Effects of birth trauma and estrogen on urethral elastic fibers and elastin expression

Abstract

Objectives: To investigate the effects of birth trauma and estrogen on urethral elastic fibers and elastin expression.

Methods: Pregnant rats were subjected to sham operation (Delivery-only), DVDO (delivery, vaginal distension and ovariectomy), or DVDO + E₂ (estrogen). At 2, 4, 8, or 12 weeks, their urethras were harvested for elastic fiber staining and reverse transcription-polymerase chain reaction analysis. Urethral cells were treated with transforming growth factor- β1 (TGFβ1) and/or estrogen and analyzed for elastin mRNA expression. Urethral cells were also examined for the activities of Smad1- and Smad3/4-responsive elements in response to TGFβ1 and estrogen.

Results: At 8 weeks post-treatment, the urethras of DVDO rats had fewer and shorter elastic fibers when compared with Delivery-only rats, and those of DVDO + E₂ rats had fewer and shorter elastic fibers when compared with DVDO rats. Elastin mRNA was expressed at low levels in Delivery-only rats and at increasingly higher levels in DVDO rats at 2, 4, and 8 weeks but at sharply lower levels in DVDO + E₂ rats when compared with DVDO rats at 8 weeks. Urethral cells expressed increasingly higher levels of elastin mRNA in response to increasing concentrations of TGFβ1 up to 1 ng/mL. At this TGFβ1 concentration, urethral cells expressed significantly lower levels of elastin mRNA when treated with estrogen before or after TGFβ1 treatment. Both Smad1- and Smad3/4-responsive elements were activated by TGFβ1 and such activation was suppressed by estrogen.

Conclusions: Birth trauma appears to activate urethral elastin expression via TGFβ1 signaling. Estrogen interferes with this signaling, resulting in improper assembly of elastic fibers.

Figure 1

Effects of DVDO and estrogen on urethral elastic fibers. Female rats were sham-operated (Delivery-only), DVDO-treated, or DVDO+E2-treated; 8 weeks later, their urethras were stained for elastic fibers. A. In the submucosa of Delivery-only rats, elastic fibers were scanty and short; in the smooth muscle, they were abundant; in the striated muscle, they were not as abundant but were longer. In DVDO-treated rats, elastic fibers were fewer and shorter in all three compartments; in DVDO+E2-treated rats, elastic fibers were fewest and shortest in all three compartments. Images in the large panels were originally photographed at 400x; those in the inserts at 1000x. Arrows point to representative elastic fibers or fragments. B. The elastic fibers in all three compartments were quantified by counting the number of pixels of positively stained elastic fibers in 5 high-power fields (HPF) per tissue sample. Statistical analysis of the average of the number of pixels/HPF of each treatment group (n=6 per group) indicated significant differences (* p<0.05), between the DVDO+E2 and DVDO groups.

Figure 2

Effects of DVDO and estrogen on urethral elastin mRNA expression. Female rats were sham-operated (Delivery-only; n=6 at each time point), DVDO-treated (n=6 at each time point), or DVDO+E2-treated (n=6 at the 8-week time point); 2, 4, 8, and 12 weeks later, their urethras were examined by RT-PCR for elastin mRNA expression. The PCR products were visualized by gel electrophoresis followed by densitometric and statistical analyses. “Relative expression” is the ratio of elastin versus β-actin expression. * indicates significant difference (p<0.05) compared to 8-week DVDO. For ease of presentation, the 12-week data, which was similar to the 8-week data, is not shown.

Figure 3

Effects of TGF-β1 and estrogen on urethral elastin mRNA expression. Urethral cells were isolated from female rats, treated with TGF-β1 and/or estrogen, and analyzed by real-time PCR. A. Dosage response of elastin mRNA expression to TGF-β1. “Relative expression” is the ratio of elastin versus GAPDH expression. B. Elastin mRNA expression in urethral cells treated with 1 ng/ml TGF-β1 (TGF), 10 nM estrogen (E2), 10 nM estrogen for 1 h and then 1 ng/ml TGF-β1 for 16 h (E2/TGF), or 1 ng/ ml TGF-β1 for 1 h and then 10 nM estrogen for 16 h (TGF/E2). “Relative expression” is the ratio of elastin versus GAPDH expression. * indicates significant difference (p<0.05) compared to TGF-β1 alone. The data are the average of 3 independent experiments.

Figure 4

Effects of TGF-β1 and estrogen on Smad responsive elements. Urethral cells were isolated from female rats, transfected with Smad1 or Smad3/4 responsive elements, treated with TGF-β1 and/or estrogen, and analyzed by luciferase assay. “TGF” denotes treatment with 1 ng/ml TGF-β1; “E2” denotes treatment with 10 nM estrogen; “TGF/E2” denotes treatment with 1 ng/ ml TGF-β1 for 1 h and then 10 nM estrogen for 16 h. “Relative activity” is the ratio of firefly luciferase activity (derived from Smad responsive element) versus Renilla luciferase activity (internal control). * indicates significant difference (p<0.05) compared to TGF-β1 alone. The data are the average of 3 independent experiments.