Vinclozolin exposure in utero induces postpubertal prostatitis and reduces sperm production via a reversible hormone-regulated mechanism

Abstract

Vinclozolin is an endocrine-disrupting chemical (EDC) that binds with high affinity to the androgen receptor (AR) and blocks the action of gonadal hormones on male reproductive organs. An alternative mechanism of action of Vinclozolin involves transgenerational effects on the male reproductive tract. We previously reported in utero Vinclozolin exposure-induced prostatitis (prostate inflammation) in postpubertal rats concurrent with down-regulation of AR and increased nuclear factor-kappaB activation. We postulated the male reproductive abnormalities induced by in utero Vinclozolin exposure could be reversed by testosterone supplementation, in contrast to the permanent modifications involving DNA methyltransferases (Dnmts) described by others. To test this hypothesis, we administered high-dose testosterone at puberty to Vinclozolin-treated rats and determined the effect on anogenital distance (AGD); testicular germ cell apoptosis, concentration of elongated spermatids, and the onset of prostatitis. Concurrently we examined Dnmt1, -3A, -3B, and -3L mRNA expression. Consistent with previous reports, in utero exposure to Vinclozolin significantly reduced AGD, increased testicular germ cell apoptosis 3-fold, reduced elongated spermatid number by 40%, and induced postpubertal prostatitis in 100% of exposed males. Administration of high-dose testosterone (25 mg/kg) at puberty normalized AGD, reduced germ cell apoptosis, and restored elongated spermatid number. Testosterone restored AR and nuclear factor-kappaB expression in the prostate and abolished Vinclozolin-induced prostatitis. Altered Dnmt expression was evident with in utero Vinclozolin exposure and was not normalized after testosterone treatment. These data demonstrate in utero Vinclozolin-induced male reproductive tract abnormalities are AR mediated and reversible and involve a mechanism independent of Dnmt expression.

Figure 1

Gross analysis of male offspring exposed in utero to control (open bar) or Vinclozolin (solid bar) and exposed at puberty to 0, 5, or 25 mg testosterone. *, Body weight denotes significance vs. 0 mg testosterone-treated groups (A) and AGD (B). *, Significance vs. matched testosterone-treated control group (P < 0.05, mean ± sem).

Figure 2

Germ cell apoptosis (A), elongated spermatid content (B) in the testis, and VP weight (C) of male offspring exposed in utero to control (open bar) or Vinclozolin (solid bar) and exposed at puberty to 0, 5, or 25 mg testosterone. Significant difference in the proportion of apoptotic cells between control (open bar) and Vinclozolin (solid bar)-treated testis per 10 × 10⁶ μm² were evident and testosterone treatment (25 mg) at puberty normalized germ cell apoptosis (A). A significant reduction in elongated spermatid content was observed in Vinclozolin-treated animals (solid bar) compared with control (open bar). VP weight in male offspring exposed in utero to control (open bar) or Vinclozolin (solid bar) (B) and exposed at puberty to 0, 5, or 25 mg testosterone (C) is shown. *, P = 0.05 vs. matched testosterone-treated control group. Mean ± sem

Figure 3

H&E of prostate specimens exposed in utero to control (A and B) or Vinclozolin and 5 mg (C and D) or 25 mg testosterone at puberty (E and F). Prostate inflammation (←) was evident in prostates exposed in utero to Vinclozolin and 0 or 5 mg testosterone (B). Bar, 20 μm .

Figure 4

Unbiased stereological analysis of the percent of epithelial cells that were AR (A) and nuclear NFκB localization (B) in tissues exposed in utero to control (open bar) or Vinclozolin (solid bar) and 0, 5, or 25 mg testosterone at puberty. A, Compared with control counterparts, epithelial expression of AR was significantly decreased in all tissues except after 25 mg pubertal testosterone treatment. B, Compared with control counterparts, nuclear NF-κB immunolocalization was increased in all specimens except after 25 mg pubertal testosterone treatment. *, Significance vs. matched testosterone-treated control groups (P < 0.05). Mean ± sem (n = 5)..

Figure 5

Dnmt mRNA and protein expression in the VP. Relative mRNA expression after in utero Vinclozolin exposure and exposed at puberty to 0, 5, or 25 mg testosterone, Dnmt1 (A), Dnmt3A (B), Dnmt3B (C), and Dnmt3L (D) normalized to β-actin. Relative protein expression after in utero Vinclozolin exposure and exposed at puberty to 0 or 25 mg testosterone, Dnmt1 (E), Dnmt3A (F), Dnmt3B (G), and Dnmt3L (H) was normalized to GAPDH. Control (open bar) and Vinclozolin (solid bar) (mean ± sem, n = 4–5 in duplicate). *, Significance vs. matched testosterone-treated control group (P < 0.05).

Figure 6

DNA methyltransferase mRNA and protein expression in the testis. Relative mRNA expression following in utero Vinclozolin exposure and exposed at puberty to 0, 5, or 25 mg testosterone, Dnmt1 (A), Dnmt3A (B), Dnmt3B (C), and Dnmt3L (D) normalized to β-Actin. Relative protein expression following in utero Vinclozolin exposure and exposed at puberty to 0 or 25 mg testosterone, Dnmt1 (E), Dnmt3A (F), Dnmt3B (G), and Dnmt3L (H) normalized to GAPDH. Control (open bar) and Vinclozolin (solid bar). Mean ± SEM (n = 4-5 in duplicate). *, Significance vs. matched testosterone-treated control group (P < 0.05).