* Division of Basic Biomedical Sciences, School of Medicine, University of South Dakota, 414 East Clark Street, Vermillion, South Dakota 57069;
School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705; and
Division of Biological Sciences, University of MissouriColumbia, Columbia, Missouri 65211
Received November 14, 2001; accepted February 4, 2002
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ABSTRACT |
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Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); prostate development; intrauterine position; testosterone; estrogen; rat.
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INTRODUCTION |
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The prostate in male rats begins developing on GD 18.5 (Timms et al., 1994). Induction of prostatic budding appears to be regulated by the underlying urogenital sinus (UGS) mesenchyme (Hayward et al., 1997
). Epithelial buds develop bilaterally in specific anatomical regions of the UGS: dorsocranial, dorsal, lateral, and ventral (Fig. 1
). By GD 20 (the time at which we examined male fetuses in this study), the initial budding process is complete. Subsequent epithelial glandular growth and branching continues postnatally (Hayashi et al., 1991
; Prins and Birch, 1997
).
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Timms and colleagues have described a 3-D reconstruction technique for studying fetal development of the prostate (Timms et al., 1994). Using this technique, the development of the prostate was found to differ in male Sprague-Dawley rat fetuses that occupied different intrauterine positions (IUPs). Male fetuses that occupied an IUP between female fetuses (2F males) had a greater mean area of prostatic buds in the dorsocranial, dorsal, and lateral regions relative to male fetuses located between other male fetuses (2M males; Timms et al., 1999
). This finding was consistent with prior studies in mice in which as adults, 2F male mice were found to have larger prostates relative to 2M males (Nonneman et al., 1992
). The enlarged prostate in 2F males was hypothesized to be mediated by an elevated level of serum estradiol in 2F male fetuses relative to 2M fetuses, due to transport of estradiol from adjacent female fetuses (Even et al., 1992
; vom Saal, 1989
). This hypothesis was confirmed in a study in which estradiol was experimentally elevated by 50% in male mouse fetuses (via maternal administration), and the estrogen-treated males showed both a significant increase in prostatic glandular buds and significantly larger buds during fetal life, as well as enlarged prostates in adulthood (vom Saal et al., 1997
).
Numerous studies have now shown that a small increase in estrogenic activity, either from estradiol, estrogenic drugs, or environmental estrogens in plastic or pesticides, in male mouse fetuses results in a permanent increase in prostate size during postnatal life (Gupta, 2000a,b
; Nagel et al., 1997
; Nonneman et al., 1992
; Thayer et al., 2001
; vom Saal et al., 1997
; Welshons et al., 1999
). The increase in prostate size is associated with an increase in prostatic androgen receptors (Gupta, 2000a
; Nonneman et al., 1992
; Thayer et al., 2001
; vom Saal et al., 1997
). It thus appears that a small increase in estrogen during the initial period of prostate development in fetal life results in an increase in the response of the prostate to androgen. In contrast, supraphysiological doses of estradiol or estrogenic chemicals can have the opposite effect of low doses and dramatically interfere with normal development of the prostate (Gupta, 2000a
; Prins, 1997
; vom Saal et al., 1997
).
In the present study we examined the effect of TCDD administration to pregnant rats on early development of the prostate in male rat fetuses, with attention being paid to the IUP of the males. The objective was to determine whether the intrauterine position of male fetuses, which is related to background levels of estradiol (elevated in 2F males) and testosterone (elevated in 2M males), would influence the response of the developing prostate to TCDD. TCDD has been shown to inhibit estrogen-induced responses in several tissues (Buchanan et al., 2000; Peterson et al., 1992
, 1993
). We report here that exposure to TCDD significantly reduced serum estradiol in 2F males but not 2M males, and also significantly interfered with initial budding and subsequent growth of the prostate (particularly in the dorsal-lateral region) in 2F but not 2M males. In sharp contrast, the seminal vesicles were larger in control 2M males than in control 2F males, similar to prior findings in mice (Nonneman et al., 1992
), and TCDD only decreased the size of the seminal vesicles in 2M males.
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MATERIALS AND METHODS |
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On the morning of GD 20, fetuses were removed by caesarian section, and the intrauterine position of each animal was noted prior to collection of blood for steroid radioimmunoassays and removal of the urogenital complex for reconstruction analysis. Male fetuses residing in utero between 2 male fetuses (2M), between a male and a female fetus (1MF), and between 2 female fetuses (2F) were examined.
Fetuses from untreated dams were also collected on the morning of GD 18, 19, 20, and 21 in a separate experiment to examine the temporal pattern of prostate morphogenesis in this strain of rat. In the latter experiment, only 1MF male fetuses were examined to control for variability due to intrauterine position (n = 2 per time point).
Estradiol radioimmunoassay. Estradiol radioimmunoassay was performed as previously described (vom Saal et al., 1990). Briefly, [125I] estradiol and antisera were obtained from ICN Biomedicals (Costa Mesa, CA), and unlabeled estradiol was obtained from Steraloids (Wilton, NH). Sensitivity of the assay was 0.5 pg. Intra-and interassay coefficients of variation were 3 and 11%, respectively. We determined the percent cross-reactivity of the estradiol antiserum with estrone to be 0.6%. Cross-reactivity with other steroids was reported by ICN to be negligible.
Testosterone radioimmunoassay. Testosterone was assayed as described (vom Saal et al., 1990). Briefly, first antibody (rabbit anti-testosterone), [125I]testosterone, and second antibody (goat anti-rabbit) were obtained from ICN Biomedicals (Costa Mesa, CA). Intra-and interassay coefficients of variation were determined to be 3 and 12%, respectively. We determined the cross reactivity of the antisera with DHT and androstenedione to be 1.3% and 10%, respectively. Cross-reactivity with other steroids was reported by ICN to be negligible.
Reconstruction analysis. Rat fetuses were euthanized by decapitation and the entire urogenital complex, which includes the bladder, UGS, and associated accessory sex glands, was fixed overnight at 4°C in Bouin's solution. Fixed tissues were processed for histological examination and serial section reconstruction (Timms et al., 1994). Epithelial outgrowths of the UGS called prostatic buds were categorized into ventral, lateral and dorsal and dorsocranial anatomical regions and analyzed by previously reported techniques (Roman et al., 1998
; Timms et al., 1999
; vom Saal et al., 1997
). Parameters measured for these regions included mean cross sectional area (CSA), total area of budding (TA), which is comparable to prostate volume, number of buds in each region, and length of budding (LB) along the UGS in a particular region of the prostate (Timms et al., 1999
). In addition to the prostate, the developing seminal vesicles associated with the proximal Wolffian ducts were also examined. For both the dorsocranial prostatic buds (also referred to as the coagulating glands) and the seminal vesicles, LB represents the mean distance in µm that the individual bud forming the organ extended from the urethra (dorsocranial buds) or Wolffian ducts (seminal vesicles); see Figures 1 and 2
.
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Morphometric Parameters |
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LB (in µm). LB was the length of UGS along which there were prostatic buds present. This was calculated from the number of sections multiplied by the thickness of sections (7 µm) of the UGS that contained buds associated with a specific region, since transverse sections through the UGS were examined. For the seminal vesicles and dorsocranial prostate (coagulating glands), the LB measure was the length of the developing structure, because the direction of growth of the individual structure forming these organs was perpendicular to the plane of section.
CSA (in µm2). The mean CSA was calculated by first summing the cross-sectional area measures for each bud identified in individual sections through a specific region, and then dividing by the number measured.
TA (in µm2). TA was calculated as the sum of all of the individual cross-sectional areas for all of the buds in a specific region. This parameter serves as an estimate of volume.
Statistical analysis. Data were analyzed by ANOVA using SAS (GLM procedure). Because each litter contributed males from different IUPs, variance due to litter (maternal) effects was assessed by including litter as a main effect variable. The F value for IUP, treatment and the interaction was divided by the F value for the litter variable to generate a corrected F to determine significance in the overall ANOVA. Planned comparisons were conducted when the overall ANOVA was statistically significant using the LS means test in SAS, again after adjusting for variance due to litter effects. Planned comparisons consisted of comparing males within each treatment group (TCDD and control) for differences due to intrauterine position, and comparing treated versus untreated males from each intrauterine position. The confidence level for statistical significance was set at p < 0.05.
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RESULTS |
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Number of Prostatic Buds
Exposure of fetal males to TCDD resulted in a significant 20% reduction in the total number of developing prostatic buds compared to untreated controls (TCDD = 76 ± 3 vs. control = 94 ± 4 [mean ± SEM]) on gestation day 20. A significant decrease in budding due to TCDD occurred in all regions (Table 1). The ventral region had the smallest population of developing buds, while the lateral region had the greatest number of developing buds.
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Morphometric Analyses
The data for the morphometric analyses for LB, mean CSA, and TA in the different regions of the prostate are presented in Table 2, and for the seminal vesicles and urethra in Table 3
.
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CSA. Ignoring IUP, there was no significant difference in mean CSA due to TCDD treatment. For the control males mean CSA was 27% greater for 2F males relative to 2M males, replicating our prior finding with Sprague-Dawley rats (Timms et al., 1999). In contrast, there was no significant difference between 2F and 2M males exposed to TCDD. Finally, TCDD had no significant effect on the mean CSA measure based on comparisons of TCDD-exposed and control males from each IUP.
TA. Ignoring IUP, there was no significant effect of TCDD on TA, but TCDD-treated 2F males showed a significant (33%) decrease in TA relative to control 2F males. Since control and TCDD-treated 2F males did not differ significantly in mean CSA, the effect of TCDD on TA was primarily due to a reduction in the length of the line of buds rather than a decrease in the cross-sectional area of the buds. In contrast to 2F males, the TCDD-exposed 1MF and 2M males did not differ significantly from control males from the same IUP on the TA measure (see also Figs. 3 and 4).
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CSA. TCDD had no significant effect on the mean CSA measurement; either based on comparisons of treated and control males from each IUP or ignoring IUP. The mean CSA of budding was slightly greater for control 2F males relative to control 2M males, but the difference was not statistically significant.
TA. Ignoring IUP, there was no significant effect of IUP or TCDD on the TA measure for the lateral prostatic region of budding. However, similar to the dorsal prostate, TCDD significantly reduced TA in 2F males but not 1MF or 2M males (see also Figs. 3 and 4).
Dorsocranial Region of the Prostate (Coagulating Glands)
Length of the paired glands. The coagulating glands in fetal rats develop as a single pair of buds located in the most cranial region of the dorsal prostatic urethra. Note that for these buds, the length measure represents the mean distance in µm that the paired buds, which form the adult coagulating glands, extend from the urethra.
Ignoring IUP, male fetuses exposed to TCDD did not show a statistically significant, decrease in the length of these bilateral dorsocranial buds relative to controls. For the comparison of males within each treatment group from different IUPs, the length of the dorsocranial pair of buds did not differ significantly as a function of IUP for control males. However, for TCDD-treated males the length of the pair of the buds was significantly greater for 2M than for 2F males. The basis for this finding is that for the TCDD-exposed 2F males, there was a significant decrease in bud length relative to control 2F males, while there was no decrease in bud length due to TCDD for 2M or 1MF males relative to controls from the same IUP. TCDD thus only decreased the length of the dorsocranial buds in 2F males.
CSA. Ignoring IUP, the mean CSA of the bilateral dorsocranial buds was greater for TCDD-treated relative to control males. There were no significant differences due to IUP for males within each treatment group in the mean CSA measure.
TA. Ignoring IUP, there was no significant difference in the TA measure between TCDD and control males. In addition, control 2F, 1MF, and 2M males did not differ in the total area of budding. However, as described above, the TCDD-treated 2F males had shorter buds relative to control 2F males, while TCDD treatment did not affect bud length in control 2M males. As a result, there was a significant difference in TA between control and TCDD-treated 2F males, but not 1MF or 2M males. In addition, there was a significant difference between 2F and 2M TCDD-treated males, with the TA measure being significantly smaller for TCDD-treated 2F males relative to 2M males.
Ventral Region
There were no significant differences in the LB, mean CSA, or TA measures based on comparisons of males from different IUPs or due to TCDD treatment in the ventral prostatic region, even though there were significantly fewer buds in TCDD-treated males relative to control males (Table 1).
Seminal Vesicles
Length of the paired glands. As was described for the length measure in the developing coagulating glands, the seminal vesicle length measure represents the length of the paired glands as they extend from the ejaculatory (Wolffian) ducts. Ignoring IUP, there was no effect of TCDD on the length, mean CSA, or TA measures for the seminal vesicles. However, for control males, the length of the seminal vesicle was significantly greater in 2M males (by 35%) relative to 2F males. TCDD treatment significantly reduced seminal vesicle length (LB) in 2M males, but not in 1MF or 2F males. As a result, for TCDD-treated males, there was no difference between 2F, 1MF, and 2M males for the LB measure.
CSA. There was no significant difference due to treatment or IUP in the mean CSA of the seminal vesicles.
TA. Ignoring IUP there was no significant effect on seminal vesicle TA due to TCDD treatment. However, the TA for control 2M males was significantly greater than for control 2F males. Maternal TCDD treatment significantly reduced TA in 2M males, but not in 1MF or 2F males. The significant decrease in TA in 2M males exposed to TCDD was due to a significant decrease in the length of the gland.
Urethra
The region of the urethra in which there were prostatic buds (prostatic urethra) was significantly longer in control 2M males (by 35%) relative to control 2F males (p < 0.01). TCDD resulted in the prostatic urethra of 2M males being significantly shorter (by 17%) relative to control 2M males (p < 0.05). Ignoring IUP, the mean CSA of the prostatic urethra tended to be larger in TCDD-treated males than in control males (p = 0.09). Furthermore, TCDD tended to increase mean CSA in 2F males (p = 0.09), but not in 1MF or 2M males. There was also a noticeable change in the shape of the urethral contour in TCDD-exposed fetuses. The prostatic sulci (Fig. 4) in TCDD-exposed males were less pronounced compared to controls.
Prostatic utricle. The prostatic utricle is the remnant of the Müllerian ducts as they enter the UGS, and this remnant persists in the dorsal region of the prostate. There was a significant decrease in the length of the utricle (LB) in TCDD-treated males (72 ± 5 µm) relative to control (92 ± 6 µm; p < 0.05). There were no differences observed relating to IUP for the utricle for control or TCDD-treated males (data not shown).
Serum Estradiol and Testosterone
In control males serum estradiol was significantly greater in 2F males than in either 1MF males or 2M males (Fig. 5). For TCDD-treated males, 2F and 2M males did not differ significantly in their serum estradiol levels. Ignoring IUP, serum estradiol in TCDD-exposed males tended to be lower than in control males (p = 0.06). Relative to control 2F males, TCDD-exposed 2F males had significantly lower serum estradiol. Similarly, relative to control 1MF males, TCDD-exposed 1MF males also had significantly lower serum estradiol. However, TCDD did not have a significant effect on serum estradiol in 2M males.
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DISCUSSION |
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The loss of prostatic buds due to TCDD exposure was primarily in the caudal region of the prostate. The findings concerning the initial pattern of bud development beginning on GD 18 (Fig. 1) suggest that the inhibitory effect of TCDD on the overall growth of prostatic buds from the UGS does not involve retardation in the rate of budding. This hypothesis is based on the fact that the first buds to form between GD 18 and 19 are in the caudal region of the prostate, followed between GD 19 and 20 by formation of buds in the cranial region. A retardation in the rate of growth by TCDD based on examination on GD 20 would thus be expected to result in a delay in the development of buds in the cranial region of the prostate, not in the caudal region. Since the prostates in TCDD-exposed males did not resemble control prostates collected on GD 19, we conclude that TCDD did not retard bud development. Instead, these findings show that the effect of TCDD on the prostate depends on the background level of estradiol in male fetuses, and that TCDD shows regional specificity in disrupting prostatic bud development and growth within the differentiating UGS.
The second major finding is that the effect of TCDD on the prostate in 2F males was associated with a significant decrease in serum estradiol in these 2F males, while their 2M male siblings were unaffected by TCDD both in terms of serum estradiol levels and dorsocranial, dorsal, and lateral prostate glandular bud number and size. These findings lead to a number of hypotheses. First, the inhibitory effect of TCDD on serum estradiol is modulated by the background level of estradiol present at the time of exposure to TCDD. In addition, the selective effect of TCDD only on the prostate of male fetuses that would otherwise have had high levels of serum estradiol (and an enlarged prostate) may have been due, at least in part, to the decrease in estradiol in addition to a likely direct effect of TCDD on the prostate. However, in the C57B1/6 mouse fetus, in utero exposure to TCDD has been shown to impair prostatic bud formation by an AhR-dependent process (Lin et al., 2000). Thus, at least some of the effects reported here are likely also due to activation of the aryl hydrocarbon receptor (AhR) in mesenchymal and/or epithelial cells of the UGS. Both AhR and the AhR nuclear translocator protein (ARNT) are expressed in the fetal rat UGS during the time that TCDD would have been acting as a result of maternal treatment on GD 15 (Sommer et al., 1999
). In the 2M fetuses, testosterone levels were high in both the TCDD and control animals, compared to 2F fetuses. There is a possibility that higher circulating levels of testosterone may provide protection for the developing prostate from the growth inhibitory effects of TCDD. If TCDD interacts with AhR and produces factors that inhibit prostate development or antagonize androgen-driven processes, then a higher level of testosterone may help to modulate this effect.
The developing prostate is primarily responsive to androgen during development (Cooke et al., 1991; Prins and Birch, 1995
). Thus, prior studies of the inhibitory effect of TCDD on prostate development in rats had focused on the possible effects of TCDD on serum testosterone (Roman and Peterson, 1998
; Roman et al., 1995
; Theobald et al., 2000a
,b
). We confirmed here that circulating testosterone is unaffected by TCDD in male rat fetuses. However, there is increasing evidence that estrogen plays an important role in both normal development and subsequent abnormal growth of the prostate gland (Ho et al., 1992
; Prins, 1997
; vom Saal and Timms, 1999
; vom Saal et al., 1997
). We previously reported that the pattern of prostatic budding from the UGS can be altered by experimentally manipulating the levels of circulating estradiol during critical periods in fetal development in mice. Specifically, increasing serum estradiol in male mouse fetuses by 50% resulted in an increase in the number and size of prostate glands, and a permanent increase in prostatic androgen receptors, relative to untreated males (vom Saal et al., 1997
). In addition, the natural phenomenon of IUP exposes developing fetuses to variable levels of circulating estradiol and testosterone. The consequences of IUP on rat prostate development, namely an increase in the size of the dorsolateral region of the developing prostate in 2F males, adds further credence to the hypothesis that estradiol enhances androgen regulation of prostatic growth in a region-specific manner (Timms et al., 1999
). In mice, 2F males also have permanently enlarged prostates relative to 2M males (Nonneman et al., 1992
).
For control male fetuses, we replicated prior findings in mice (vom Saal, 1989) and Mongolian gerbils (Clark et al., 1991
) that serum estradiol is higher in 2F than in 2M male fetuses, while serum testosterone is higher in 2M than 2F male fetuses. This difference in serum hormone levels due to being positioned between either male or female fetuses has been shown to be mediated by transport through the amniotic fluid and across the amniotic and chorionic (fetal) membranes surrounding each fetus; the fetal membranes are pressed against each other toward the end of gestation (Even et al., 1992
). In contrast to primates, where the placenta contains aromatase and secretes estrogen (Solomon, 1994
), the rat placenta secretes androgen (androstenedione and testosterone) but not estrogen (Jackson and Albrecht, 1985
; Sridaran and Gibori, 1987
; Warshaw et al., 1986
). It is interesting that TCDD did not significantly decrease serum testosterone, but did significantly lower serum estradiol in male fetuses. Why serum estradiol was selectively not reduced in 2M males, while there was a significant reduction in estradiol in 1MF and 2F males whose mothers were treated with TCDD, remains to be determined.
In previous comparisons of 2F and 2M male mice, while 2F males with elevated serum estradiol during fetal life were found to have a permanently enlarged prostate, these same males had seminal vesicles that were permanently reduced in size (Nonneman et al., 1992). Subsequent studies have revealed that the small seminal vesicles in 2F males were due to lower 5
-reductase activity relative to 2M males (Ganjam, Welshons, and vom Saal, unpublished observation), while no difference in seminal vesicle androgen receptors was observed (Nonneman et al., 1992
). In addition, administration of estrogenic chemicals, such as bisphenol A, to pregnant mice resulted in a decrease in seminal vesicle and epididymis size in male offspring and an increase in prostate size relative to controls (Gupta, 2000a
; vom Saal et al., 1998
). These findings show that during fetal life, gonadal steroids have opposite effects on the development of organs that differentiate from the Wolffian ducts and the UGS. Our finding here that 2M male Holtzman rats had larger seminal vesicles than 2F males is consistent with these prior findings in mice. Interestingly, this is not consistent with our prior comparison of 2F and 2M Sprague-Dawley rats (Timms et al., 1999
). In addition, the fact that TCDD disrupted development of the seminal vesicle in 2M males but not 2F males suggests that there is a marked difference in the interaction between TCDD and gonadal steroids in influencing seminal vesicle development in contrast to prostate development.
The mean cross sectional area of the lumen of the urethra associated with prostatic buds (prostatic urethra) was decreased in a prior study in which serum estradiol was experimentally increased in male mouse fetuses (vom Saal et al., 1997). In this study, ignoring IUP, TCDD tended to increase the mean cross sectional area of the prostatic urethra, and this was associated with a decrease in serum estradiol in these males. Development of the urethra is thus sensitive to changes in estrogen. In addition, TCDD treatment resulted in a change in the shape of the dorsal portion of the UGS, such that there was a noticeable decrease in the depth of the prostatic sulcus (Fig. 4
). This portion of the fetal UGS is the region from which the buds that form the dorsal prostate differentiate. This change in shape could thus be involved in the loss of dorsal prostatic buds from the UGS. In addition, changes in the morphology of the urethra could result in subsequent abnormalities in urethral function, which remain to be examined.
Taken together, our findings demonstrate that in utero exposure to TCDD disrupts the development of the prostate, but this disruption depends on an interaction with background levels of estradiol. There are numerous factors that influence levels of estradiol in human pregnancy, such as race, birth order, fetal body weight, and singleton versus twin pregnancy (Batra et al., 1978; Bernstein et al., 1986
; Gerhard et al., 1987
; Henderson et al., 1988
). Our findings suggest that variation in estradiol in human fetuses might also be a factor that influences the response to TCDD in humans. The interaction between TCDD and estradiol leads to the hypothesis that the effects of TCDD might also be altered by simultaneous exposure to estrogenic chemicals, which are found in plastics, pesticides, and other products. The potential for interactive effects of these chemicals on prostate growth and development requires that studies of mixtures of these chemicals be conducted.
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ACKNOWLEDGMENTS |
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NOTES |
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