* School of Pharmacy and
Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53705
Received April 3, 2002; accepted May 28, 2002
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ABSTRACT |
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Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; ventral, dorsolateral, and anterior prostate; seminal vesicles; gene expression; development; in utero and lactational TCDD exposure; critical windows of vulnerability; mice.
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INTRODUCTION |
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Our primary interest is in male reproductive system development. Absolute and relative testis and epididymis weights in C57BL/6 mice were reduced by AhR null mutation, indicating that the AhR plays a role in the normal development of these organs. In utero and lactational TCDD exposure (5 µg/kg maternal dose on GD 13) reduced absolute and relative testis and epididymis weights in wild-type but not AhRKO mice, whereas absolute (though not relative) testis and epididymis weights were slightly increased by TCDD in AhRKO mice at one of the 3 times examined. These results demonstrate that TCDD causes an AhR-dependent inhibition of testis and epididymis growth, and suggest that TCDD may possibly stimulate growth of these organs by an AhR-independent mechanism. In contrast, neither AhR null mutation nor in utero and lactational TCDD exposure, alone or in combination, affected daily sperm production or cauda epididymal sperm numbers (Lin et al., 2001a).
Effects on accessory sex organ development were also investigated (Lin et al., 2002a). The AhR was necessary for normal dorsolateral prostate, anterior prostate, and seminal vesicle development but apparently not for ventral prostate development. TCDD severely inhibited ventral prostate growth and the expression of mRNA for MP25, its major androgen-dependent secretory glycoprotein in the mouse (Mills et al., 1987
). Yet mRNA for PSP94, a secretory protein normally produced primarily by the lateral prostate, was highly expressed in TCDD-exposed ventral prostate. These results suggest that TCDD may have caused a respecification of gene expression in the ventral prostate towards that characteristic of the lateral prostate. TCDD reduced dorsolateral prostate weight but had relatively little effect on expression of mRNA for PSP94 or for probasin, a major androgen-dependent secretory protein in mice produced by the dorsolateral and anterior prostate (Johnson et al., 2000
). Anterior prostate weight was reduced, and expression of mRNA for renin-1, its major androgen-dependent secretory protein (Fabian et al., 1993
) was greatly reduced. These effects were predominantly AhR-dependent, i.e., TCDD caused these effects in wild-type but not AhRKO mice. In contrast, although seminal vesicle weight was reduced by in utero and lactational TCDD exposure in wild-type mice at both times tested, absolute and relative weight were increased by TCDD in AhRKO mice on postnatal day (PND) 35, and relative weight was decreased by TCDD on PND 90. Despite substantial changes in organ weights and gene expression, each organ in each treatment group appeared to be histologically normal.
In order to gain insights into the mechanisms by which TCDD impairs prostate and seminal vesicle development in wild-type mice, we have now investigated the developmental stages at which these organs are most sensitive to TCDD. These stages are summarized below.
The initial visible step in prostate formation is the outgrowth of buds from the urogenital sinus into surrounding mesenchyme. In control mice, bud formation is reported to begin on GD 17 (GD 0 = plug positive) and to be complete before birth. Buds elongate and coalesce into the various prostate lobes, which then begin to form ductal structures. Seminal vesicles develop from the Wolffian ducts and are reported to first appear on about GD 16 (Raynaud, 1962; Cunha, 1972
; Cunha et al., 1987
). Our observations on ventral and lateral bud formation in C57BL/6 mice are in agreement with these findings (i.e., that they appear on GD 17): in contrast, we find that dorsal buds appear on GD 16 and anterior buds appear on GD 15 (Lin et al., 2001b
). We also observed seminal vesicle buds on GD 14 (Tien-Min Lin, unpublished observations).
Accessory sex organs in control mice are rudimentary at birth and their morphogenesis occurs largely in the postnatal period. At birth, however, the right and left ventral prostate lobes are reported to already have one to three branched main ducts (though we observe no branching in mice from our colony), and the dorsolateral prostate has about ten unbranched main ducts on each side. In contrast, the anterior prostate and seminal vesicles have no ductal structure at birth. Ductal canalization, most ductal branching morphogenesis, structural cytodifferentiation, and secretory cytodifferentiation occur postnatally (Lung and Cunha, 1981; Podlasek et al., 1997
; Sugimura et al., 1986
).
In the experiments described herein, pregnant mice were dosed with 5 µg TCDD/kg or vehicle on GD 13, and their pups were fostered at birth to dams of the same treatment or cross-fostered to dams of the opposite treatment. Consequently, some males were born to and nursed by dams dosed only with vehicle, others had both in utero and lactational TCDD exposure (from GD 13 through PND 21), others had in utero TCDD exposure alone, and still others had lactational TCDD exposure alone. A fifth group of males had in utero and lactational TCDD exposure, but TCDD was not administered to their mothers until GD 16. All measurements were made when males were 35 days of age. Comparison of results from these various exposure regimens provided insights into the developmental steps that TCDD inhibits.
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MATERIALS AND METHODS |
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Mice were housed in clear plastic cages with heat-treated chipped aspen bedding, in rooms kept at 24 ± 1°C and 35 ± 4% relative humidity and lighted from 0600 to 1800 hours. Feed (5015 Mouse Diet, PMI Nutrition International, Brentwood, MO) and tap water were available ad libitum. All procedures were approved by the University of Wisconsin Animal Care and Use Committee.
Experimental design.
To obtain the mice whose reproductive organs were studied, individually housed heterozygous (Ahr+/-) females, between 90 and 120 days old, were paired overnight with Ahr+/- males. The next day was considered to be GD 0. Pregnant mice were given a single oral dose of TCDD (5 µg/kg) or vehicle (95% corn oil/5% acetone, 5 ml/kg) on GD 13. At birth, pups were fostered to dams from the same treatment group or cross-fostered to dams from the opposite treatment group. Additional dams were given a single oral dose of TCDD (5 µg/kg) on GD 16; their pups were kept with them. Genotyping was done by polymerase chain reaction analysis of ear punch tissue taken at 1016 days of age, as previously described (Benedict et al., 2000). All pups were weaned on PND 21, at which time they were housed by sex, typically 4 per cage. All results presented in this report are from wild-type mice.
Mice were euthanized by CO2 overdose on PND 35. Accessory sex organs were identified as described by Sugimura et al.(1986), removed, weighed, and frozen in polypropylene vials placed in liquid nitrogen. mRNA expression for cyclophilin, cytokeratin 8, MP25, probasin, and renin-1 was determined by real-time reverse transcription-polymerase chain reaction mRNA quantification using a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN), as previously described (Lin et al., 2002a).
Statistical analysis.
Analyses were conducted with the litter as the experimental unit. Parametric analyses were conducted on untransformed data and on log, square root, and inverse transforms as well as on ranked data. For data that passed Levene's test for homogeneity of variance and which appeared to be normally distributed, analysis of variance (ANOVA) was conducted. If a significant effect was found, the least-significant-difference test was used to determine which group(s) differed from the appropriate control group. Data were also analyzed by non-parametric methods (Kruskal-Wallis ANOVA and median test, with post hoc testing by the distribution-free multiple comparison test), but no additional significant differences were found. Significance was set at (p < 0.05). Results are presented as the mean ± SEM.
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RESULTS |
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DISCUSSION |
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Ventral Prostate
Effects of TCDD on ventral prostate development are due primarily to in utero exposure. Reductions in organ weight and MP25 mRNA expression were as severe in response to in utero exposure alone as to in utero and lactational exposure that began on GD 13. Consequently, the developmental processes most vulnerable to TCDD are those that occur prenatally, i.e., urogenital sinus development, ventral bud formation and growth, and/or the initial stages of duct formation. Lactational TCDD exposure also reduced organ weight and MP25 mRNA expression, but the magnitude of the effects was far smaller. These effects of lactational exposure demonstrate that TCDD can inhibit growth and secretory cytodifferentiation of the ventral prostate even if exposure does not begin until bud formation and elongation are complete and duct formation has already begun.
Delaying the start of in utero and lactational TCDD exposure by 3 days (from GD 13) had no effect on MP25 mRNA expression by the ventral prostate but reduced the magnitude of the reduction in organ weight. This delay also increased the proportion of luminal epithelial cells that are fully differentiated structurally, as shown by the increased cytokeratin 8:cyclophilin mRNA ratio. Consequently, effects of TCDD during GD 1316 contribute to the inhibition of ventral prostate development. Because ventral budding does not begin until GD 17, these results strongly suggest that the inhibition of ventral prostate development is due, in part, to effects of TCDD on development of the ventral portion of the urogenital sinus. It appears that fetal exposure during both GD 1316 and GD 1619 is important for the full effect of TCDD on the ventral prostate.
Dorsolateral Prostate
In contrast to the ventral prostate, the inhibition of dorsolateral prostate development by TCDD appears to be due approximately equally to in utero and to lactational exposure, because each (alone) contributed comparably to the inhibition of growth and probasin mRNA expression. Delaying the start of in utero and lactational exposure from GD 13 to GD 16 had no detectable effect on the severity of the inhibition. Consequently, the window of maximum vulnerability to TCDD for the dorsolateral prostate begins on GD 16 or later, and extends into the postnatal period. Because dorsal buds first appear on GD 16, these results indicate that TCDD does not inhibit the pre-bud formation stages of dorsal urogenital sinus development. Similarly, because lateral buds first appear on GD 17, TCDD apparently does not inhibit most of the pre-bud formation stages of lateral urogenital sinus development. Instead, TCDD must inhibit one or more of the later prenatal processes (bud formation, bud elongation, and/or duct formation) that lead to dorsolateral prostate development. Although the cytokeratin 8 mRNA expression pattern indicates that structural cytodifferentiation of luminal epithelial cells was not inhibited, the inhibition of probasin mRNA expression demonstrates that both in utero and lactational TCDD exposure inhibit secretory cytodifferentiation of the dorsolateral prostate. Whether this is due to an inhibition of luminal epithelial cell responsiveness to androgenic stimulation remains to be determined.
Anterior Prostate
Anterior prostate growth was dependent on both in utero and lactational TCDD exposure, each of which (alone) had less effect on its weight than continuous exposure from GD 13 until weaning. In utero TCDD exposure, alone, had a greater effect than lactational exposure, alone. When in utero and lactational TCDD exposure began on GD 16, the inhibition of growth was less severe than when dams were dosed 3 days earlier. Consequently, it appears that the most vulnerable time for anterior prostate growth begins before GD 16 and continues into postnatal life. Because we find anterior buds in C57BL/6 mice on GD 15, these results do not indicate whether TCDD inhibits pre-budding development of the anterior portion of the urogenital sinus or acts only after buds begin to form. Results described above suggest that TCDD inhibits multiple processes in anterior prostate development, both prenatally and postnatally.
In contrast to the ventral and dorsolateral prostate, where mRNA expression patterns were similar to organ weight patterns, renin-1 mRNA expression results differed greatly from anterior prostate weight results. Renin-1 mRNA expression was substantially inhibited regardless of the timing of TCDD exposure, indicating that the inhibition of secretory cytodifferentiation seen on PND 35 is due to effects of TCDD on the anterior prostate after birth rather than before. In contrast, the lack of an effect on cytokeratin 8 mRNA expression suggests that structural cytodifferentiation of luminal epithelial cells in the anterior prostate was not inhibited, regardless of the timing of TCDD exposure.
Seminal Vesicles
Seminal vesicle growth was essentially unaffected by in utero exposure alone or by lactational exposure alone, but was significantly inhibited by combined in utero and lactational TCDD exposure, regardless of whether dams were dosed on GD 13 or GD 16. It appears that prenatal and postnatal exposures interact to inhibit seminal vesicle development, possibly in a synergistic fashion. Yet significant vulnerability to TCDD does not begin until at least GD 16, 2 days after rudimentary seminal vesicles have already appeared.
Spectrum of Effects of TCDD on Accessory Sex Organ Development
The time during which each of the 4 organs studied is most vulnerable to TCDD varies from one organ to the other. The developmental processes inhibited by TCDD also vary to some extent from one organ to another. There was no obvious correlation between the time prostatic or seminal vesicle buds first appeared and the severity of the effects of TCDD on these organs. Nor did there appear to be a connection between whether uncanalized "ducts" appeared before (ventral prostate and dorsolateral prostate) or after birth (anterior prostate and seminal vesicles) and the severity of the effects. The possibility that regional differences in temporal/spatial AhR expression in the urogenital sinus may account for differences in the effects of TCDD from one organ to another is currently under investigation.
We had previously investigated the relative contributions of in utero versus lactational TCDD exposure to developmental abnormalities in Holtzman rats (Bjerke and Peterson et al., 1994), though analysis of effects on accessory sex organs was more limited than in the present study. Effects of in utero exposure on ventral prostate and seminal vesicle weight, protein content, and DNA content were comparable in magnitude to effects of lactational exposure (possible effects on the dorsolateral and anterior prostate were not investigated). Effects of each exposure (alone) on the ventral prostate were smaller than effects of combined in utero and lactational exposure, whereas effects of in utero or lactational exposure on the seminal vesicles were comparable in magnitude to the effects of combined exposure. Ohsako et al.(2002) recently reported that maternal TCDD dosing on GD 15 reduced ventral prostate and total accessory sex organ weights in Sprague-Dawley rats but that maternal dosing on GD 18 or direct dosing of pups on PND 2 had no such effects. Results of the present study demonstrate that critical windows of exposure to TCDD are somewhat different in the C57BL/6 mouse than in Holtzman or Sprague-Dawley rats.
The observation that in utero TCDD exposure inhibited ventral prostate development in mice as severely as combined in utero and lactational exposure is particularly significant in light of the substantial disparity between prenatal and postnatal TCDD transfer to offspring. Studies on the disposition of TCDD given orally to pregnant mice demonstrate that the vast majority of the TCDD transmitted to pups by their mothers is delivered in milk rather than across the placenta (Abbott et al., 1989; Nau and Bass, 1981
; Nau et al., 1986
; Weber and Birnbaum, 1985
). Yet in utero TCDD exposure alone was sufficient to severely inhibit ventral prostate growth and almost completely inhibit the expression of mRNA for its major androgen-dependent secretory protein. In utero exposure was also an essential component of the inhibitory effects on dorsolateral prostate, anterior prostate, and seminal vesicle development, demonstrating that each organ studied is sensitive to the exceedingly small amounts of TCDD that cross the placenta.
The mechanisms responsible for inhibited prostate development in TCDD-exposed mice are not well understood. Jana et al.(1999) reported that TCDD inhibited androgen-dependent gene expression in LNCaP prostate cancer cells, but we were unable to replicate their results. Ohsako et al.(2002) observed significant decreases in androgen receptor mRNA expression in rat ventral prostate, but we found no such changes in any prostate lobe in mice (Lin et al., 2002a). Effects of TCDD on prostate development in mice do not appear to be due to differences in androgen concentrations: we found no effect of maternal TCDD treatment (5 µg/kg on GD 13) on testicular testosterone content on GD 16 or 18 or on testicular testosterone or 5
-androstane-3
,17ß-diol content on PND 1, 7, or 14 (Ko and Lin, unpublished observations). Nor were serum testosterone concentrations significantly altered on PND 35 or 90 (Lin et al., 2002a
). The only significant effect on androgens we have seen in TCDD-exposed mice is a modest reduction in serum 5
-androstane-3
,17ß-diol concentrations on PND 21 (Lin et al., 2002a
). To increase our understanding of the mechanisms by which TCDD inhibits prostate development, we are currently examining the effects of TCDD on region-specific urogenital sinus bud formation (Lin et al., 2001b
), region-specific gene expression in the urogenital sinus (Lin et al., 2002b
), and ductal branching morphogenesis in each prostate lobe (Ko et al., 2001
). Results of these experiments will be described elsewhere.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at the School of Pharmacy, University of Wisconsin, 777 Highland Ave., Madison, WI 53705. Fax: (608) 265-3316. E-mail: repeterson{at}pharmacy.wisc.edu.
Portions of this research were presented at the 33rd Annual Meeting of the Society for the Study of Reproduction, Madison, WI, July 1518, 2000, the 40th annual meeting of the Society of Toxicology, San Francisco, CA, March 2529, 2001, and the 21st International Symposium on Halogenated Environmental Organic Pollutants and POPs (Dioxin 2001), Gyeongju, Korea, September 914, 2001. This article is Contribution 341 from the Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53705.
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