Levonorgestrel causes feminization and dose-dependent masculinization in medaka fish (Oryzias latipes): Endocrine-disruption activity and its correlation with sex reversal.

The effect of a synthetic progestin, levonorgestrel (LNG), on the sex of exposed embryos was examined in medaka fish (Oryzias latipes). The aims of this study are to clarify the dual effect of LNG on sex and the correlation with its androgenic/estrogenic potential in medaka. LNG exposure causes significant dose-dependent masculinization (0.1-100 µg/L), whereas a decrease in the masculinization ratio is observed at 100 µg/L. LNG also causes significant feminization at 1-100 µg/L, but not in a dose-dependent manner. Exposure of estrogen-responsive gene (choriogeninH-EGFP) transgenic embryos to 100 µg/L LNG produced significant fluorescent signals in hatched fry. In vitro transcriptional assays indicated that LNG at 10-7-10-5 M induced significant activity for estrogen receptor (ESR)2a and ESR2b, but not for ESR1. In pre-self-feeding fry at 5 days post hatching (dph), 1-100 µg/L LNG caused a significant increase in the mRNA of choriogeninH, irrespective of genetic sex. Moreover, LNG (10-10-10-5 M) also caused a significant increase in the transcriptional activity of androgen receptor (AR) α and ARß in vitro, and 0.1 µg/L LNG significantly increased the mRNA levels of a testis-differentiation initiation factor, gonadal soma-derived factor (gsdf), as an androgen-upregulated and estrogen-downregulated gene, in 5 dph XX fry to levels similar to those in the control XY fry. However, 100 and 10 µg/L LNG suppressed or did not induce gsdf mRNA expression in XY and XX fry, respectively. Together, these findings show that LNG exerts estrogenic and androgenic activities in different concentration ranges, which correlate with the ratio of LNG-induced sex reversal. These results suggest for the first time, that medaka exposure to LNG can induce masculinization and feminization, based on the balance between androgenic and estrogenic activities, and the protocol applied in this study represents an alternative to the traditional animal model used to screen for endocrine-disrupting potential.

Inter- and Intraspecific Variation in Sex Hormone-Induced Sex-Reversal in Medaka, Oryzias latipes and Oryzias sakaizumii.

We compared sex-reversal ratios induced by 17α-methyltestosterone (MT) and 17ß-estradiol (E2) exposure in two inbred medaka strains: Hd-rR derived from Oryzias latipes and HNI-II from O. sakaizumii. All MT exposures (0.2-25 ng mL-1) induced complete XX sex-reversal in HNI-II. Although MT exposure at 0.2 ng mL-1 induced XX sex-reversal at > 95% in Hd-rR, other concentrations tested caused XX sex-reversal at lower frequencies (<50%). MT exposure at 1, 5, and 25 ng mL-1 induced XY sex-reversal in Hd-rR, but not in HNI-II. In Hd-rR, E2 exposure induced XY sex-reversal at > 10 ng mL-1, and in all fish feminization occurred 500 ng mL-1. In HNI-II, E2 induced XY sex-reversal at 50 and 250 ng mL-1, but only at rates below 20%. To clarify whether the strain differences in sex hormone-induced sex-reversal are characteristic of each species, we examined the effects of MT and E2 exposure on sex differentiation in five and two additional strains or wild stocks/populations of O. latipes and O. sakaizumii, respectively. MT exposure induced low XX and high XY sex-reversal rates in O. latipes, except in the Shizuoka population, but the trend was reversed in O. sakaizumii. Furthermore, E2-induced XY sex-reversal rates varied intraspecifically in O. latipes. Our results demonstrated that sensitivity to MT and E2 varied within O. latipes species. To evaluate the ecological impacts of environmental chemicals using medaka, it is important to define not only the species, but the strains, stocks, and populations to obtain accurate results.