Region-Specific Inhibition of Prostatic Epithelial Bud Formation in the Urogenital Sinus of C57BL/6 Mice Exposed in Utero to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Abstract

In utero 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure causes abnormal ventral, dorsolateral, and anterior prostate development in wild-type but not aryl hydrocarbon receptor (AhR) null mutant C57BL/6 mice. Experiments have now been conducted to test the hypothesis that TCDD causes an AhR-dependent inhibition of the earliest visible stage of prostate development, the formation of prostatic buds by urogenital sinus (UGS) epithelium. A novel method for viewing budding was developed that uses scanning electron microscopy of isolated UGS epithelium instead of three-dimensional reconstruction of serial histological sections of intact UGS. In the initial experiment, the time course for prostatic epithelial bud formation in vehicle- and TCDD-exposed wild-type C57BL/6J mice was determined. A single maternal dose of TCDD (5 mug/kg) on gestation day 13 delayed the appearance of dorsal, lateral, and anterior buds by about one day, reduced dorsolateral bud number, and prevented ventral buds from forming. No such effects were seen in TCDD-exposed AhR null mutant fetuses, while AhR null mutation, alone, had no detectable effect on budding. Treatment of wild-type dams with sufficient 5alpha-dihydrotestosterone (DHT) to masculinize female fetuses failed to protect against the inhibition of budding caused by TCDD. These results demonstrate that in utero TCDD exposure causes an AhR-dependent inhibition of prostatic epithelial bud formation commensurate with its inhibitory effects on ventral and dorsolateral prostate development, and that the inhibition of budding is not due to insufficient DHT. Inhibited bud formation appears to be the primary cause of abnormal prostate development in TCDD-exposed mice.

Retinoic acid induces prostatic bud formation

“…Since transgenic loss of Hoxd13 has been shown to decrease the number of dorsolateral buds (Podlasek et al, 1997), it seems quite plausible that RA-induced changes in Hox gene expression and UGS patterning could result in additional or expanded domains of budding and supernumerary bud formation. Lin et al (2003) demonstrated the C67BL/6J male mouse UGS reproducibly displays 8 ventral buds, an average of 52 dorsolateral buds and 5-6 anterior buds on E18.5, but since the three-dimensional UGS shape is distorted during incubation in UGS organ culture, the regional identity of buds cannot be reliably determined. Therefore, it was not possible to determine from the present study whether the induction of supernumerary prostatic buds by RA was restricted to specific lobes, interlobular boundary regions, or a generalized effect on the UGS as a whole.…”

“…UGS epithelium was then fixed in 2.5% glutaraldehyde and evaluated by scanning electron microscopy (SEM). SEM was performed as previously described (Lin et al, 2003). UGS specimens were imaged from 4 angles to visualize budding across the entire UGS surface.…”

“…Reverse transcription was carried out for 1 hr at 42°C in 1ϫ first-stand buffer containing 500 ng total RNA, 0.5 M dNTPs, 150 ng random hexamers, 5 mM DTT, and 200 U Superscript II reverse transcriptase (Invitrogen). PCR and real-time PCR reactions were assembled as described previously (Lin et al, 2003) and thermocylcing was performed using the GeneAmp 9600 (Perkin Elmer, Norkwalk, CT) or Roche LightCycler (Roche Molecular Biochemicals, Indianapolis, IN). PCR primers were as follows: Cyp26b1: 5Ј-ATGACTGGGT-GGAAGACGAG-3Ј and 5Ј-TGTTCT-GTGTGGCTGGTGAG-3Ј, Bmp4: 5Ј-AACTGCCGTCGCCATTCACTATAC-3Ј and 5Ј-TGCACAATGGCATGGTTG-GTTGAG-3Ј, Nr2f2: 5Ј-GGGCTGGT-GACAGAGGTGA-3Ј and 5Ј-GG-GAAGCTGAGGGTCAGATAAAG-3Ј, and Ppia: 5Ј-TCTCTCCGTAGATG-GACCTG-3Ј and 5Ј-ATCACGGC-CGATGACGAGCC-3Ј, Rara: 5Ј-CGACCAGATCACCCTCCTC-3Ј and 5Ј-CAGTCCAGTCTCAGCATCGTC-3Ј, Rarb: 5Ј-GTAGCCCAGTGTCCT-TGCTG-3Ј and 5Ј-CTTGAGTCT-GTCAACCACTCATTC-3Ј, Rarg: 5Ј-GCAGGGCCCTCCTACACTAC-3Ј and 5Ј-GCCTGCAGGAATCTTATT-TGG-3Ј, Rxra: 5Ј-TCCTGTGGT-CAGCTCCAGTC-3Ј and 5Ј-AGAAG-CTGGGTGCAGGAAAG-3Ј, Rxrb: 5Ј-CATTTGGCCTCACTCCCTTC-3Ј and 5Ј-AGAGCAATGGGTTCCTCCACЈ, Rxrg: 5Ј-GCCCTACCCTGCACACT-CTC-3Ј and 5Ј-TGGCCACTCTTC-CCAAGAAC-3Ј, Shh: 5Ј-GTGGAAG-CAGGTTTCGACTG-3Ј and 5Ј-GGTC-CAGGAAGGTGAGGAAG-3Ј.…”

“…Prostatic buds emerge in a spatially defined pattern that establishes the position of mature dorsal, lateral, ventral, and anterior prostate lobes (Cunha et al, 1987). Buds that give rise to dorsal and anterior prostate ducts are the first to form in the mouse UGS (E16.5) and lateral and ventral prostatic buds form about one day later (Lin et al, 2003). A finite quantity of prostatic buds are formed in each region of the UGS during in utero mouse development: there is an average of 52 dorsal prostate buds, 5-6 anterior buds, 6 lateral buds, and 8 ventral buds (Lin et al, 2003).…”

“…Buds that give rise to dorsal and anterior prostate ducts are the first to form in the mouse UGS (E16.5) and lateral and ventral prostatic buds form about one day later (Lin et al, 2003). A finite quantity of prostatic buds are formed in each region of the UGS during in utero mouse development: there is an average of 52 dorsal prostate buds, 5-6 anterior buds, 6 lateral buds, and 8 ventral buds (Lin et al, 2003). Most of the prostate buds that form during the initial stage of prostate development continue to elongate and then undergo postnatal branching morphogenesis, canalization, and differentiation.…”