Cortisol is involved in temperature-dependent sex determination in the Japanese flounder.

In vertebrates, sex is normally determined by genotype. However, in poikilothermal vertebrates, including reptiles, amphibians, and fishes, sex determination is greatly influenced by environmental factors, such as temperature. Little is known about the molecular mechanisms underlying environmental sex determination in these species. The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. However, XX flounder can be induced to develop into predominantly either phenotypic females or males, by rearing at 18 or 27 C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that an aromatase inhibitor, an antiestrogen, and 27 C treatments cause masculinization of XX flounder, as well as suppression of mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, estrogen administration completely inhibits masculinization by these treatments, suggesting suppression of cyp19a1 mRNA expression, and the resultant estrogen biosynthesis may trigger masculinization of the XX flounder induced by high water temperature. Here, we demonstrated that cortisol causes female-to-male sex reversal by directly suppressing cyp19a1 mRNA expression via interference with cAMP-mediated activation and that metyrapone (an inhibitor of cortisol synthesis) inhibits 27 C-induced masculinization of XX flounder. Moreover, cortisol concentrations in 27 C-reared juveniles were significantly higher than in 18 C-reared fishes during sexual differentiation. These results strongly suggest that masculinization by high water temperature is ascribable to elevation of cortisol concentration during gonadal sex differentiation in the flounder.

Mullerian inhibiting substance is required for germ cell proliferation during early gonadal differentiation in medaka (Oryzias latipes).

Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-beta superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2'-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.

Tamoxifen induces masculinization of genetic females and regulates P450 aromatase and Müllerian inhibiting substance mRNA expression in Japanese flounder (Paralichthys olivaceus).

Japanese flounder, Paralichthys olivaceus, provides an excellent model to elucidate the roles of sex steroid hormones in gonadal sex differentiation because the sex is easily altered by sex steroid treatments or water temperature control during the sex differentiation. We have previously shown that high water temperature, an aromatase inhibitor (fadrozole), or 17alpha-methyltestosterone treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (P450arom), which is a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that treatment of the genetic females with anti-estrogen (tamoxifen) leads to their masculinization, suppresses P450arom mRNA expression, and induces mRNA expression of Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta (TGF-beta) superfamily, while it has no effect on mRNAs expression of estrogen receptor-alpha (ERalpha) and ERbeta. In contrast, 17beta-estradiol counteracted masculinization of the genetic females by tamoxifen or high water temperature treatment, up-regulated P450arom mRNA expression, and down-regulated MIS mRNA expression. These results strongly suggest that estrogen signaling through ERs dramatically influences the gonadal sex differentiation by regulating P450arom and MIS mRNA expression.

Sexually dimorphic expression of a teleost homologue of Müllerian inhibiting substance during gonadal sex differentiation in Japanese flounder, Paralichthys olivaceus.

Müllerian inhibiting substance (MIS), also known as anti-Müllerian hormone, is a glycoprotein belonging to transforming growth factor beta superfamily. In mammals, MIS is responsible for regression of Müllerian ducts, anlagen of the female reproductive ducts, in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fishes, which do not have the Müllerian ducts, has yet to be clarified. To address the role of MIS on gonadal sex differentiation in fishes, we isolated a MIS cDNA from the Japanese flounder testis and examined the expression pattern of MIS mRNA in gonads of both sexes during sex differentiation period. In this study, we present the first demonstration of sexually dimorphic expression of MIS mRNA during sex differentiation in teleost fishes, similarly to amniote vertebrates which possess the Müllerian ducts.