Hirofumi Yokota

Effect of 17β-estradiol on the reproduction of Japanese medaka (Oryzias latipes)

Abstract Estrogenic compounds such as 17β-estradiol (E2) and its analogs are present in the aquatic environment and can adversely affect the reproductive systems of aquatic organisms. Although E2 has been detected at a maximum concentration of 64 ng/l in effluents of sewage treatment works (STWs), few reports address the associated effects on reproduction in fish. Therefore, we exposed adult medaka (Oryzias latipes) to mean measured E2 concentrations of 29.3, 55.7, 116, 227, and 463 ng/l for 21 d and assessed the effects on the egg number and fertility of paired medaka during the exposure period. In addition, we determined the hepatic vitellogenin (Vtg) concentration and histologically assessed the gonads of these fish. The number of egg produced and fertility of the paired medaka exposed to 463 ng/l E2 were significantly less compared with those of the control fish. Males in all treatment groups had developed testis–ova. Males treated with E2 concentrations=55.7 ng/l contained relating great concentrations of hepatic Vtg. Therefore, although only the greatest E2 concentration tested in our study affected fecundity and fertility, effects of E2 were observed on induction of Vtg and testis–ova in male medaka exposed to environmentally relevant concentrations of E2.

Effects of 4‐nonylphenol on reproduction of japanese medaka, Oryzias latipes

We exposed mature Japanese medaka (Oryzias latipes) to 4-nonylphenol (4-NP) to elucidate its reproductive effects and estrogenic responses. For each treatment, eight breeding pairs were exposed for 3 weeks at measured 4-NP concentrations of 24.8, 50.9, 101, and 184 micrograms/L. Egg production of the pairs during the third week of exposure (15-21 d) was significantly decreased in the > or = 101-microgram/L treatment group, and fertility was significantly decreased in the 184-microgram/L group. Induction of testis-ova was observed in male fish at all 4-NP concentrations, whereas abnormality of spermatogenesis was found only in those receiving the 184-microgram/L treatment. The gonadosomatic index of the males in the 184-microgram/L group was also remarkably reduced. Hepatic vitellogenin (VTG) levels were significantly increased in both sexes receiving 4-NP treatments of > or = 50.9 micrograms/L. Overall, the lowest-observed effective concentrations (LOECs) of 4-NP for decreasing reproductive capacity (fecundity and fertility) and eliciting estrogenic responses (induction of testis-ova and VTG) were 101 and < or = 24.8 micrograms/L, respectively. The LOEC for reduced reproduction in medaka was at least four-fold greater than that required to elicit estogenic responses in male fish. Thus, we suggest that the estrogenic responses expressed in male fish are not responsible for the reduction in reproductive capacity.

Fish full life-cycle testing for androgen methyltestosterone on medaka (Oryzias latipes).

Abstract-We studied the chronic effects of methyltestosterone (MT) on reproductive status of medaka (Oryzias latipes) over two generations under continuous exposure to verify the applicability of the fish full life-cycle test (FFLC) for this androgen with this species. The exposure of parental (F0) medaka to MT was begun on embryos within 12 h postfertilization and continued for up to 101 d; assessment endpoints included embryological development, hatching, posthatch survival, growth, sexual differentiation, reproduction, and hepatic vitellogenin (VTG) levels under flow-through exposure to MT at each mean measured concentration of 0.35, 1.09, 3.29, 9.98, and 27.75 ng/L. Eggs (F1) spawned from the F0 fish at 98, 99, and 100 d posthatch were examined for hatchability, survival after hatching, growth, sexual differentiation, and hepatic VTG level until 60 d posthatch. In the FFLC with medaka, MT induced masculinization of both secondary sex characteristics and gonads. We observed that all F0 fish in the 27.75-ng/L treatment group showed male secondary sex characteristics in which no fish with ovary could be discerned. Several fish with ovaries in F0 and F1 generations treated with 9.98 ng/L showed male secondary sex characteristics. We also observed swollen abdomens in the F0 and F1 female fish in the 9.98-ng/L treatment group. These swollen abdomens were induced by enlarged ovaries and were accompanied with declined fecundity and fertility in the F0 generation. These results indicate that MT reduces the reproductive potential of medaka and that the FFLC with this species is applicable to the evaluation of androgens.

Fish full life‐cycle testing for 17β‐estradiol on medaka (Oryzias latipes)

We studied the chronic effects of 17beta-estradiol (E2) on the reproductive status of medaka (Oryzias latipes) over two generations under continuous exposure to assess its hazard on this species. The exposure of parental (F0) medaka to E2 was begun on embryos within 12 h postfertilization and continued for up to 101 d; the assessment endpoints included embryological development, hatching, posthatch survival, growth, sexual differentiation, reproduction, and hepatic vitellogenin (VTG) levels under flow-through exposure to E2 at each mean measured concentration of 0.939, 2.86, 8.66, 27.9, and 92.4 ng/L. Eggs (F1) spawned from the F0 fish at 98, 99, and 100 d posthatch were also examined for hatchability, survival after hatching, growth, sexual differentiation, and hepatic VTG level until 59 d posthatch. In the F0 medaka, no concentration-related effect of E2 was observed on hatching, posthatch mortality, and growth. However, we observed abnormal sex differentiation, induction of VTG, and reproductive impairment in a dose-dependent manner. The lowest-observed-effect concentration and no-observed-effect concentration of E2 for these effects were 8.66 and 2.86 ng/L, respectively. In the F1 medaka treated with E2 at < or = 8.66 ng/L, however, dose-dependent effects were not observed in any parameter. The results indicate that E2 can have significant effects on reproductive potential of medaka at concentrations as low as 8.66 ng/L, suggesting that the ecological risk of E2 is higher than that of weak estrogens, such as nonylphenol and octylphenol.

The effects of methyltestosterone on the sexual development and reproduction of adult medaka (Oryzias latipes)

We examined the effects of methyltestosterone (MT) on the reproduction of medaka (Oryzias latipes). Six mating pairs of medaka were exposed for 3 weeks to MT at the measured concentrations of 22.5, 46.8, 88.1, 188, and 380 ng/L. The fecundity and fertility of paired medaka were significantly decreased in the 46.8, 88.1, 188, and 380 ng/L MT groups compared with those of controls. The gonadosomatic indices of female fish exposed to >or=46.8 ng/L were increased significantly. Gonadal histology showed degeneration of oocytes in the ovaries of fish in all MT treatment groups. Hepatic vitellogenin concentrations were significantly decreased in female fish treated with MT at 188 and 380 ng/L, and the hatchability and survival rate of the offspring were decreased in all MT treatment groups. These results clearly demonstrate that MT at >or=46.8 ng/L inhibited gonadal development and adversely affected the reproduction of medaka.