Comparative Evaluation of Sex Reversal Effects of Natural and Synthetic Estrogens in Sex Reversal Test Using F1 (AWE×WE) Japanese Quail Embryos

Sex reversal effects of 17 beta-estradiol (E2), diethylstilbestrol (DES) and ethynylestradiol (EE2) on male gonads in F1 (AWE×WE) Japanese quail (Corturnix japonica) embryos were comparatively evaluated in a newly developed in vivo screening model called as the sex reversal test. Male and female offspring of F1 (AWE×WE) Japanese quail exhibit exactly wild and albino plumage colors, respectively, ruled by a criss-cross inheritance. The natural and synthetic estrogens were injected into egg white just before the incubation. At 16 days of incubation, embryos were subjected by a complete necropsy and their gonads were grossly observed and examined histopathologically and morphometrically. Grossly, genetic sex confirmed by plumage colors coincided completely with external sex phenotype of the gonads in all embryos of the control group and E2 and DES-treated groups. However, several male embryos with wild plumage in the EE2 2000ng group possessed an ovary-like gonad in the left side and a vestigial right gonad. Histopathologically, E2, DES and EE2 exposures induced a dose-dependent sex reversal effect, i.e. ovotestis development, in the left testis. The left testes showing an ovary-like morphology in the EE2 2000ng group consisted of the most of area replaced with ovarian tissue and the small area of remaining testicular cords. The incidence and morphometric analysis of the ovotestis revealed that the order of potency of sex reversal effect in Japanese quail embryos was EE2>DES>E2. E2, DES and EE2 exposures induced no noticeable changes in the ovaries of any embryos. The present study suggests that the sex reversal test using F1 (AWE×WE) Japanese quail embryo is possible to evaluate feminization effects of endocrine disrupting chemicals with estrogenic activities in avian male embryos

mRNA Expression of Cytochrome P450 17αhydroxylase, Cytochrome P450 Aromatase, Anti-Müllerian Hormone, Estrogen Receptor α, and Androgen Receptor in Developing Gonads of Japanese Quail

Japanese quail (Coturnix coturnix japonica) is widely used in avian reproduction tests and avian sex reversal screening tests for evaluating adverse effects of endocrine disrupting chemicals (EDCs). However, there are few reports on the molecular biological profiles during the development and sexual differentiation of the gonads in Japanese quail. In the present study, we investigated mRNA expression of cytochrome P450 17αhydroxylase (P450c17), cytochrome P450 aromatase (P450arom), anti-Müllerian hormone (AMH), estrogen receptor α (ERα) and androgen receptor (AR) in the embryonic and post-hatching gonads of Japanese quail by using real-time RT-PCR. The left gonads of male and female Japanese quail at 16 days of incubation and 3, 7 and 14 days after hatching were used in the study. P450c17 mRNA expression in females was significantly higher than that in males at 16 days of incubation, 3 and 7 days after hatching. P450arom mRNA expression in females was markedly higher than that in males and there were significant differences at 3, 7 and 14 days after hatching. AMH mRNA expression in males was apparently higher than that in females and there were significant differences at 16 days of incubation and 3 days after hatching. ERα mRNA was highly expressed not only in females but also in males at 16 days of incubation. There were no meaningful trends of AR mRNA expressions in male and female gonads during the observation periods. It is likely that the profiles of mRNA expression of P450c17, P450arom, AMH, ERα and AR in the embryonic and post-hatching gonads of normal Japanese quail in the present study provide basic and useful information