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.     

Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus)

The sex of Japanese flounder (Paralichthys olivaceus) is easily altered by water temperature or sex steroid hormone treatment during the period of sex determination. We have previously shown that rearing the genetically female larvae at high water temperature caused the suppression of P450 aromatase (P450arom) gene expression in the gonad and phenotypic sex-reversal of the individuals to males (Kitano et al. 1999. J Mol Endocrinol 23:167-176). In the present study, we show that treatment of genetically female larvae with fadrozole (aromatase inhibitor) or 17alpha-methyltestosterone induces sex-reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co-administration of estradiol-17beta. Effective periods for fadrozole treatment to induce sex-reversal were similar to those for high water temperature treatment. RT-PCR did not detect P450arom mRNA in gonad of the sex-reversed, phenotypic males. These results indicate that sex-reversal of the genetically female larvae by aromatase inhibitor (or 17alpha-methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen.     

Dax1 suppresses P450arom expression in medaka ovarian follicles

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.     

Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary

Recent studies point to a key role for the estrogen synthesizing enzyme P450 aromatase (P450 arom) in ovary determination in fish, birds and reptiles. It is unclear whether estrogen synthesis is important in sex determination of Xenopus gonad. To determine whether the aromatase gene is transcribed in the gonads of Xenopus tadpoles during the sex determination, we cloned a P450 arom cDNA and examined the level of P450 arom and estrogen receptor (ER) gene expression in association with estrogen activity. cDNA clones for P450 arom were isolated from a Xenopus ovarian cDNA library. There was an open reading frame (ORF) of 1500 bp from the ATG start to TAA stop codons encoding 500 predicted amino acids. cDNAs for P450 arom have previously been cloned from various vertebrates. The homology between the Xenopus P450 aromatase and the human P450 arom was higher. The expression of the P450 arom gene was mainly limited to reproductive organs. To determine the beginning of estrogen activity in gonads of embryos, expression of the aromatase and ER gene was also examined by RQ-RT-PCR. Both Xenopus aromatase and ER mRNA was detected at stage 51 in gonads. These observations are consistent with estrogens having a key role in ovarian development in various other vertebrates.     

黄鳝P-450芳香化酶基因的克隆及序列分析

P-450芳香化酶(P450arom)是催化雄激素生物合成雌激素的关键酶。本文采用RT-PCR和RACE(Rapid amplifi- cation of cDNA ends)法,首次分离和克隆了雌雄同体鱼黄鳝卵巢中P450 arom基因。该基因cDNA全长1802bp(不包 括poly(A)),5'端非翻译区有49bp,3'端202bp(不包含poly(A)),阅读框(Open reading frame,ORF)1551bp,翻译成517 个氨基酸,计算的蛋白质分子量58．2kDa。同源性分析显示,黄鳝卵巢P450arom的氨基酸序列与其他鱼卵巢 P450arom具有63％-80％同源性,与其他鱼脑P450arom为58％-60％同源,与人胚盘和鸡卵巢P450arom则为 50％-52％同源;但在芳香化酶高保守区(包括1-螺旋区,芳香化酶特异保守区和血红素结合区)的同源性高达 76％-92％。系统发育分析表明芳香化酶基因是单起源,黄鳝卵巢芳香化酶基因与鳉鱼卵巢的关系最近,与鱼类卵 巢P450arom属于同一分支的,与鱼类脑及鸡和人的属于不同分支。     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶(P450aromB)在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型P450arom的同源性较高(48.3%-66.1%),与性腺型P450arom的同源性较低(34.2%-49.9%),与自身的性腺型芳香化酶同源性为45.1%。RT-PCR分析表明:P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder

The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C 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 cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.     

P450 aromatase expression in the temperature-sensitive sexual differentiation of salamander (Hynobius retardatus) gonads

Sex differentiation of gonads in amphibians is believed to be controlled genetically, but altered epigenetically or environmentally. When larvae of the salamander Hynobius retardatus were reared at defined temperatures from hatching to metamorphic stages, a high temperature (28 degrees C) induced exclusively female gonads (ovaries), whereas intermediate (20 and 23 degrees C) or lower (16 degrees C) temperatures produced a 1:1 sex ratio of the morphological gonads. The thermosensitive period was determined to be restricted from 15 to 30 days after hatching, just before or when sexual differentiation occurred. Hynobius P450 aromatase (P450arom) cDNA was isolated from adult gonads and the partial nucleotide or deduced amino acid sequences were determined, showing a high level of identity with various vertebrate species. The P450arom gene was expressed predominantly in the adult ovary and brain, weakly in testis, but not in other somatic organs. A typical sexual dimorphism in P450arom expression was detected in normally developing larvae by a quantitative competitive RT-PCR; strong expression in the female gonads but very weak in male gonads. The dimorphism was detected much earlier than the morphological sexual differentiation of the gonads. When larvae were reared at the female-producing temperature (28 degrees C), strong expression was detected in all the temperature-treated larvae, suggesting that P450arom was up-regulated, even in genetic males. Our results confirm the importance of the P450arom regulation in the sexual differentiation of gonads and demonstrate that an up-regulation of P450arom is involved in the process of temperature-sensitive sex reversal in this species.     

Induction of XY sex reversal by estrogen involves altered gene expression in a teleost, tilapia

To clarify the importance of endogenous estrogens during sex differentiation in a teleost fish, the Nile tilapia, we examined the target events for endogenous estrogens and their role during gonadal sex differentiation. The expression of CYP19a (P450arom) precedes any morphological gonadal sex differentiation. Further to these findings, the treatment of XX fry with non-steroidal aromatase inhibitor (AI), Fadrozole, from seven to 14 days after hatching caused complete sex reversal to functional males. The XX sex reversal induced by AI was rescued completely with simultaneous estrogen treatment. We also found that XY fry treated with estrogen, before the appearance of morphological sex differences, caused complete sex reversal from males to females. Taken together, these results suggest that endogenous estrogens are required for ovarian differentiation. To identify the down-stream gene products of estrogen during ovarian differentiation, we performed subtractive hybridization using mRNA derived from normal and estrogen treated XY gonads. Two out of ten gene products were expressed in germ cells, whereas the others were expressed in somatic cells.     

Molecular aspects of brain aromatase cytochrome P450

Aromatase cytochrome P450 (P450arom) enzyme activity catalyses the conversion of androgens to estrogens in specific brain areas. During central nervous system (CNS) development local estrogen formation influences sexual differentiation of neural structures, regulates neuroendocrine functions and sexual behavior. A proposed mechanism (and re-examination) of the sexual differentiation of the rodent brain is presented. The metabolic pathway of androgen metabolism by P450arom was characterized in the medial basal hypothalamic (MBH) tissue from male rats during various prenatal and postnatal developmental intervals. The P450arom enzyme activity was determined using a saturating concentration of [³H]testosterone as the substrate, and the rates were quantified by scintillation counting. The MBH P450arom activity was highest during prenatal development (i.e. 3–6 pmol/h/mg protein), declined to moderate levels in newborns and infantile animals (approximately 1 pmol/h/mg protein) and then continued to decline to low activity rates in adult animals (approximately 80 fmol/h/mg protein). Regulation of the P450arom gene was characterized by a series of molecular biology studies where the controlling mechanism for brain P450arom was determined in MBH and amygdaloid tissue sites. Evidence for brain P450arom-specific mRNA in perinatal rats is presented as well as comparisons with rat ovary, a rat Leydig tumor cell line (R2C) and human fetal brain P450arom. Specifically, P450arom gene expression is driven in perinatal rat brain tissue by a different promoter compared to rat ovarian tissue or a R2C cell line, whereas human fetal brain tissue utilizes an almost identical promoter segment to that observed in the rodent. These findings provide an insight into the regulation of brain P450arom gene expression and suggest that there is an additional level of control for the expression of this gene during perinatal development. However, further study is necessary to understand the molecular basis of this complex developmental pattern of brain P450arom expression.     

Transcriptional regulation of aromatase in placenta and ovary

Our goal is to define the cellular and molecular mechanisms for tissue- and cell-specific, developmental and hormonal regulation of the human CYP19 (aromatase P450/P450arom) gene in estrogen-producing cells. In this article, we review studies using transgenic mice and transfected cells to identify genomic regions and response elements that mediate CYP19 expression in placenta and ovary, as well as to define the molecular mechanisms for O₂ regulation of differentiation and CYP19 gene expression in human trophoblast cells in culture. We also highlight recent findings regarding LRH-1 versus SF-1 mRNA expression and cellular localization in the mouse ovary during the estrous cycle and various stages of pregnancy. Spatial and temporal expression patterns of mRNAs encoding these orphan nuclear receptors in comparison to those of P450arom and 17-hydroxylase/17,20-lyase mRNAs, suggest an important role of LRH-1 together with SF-1 in ovarian steroidogenesis.     

Sex differentiation and mRNA expression of P450c17, P450arom and AMH in gonads of the chicken

The present study was conducted to reveal effects of in ovo injection of nonsteroidal aromatase inhibitor (Fadrozole) or estradiol at day 3 of incubation on mRNA levels of P45017alphahydroxylase (P450c17), P450 aromatase (P450arom) and anti-Müllerian hormone (AMH) in the chicken gonads. The mRNA levels in the gonads at days 4-8 of incubation were assessed by in situ hybridization analysis using digoxigenin labeling method. The in situ hybridization data were analyzed by relative expression of specific hybridizable signals of each mRNA corrected by the non-specific background by employing an image analyzer. P450c17 mRNA expression increased rapidly at day 6 of incubation in the male but decreased thereafter. In contrast to the transient expression in the male, the expression was gradually increased in the female. P450arom mRNA was not expressed in the male but was detectable in the female as early as day 6 and increased subsequently with days of incubation. AMH mRNA was expressed as early as day 5 of incubation followed by a sharp increase on day 6, which was maintained in the male thereafter. In contrast, the female showed very little expression. The injection of Fadrozole caused no effect on P450c17 mRNA expression, while it suppressed P450arom mRNA expression but increased AMH mRNA expression in the female. In contrast, the injection of estradiol induced P450arom mRNA expression significantly but suppressed AMH mRNA expression in the male. These results indicate that expression of P450arom and AMH is sexually dimorphic and is reciprocally regulated during early ontogenic life in chicken gonads.