Molecular and functional characterization of a novel aryl hydrocarbon receptor isoform, AHR1ß, in the chicken (Gallus gallus).

Dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause toxic effects through activation of the aryl hydrocarbon receptor (AHR)-mediated signaling pathway. Our previous studies have investigated the function of 2 AHR isoforms (AHR1 and AHR2) in avian species and identified a third AHR in the chicken (Gallus gallus) genome. Knowledge of multiple avian AHRs is indispensable to understand molecular mechanisms of AHR-mediated toxic effects and establish risk assessment framework for environmental AHR ligands in avian species. In this study, we successfully isolated a third novel AHR1-like cDNA from chicken and designated it as chicken AHR1 beta (ckAHR1ß). The mRNA expression of ckAHR1ß was primarily detected in the liver, and the hepatic protein expression was confirmed by Western blotting. Although mRNA expression of ckAHR1ß was not altered by in ovo TCDD exposure, ckAHR1ß exhibited specific binding to [(3)H]TCDD, TCDD-dependent nuclear translocation, and interaction with xenobiotic responsive elements (XREs) and AHR nuclear translocators (ARNTs). In vitro XRE-driven reporter gene assays revealed ckAHR1ß-mediated transactivation of TCDD in a dose-dependent manner, showing a 10-fold reduced sensitivity (high EC50) compared with that mediated by ckAHR1. The mutation of Val(371) to Ser(371) in the ligand-binding domain of ckAHR1ß shifted the TCDD-EC50 toward the value observed in ckAHR1, indicating the critical roles of the amino acid in sensitivity. Furthermore, ckAHR1ß-mediated transactivation of TCDD was enhanced by 17ß-estradiol (E2)-activated chicken estrogen receptor α (ckERα), suggesting a positive cross talk between ckERα and ckAHR1ß signaling pathway. Both TCDD-induced and its enhanced activities by E2 were suppressed by the ckAHR repressor in a manner similar to ckAHR1. Collectively, our findings discover the role of ckAHR1ß in dioxin toxicity and give an insight into the evolutionary history of the AHR signaling pathway.

Molecular and functional characterization of Aryl hydrocarbon receptor repressor from the chicken (Gallus gallus): interspecies similarities and differences.

The aryl hydrocarbon receptor (AHR) repressor (AHRR) has been recognized as a negative feedback modulator of AHR-mediated responses in fish and mammals. However, the repressive mechanism by the AHRR has not been investigated in other animals. To understand the molecular mechanism of dioxin toxicity and the evolutionary history of the AHR signaling pathway in avian species, the present study addresses chicken AHRR (ckAHRR). The complementary DNA sequence of ckAHRR encodes an 84-kDa protein sharing 29-52% identities with other AHRRs. High levels of ckAHRR messenger RNA were recorded in the kidney and intestine of nontreated chicks. In hepatoma LMH cells, the 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) 50% effective concentration value for ckAHRR induction (0.0016nM) was the same as that for chicken cytochrome P450 1A5 (ckCYP1A5), implying a shared transcriptional regulation of ckAHRR and ckCYP1A5 by chicken AHR (ckAHR). In ckAHRR transient transfection assays, ckAHRR repressed both ckAHR1- and ckAHR2-mediated transcriptional activities. Deletion and mutation assays revealed that basic helix-loop-helix/Per-ARNT-Sim A domains of ckAHRR, particularly 217-402 amino acid residues, are indispensable for the repression, but the AHR nuclear translocator sequestration by ckAHRR and SUMOylation of ckAHRR are not involved in its repressive mechanism. Additionally, subcellular localization assay of ckAHR1-enhanced green fluorescent protein fusion protein showed that ckAHRR did not affect nuclear translocation of the ckAHR1. Furthermore, ckAHRR inhibited the TCDD- and 17ß estradiol-enhanced ckCYP1A5 transcription through AHR-estrogen receptor α (ERα) cross talk. Taken together, the function of AHRR is conserved in chicken in terms of the negative regulation of AHR and ERα activities, but its functional mechanism is likely distinct from those of the mammalian and fish homologues.

Short-term storage and transport at cold temperatures of 2-cell mouse embryos produced by cryopreserved sperm.

At refrigerated temperatures, mouse embryos can maintain developmental ability for short periods. Previously, we succeeded in transporting vitrified and warmed 2-cell mouse embryos while maintaining developmental ability at refrigerated temperatures for 50 h. Transport of nonfrozen embryos is an easier and more useful means of exchanging genetically engineered mice between laboratories than is transport of cryopreserved embryos. Here we examined the developmental ability of transported 2-cell embryos that were produced through in vitro fertilization using cryopreserved sperm. Results show that 2-cell embryos produced by cryopreserved sperm can develop into blastocysts after cold storage for 24, 48, and 72 h. Transported 2-cell embryos produced by cryopreserved sperm yielded a favorable number of pups in all of the receiving laboratories after transport lasting 48 to 52 h. In summary, cold storage and transport of 2-cell embryos derived from cryopreserved sperm at refrigerated temperatures provides a novel means of transporting genetically engineered mice as an alternative to the transport of cryopreserved embryos and sperm.

Birth of mice from vitrified/warmed 2-cell embryos transported at a cold temperature.

Cryopreservation of 2-cell embryos is an effective technology for storage of genetically engineered mouse strains. Transport of genetically engineered mice between laboratories has frequently been performed using such cryopreserved 2-cell embryos. However, the receiving laboratory requires proficient skills and special instruments to obtain live young from cryopreserved and transported embryos. Therefore, in this study, we tried to address the storage and transport of vitrified/warmed 2-cell embryos at a cold temperature. In cold storage experiments, the development rates of 2-cell embryos stored in M2 medium for 24, 48 and 72 h into blastocysts were relatively high (83%, 63% and 43%, respectively). Although, 2-cell embryos stored in PB1 and mWM maintained the developmental potency for 24h, the rates were markedly decreased to low levels after 48 h (PB1: 0%; mWM: 5%). In transport experiments, many pups were obtained from vitrified/warmed 2-cell embryos transported at a cold temperature in all receiving laboratories (incidence of successful development: 49%; 249/511). In summary, short-term storage and transport of vitrified/warmed 2-cell embryos in M2 medium at a cold temperature can maintain their ability to develop into live young.