Sex- and tissue-specific expression of P450 aromatase (cyp19a1a) in the yellowtail clownfish,Amphiprion clarkii

To investigate the role of estrogen in the gonad of yellowtail clownfish Amphiprion clarkii, we isolated cDNA encoding cytochrome P450 aromatase (Cyp19a1a) from the adult ovary. The full-length cDNA of clownfish cyp19a1a is 1928-bp long and encodes 520 amino acids. Real-time quantitative RT-PCR analysis showed that cyp19a1a was expressed mainly in the ovary of female-phase fish. In situ hybridization and immunohistochemical observations showed that positive signals were restricted to the ovarian follicle of the female-phase fish. In contrast, Cyp19a1a signal was not detected in the ambisexual gonad of the male-phase fish. These findings suggest that Cyp19a1a is involved in oogenesis in the female-phase fish, but not in the ambisexual gonad of male-phase fish.     

稀有鮈鲫脑芳香化酶cDNA片段的克隆与表达分析

芳香化酶（P450arom）是雌激素合成过程中的关键酶,在性别分化中起重要作用。鱼类存在卵巢性和脑型两种芳香化酶,分别由Cyp19a和Cyp19b编码。稀有鮈鲫作为我国特有的实验动物,尚无其芳香化酶的有关资料,其性别分化机制亦不清楚。本研究采用RT-PCR的方法以简并引物扩增了稀有鮈鲫脑型芳香化酶基因Cyp19b的部分序列,其长度为1070 bp, 编码357个氨基酸残基,具有典型的芳香化酶结构域。RT-PCR分析发现该基因主要在稀有鮈鲫的脑中表达,在性腺、肠、肾中也有表达;其在雌、雄脑中的表达差异不显著。在胚胎发育阶段,Cyp19b的表达从囊胚期开始,至神经胚期达到较高水平,随后下降,孵化期又有所增强,孵化10天后维持在高水平。这些结果说明Cyp19b以脑中表达为主,可能在稀有鮈鲫神经系统发育和脑的性别分化中有重要作用。     

From molecule to behavior: Brain aromatase (cyp19a1b) characterization,expression analysis and its relation with social status and male agonistic behavior in a Neotropical cichlid fish

The enzyme aromatase, responsible for the conversion of C19 androgens to C18 estrogens, exists as two paralogue copies in teleost fish: Cyp19a1a mostly expressed in the gonads, referred as gonadal aromatase, and Cyp19a1b, mostly expressed in the brain, accordingly known as brain aromatase. The neural localization of Cyp19a1b is greatly contained within the social behavior network and mesolimbic reward system in fish, suggesting a strong role of estrogen synthesis in the regulation of social behavior. In this work we aimed to analyze the variation in cyp19a1b expression in brain and pituitary of males of a highly social cichlid, Cichlasoma dimerus (locally known as chanchita), and its relation with inter-individual variability in agonistic behavior in a communal social environment. We first characterized chanchita's cyp19a1b mRNA and deduced amino acid sequence, which showed a high degree of conservation when compared to other teleost brain aromatase sequences, and its tissue expression patterns. Within the brain, Cyp19a1b was solely detected at putative radial glial cells of the forebrain, close to the brain ventricles. We then studied the relative expression levels of cyp19a1b by Real Time PCR in the brain and pituitary of males of different social status, territorial vs. non-territorial, and its relationship with an index of agonistic behavior. We found that even though, brain aromatase expression did not differ between types of males, pituitary cyp19a1b expression levels positively correlated with the index of agonistic behavior. This suggests a novel role of the pituitary in the regulation of social behavior by local estrogen synthesis.     

Characterization of duplicated zebrafish cyp19 genes.

The zebrafish has recently been developed as a good genetic model system. We report here the use of zebrafish to study the regulation of estrogen biosynthesis. The CYP19 gene encodes cytochrome P450 aromatase, which catalyzes the synthesis of estrogens. Two cyp19 genes, termed cyp19a and cyp19b, have been isolated from zebrafish. Sequence comparison shows that Cyp19a and Cyp19b belong to two separate Cyp19 subfamilies. The cyp19a gene is expressed in the ovary, whereas cyp19b is expressed in the brain. The cyp19a and cyp19b genes are located on zebrafish chromosomes LG 18 and 25, respectively. Our data indicate that these gene loci arose through an ancient chromosomal duplication event. The expression of duplicated genes in distinct tissues may have evolutionary significance.     

Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus

Fish have diverse sex determination and differentiation. DMRT1 and aromatase are conserved in the phyla and play pivotal roles in sex development. Gobiocypris rarus is a small fish used as a model in aquatic toxicology in China and has been used to study the effects of environmental endocrine disruptors on gene expression, but its sexual development remains elusive. Here, we report the full-length cDNA of G. rarus dmrt1 and its expression along with the expression of cyp19a1a and cyp19a1b, two genes encoding gonad and brain type aromatases, in adults and during ontogenesis. Both cyp19a1a and dmrt1 are expressed in the ovary and testis but show sexual dimorphism. Expression of cyp19a1a in the ovary is higher than in testes and dmrt1 follows the opposite pattern. Juvenile gonad histology changes at 15 days after hatching. The dimorphic expression of dmrt1 and cyp19a1a appears from 5 days after hatching, which is earlier than histological change. cyp19a1b is expressed coordinately with cyp19a1a until 15 days after hatching. These results show that dmrt1 and cyp19a1a play important roles in sex determination and sex differentiation in G. rarus.     

两种泥鳅芳香化酶基因的克隆与时空表达

鱼类的性别分化易受发育环境的影响。向性成熟的泥鳅和大鳞副泥鳅个体注射绒毛膜促性腺激素,获得卵子和精子进行人工授精。把胚胎分别置于20℃、25℃和30℃条件下,使其发育。经性腺检查发现随着温度的升高两种泥鳅中雄性个体所占的比例明显升高,获得明显的偏雄比率群体。根据已知细胞色素P450芳香化酶CYP19 b基因序列设计嵌套简并引物用巢式PCR扩增并克隆出了两种泥鳅的CYP19 b的DNA片段。MaCYP19 b片段和Pd-CYP19 b片段分别长1337bp和1473bp。在此基础上用各自的特异引物克隆出两种泥鳅CYP19 b的相应cDNA片段。通过基因组DNA和cDNA序列的比较证明两种泥鳅的CYP19 b基因均包含三个内含子和四个外显子,编码的蛋白质序列长145氨基酸残基。以GAPDH基因为对照,分别对两种泥鳅成体组织和不同发育阶段的胚胎的CYP19 b进行了半定量RT-PCR表达分析,结果表明泥鳅CYP19 b基因只在成体泥鳅卵巢、肾以及原肠胚和神经胚中表达。大鳞副泥鳅CYP19 b基因在成体的脑、卵巢和肾以及神经胚和卵黄吸收期表达。这些结果为揭示细胞色素P450芳香化酶基因与环境性别决定机制的关系奠定了基础。       

Overexpression of Aromatase Alone is Sufficient for Ovarian Development in Genetically Male Chicken Embryos

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.     

Two Cyp19 (P450 aromatase) genes on duplicated zebrafish chromosomes are expressed in ovary or brain

Cytochrome P450 aromatase (Cyp19) is an enzyme catalyzing the synthesis of estrogens, thereby controlling various physiological functions of estrogens. We isolated two cyp19 cDNAs, termed cyp19a and cyp19b, respectively, from zebrafish. These genes are located in linkage groups 18 and 25, respectively. Detailed gene mapping indicated that zebrafish linkage groups 18 and 25 may have arisen from the same ancestral chromosome by a chromosome duplication event. Cyp19a is expressed mainly in the follicular cells lining the vitellogenic oocytes in the ovary during vitellogenesis. Cyp19b is expressed abundantly in the brain, at the hypothalamus and ventral telencephalon, extending to the olfactory bulbs. The expression of duplicated cyp19 genes at two different tissues highlights the evolutionary significance of maintaining two active genes on duplicated zebrafish chromosomes for specific functions in the ovary and the brain.     

Activation of brain steroidogenesis and neurogenesis during the gonadal differentiation in protandrous black porgy,Acanthopagrus schlegelii

The early brain development, at the time of gonadal differentiation was investigated using a protandrous teleost, black porgy. This natural model of monosex juvenile fish avoids the potential complexity of sexual dimorphism. Brain neurogenesis was evaluated by histological analyses of the diencephalon, at the time of testicular differentiation (in fish between 90 and 150 days after hatching). Increases in the number of both Nissl‐stained total brain cells, and Pcna‐immunostained proliferative brain cells were observed in specific area of the diencephalon, such as ventromedialis thalami and posterior preoptic area, revealing brain cell proliferation. qPCR analyses showed significantly higher expression of the radial glial cell marker blbp and neuron marker bdnf. Strong immunohistochemical staining of Blbp and extended cellular projections were observed. A peak expression of aromatase (cyp19a1b), as well as an increase in estradiol (E₂) content were also detected in the early brain. These data demonstrate that during gonadal differentiation, the early brain exhibits increased E₂ synthesis, cell proliferation, and neurogenesis. To investigate the role of E₂ in early brain, undifferentiated fish were treated with E₂ or aromatase inhibitor (AI). E₂ treatment upregulated brain cyp19a1b and blbp expression, and enhanced brain cell proliferation. Conversely, AI reduced brain cell proliferation. Castration experiment did not influence the brain gene expression patterns and the brain cell number. Our data clearly support E₂ biosynthesis in the early brain, and that brain E₂ induces neurogenesis. These peak activity patterns in the early brain occur at the time of gonad differentiation but are independent of the gonads. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 121–136, 2016     

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.