Effects of 4-tert-pentylphenol on the gene expression of P450 11β-hydroxylase in the gonad of medaka (Oryzias latipes)

Alkylphenols including 4-tert-pentylphenol (4-PP) have been shown to alter sexual differentiation in fish due to their estrogenic properties. Medaka (Oryzias latipes) is so sensitive to these substances because morphological sex reversal and testis-ova induction are well developed in the exposed males. However, little work has been done to characterize the molecular effects of estrogenic substances on the morphological and gonadal feminization in male fish. Cytochrome P450 11β-hydroxylase (P45011β) is a key steroidogenic enzyme in production of 11-ketotestosterone which is the predominant androgen in male fish. In this study, we cloned a cDNA encoding medaka testicular P45011β, and then investigated the gene expression of P45011β in the testes of genetically male medaka exposed to 4-PP. The cDNA contains 1740 nucleotides that encode a protein of 543 amino acids, which shares 68.9% and 73.4% homology with testicular P45011βs from Japanese eel (Anguilla japonica) and rainbow trout (Oncorhynchus mykiss), respectively. HeLa cells transfected with an expression vector containing the open reading frame of medaka P45011β cDNA showed 11β-hydroxylating activity in the presence of exogenous testosterone. Analysis of tissue distribution by RT-PCR showed great abundance of P45011β mRNA in testis. In the partial life-cycle exposure with 4-PP, morphologically sex-reversal was observed in XY medaka exposed to 4-PP concentrations of ≥ 238 μg/L. Furthermore, exposure to 4-PP completely inhibited P45011β mRNA expression in the gonads of sex-reversed XY fish at 60-day posthatch. These results suggest that xeno-estrogen 4-PP may have inhibitory effects on the synthesis of testicular 11-oxygenated androgens through downregulation of P45011β expression in the genetically male fish.