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Androgenic regulation of ventral epithelial bud number and pattern in mouse urogenital sinus - PubMed

Comparative Study

Androgenic regulation of ventral epithelial bud number and pattern in mouse urogenital sinus

Sarah H Allgeier et al. Dev Dyn. 2010 Feb.

Abstract

The ventral urogenital sinus (UGS) of control male mice has two rows of 3-4 prostatic buds at birth, but how androgens regulate ventral bud (VB) number and patterning is unclear. VBs in both sexes appeared to be a mixture of prostatic and urethral buds. UGSs from Tfm male and antiandrogen (flutamide)-exposed mice had small VBs, suggesting that initiation of some VBs is androgen independent. Tfm male mice are widely considered completely androgen insensitive yet their UGSs were 5alpha-dihydrotestosterone (DHT)- responsive. VBs (6-8) were generally distributed bimodally on the left-right axis at both minimal and normal male androgen signaling. Yet control females and DHT-exposed Tfm males had 13-14 VBs, whose left-right distribution was fairly uniform. These results suggest that VB number and distribution respond biphasically as androgen signaling increases from minimal, and that androgens regulate bud specification. Complete VB agenesis by the selective budding inhibitor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) required high androgen signaling.

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Figures

Fig. 1
Fig. 1

Scanning electron micrographs of UGS epithelium (UGE) from control wild-type male (A), control wild-type female (B), and control Tfm male (C) mice on the day of birth. All images are lateral views, taken with samples in the same orientation and at the same nominal magnification; the craniocaudal axis is as indicated. The ventral budding region is contained within the white dotted rectangle. Small buds on the ventral UGE surface are tinted orange, while the large ventral prostatic buds found in control males are tinted blue. BN: Bladder neck; ED: ejaculatory duct; PU: pelvic urethra; VAP: vaginal attachment point.

Fig. 2
Fig. 2

The degree of androgen signaling appears to regulate ventral UGS bud number and distribution. A–C: SEM images of ventral UGE from control mice on the day of birth. Samples are from a wild-type male (A), wild-type female (B), and Tfm male (C). Ventral prostatic buds are tinted blue, while small ventral buds are tinted orange. One prostatic bud broke off during sample preparation; its base is outlined in black. All images are ventral views, taken with samples in the same orientation and at the same nominal magnification. D–F: Bud count data and histograms of ventral bud distribution on the left-right axis. Histograms were prepared by plotting the total number of ventral buds, from all replicate samples, present in each of 20 equal segments along the left-right UGE axis. For wild-type males (D), the blue histogram is for ventral prostatic buds and the white histogram is for the markedly smaller ventral buds. Ventral bud counts are shown as mean ± SE, with the number of replicate samples in parentheses. The asterisk indicates a mean bud number significantly different from the wild-type male group (p < 0.05).

Fig. 3
Fig. 3

Nkx3.1 expression does not distinguish prostatic buds from urethral buds. Photomicrographs (lateral views, bladder neck to the right) of UGE from a control wild-type male (A) and a control wild-type female (B) on the day of birth. Nkx3.1 expression (dark blue staining) was determined by in situ hybridization. The ventral budding regions are contained within the black dotted rectangles. PU: pelvic urethra.

Fig. 4
Fig. 4

In utero DHT exposure reduces ventral UGS bud number and masculinizes their distribution pattern in wild-type females, while increasing ventral bud number in Tfm males. A,B: SEM images of ventral UGE from DHT-exposed mice on the day of birth. Samples are from a wild-type female (A) and a Tfm male (B). Ventral buds are tinted orange. Both images are ventral views, taken with samples in similar orientations and at the same nominal magnification. C,D: Bud count data and histograms of ventral bud distribution on the left-right axis. Histograms were prepared by plotting the total number of ventral buds, from all replicate samples, present in each of 20 equal segments along the left-right UGE axis. For purposes of comparison, ventral bud distribution histograms from control as well as DHT-exposed mice are shown. Ventral bud numbers for the DHT-exposed groups are shown as mean ± SE, with the number of replicate samples in parentheses. The dagger indicates a mean bud number significantly different from the corresponding control group (p < 0.05).

Fig. 5
Fig. 5

UGSs from Tfm male mice are DHT responsive. E14.5 UGSs from wild-type males (positive control) and Tfm males were cultured for three days in media containing 0, 1, 10, 100, or 1000 nM DHT. Mesenchyme was then removed and UGE was visualized by SEM. A: Representative samples of wild-type male and Tfm male UGEs after incubation with vehicle (left) or 1000 nM DHT (right). Prostatic buds are highlighted in blue-green. PU: pelvic urethra, BN: bladder neck. B: Prostatic bud counts are presented as the mean ± SE (n ≥ 3). An asterisk indicates that a mean is significantly different from the corresponding control group (p < 0.05).

Fig. 6
Fig. 6

In utero flutamide exposure inhibits bud elongation but has little if any effect on bud specification or initiation in the ventral UGS of wild-type males. A,B: SEM images of ventral UGE from a control (A) and a flutamide-exposed (B) mouse on the day of birth. Ventral buds are tinted orange. One prostatic bud broke off during sample preparation; its base is outlined in black. Both images are ventral views, taken with samples in the same orientation and at the same nominal magnification. C: Bud count data and histograms of ventral bud distribution on the left-right axis. Histograms were prepared by plotting the total number of ventral buds, from all replicate samples, present in each of 20 equal segments along the left-right UGE axis. For purposes of comparison, ventral bud distribution histograms from both control and flutamide-exposed mice are shown. Ventral bud numbers for the flutamide-exposed group are shown as mean ± SE, with the number of replicate samples used to determine bud number in parentheses. Bud distribution was plotted from fewer samples (n = 6) because 2 UGEs were not oriented favorably.

Fig. 7
Fig. 7

The selective UGS budding inhibitor TCDD prevents buds from forming in the ventral UGS of wild-type males. Dams were dosed orally with vehicle (A) or TCDD (B; 5 µg/kg) on E15.5, and UGSs were collected on the day of birth. SEM images of ventral UGE are shown. Ventral prostatic buds are tinted blue, while the small ventral bud is tinted orange. One prostatic bud broke off during sample preparation; its base is outlined in black. Both images are ventral views, taken with samples in similar orientations (cranial at the top and caudal at the bottom) and at the same nominal magnification. Ventral bud numbers are shown as mean ± SE, with the number of replicate samples in parentheses. The asterisk indicates a mean bud number significantly different from the control group (p < 0.05).

Fig. 8
Fig. 8

DHT sensitizes the ventral UGS to budding inhibition by TCDD. Some dams were implanted with sustained-release DHT pellets on E13.5, and all dams were dosed orally with vehicle or TCDD (5 µg/kg) on E15.5. UGSs were collected on the day of birth. A–F: SEM images of ventral UGE. Samples are from wild-type females exposed to vehicle/placebo (A), TCDD (B), or DHT + TCDD (C), and from Tfm males exposed to vehicle/placebo (D), TCDD (E), or DHT + TCDD (F). All images are ventral views, taken with samples in the same orientation (cranial at the top and caudal at the bottom) and at the same nominal magnification. Ventral buds are tinted orange. G: Ventral bud counts (mean ± SE). An asterisk indicates a significant difference from the control group, and a dagger indicates a significant difference from the TCDD alone group (p < 0.05). There were 3–7 replicates per group.

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References

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