A mechanism linking perinatal 2,3,7,8 tetrachlorodibenzo-p-dioxin exposure to lower urinary tract dysfunction in adulthood

Abstract

Benign prostatic hyperplasia/lower urinary tract dysfunction (LUTD) affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging paradigms about when this disease process begins. We delivered a single dose of a widespread environmental contaminant present in the serum of most Americans [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 1 µg/kg], and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues, and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood.

Keywords: Artemin; Dioxin; Lower urinary tract dysfunction; Noradrenergic; Prostate; Smooth muscle.

Fig. 1.

A single dose of TCDD, delivered to male mice during development, changes urodynamic voiding behavior in adulthood. C57BL/6J mouse fetuses were exposed to a single dose of TCDD [1 µg/kg maternal dose, orally (po)] or vehicle (5 ml/kg corn oil, po, control) on E13 and were evaluated by cystometry between P90 and P98. Mice were anesthetized, a cystostomy catheter was passed through the bladder dome, and saline was infused at a rate of 1.5 ml/h while continuously measuring bladder pressure in response to filling and emptying. The left panel shows representative pressure versus time traces (top, vehicle; bottom, TCDD), with each peak indicating bladder contraction during a voiding event. The y-axes indicate changes in intravesical pressure (mmHg). Three to five consecutive voids were used to quantify mean responses. In utero and lactational TCDD exposure decreases the intervoid interval. Results are from five mice per group, representing at least three independent litters. Scale bars: 2.5 min. The intervoid intervals are quantified in the right panel. Unpaired Student's t-test was used to identify differences between groups after a log transformation to normalize distribution. Results are from five mice per group, representing at least three independent litters.

Fig. 2.

A single dose of TCDD, delivered to male mice during the perinatal period, increases adult prostatic smooth muscle responsiveness to field stimulation. E13 Myh11^cre/+;GCaMP5g/+ male mice were exposed to TCDD (1 µg/kg maternal dose, po) or vehicle (5 ml/kg, po, control). For A-D, prostates were collected between P90 and P98 and placed in a perfusion chamber with 37°C HEPES buffer. (A) Representative fluorescent images were captured at baseline and (contracted) 40 s after a 0.1 Hz stimulus. (B,C) In utero and lactational TCDD exposure of Myh11^cre/+;GCaMP5g/+ mice increases the percentage of prostatic ducts that elicit a change in diameter (contraction) in response to a 0.1 Hz stimulus (B) and reduces the time needed to reach peak fluorescence (C). (D-F) In separate experiments, field stimulation was applied to tensioned adult dorsal prostate tissue incubated in 37°C Krebs buffer. (D) In utero and lactational TCDD exposure increases the maximum response to 0.1 Hz stimulation compared to vehicle-exposed dorsal prostate tissue. (E) In utero and lactational TCDD exposure increases the contractile response to 0.1, 10 and 60 Hz stimuli compared to control with a repeated measures two-way ANOVA (*P≤0.05). (F) Pretreatment with 10 µM guanethidine decreased the maximal response of TCDD-exposed tissues to field stimulation. The peak amplitude at 10 Hz is shown as an example indicating that axons are responsible for TCDD-induced sensitization. Results are from six to ten mice per group, representing at least three independent litters. Unpaired Student's t-test was used to identify differences between groups.

Fig. 3.

A single perinatal dose of TCDD increases noradrenergic axon density in the prostatic periductal region beginning in the fetal period and persisting into adulthood. (A-C) Male mice were exposed to TCDD (1 µg/kg, po) or vehicle (5 ml/kg, po, control) at E13.5, and noradrenergic axon density was assessed immunohistochemically in a dorsal prostate tissue sections (three non-serial sections per animal) at P90 (seven mice per group) (A), P9 (four to five mice per group) (B) and E17.5 (five mice per group) (C). Results are from at least three independent litters per group. An antibody against tyrosine hydroxylase (TH; green) was used to identify noradrenergic axons and an antibody against cadherin 1 (CDH1; magenta) was used to localize prostatic epithelium. TH⁺ axons were quantified in the area extending 10 µm from prostatic epithelium. Vehicle groups at P9-P90 differ in density due to different tissue structures in neonatal versus adult prostate. Scale bars: 50 µm for P50 and P9, 100 µm for E17.5. Unpaired Student's t-test was used to identify differences between groups, and P≤0.05 was considered significant. Results are from four to seven mice per group, representing at least three independent litters.

Fig. 4.

In utero TCDD exposure, coinciding with the beginning of prostatic neuroanatomical development, increases mRNA abundance of the GDNF member Artn in the fetal prostate. Male mice were exposed to TCDD (5 µg/kg, po maternal dose) or vehicle (5 ml/kg, po maternal dose, control) on E13.5, and urogenital sinus (UGS) epithelium was collected for RNA-seq on E16.75. (A) Volcano plot showing significantly up- and downregulated genes from TCDD exposure. Red indicates upregulated genes, blue indicates downregulated genes, and Artn is identified in black (n=3-4, FDR-adjusted P≤0.05). (B) The top 20 differentially expressed genes, ordered by FDR-adjusted P-value, included Artn. (C) In situ hybridization localized Artn mRNA (brown staining) to UGS epithelium and periprostatic bud mesenchyme of E17.5 (control) C57BL/6J male fetuses. BL, bladder; SV, seminal vesicle; arrowhead, prostatic bud; the dashed line indicates the boundary between UGS epithelium and mesenchyme. (D) Real-time RT-PCR indicated that a single maternal dose of TCDD (1 µg/kg, po) on E13.5 significantly increased Artn mRNA abundance in the E17.5 male UGS compared to vehicle. Scale bar: 250 µm. Unpaired Student's t-test was used to assess differences in RT-PCR data, and differential expression of genes was determined using functions from edgeR, the Cox–Reid profile-adjusted likelihood method to calculate dispersions, empirical Bayes quasi-likelihood F-tests, and a version of the t-tests relative to a threshold (TREAT) method. Results are from eight to 11 male fetuses per group, representing three independent litters, and P≤0.05 was considered significant.

Fig. 5.

Proposed mechanism underlying TCDD-induced urinary dysfunction in mice. TCDD increases the abundance of the neurotrophic factor Artn in the developing prostate and enhances noradrenergic axon growth. These TCDD actions increase prostatic noradrenergic density in the fetus, neonate and adult, and lead to prostatic smooth muscle hyperactivity and urinary dysfunction in adulthood.