 162-0826, Japan; and
§ Regional Environment Division, National Institute for Environmental Studies, 162 Onogawa, Tsukuba, Ibaraki 305-0053, Japan
Received September 7, 2000; accepted December 6, 2000
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ABSTRACT |
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Key Words: TCDD; male reproduction; ventral prostate; androgen receptor; 5-alpha-reductase.
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INTRODUCTION |
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Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes irreversible changes in the reproductive systems of offspring. The lowest-observed-effect level (LOEL) of maternal TCDD exposure is much lower than those estimated in the experiments using adult animals (Chahoud et al., 1989; McConnell et al., 1978
; Tofilon and Piper, 1982
). Therefore, the effects of TCDD on human endocrine functions during the developmental stage are a highly important issue in terms of human health risk assessment (Birnbaum, 1994
; Peterson et al., 1993
). Many researchers have reported that in utero and lactational exposure to TCDD results in adverse effects on the male reproductive system, i.e., reduced sperm count (Faqi et al., 1998
; Gray et al., 1995
, 1997
; Mably et al., 1992c
; Sommer et al., 1996
; Wilker et al., 1996
); reduced size of reproductive organs (Bjerke and Peterson, 1994
; Gray et al., 1997
; Mably et al., 1992a
; Roman et al., 1995
, 1998a, Roman et al., b
), and feminized behavior or brain (Bjerke et al., 1994a
; Faqi et al., 1998
; Mably et al., 1992b
).
Peterson and coworkers reported a series of extensive, carefully designed studies with male Holtzman rat offspring born from mothers that were administered a single oral dose of TCDD (0, 64, 160, 400, or 1000 ng/kg bw) on gestational day (GD) 15 (Mably et al., 1992a, b
, c
). Statistically significant reductions in the weight of the testes and epididymis, and in daily sperm production (DSP) and epididymal sperm number were detected by administration of as low a dose as 64 ng TCDD/kg bw. Some of the animals showed reduced fertility at higher doses (400 and 1000 ng TCDD/kg bw), suggesting that maternal TCDD exposure induces defects in sperm production and causes male infertility. Gray and colleagues (Gray et al., 1997
) also reported that maternal TCDD exposure (50, 200, or 800 ng TCDD/kg bw) on GD 15 induced changes in the reproductive system of male Long Evans (LE) rats. Reductions in the epididymal and ejaculated sperm number occurred in the same manner as in Peterson's study. In the study by Gray et al.(1997), however, neither testicular weight nor DSP was affected at any of the doses used, and fertility was found to be normal. In a study using Sprague-Dawley rats, another research group reported that there was no significant change in DSP by a single injection of TCDD on GD 15, even at the highest dose (2 µg/kg bw) (Wilker et al., 1996
). In addition, Chahoud and coworkers used a different protocol in which mothers were given an initial loading dose at 2 weeks prior to mating (25, 60, or 300 ng TCDD/kg bw) followed by a weekly maintenance dose (5, 12, or 60 ng TCDD/kg bw). No effects were observed on testicular weights of male offspring; however, DSP was significantly reduced at all doses tested (Faqi et al., 1998
). Taking these results together, it is difficult to draw a conclusion concerning the consistent effects of TCDD on testes and sperm production.
Another potentially sensitive index of TCDD exposure is the ejaculated sperm count (Gray et al., 1995, 1997
), in conjunction with the reduction in size of sex-accessory glands and reduction in anogenital distance (Gray et al., 1997
; Mably et al., 1992a
; Roman et al., 1995
, 1998a, Roman et al., b
). However, the ejaculated sperm number is a less reliable index than testicular weight, DSP, or the weight of other reproductive organs, because the protocol for determining the ejaculated sperm count is highly complex and difficult to carry out.
In the present paper, we used Holtzman rats, which can be classified as highly responsive to TCDD in terms of CYP1A1 induction (Jana et al., 1998). The aims of our experiment were to investigate the effects of a low dose of TCDD on the male reproductive system, particularly with respect to decreases in testicular weight or DSP, and to determine the most sensitive and reliable indexes for risk assessment. Anogenital distance and ventral prostate weight were revealed to be the most sensitive indexes in our experiment. Moreover it was demonstrated by semiquantitative RT-PCR with total RNA in the ventral prostate from pubertal rats, that content of androgen receptor (AR) transcript was highly down-regulated and 5
-reductase type 2 (5
R-II) was upregulated by maternal TCDD administration in a dose-dependent manner.
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MATERIALS AND METHODS |
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Animals.
Male and female Holtzman rats were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and bred in our own facility. They were maintained in a controlled environment with temperature at 24 ± 1°C, humidity at 45 ± 5%, and a 12/12-h light/dark cycle. They were given food and distilled water ad libitum. Ten-week-old female rats in proestrus were mated 1:1 with males overnight, and females that had a vaginal plug the following morning were designated as being at Day 0 of gestation. Dams were housed individually in clear plastic cages with heat-treated wood chips as bedding.
TCDD administration.
The treatment and necropsies described below were all performed in the hazardous chemical regulation area at our institute. TCDD was dissolved first in nonane, followed by further dilution in corn oil to make a TCDD concentration of 20 µg/ml. On GD 15, pregnant rats were given a single dose (po) of TCDD (12.5, 50, 200 or 800 ng/kg; 2.5 ml/kg) or an equivalent volume of vehicle (6 pregnant rats per each dose). On PND 2, litters were randomly culled to 8 pups, with 5 males and 3 females, when possible. After weaning, the 5 males of each litter were housed in a separate stainless steel cage. Two male offspring per litter were sacrificed under diethylether anesthesia on PNDs 49 and 120.
Sample collection and processing.
The length between the base of the genital tubercle and the anterior edge of the anus was measured with a caliper as the anogenital distance (AGD). The testis and epididymis of both sides were excised from the abdomen and the surrounding adipose tissue was carefully removed. After removing the urine from the bladder, the deferent ducts were cut at the base of the bladder and the anterior end of the urethra was cut to excise the urogenital complex, which was then measured, followed by dissection and re-weighing of the seminal vesicle and ventral prostate. The tissue-weight data were expressed as a percentage of the body weight of each animal. All tissue samples were frozen by liquid nitrogen immediately after dissection and kept at 80°C until measurement of daily sperm production and sperm reserve, or RNA extraction.
Daily sperm production and epididymal sperm reserve.
The testes and cauda epididymis were homogenized in phosphate-buffered saline by a polytoron homogenizer. Homogenization-resistant spermatid nuclei were counted by a hemocytometer. The numbers of homogenization-resistant spermatid nuclei per testis were calculated and then divided by 6.1 days to convert them to testicular daily sperm production (DSP) (Robb et al., 1978). The numbers of homogenization-resistant sperm head per cauda epididymis were defined as the cauda epididymal sperm reserve (SR).
Histopathology.
Testes were fixed with Bouin's fixative and embedded in paraffin. Sections (5-µm thickness) were deparaffinized and stained with hematoxylin and eosin. The stage of the rat seminiferous epithelium was evaluated by using the STAGES 2.2 software developed by Dr Rex A. Hess of the University of Illinois (Urbana, IL).
Gas chromatogragh-mass spectrometry.
The tissue content of TCDD was determined according to the methods described previously (Miyabara et al., 1999). Briefly, a tissue sample (0.11.0 g) was spiked with 20 pg of (13C) 2,3,7,8-TCDD as an internal standard and then digested in 2 mol/l potassium hydroxide solution for 12 h. The TCDD in the digested material was extracted with n-hexane. The solution was applied to a silica gel column that was connected to an activated carbon-silica gel column, and then eluted with toluene. The final eluate was concentrated and the residue was dissolved in n-hexane. The GC-MS analysis was performed in the selected ion mode on a JMS700 high performance double focusing mass spectrometer (JEOL, Japan). The area of mass profile peaks of the quantification ions was used for the quantitative analysis of TCDD.
Hormone assay.
Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels were determined using an enzyme immunoassay (EIA) system (Amersham Life Sciences, Buckinghamshire, UK). Testosterone levels were determined using a radioimmunoassay (RIA) kit (Diagnostic Products Corporation, Los Angeles, CA) according to the manufacturer's instructions.
Semiquantitative RT-PCR.
Total RNA was extracted from ventral prostates and caput epididymes (n = 3) by the protocol of Chomczynski and Sacchi (1987). The RNA samples (4 µg) were reverse-transcribed for 50 min at 42°C in a 20-µl reaction with 200 units of SuperScriptTM II reverse transcriptase and 0.5 µg of oligo(dT)1218 primer by the standard protocol of the supplier. For AR mRNA amplification, 200-µl of a PCR mixture containing 4 µl of the reverse transcriptase reaction, 10 units of TaKaRa LA TaqTM polymerase, 1x GC Buffer I, 0.4 mM of each dNTP mixture, and 0.4 µM of each primer, was equally divided into 4 tubes. The primers were designed to amplify a 630-base pair (bp) fragment for rat androgen receptor (AR) (forward, ATCGAGGAGCGTTCCAGAATCTG; reverse, ATATGGTCGAATTGCCCCCTAGG). PCR was subsequently performed using an optimized protocol consisting of between 16 and 50 cycles. Each cycle consisted of the following: 94°C, 30 s; 63°C, 30 s; 72°C, 1.5 min. For 5R-II and cyclophilin mRNA amplification, 200 µl of PCR mixture containing 4 µl of the reverse transcriptase reaction, 5 units of TaKaRa Ex TaqTM polymerase, 1x Ex TaqTM Buffer, 0.2 mM of each dNTP mixture, and 0.4 µM of each primer, was equally divided into 4 tubes. The primers were designed to amplify a 496-bp cDNA fragment for rat 5
R-II (forward, ATCCTGTGCTTAGGGAAAC; reverse, CATACGTAAACAAGCCACC) and a 524-bp fragment for cyclophilin (forward, TCTGAGCACTGGGGAGAAAG; reverse, AGGGGAATGAGGAAAATATGG). PCR was subsequently performed by several cycles. Cycling parameters were as follows: 94°C, 30 s; 63°C, 30 s; 72°C and 1 min for 5
R-II; and 94°C, 30 s; 55°C, 30 s; and 72°C, 45 s for cyclophilin. The PCR products were separated by 2% agarose gel. The relative amounts of RT-PCR products for AR and 5
R-II were then quantified by standardizing with the PCR products of cyclophilin using Scion Images software (Scion Corporation, Frederick, MD). PCR products for AR were evaluated using 25 cycles for PND 120 ventral prostate, 24 cycles for PND 49 caput epididymis, and 30 cycles for PND 120 ventral prostate. 5
R-II was evaluated using 25 cycles for PND 49 ventral prostate, 19 cycles for PND 49 caput epididymis, and 30 cycles for PND 120 ventral prostate. The PCR product for cyclophilin at 20 cycles was used as an internal standard. Each amplification reaction was performed at least 3 times and confirmed for the reproducibility.
The PCR products for rat AR, 5R-II, and cyclophilin were subcloned into pGEM-T Easy vectors and sequenced by the dideoxynucleotide chain termination method using the ABI Prism BigDye terminator cycle sequencing kit (PE-Biosystems, Foster City, CA).
Statistical analysis.
For statistical analysis, StatView for Windows version 5.0 (SAS Institute, Cary, NC) was used. All results represented are the means ± SE. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test as a post hoc test for comparison of means for tissue weights and AGD. Fisher's PLSD test was used for band intensity in semiquantitative RT-PCR analysis. p Values less than 0.05 were considered to indicate statistical significance.
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RESULTS |
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Ventral prostate on PND 49.
The maternal TCDD administration resulted in a dose-dependent increase in the amounts of 5R-II transcripts in the ventral prostates on PND 49 (Fig. 8
). For the purpose of comparison, relative arbitrary units were calculated by dividing the intensity of the band of the 5
R-II PCR product by the corresponding band intensity of cyclophilin using the same cDNA template. Multiple comparison analysis showed that there were statistically significant differences at doses as low as 200 ng/kg (Fig. 8B
). On the other hand, a clear tendency for TCDD to cause a dose-related decrease in concentrations of AR mRNAs was observed. Statistically significant differences were detected at doses of more than 12.5 ng/kg by measuring the band intensity.
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DISCUSSION |
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Maternal Exposure to A Low Dose of TCDD Does Not Affect Epididymal Development or Sperm Number
No statistically significant difference in epididymal weight or SR was observed between the highest dose group (800 ng TCDD/kg) and the control group (Fig. 5). This observation is in contrast to that of Mably et al. (1992c), who reported that TCDD treatment reduced the size of the epididymis and the SR at doses larger than 400 ng/kg and 64 ng/kg, respectively. Furthermore, Gray et al. (1997) observed a statistically significant reduction of epididymal weight and SR in LE rats only at the highest dose of 800 ng TCDD/kg. In the present study, the cauda epididymal weight and SR of the highest TCDD dose (800-ng/kg) groups on both PNDs 49 and 120 showed a clear trend of reduction, although the difference was not statistically significant. In addition, in our current experiment using SD rats, dramatic hypoplasia of the epididymis was detected in some male offspring dosed maternally to 1 µg TCDD/kg (unpublished observation). Thus, the threshold of effects on epididymal development is thought be around 800 ng/kg by a single oral dose in the case of rats.
Reduction of ejaculated sperm number (ESN) is documented to be the most sensitive endpoint by Gray et al. (1997). They reported that a dose of only 50 ng TCDD/kg led to a significant decrease in ESN, and that this decrease was much greater than that of SR. To explain this phenomenon, Peterson and co-workers (Sommer et al., 1996) hypothesized that an increase in the phagocytotic activity of epithelial cells of epididymal and efferent ducts might be involved in the reduction of ESN. Because of the lack of experimental evidence on ESN, we cannot discuss the mechanism of the decrease of sperm count in the epididymis and ejaculates.
Low-Dose TCDD Reduced the Size of Sex-Accessory Glands and the Anogenital Distance
At TCDD doses larger than 200 ng/kg, the weight of the urogenital complex showed a clear pattern of dose-dependent decreases, and these decreases were significantly different from the control values. This reduction is attributable to the decreased size of the ventral prostate. Moreover, the AGD of male rats showed a dose-dependent reduction on PND 120, and the lowest-observed-adverse-effect level (LOAEL) was estimated to be 50 ng TCDD/kg in this protocol. Based on the above results, we concluded that ventral prostate weight and AGD are much more sensitive endpoints to TCDD exposure than testicular weight, DSP, epididymal weight, or SR. The reports by Mably et al. (1992a,b,c) also documented that maternal TCDD exposure significantly reduced AGD on PND 1 and PND 4. The ventral prostate weight on PND 49, 63, and 120 was reduced in a dose-dependent manner, and a statistically significant difference was detected at more than 160 ng/kg. At the highest dose of 800 ng/kg, the ventral prostate weight was reduced less than 50% in size. Gray et al. also reported the same effects on AGD and ventral prostate (Gray et al., 1995, 1997
).
A Low Dose of TCDD Reduced Ventral Prostate Size without Affecting Serum Gonadotropins or Testosterone Levels
Since the growth of these organs is dependent upon androgen, Peterson and coworkers measured the concentrations of plasma testosterone (T), 5-dihydrotestosterone (DHT), and luteinizing hormone (LH) (Mably et al., 1992a
; Roman et al., 1995
), and observed a tendency toward reduction of T and DHT during stages of development. Cooke et al. (1998) also found significantly lower concentrations of total serum androgen on PND 45 in TCDD-exposed rats than in control rats. However, a detailed examination concerning circulating and intratesticular androgen levels and in vitro androgen production of TCDD-treated rats revealed that the decreased levels were not large enough to explain the reductions of androgen (Roman et al., 1995
). In the present study, we also failed to find significant differences in serum gonadotropins (LH and FSH) and T (Table 1
). It would thus seem that maternal exposure to TCDD did not alter the productivity of androgen in male offspring.
When the castrated male rats that had been maternally exposed to 700 ng TCDD/kg were implanted with T-filled silastic capsule on PND 63, ventral prostate weight and protein content did not respond to testosterone by growing as much as those of vehicle-treated control rats (Bjerke et al., 1994b). Moreover, it has been reported that a set of androgen-responsive genes, e.g., probasin and 20-kDa cystatin-like protein, which are all major secretory proteins of ventral prostate, were expressed at significantly lower levels in the prostates of TCDD-treated rats than in the prostates of vehicle-treated rats (Roman et al., 1998a). Based on these investigations, Peterson and coworkers concluded that TCDD reduces the androgen responsiveness of the prostate without inhibiting androgen production. In our study, we also suggest that maternal TCDD exposure caused the reduced androgen responsiveness of the prostate without inhibiting androgen production during the pubertal period.
5R-II mRNA Level in the Ventral Prostate Is Increased by A Low Dose of TCDD
5-Reductase type 2 (5
R-II), one of the two isoenzymes for steroid-5
-reduction (Mahendroo and Russell, 1999
; Normington and Russell, 1992
), was reported to be responsible for prostate growth (George, 1997
). The ventral prostate and AGD seem to be more responsive to TCDD than the testis and epididymis, and their growth is known to be more sensitive to DHT, a metabolite of T, than is the growth of the internal reproductive organs, testis, and epididymis (Iguchi et al., 1991
; Imperato-McGinley et al., 1992
; Tsuji et al., 1994
). Accordingly, we here investigated the association between the 5
-reductase enzyme and growth of the TCDD-affected ventral prostate. In a preliminary experiment using RT-PCR, we confirmed that 5
R-II mRNA was expressed in the ventral prostate, seminal vesicle, and epididymis, but not in the testis of adult Holtzman rats. If the decreased size of the TCDD-exposed prostate was due to reduction of DHT production, then we would expect that the 5
R-II mRNA level would be reduced. Surprisingly, however, semiquantitative RT-PCR analysis revealed that the expression level of 5
R-II mRNAs on PND 49 was increased in a TCDD-dose-dependent manner (Fig. 8
). This result revealed that the reduction in ventral prostate size was not accompanied by a decrease in 5
R-II mRNA. More recently, Peterson and coworkers examined 5
R-II enzyme activity in the ventral prostate on PNDs 14, 21, and 32 of rats that were exposed maternally to 1 µg TCDD/kg, and found that the activity was increased by TCDD, which is consistent with our current results on 5
R-II mRNA (personal communication).
In the control rats, the 5R-II mRNA levels in the prostate were higher on PND 49 than on PND 120 (Fig. 11
). In contrast, the average weights of the ventral prostate on PNDs 49 and 120 were around 280 mg and 1000 mg, respectively. Therefore, although the regulatory mechanism of 5
R-II expression is not clear, it appears that the 5
R-II mRNA levels are inversely correlated with the size of the prostate. Because the size of the prostate is reduced by exposure to TCDD, the higher level of 5
R-II mRNA in the TCDD-exposed prostate might reflect in normal expression level. The present results suggest that TCDD exposure results in permanent alteration of the prostate via an as-yet-unknown mechanism, and that this mechanism is related to the above-described decrease of androgen responsiveness in the pubertal stages (Bjerke et al., 1994b
).
A Low Dose of TCDD Decreased AR mRNA Level in the Ventral Prostate
Gray et al. (1995) reported that the number of AR in the TCDD-exposed ventral prostate and seminal vesicles were similar to those of control organs. More recently, in a study using competitive RT-PCR, Hamm et al. (2000) reported that maternal administration of a single dose of 1 µg TCDD/kg to LE rats did not result in any significant reduction in the number of AR mRNA molecules in seminal vesicles on either PND 25 or PND 32. In the present study, however, a dose-dependent decrease of PCR product for AR mRNA was detected in TCDD-exposed ventral prostates on PND 49 by the use of semiquantitative RT-PCR, and a statistical significance was detected even at a dose of 12.5 ng TCDD/kg, suggesting that maternal exposure to a low dose of TCDD decreases AR mRNA level. We speculate that this reduction of AR might be involved in the decrease of androgen responsiveness.
The AR gene itself is a target for androgen, and androgen regulates AR mRNA expression in a tissue-specific fashion (Grad et al., 1999). In the normal ventral prostate of adult rats, AR mRNA levels have been shown to be down-regulated by androgens (Lubahn et al., 1989
; Prins et al., 1995). As shown in Figure 11
, the AR mRNA level in the ventral prostate on PND 120 was lower than that on PND 49 and the level of AR mRNA was inversely correlated with the size of the prostate. This decreased level of AR mRNA might be the result of the elevated T level during puberty (Zanato et al., 1994
). From the standpoint of this T-mediated AR down-regulation, it should be considered that AR mRNA levels tend to be elevated in the TCDD-exposed prostate on PND 49 because of the previous reports that T concentrations on PND 49 tended to be reduced by TCDD (Cooke et al., 1998
; Mably et al., 1992a
; Roman et al., 1995
). In the present study, however, we failed to observe any indication of an increased T level; this makes it difficult to explain the probable reasons for this decrease of AR mRNA level.
With respect to age-related changes of the prostate, the apparent discrepancy between our data and those from Peterson's laboratory may be attributable to one or more of the possible causes below. In their reports, the difference in prostate weight between the TCDD-treated and control groups was more conspicuous at the prepubertal or pubertal stages (PND 46 and PND 63) than at the mature stage (PND 120). That is, the magnitude of the size reduction became smaller as development progressed (Mably et al., 1992a). This suggests that male reproductive effects by maternal TCDD exposure may be reversible. In contrast, our data on the ventral prostate and AGD showed that the effects were more evident on PND 120, suggesting that the effects were irreversible. However, in RT-PCR analysis on PND 120, AR mRNA levels in rats treated at a TCDD dose of 800 ng/kg were higher than those in controls, although the difference was not statistically significant. This was the reverse of the pattern seen on PND 49, suggesting that the androgen receptor expression of the TCDD-exposed prostate on PND120 appeared to be differently regulated from that on PND 49. Further investigations focusing on DHT production and the relationship between AR mRNA number and androgen responsiveness in the ventral prostate of normal male rats will be needed.
Conclusion
Maternal exposure to a relatively low dose of TCDD affected the development of the ventral prostate and external genital organs as well as the AGD, more than it affected the development of the testis and other internal genital organs. The decreased size of the ventral prostate by maternal TCDD exposure is thought to be due to decreased responsiveness of the prostate to androgen; that is, it is thought to be derived from insufficient expression of androgen receptor mRNA during puberty.
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ACKNOWLEDGMENTS |
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NOTES |
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