2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a global environmental contaminant and the prototypical ligand for investigating aryl hydrocarbon receptor (AHR)-mediated toxicity. Environmental exposure to TCDD results in developmental and reproductive toxicity in fish, birds and mammals. To resolve the ecotoxicological relevance and human health risks posed by exposure to dioxin-like AHR agonists, a vertebrate model is needed that allows for toxicity studies at various levels of biological organization, assesses adverse reproductive and developmental effects and establishes appropriate integrative correlations between different levels of effects. Here we describe the reproductive and developmental toxicity of TCDD in feral fish species and summarize how using the zebrafish model to investigate TCDD toxicity has enabled us to characterize the AHR signaling in fish and to better understand how dioxin-like chemicals induce toxicity. We propose that such studies can be used to predict the risks that AHR ligands pose to feral fish populations and provide a platform for integrating risk assessments for both ecologically relevant organisms and humans.

Natural luteolysis involves multiple pulses of prostaglandin F2alpha (PGF) released by the nonpregnant uterus. This study investigated expression of 18 genes from five distinct pathways, following multiple low-dose pulses of PGF. Cows on Day 9 of the estrous cycle received four intrauterine infusions of 0.25 ml of phosphate-buffered saline (PBS) or PGF (0.5 mg of PGF in 0.25 ml of PBS) at 6-h intervals. A luteal biopsy sample was collected 30 min after each PBS or PGF infusion. There were four treatment groups: Control (n = 5; 4 PBS infusions), 4XPGF (4 PGF infusions; n = 5), 2XPGF-non-regressed (2 PGF infusions; n = 5; PGF-PBS-PGF-PBS; no regression after treatments), and 2XPGF-regressed (PGF-PBS-PGF-PBS; regression after treatments; n = 5). As expected, the first PGF pulse increased mRNA for the immediate early genes JUN, FOS, NR4A1, and EGR1 but unexpectedly also increased mRNA for steroidogenic (STAR) and angiogenic (VEGFA) pathways. The second PGF pulse induced immediate early genes and genes related to immune system activation (IL1B, FAS, FASLG, IL8). However, mRNA for VEGFA and STAR were decreased by the second PGF infusion. After the third and fourth PGF pulses, a distinctly luteolytic pattern of gene expression was evident, with inhibition of steroidogenic and angiogenic pathways, whereas, there was induction of pathways for immune system activation and production of PGF. The pattern of PGF-induced gene expression was similar in corpus luteum not destined for luteolysis (2X-non-regressed) after the first PGF pulse but was very distinct after the second PGF pulse. Thus, although the initial PGF pulse induced mRNA for many pathways, the second and later pulses of PGF appear to have set the distinct pattern of gene expression that result in luteolysis.

To use the zebrafish (Danio rerio) as a model to study 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) developmental toxicity, it is essential to know which proteins are involved in mediating toxicity. Previous work has identified zfAHR2 as the receptor that binds TCDD mediating downstream responses. Although zfARNT2b can form a functional heterodimer with zfAHR2 in vitro, zfarnt2 null mutants show no protection against endpoints of TCDD developmental toxicity, demonstrating that zfARNT2b cannot be the physiological dimerization partner for zfAHR2 mediating responses to TCDD in zebrafish embryos. The purpose of the current study was to identify an alternate dimerization partner(s) for zfAHR2 that may function to mediate TCDD developmental toxicity. By searching zebrafish genomic sequence and using the polymerase chain reaction-based rapid amplification of cDNA ends technique, three forms of cDNA that seem to be alternate mRNA splice variants of a zebrafish homolog of ARNT1 were detected. Analysis of the zfARNT1 proteins in vitro demonstrates that the two longest forms of zfARNT1, zfARNT1b and zfARNT1c, can form functional heterodimers with zfAHR2. However, the shortest form, zfARNT1a, seems to be nonfunctional with zfAHR2 in vitro. To determine whether a zfARNT1 protein functions with zfAHR2 in vivo, a morpholino targeted against the 5Ј end of zfARNT1 (zfarnt1-MO) was used. Injection of the zfarnt1-MO before TCDD treatment significantly decreases the induction of zfCYP1A mRNA and protein. In addition, zfarnt1 morphants show complete protection against TCDD-induced pericardial edema and show partial protection against reduced blood flow and craniofacial malformations caused by TCDD, demonstrating the role of zfARNT1 proteins in mediating these responses.

Prostate development is influenced by β-catenin signaling, but it is unclear which β-catenin activators are involved, where they are synthesized, and whether their mRNA abundance is influenced by androgens. We identified WNT/β-catenin-responsive β-galactosidase activity in the lower urogenital tract (LUT) of transgenic reporter mice, but β-galactosidase activity differed among the four mouse strains we examined. We used in situ hybridization to compare patterns of Wnts, r-spondins (Rspos, co-activators of β-catenin signaling), β-catenin-responsive mRNAs, and an androgen receptor-responsive mRNA in wild type fetal male, fetal female, and neonatal male LUT. Most Wnt and Rspo mRNAs were present in LUT during prostate development. Sexually dimorphic expression patterns were observed for WNT/β-catenin-responsive genes, and for Wnt2b, Wnt4, Wnt7a, Wnt9b, Wnt10b, Wnt11, Wnt16, and Rspo3 mRNAs. These results reveal sexual differences in WNT/β-catenin signaling in fetal LUT, supporting the idea that this pathway may be directly or indirectly responsive to androgens during prostate ductal development.

Epithelial-stromal interactions in the lower urogenital tract (LUT) are integral to prostatic and seminal vesicle development in males, vaginal and uterine development in females, and urethral development in both sexes. Gene expression profiling of isolated LUT stroma and epithelium has unraveled mechanisms of LUT development, but such studies are confounded by heterogeneous and ill-defined cell sub-populations contained within each tissue compartment. We used in situ hybridization to synthesize a high-resolution molecular atlas of 17 days post coitus fetal mouse LUT. We identified mRNAs that mark selective cell populations of the seminal vesicle, ejaculatory duct, prostate, urethra and vagina, subdividing these tissues into 16 stromal and 8 epithelial sub-compartments. These results provide a powerful tool for mapping LUT gene expression patterns and also reveal previously uncharacterized sub-compartments that may play mechanistic roles in LUT development of which we were previously unaware.