In Utero and Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the C57BL/6J Mouse Prostate: Lobe-Specific Effects on Branching Morphogenesis

Kinarm Ko*, H. Michael Theobald{dagger} and Richard E. Peterson*,{dagger},{ddagger},1

* Endocrinology-Reproductive Physiology Program, {dagger} School of Pharmacy, and {ddagger} Molecular and Environmental Toxicology Center, University of Wisconsin, 777 Highland Avenue, Madison, Wisconsin 53705

Received July 1, 2002; accepted August 27, 2002

ABSTRACT

Branching morphogenesis is an essential component of prostate development. This study was conducted to test the hypothesis that in utero and lactational 2,3,7,8-tetrachlorodibenzo– p-dioxin (TCDD) exposure differentially inhibits branching morphogenesis and ductal canalization in the ventral, dorsal, lateral, and anterior mouse prostate. Pregnant C57BL/6J mice were given TCDD (5 µg/kg, orally) or vehicle on gestation day (GD) 13 and their pups examined at 1, 7, 14, 21, 35, and 90 days of age. Prostate lobes were microdissected after incubation in 0.5% collagenase and the numbers of ductal tips, main ducts, and ductal tips per main duct were determined by examining photographs of microdissected, whole-mount specimens. Ductal canalization was determined using histological sections of the dorsolateral and anterior prostate lobes. TCDD inhibited branching morphogenesis in all prostate lobes. The ventral prostate (VP) was extremely small throughout development and never developed any ductal structure. TCDD reduced the numbers of ductal tips and main ducts in the dorsal (DP) and lateral prostate (LP), but reductions in ductal tip numbers appeared to result entirely from reductions in the number of main ducts. Dorsolateral prostate (DLP) weights were slightly reduced by TCDD, but there was no effect on ductal canalization in the dorsal, lateral, or anterior lobes. TCDD had no effect on main duct number in the anterior prostate, but weight, ductal tip number, and the number of ductal tips per main duct was substantially reduced. These results demonstrate that the severe inhibition in ventral prostate development caused by in utero and lactational TCDD exposure is accompanied by a complete absence of branching morphogenesis. The impairment in dorsal, lateral, and anterior prostate (AP) development is associated with a lobe-specific inhibition of the various processes involved in duct formation.

Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD; ventral, dorsolateral, and anterior prostate; branching morphogenesis, canalization; in utero and lactational TCDD exposure; prostate lobe-specific effect; C57BL/6J mouse; developmental toxicity.

Ductal branching morphogenesis is an important process in prostatic development (Cunha et al., 1987Go). Due to the inhibition of prostatic growth and development caused by in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in rats and mice (Lin et al., 2002aGo,bGo; Mably et al., 1992Go; Roman et al., 1995Go, 1998Go; Sommer and Peterson, 1997Go; Theobald and Peterson, 1997Go), this study was conducted to determine whether prostatic ductal morphogenesis is adversely affected by in utero and lactational TCDD exposure in the mouse. Shortly after birth, epithelia of the prostatic main ducts undergo rapid branching, such that the branching patterns are distinctive for each prostatic lobe (Cancilla et al., 2001Go; Lamm et al., 2001Go; Sugimura et al., 1986Go). The development of rounded acinar structures in the prostate epithelium can be visualized in histologic cross-section, whereas the three-dimensional ductal arrays that form in ventral prostate (VP), dorsal prostate (DP), lateral prostate (LP), and anterior prostate (AP) epithelium can be readily displayed in a whole mount preparation and quantified by morphometic image analysis.

At birth, the VP consists of 1–3 main ducts per side, with secondary and tertiary branches. No new main ducts form after birth; however, during the first 15 days of life, numerous branches arise from each main duct so that there is a marked increase in the number of ductal tips (Sugimura et al., 1986Go). About 80% of VP ductal tips are formed by postnatal day (PND) 15. The branching pattern in the LP is similar to that of the VP. In contrast, the DP consists of about 10 main ducts per lobe that do not start branching until after PND 5. About 70% of combined dorsal and lateral prostate (dorsolateral prostate, DLP) ductal tips are present by PND 15 (Sugimura et al., 1986Go). The AP consists of 2 main ducts per side, which begin branching just after birth (Lung and Cunha, 1981Go).

Canalization is the process of spatial organization inside the branches that occurs concurrently with the development of distinct epithelial cell types (Hayward et al., 1996Go). Before canalization, prostatic ducts are composed of undifferentiated basal epithelial cells, some of which differentiate into secretory luminal cells during the formation of acini. Prostatic ductal canalization begins in the proximal regions near the urethra or main duct and extends distally to the ductal tips, resulting in the formation of a functional secretory gland.

During postnatal development, androgens play a limited role in ductal-branching morphogenesis. Neonatal castration significantly reduces the number of ductal tips in the VP and DLP (Donjacour and Cunha, 1988Go). However, neonatal castration does not completely block ductal branching morphogenesis, suggesting that androgens may be required to trigger the process, but not necessarily to maintain it. After birth, androgen receptors are expressed in both the mesenchyme and the epithelium; however, during gestation, only the mesenchyme contains functional androgen receptors (Hayward et al., 1996Go; Takeda and Chang, 1991Go). Therefore, androgenic effects on prostatic epithelial development may be elicited through prenatal mesenchymal-epithelial interactions as well as by epithelial androgen-receptor signaling pathways postnatally. For example, epithelial androgen receptor is thought to be involved in the postnatal process of ductal canalization (Hayward et al., 1996Go).

In the male C57BL/6 mouse, in utero and lactational TCDD exposure reduces VP, DLP, and AP weight on PNDs 35 and 90 (Lin et al., 2002aGo,bGo). These effects of TCDD are not associated with decreased plasma or tissue androgen concentrations in Holtzman rats and ICR mice (Roman et al., 1995Go; Theobald and Peterson, 1997Go), but are associated with prenatal effects on prostatic bud formation. More specifically, in utero TCDD exposure completely inhibits VP bud formation from the urogenital sinus in the C57BL/6 mouse fetus and also reduces the number of buds in the region of the urogenital sinus that gives rise to the DLP (Lin et al., 2001aGo). Prostatic bud formation, which occurs in utero, precedes bud elongation, formation of main ducts, and branching morphogenesis. The latter process occurs after birth and is largely complete by PND 21 (Cunha et al., 1987Go; Donjacour and Cunha, 1988Go; Sugimura et al., 1986Go).

Our objective was to determine if in utero and lactational exposure to TCDD alters the pattern of branching morphogenesis of the prostate in a lobe-specific manner. This seemed probable because TCDD exposure only during lactation decreased VP and AP weights in rats and mice (Bjerke et al., 1994Go; Lin et al., 2002bGo). Therefore, effects of TCDD on ductal branching morphogenesis were evaluated in the VP, DP, LP, and AP, respectively. Mouse offspring were exposed to a single maternal dose of TCDD (5 µg/kg, orally) on GD 13, which is less than 1/30th of the LD50 for TCDD in non-pregnant C57BL/6J mice (Birnbaum et al., 1990Go). This dose is not large enough to cause overt signs of maternal toxicity, prenatal or postnatal mortality, or unacceptable reductions in offspring body weight in C57BL/6J and C57BL/6N mice (Couture et al., 1990Go; Lin et al., 2001bGo).

MATERIALS AND METHODS

Animals.
C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were used in all experiments. Mice were maintained in a 12-h light/dark cycle controlled environment (lights on at 0600 h, off at 1800 h) with temperature 22 ± 1°C and humidity 35 ± 5%. Mice were housed in clear plastic cages on heat-treated aspen chip bedding. Mouse feed (5015 Mouse Diet, PMI Nutrition International, Brentwood, MO) and water were supplied ad libitum. All animal procedures were conducted under protocols approved by the University of Wisconsin Animal Care and Use Committee.

To produce timed pregnant females, groups of three 90-120-day-old dams were housed with one male per cage overnight. The day when sperm plugs were found was considered GD 0. Pregnant females were given a single oral dose of either vehicle (95% corn oil/5% acetone, v/v), or 5 µg TCDD/kg by gavage on GD 13. The number of male pups per litter varied between litters; however, only one pup per litter was designated for any particular time. Therefore, litter independence was maintained throughout all assessment times in the study. To terminate lactational TCDD exposure, the offspring were weaned on PND 21 and housed 4 males per cage thereafter. Male offspring were sacrificed by CO2 overdose and the various prostate lobes were removed and prepared for evaluation as whole-mount or histology specimens.

Hormone assays.
Steroids were extracted from testes with ethyl acetate. Diethyl ether was used for extraction of serum steroids. Organic phases were evaporated to dryness under a gentle nitrogen stream and reconstituted in 100% ethanol. Testicular testosterone content and serum testosterone concentrations were measured by enzyme immunoassay (EIA), according to the protocol of the kit supplier (Assay Designs, Inc.). The EIA assay range was 7.81-2000 pg testosterone/ml. Testicular 5{alpha}-androstane-3{alpha},17ß-diol (3{alpha}-diol) content and serum 3{alpha}-diol concentrations were measured by radioimmunoassay (RIA), according to the protocol of the antibody supplier (Endocrine Science, Calabasas Hills, CA). The RIA assay range was 30-800 pg 3{alpha}-diol/tube. 3{alpha}-Diol used for standards was purchased from Steraloids (Wilton, NH), testosterone was purchased from Sigma Chemical Co. (St. Louis, MO), and [3H]-labeled steroids were purchased from DuPont-New England Nuclear (Boston, MA).

Prostate histology.
On PNDs 7, 14, 21, and 35 the entire prostate, seminal vesicles, part of the urethra, and the urinary bladder were dissected as a single unit. After removing the seminal vesicles under a dissection microscope, the remaining tissues were trimmed of fat, fixed in Z-5 fixative (Anatech, Ltd., Battle Creek, MI), and stored in 70% ethanol. Fixed tissues were dehydrated in a graded series of ethanol concentrations, laid flat, oriented, embedded in paraffin, sectioned at 5 µm intervals, and stained with hematoxylin and eosin. Similarly oriented tissue sections from vehicle- and TCDD-exposed offspring, cut from approximately the same depth of tissue, were then examined microscopically.

Ductal branching morphogenesis.
To examine prostatic morphogenesis, male offspring were sacrificed in groups of 5 at 1, 7, 14, 21, 35, and 90 days of age. The VP, DLP, and AP were carefully separated in chilled Hank’s balanced salt solution (HBSS; Ca2+ and Mg2+ free) with microdissecting scissors and forceps under a dissecting microscope. Body and organ weights were obtained on PNDs 35 and 90. One lobe of the VP, DLP, and AP was incubated in 0.5% collagenase in HBSS for 25 min at 35°C in order to remove the stroma. The numbers of ductal tips and main ducts were determined by examining photographs of microdissected, whole-mount specimens of the isolated epithelium. These were obtained by using a Nikon Optiphot-2 microscope equipped with a Sony 3CCD color video camera. The software package Image Pro Plus (Media Cybernetics, Silver Spring, MD) was used for data analysis.

Ductal canalization.
The entire prostate, seminal vesicles, urethra, and bladder were removed from male pups as a single unit. The VP, seminal vesicles, bladder, and part of the urethra were dissected away. The remaining DLP and AP, attached to the urethra, were flattened on a microscope slide in order to orient the DLP epithelial ducts in a common direction. These tissues then remained flattened during fixation in Z-5. After washing with graded concentrations of 50% and 70% ethanol, tissues were embedded in paraffin blocks, and cross-sectioned from the distal to the proximal region of the prostatic duct at 5 µm intervals. Every 5th cross-section was stained with hematoxylin and eosin, and ductal canalization was evaluated by image analysis. The degree of canalization in the DLP was quantified as the percentage of ducts that were open in each serial sectioned distal, intermediate I, intermediate II, and proximal regions on PND 7. A preliminary experiment indicated that ductal canalization of the DLP was not complete by PND 7 in control male pups, and this was the rationale for selecting this time. AP canalization was determined by examining every 5th, proximal to distal, serial, cross-section for the percentage of open main ducts at PND 7.

Statistical analysis.
Student’s t-test was used to compare the number of ductal tips, main ducts, ductal tips/main duct, and canalized ducts in vehicle- and TCDD-exposed prostates on a given postnatal day. Differences were considered statistically significant at p < 0.05.

RESULTS

Prostate weight.
Growth of the VP was totally inhibited by in utero and lactational exposure to a single maternal dose of 5 µg TCDD/kg (Fig. 1Go). On PND 35 there was no glandular structure in the VP epithelium of TCDD-exposed mice. Only rounded blebs of tissue that were too small to weigh accurately could be found on the left and right sides of the normal VP anatomical location. Little or no growth of this tissue was observed at PND 90 and morphological assessment of the tissue at this time revealed that it still contained no discernable glandular epithelial structure.



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FIG. 1. Relative ventral, dorsolateral, and anterior prostate weights in vehicle- and TCDD-exposed male offspring. Each bar represents the mean ± SE of 5 vehicle-exposed (open bars), or TCDD-exposed (filled bars) male offspring. *Significantly different from vehicle-exposed mice (p < 0.05). ND, not determined.

 
In utero and lactational exposure to TCDD significantly decreased relative DLP weight by 31%on PND 35 (Fig. 1Go). However, by PND 90, DLP weight in TCDD-exposed mice recovered to the level of the vehicle-exposed animals. In addition, unlike the situation with the VP, secretory ductal epithelial structure clearly developed in the DLPs of TCDD-exposed male offspring.

There was a 54% reduction of relative AP weight on PND 35 (Fig. 1Go). Unlike the DLP, there was little recovery in relative AP weight in TCDD-exposed mice, as in utero and lactational TCDD exposure caused a 31% relative weight reduction on PND 90. Despite the weight reductions, glandular structure was present in the AP epithelium on PNDs 35 and 90 after in utero and lactational TCDD exposure.

Androgen concentrations.
To determine whether in utero and lactational TCDD exposure could alter prostate branching morphogenesis by decreasing androgen levels, testicular androgen content and serum androgen concentrations were evaluated. No TCDD-induced decrease in testicular testosterone or 3{alpha}-diol content was found on PNDs 1, 7, and 14 (Fig. 2AGo). The slight TCDD-induced increase in testicular 3{alpha}-diol content on PND 1 was not statistically significant. As expected, serum testosterone levels increased, whereas those of 3{alpha}-diol decreased in vehicle- and TCDD-exposed mice during the post-pubertal interval from PND 35 to 90 (Fig. 2BGo). However, there was no effect of in utero and lactational TCDD exposure on these serum androgen concentrations. The apparent TCDD-induced decrease in serum testosterone and increase in serum 3{alpha}-diol concentration on PND 35 were not significant.



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FIG. 2. Effects of in utero and lactational TCDD exposure on (A) testicular content and (B) serum concentration of testosterone and 5{alpha}-androstane-3{alpha},17ß-diol (3{alpha}-diol) in male offspring on different postnatal days. Each bar represents mean ± SE of 5 vehicle-exposed (open bars), or TCDD-exposed (filled bars) mice.

 
Histological assessment of early postnatal ventral prostate development.
In vehicle-exposed mouse offspring, the development of secretory VP ductal acini could clearly be seen in hematoxylin and eosin-stained cross-sections. By PND 7, islands of VP epithelia surrounded by stroma, some of which already appeared to be lumenized, formed near the ventral surface of the urethra near the bladder neck (Fig. 3Go). By PND 14, virtually all VP ducts were canalized, consistent with the differentiation of secretory luminal cells around the ductal lumens. The secretory lumens were larger on PND 21 than they were at the previous times, and by PND 35, many of them were filled with secretory material. Similar to the VP, the DP and LP also developed acinar structures in vehicle-exposed offspring.



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FIG. 3. Agenesis of VP epithelial development in TCDD-exposed male offspring. Histologic cross-sections of the region between the bladder neck and urethra were stained with hematoxylin and eosin. Cross-sections of 5 µm were cut from a similar depth of the embedded tissue from vehicle- and TCDD-exposed male offspring on each postnatal day. Ventral prostate (VP), dorsal prostate (DP), lateral prostate (LP), urinary bladder (BL), and urethra (UR). Original magnification x40.

 
An entirely different time course of VP development was revealed in similar hematoxylin and eosin-stained cross-sections from TCDD-exposed offspring. No epithelial development was visible in the center of the ventral region between the urethra and the bladder neck (Fig. 3Go). On PND 7, two small loci of apparently undeveloped epithelial cells were present near the right and left ventrolateral aspect of the urethra. These grew larger until PND 21, but there was still no apparent secretory (luminal) epithelial cell differentiation, acini formation, or ductal development. After PND 21, these small, undeveloped tissue blebs tended to diminish in size, and appeared at dissection to be smallest on PND 90.

Branching morphogenesis in the ventral, dorsolateral, and anterior prostate.
Adult prostatic epithelial ductal structures in 90-day-old vehicle- and TCDD-exposed offspring are shown in Figure 4Go. Stroma have been removed from each specimen by gentle digestion with collagenase and by teasing the tissue with a fine forceps. However, this preparation has preserved the tree-like branching structure of the glandular epithelium that is present in all regions of the mouse prostate. Each image shows a single paired lobe from the VP, DP, LP, or AP. The most complicated branching pattern was observed in the VPs of vehicle-exposed male offspring. An average of three main ducts emerge from a central stalk on each side of the VP. These give rise to primary, secondary, and tertiary branches resembling an elm tree (Sugimura et al., 1986Go). As a result, the number of VP ductal tips can be more than 30 times more numerous than the number of main ducts. In contrast to the abundant VP epithelial growth in vehicle-exposed mice, there was no development of epithelial glandular structure in VP obtained from TCDD-exposed male offspring. This illustrates the nearly complete agenesis displayed by the VP epithelium in C57BL/6J mice exposed to a single maternal dose of 5 µg TCDD/kg in utero and via lactation.



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FIG. 4. Effects of in utero and lactational TCDD exposure on epithelial morphogenesis in the ventral, dorsolateral, and anterior prostate of male offspring. Intact prostatic regions were removed from vehicle- and TCDD-exposed mice on PND 90 and separated into their right and left lobes. One lobe from each region was digested briefly with collagenase to remove the stroma, and the individual ducts were gently teased apart with a fine forceps. Representative microscopic whole-mount images of the isolated epithelial structure of each prostate lobe were photographed as in Materials and Methods. Original magnification x9.

 
The DLP consists of two distinct subdivided prostate lobes (the DP and the LP). In vehicle-exposed mice, the LP has an average of 2-3 main ducts per lobe and has a ductal branching pattern similar to that of the VP, whereas the DP averages 20-25 main ducts per lobe that branch into 3-5 ductal tips per main duct in a pattern that resembles a palm tree (Sugimura et al., 1986Go). In comparison to the LP and DP of vehicle-exposed mice, those from TCDD-exposed offspring had smaller branched epithelial structures (Fig. 4Go). The primary branches of DP and LP epithelial ducts in TCDD-exposed offspring appeared to be less elongated than those from vehicle-exposed mice.

In vehicle-exposed mice, each right or left half of the AP epithelium had two main ducts that formed numerous outgrowths of primary branches (Fig. 4Go). Similarly, APs from TCDD-exposed mice also contained two main ducts per side. However, these main ducts were less branched than those from vehicle-exposed mice, and this resulted in the formation of fewer ductal tips compared to vehicle-exposed offspring. In addition, the primary branches in TCDD-exposed AP epithelium were shorter than those in vehicle-exposed mice.

Morphometric analysis of branching.
Digital photographs such as those in Figure 4Go were obtained for each specimen of isolated mouse prostate epithelium. Morphometric analysis was then done to quantify the number of ductal tips, main ducts, and ductal tips per main duct in the VP, DP, LP, and AP of vehicle- and TCDD-exposed offspring. In utero and lactational TCDD exposure significantly decreased the number of epithelial ductal tips in the VP, DP, and AP, beginning within the first few days of life and persisting at least up to PND 90 (Fig. 5Go). In contrast, TCDD exposure did not reduce the number of LP ductal tips until PND 35.



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FIG. 5. Effects of in utero and lactational TCDD exposure on the number of ductal tips in the ventral, dorsal, lateral, and anterior prostate epithelium of male offspring. Photomicrographs similar to those in Figure 3Go were evaluated for the number of ductal tips in each prostate region. Every symbol represents the mean ± SE of 5 vehicle-exposed (open squares), or TCDD-exposed (filled squares) mice. *Significantly different from vehicle-exposed at the indicated time point. **Significantly different from vehicle-exposed at all time points (p < 0.05).

 
The maximal number of ductal tips on one side of the VP in vehicle-exposed mice was approximately 70. In TCDD-exposed VP on the other hand, epithelial glandular structure was completely undeveloped and no ductal tips were present (Fig. 5Go). In utero and lactational TCDD exposure decreased the number of epithelial ductal tips in the DP slightly more than in the LP. The AP was affected by TCDD exposure to an even greater extent than the dorsal and lateral regions. TCDD-exposed APs developed only about one-third the number of ductal tips that were present in the APs of vehicle-exposed mice. In contrast to the effect of TCDD exposure on the other prostate lobes, the decrease in LP ductal tip number was significant only later in development, just prior to puberty on PND 35, and in adulthood on PND 90.

There was a maximum of about 2-3 main ducts per lobe of VP epithelium in vehicle-exposed offspring, whereas VP main duct formation was absent in TCDD-exposed mice (Fig. 6Go). Main ducts were abundant in the DP, where a single lobe from each vehicle-exposed mouse could contain more than 20 main ducts by PND 35. In utero and lactational TCDD exposure resulted in a 40% reduction in the number of DP main ducts, as an average maximum of 12 main ducts developed in each paired lobe of DP from TCDD-exposed animals. This reduction in DP main-duct formation was apparent just after birth, and it persisted through PND 90. In utero and lactational TCDD exposure caused a slightly less extensive reduction in the number of LP main ducts. Unlike the DP, this reduction was not significant until PND 35, but once it occurred it persisted through PND 90. While it was difficult to distinguish the LP from the DP until after PND 1, LP epithelial development appeared to be less inhibited by in utero and lactational TCDD exposure until PND 35. At that time the reduction in ductal tip number corresponded to the reduction in the number of main ducts.



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FIG. 6. Effects of in utero and lactational TCDD exposure on the number of ventral, dorsal, and lateral prostate main ducts of male offspring. Each symbol represents the mean ± SE of 5 vehicle-exposed (open squares), or TCDD-exposed (filled squares) mice. *Significantly different from vehicle-exposed at the indicated time. **Significantly different from vehicle exposed at all times (p < 0.05).

 
Only two main ducts normally develop on the right and left sides of the AP. In contrast to the effect of in utero and lactational TCDD exposure on epithelial main duct formation in the VP, DP, and LP, there was no reduction in the number of main ducts in the AP on any of the PNDs evaluated.

To determine whether in utero and lactational TCDD exposure affected the extent of prostate-branching morphogenesis, the number of ductal tips per main duct in each prostate lobe was evaluated. In the VP, the number of ductal tips per main duct in vehicle-exposed offspring reached a maximum of about 30, early on PND 14, and was unchanged at later times (Fig. 7Go). Since there was no VP epithelium in TCDD-exposed mice, the number of ductal tips per main duct was zero.



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FIG. 7. Effects of in utero and lactational TCDD exposure on the number of ductal tips per main duct of the ventral, dorsal, lateral and anterior prostate of male offspring. Each symbol represents the mean ± SE of 5 vehicle-exposed (open squares), or TCDD-exposed (filled squares) mice. *Significantly different from vehicle-exposed at the indicated time. **Significantly different from vehicle-exposed at all times (p < 0.05).

 
Morphometric analysis demonstrated that in utero and lactational TCDD exposure did not inhibit branching morphogenesis, per se, in the dorsal and lateral prostate regions that was essentially completed by PND 21 in vehicle- and TCDD-exposed offspring. Each DP main duct contained 3–4 ductal tips on PNDs 35 and 90 (Fig. 7Go). Branching morphogenesis was relatively more extensive in the LPs of vehicle- and TCDD-exposed mice where there were 14-16 ductal tips per main duct, regardless of TCDD exposure.

In the APs of vehicle-exposed offspring, the number of ductal tips per main duct increased up to PND 21, so that there were about 30 ductal tips per main duct at all subsequent time points (Fig. 7Go). AP branching morphogenesis in TCDD-exposed mice followed a similar time course, in that it was complete by weaning on PND 21. However, in utero and lactational TCDD exposure caused a significant reduction in the number of AP ductal tips per main duct. This reduction was apparent just after birth, and it persisted through PND 90.

Ductal canalization.
The degree of canalization in the DLP was evaluated by determining the percentage of opened ducts in the proximal, intermediate I, intermediate II, and distal regions of the epithelial ducts on PND 7 (Fig. 8Go). In vehicle-exposed offspring, there was a proximal to distal decrease in the number of opened ducts, so that 57% of ducts were canalized in the proximal region and 20% in the distal region. The percentage of opened ducts in each DLP region in TCDD-exposed offspring was similar to that in vehicle-exposed offspring. Therefore, in utero and lactational TCDD exposure did not inhibit or delay ductal canalization in the DLP.



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FIG. 8. Effects of in utero and lactational TCDD exposure on ductal canalization of dorsolateral prostate of male offspring. The progression of ductal canalization from the proximal to the distal region is shown as the percentage of open ducts in each region on PND 7. Each bar represents mean ± SE of 5 vehicle-exposed (open bars), or TCDD-exposed (filled bars) mice.

 
Since there was such extensive branching of the AP epithelium, it was not possible to analyze ductal canalization in different regions of each individual branched duct. Therefore, canalization was evaluated in the main ducts on one side of the AP from each mouse. Morphometric analysis demonstrated that canalization of these main ducts was completed by PND 7 in vehicle- and TCDD-exposed offspring. Therefore, similar to the DLP, any effect of TCDD exposure on main duct canalization in the AP, if present at earlier times, did not persist to PND 7.

DISCUSSION

TCDD impairs prostate development.
In utero and lactational TCDD exposure inhibits VP, DLP, and AP development in rats (Gray et al., 1995Go, 1997Go; Mably et al., 1992Go; Ohsako et al., 2002Go; Roman et al., 1995Go, 1998Go), ICR mice (Sommer and Peterson, 1997Go; Theobald and Peterson, 1997Go), and C57BL/6 mice (Lin et al., 2002aGo,bGo). In male C57BL/6 mouse offspring, exposure to a single maternal 5 µg TCDD/kg dose administered on GD 13 causes a reduction in prostate weight (Lin et al., 2002aGo,bGo). Effects of in utero and lactational TCDD exposure are prostate lobe-specific and are mediated by the aryl hydrocarbon receptor (AHR) (Lin et al., 2002aGo). The VP is the lobe that is most sensitive to TCDD-induced growth inhibition in wild-type C57BL/6 mice, and this effect of TCDD exposure is absent in AHR knockout mice with a C57BL/6 genetic background (Lin et al., 2002aGo). Reductions in serum androgen concentration and testicular androgen content do not appear to play a role in the TCDD exposure-induced inhibition of prostate growth. Rather, it is more likely that TCDD exerts its influence on prostate development by interaction with the AHR within the nascent prostate. The critical window for the growth-inhibitory effects of TCDD on all three prostatic lobes occurs during the prenatal period (Lin et al., 2002bGo). Nevertheless, lactational TCDD exposure alone can reduce VP, DLP, and AP growth, but the magnitude of these reductions is smaller than those which occur after in utero, or in utero and lactational TCDD exposure (Lin et al., 2002bGo).

Since the branching and canalization processes occur mostly between birth and PND 15, it is possible that postnatal effects of TCDD exposure on prostate weight may be caused by inhibition of these processes. The present study demonstrates that in utero and lactational TCDD exposure inhibits morphological development of each prostate lobe in a lobe-specific manner with respect to TCDD sensitivity and morphological endpoints affected. The results of this study provide insights into the mechanism by which TCDD affects prostate development, because they confirm that the greatest effects of TCDD exposure on the VP, DP, and LP occur prior to birth. However, after a single maternal TCDD dose administered on GD 13, effects on AP development do not become manifest until after birth.

Relevance to risk assessment.
Hydronephrosis has been previously regarded as the most sensitive developmental response elicited by TCDD in the mouse (Peterson et al., 1993Go). A cumulative maternal dose of 5 µg TCDD/kg causes hydronephrosis when mouse offspring are exposed to 0.5 µg TCDD/kg/day on GDs 6 through 15 (Peterson et al., 1993Go). Hydronephrosis also results from a one-time, 3 µg TCDD/kg dose administered to C57BL/6N mice on GD 12 (Couture et al., 1990Go). The single maternal 5 µg TCDD/kg dose used in the present study is similar to the minimal single and cumulative TCDD doses that cause hydronephrosis. Therefore, the adverse effects of perinatal TCDD exposure on prostate development reported in this study, combined with those reported by Lin et al. (2002aGo,bGo), may be relevant to risk assessment.

Ventral prostate.
In utero and lactational TCDD exposure in the C57BL/6J mouse caused permanent agenesis of the VP at a single maternal dose of 5 µg TCDD/kg. This mouse strain is very sensitive to TCDD toxicity in the VP. The same degree of inhibition of VP growth, in response to a maternal dose of 5 µg TCDD/kg, is not seen in ICR mice (Sommer and Peterson, 1997Go; Theobald and Peterson, 1997Go), or in homozygous Ah receptor wild-type (Ahr+/+) mice obtained from an Ah receptor knockout (AhRKO) colony (Lin et al., 2002aGo). These "AhRKO colony" mice were backcrossed with C57BL/6J mice for 9–11 generations before being used by Lin et al.(2002a)Go, at which time a maternal dose of 5 µg TCDD/kg reduced VP weight by more than 80% but failed to cause complete VP epithelial agenesis (Lin et al., 2002aGo) as it did in progeny of authentic C57BL/6J mice. With continued backcrossing, this dose is now causing complete agenesis of the VP (Lin, unpublished results).

As a result of in utero and lactational TCDD exposure, the VP in TCDD-exposed C57BL/6J male offspring never developed any branched or canalized epithelial structure. Microscopic tissue histology revealed no VP ductal development in TCDD-exposed offspring at any time from PND 1–90. When dissected it was not clear that epithelium within the presumed VP structures originated from VP buds. In addition, the right and left "VPs" were not joined in the middle as are the normal VP lobes in vehicle-exposed mice. However, mitigating against the possibility that this tissue is not VP are the facts that the VP and adjacent LP were encapsulated separately, and that acinar structures clearly developed in the adjacent LP, as well as in the DP (Fig. 3Go, PND 14, PND 21, and PND 35). This last point is indicative of the difference between prostate lobes with respect to their TCDD sensitivity.

In utero exposure of C57BL/6J mice to the same 5 µg TCDD/kg dose that was used in this study, causes a complete absence of VP epithelial bud formation in the urogenital sinus (Lin et al., 2001aGo,cGo). While this correlates with the lack of postnatal epithelial development in the VP, it is not known if the TCDD dose response for inhibiting epithelial budding from the ventral urogenital sinus is the same or different from that for inhibiting VP main duct formation or VP branching morphogenesis.

Dorsolateral prostate.
The magnitude of DP and LP developmental inhibition caused by in utero and lactational TCDD exposure was far less than that in the VP. Effects of TCDD exposure on branching morphogenesis in the DP and LP were evaluated separately, because the branching pattern of the two lobes is different. TCDD inhibition of branching morphogenesis followed dissimilar time courses in the DP and LP. Reductions in the numbers of main ducts and ductal tips were observed just after birth in the DP, but were not significant until just before puberty in the LP. In contrast, similar numbers of ductal tips per main duct were found in vehicle- and TCDD-exposed male offspring in each of these regions. Therefore, TCDD inhibited DP and LP main duct formation, but there was little or no TCDD exposure-induced inhibition of branch formation. The reduction in the number of main ducts in the DP and LP may have resulted from a TCDD-induced decrease in the number of dorsolateral buds during gestation (Lin et al., 2001cGo; Roman et al., 1998Go; Timms et al., 2002Go).

Consistent with this interpretation, in utero and lactational TCDD exposure did cause a numerical decrease in the number of LP main ducts at all time points, but this turned out to be statistically significant only at the later times. While there is not a one-to-one correspondence between the number of buds and the number of main ducts, the eventual reduction in LP main duct number may have originated as a decrease in the number of buds, but this remains to be proven. Alternatively, TCDD may have inhibited the maturation of LP buds into main ducts, even though it apparently did not do this in the DP where the number of main ducts was significantly reduced at all times.

The proximal to distal progression of canalization in the DLP was not affected by TCDD exposure when assessed on PND 7. Therefore, TCDD had no lasting effect on epithelial acini formation in the DLP. This indicates that the morphological structure for prostate function develops normally in the DLP of TCDD-exposed offspring. While the DLPs from TCDD-exposed mice were reduced in size on PND 35, they contained histologically normal epithelial ducts in which secretory luminal cells were present.

Anterior prostate.
In utero and lactational exposure of C57BL/6 mice to TCDD inhibited relative AP weight more than that of the DLP, but less than that of the VP (Lin et al., 2002aGo). This result is consistent with the ability of TCDD exposure to inhibit AP and VP weight, but not DLP weight in the less TCDD-responsive ICR mouse (Theobald and Peterson, 1997Go). In addition, the extent of TCDD-induced effects on AP epithelial branching was greater than that in the DLP.

Branched ductal tips were found on AP main ducts in vehicle-exposed male offspring on PND 1. Therefore, the branching processes in the AP appear to begin at about the time of birth. In utero and lactational TCDD exposure caused an apparently permanent significant reduction in the number of AP ductal tips beginning on PND 1. Unlike the DLP, however, the number of AP main ducts was not affected by TCDD exposure. As a result, TCDD exposure appeared to inhibit the formation of new ductal tips during branching morphogenesis. Consistent with the lack of an effect on main duct formation in the mouse AP, in utero TCDD exposure beginning on GD 15 did not have a prior effect on the formation of AP epithelial buds in the rat (Roman et al., 1998Go).

In utero and lactational TCDD exposure did not inhibit canalization of the AP main ducts. Since such TCDD exposure can significantly inhibit secretory function in the AP (Lin et al., 2002aGo), it is possible that the decreases in the number of main ducts, ductal tips, and secretory acini could play a role in this effect.

Possible mechanisms.
Regional differences in the urogenital sinus mesenchyme are responsible for determining lobe-specific ductal morphology, and for the instructive induction of secretory products that are uniquely characteristic of the VP, DLP, and AP (Hayashi et al., 1993Go). Differences between the different prostate lobes with respect to their TCDD sensitivity may have their origins in the particular heterogeneous forms of mesenchymal and/or stromal induction. For example, bone morphogenetic protein 4 (Bmp4) is a mesenchymal factor that causes lobe-specific inhibition of ductal budding and ductal branching morphogenesis in the mouse prostate (Lamm et al., 2001Go). Consistent with the lobe-specific inhibitory effects of Bmp4, its haploinsufficiency results in an increased number of ductal tips in the VP and AP, but not in the DLP. This pattern of responsiveness to Bmp4 is similar to the lobe-specific sensitivity to TCDD exposure during prostate development. Whatever their cause, lobe-specific differences in sensitivity to TCDD exposure can occur during epithelial bud formation, the maturation of elongated buds into main ducts, or during ductal branching morphogenesis. Another member of the TGF-ß superfamily that may be involved in the regulation of prostate branching morphogenesis is activin A (Ball and Risbridger, 2001Go; Cancilla et al., 2001Go). Essentially, nothing is known about the potential interactions that TCDD may have with the actions of Bmp4 and activin A. Podlasek et al. (1999)Go have shown that another gene involved in prostate development is the sonic hedgehog gene, but whether the inhibitory effect of TCDD exposure on ductal development in the various prostate lobes is caused by disruption of sonic hedgehog signaling is unknown. Finally, the epidermal growth factor receptor (EGFR) signal transduction pathway regulates cellular proliferation and differentiation and is affected by TCDD in some tissues, but whether such interactions occur during prostate epithelial development has only begun to be investigated (Rasmussen et al., 2002Go).

The lobe-dependent effects of TCDD exposure could be due to differences in the timing of epithelial bud formation and branching morphogenesis relative to the time of TCDD dosing. In terms of bud formation, AP buds can be found on GD 15, whereas they do not occur until GD 16 in the DP, and GD 17 in the LP (Lin et al., 2002bGo). VP, the last prostate lobe to form epithelial buds, does not do so until GD 17, but unlike the DLP branching, morphogenesis begins before birth in the VP (Sugimura et al., 1986Go). It is interesting that the VP, which buds last and branches first is the lobe most severely affected by in utero and lactational TCDD exposure. In this regard, the previously reported critical window for TCDD exposure in the rat and mouse, GDs 13 through 16, is consistent with the idea that TCDD exerts its effect on the ventral region of the UGS prior to the formation of any distinctive morphologic VP (Clark et al., 1993Go; Lin et al., 2002bGo; Ohsako et al., 2002Go). Critical windows in the other lobes of the mouse prostate suggest that the effects of TCDD begin to occur either during or after the initiation of budding (Lin et al., 2002bGo). As a result of the interplay between the GD 13 initiation of TCDD exposure and the lobe-specific branching patterns, in utero and lactational TCDD exposure can completely block VP duct formation, whereas it inhibits epithelial bud formation and the formation of main ducts in the DLP, and branch formation in the AP.

The underlying mechanism of how prostatic buds become main prostatic ducts is unknown. However, the complete absence of VP main ducts in C57BL/6 mice is consistent with a complete absence of ventral buds in this strain (Lin et al., 2001aGo). In addition, the observed decrease in DP and LP main ducts is consistent with decreased numbers of dorsolateral buds (Lin et al., 2001cGo). In contrast, bud formation in the anterior region of the urogenital sinus, which occurs earlier than that in the dorsolateral region, appears to be unaffected by GD 13 TCDD exposure. Unlike the other lobes, there was no effect of TCDD exposure on the number of AP main ducts, but the subsequent formation of new ductal tips was uniquely inhibited. Therefore, in utero exposure to TCDD eliminated prostate duct formation only in the VP and decreased ductal branching only in the AP. Branching morphogenesis of the main ducts in the VP and AP is initiated earlier than that in the DP and LP (Sugimura et al., 1986Go), suggesting that effects related to the timing of the initial TCDD exposure may have played a role in producing these results.

ACKNOWLEDGMENTS

This work was supported by NIEHS grant ES01332 (R.E.P.) and by an NIEHS Developmental and Molecular Toxicology Center Grant P30 ES09090. We thank Dr. Robert W. Moore for his discussion and ideas with respect to the planning of these experiments.

NOTES

Portions of this research were presented at the 40th Annual Meeting of the Society of Toxicology, March 25–29, 2001, San Francisco, CA. This article is contribution 343 from the Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706.

1 To whom correspondence should be addressed. Fax: (608) 265-3316. E-mail: repeterson{at}pharmacy.wisc.edu. Back

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