Full-length CDNA Cloning Of Androgen Receptor Gene From Rare Minnow (Gobiocypris Rarus) And Effect Of Endocrine Disrupting Chemicals On Its MRNA Expression

Androgens play key roles in sex differentiation, gonad maturation and reproductive behaviors and their actions are generally mediated through androgen receptor (AR). Upon hormone binding, the AR dissociates from accessory proteins, translocates into the nucleus and dimerizes, thus stimulating transcription of androgen responsive genes. Androgen receptor is a ligand-activated transcription factor that belongs to a large family of nuclear receptors. This family of receptors share a molecular structure that consists four main functional domains.In the present study, RT-PCR and RACE (Rapid Amplification cDNA Ends) methods were used for the isolation of the cDNA of AR gene from testis of Gobiocypris rarus. We show for the first time in fish, different mRNA AR expression levels between males and females in five different tissues, suggesting that AR is involved in many physiological functions in fish.With the frequency of human production and life, much attention has been focused on the potential health threats of endocrine disrupting chemicals capable of disrupting the endocrine systems of animal and human population. Research mostly focused on estrogenic compounds. In this report, we use EE2, NP and BPA as estrogenic model compounds ,after 3 days exposure of rare minnow juveniles using real-time PCR. We discuss whether AR gene can be sensitive as a molecular biomarker in assessing the potential impact of estrogenic compounds using this specie as a model system. The main results and conclusions are as follows:1. RT-PCR and RACE methods were used for the isolation of the whole cDNA of AR gene from the testis of Gobiocypris rarus. We cloned cDNA fragments of 600bp, 570bp, 731bp, 979bp and 146bp in 3′end and 492 bp in 5′end. The full-length cDNA sequences of AR gene have been formed by joint for the first time, consisting of a 103bp 5'untranslated region (UTR), a 2535bp open reading frame (ORF) encoding 844 amino acid residues and a 492bp 3'UTR. The Genbank accession number is GU226857.2. The deduced amino acid sequence of rare minnow AR was aligned with known full-length AR amino acid sequences from teleost fish fathead minnow and goldfish using Blastp. The identity of rare minnow AR was with fathead minnow and goldfish with 86%. Multiple amino acids sequence alignment indicated AR has three main functional domain, TAD, DBD and LBD. DBD and LBD are highly conserved among the cyprinid fish species, whose identity ranged from 98% to 99%. The identity of LBD is about 97% to 98%. TAD is the most variable domain, whose identity fluctuated from 75% to 82%.3. AR mRNA expression was detected using semiquantitative RT-PCR in gonad, liver, brain, intestine, and muscle. AR mRNA was expressed at high levels in the testis and liver, low levels in the brain, intestine, muscle of male. However it was expressed at moderate levels in all the tissues except the muscle of female.4. Exposure to 0.01 and 0.1 nmol/L ethynylestradiol (EE2) for 3 days caused nonsignificant and significant increase of AR mRNA expression in rare minnow juveniles, respectively. On the contrary, 1nmol/L EE2 caused nonsignificant decrease in its expression. 0.01μmol/L nonylphenol (NP) caused significant decrease of AR gene expression. While AR mRNA expression had downregulation tendency in both 0.1 and 1μmol/L NP exposed groups. Bisphenol (BPA) 0.1,1 and 10 nmol/L caused significant decrease of AR gene expression.Therefore AR gene expression was differentially modulated by various classes of EDCs and their different exposure concentrations.