Phylogeny, expression and enzyme activity of zebrafish cyp19 (P450 aromatase) genes

The cyp19 encodes P450 aromatase, the enzyme catalyzing the conversion of estrogens from androgens. Estrogens affect the dimorphic, anatomical, functional and behavioral aspects of development of both males and females. In zebrafish, two cyp19 genes, cyp19a and cyp19b were found. They are expressed in ovary and brain, respectively. Expression of cyp19b can be detected by 11 days post-fertilization (dpf) by in situ hybridization in the olfactory bulbs, ventral telencephalic region and the hypothalamus of the brain in both male and female, where it is generally known to be affecting the reproductive function and sexual behavior. COS-1 clones permanently expressing the enzymes have been isolated. Both aromatase enzymes encoded by these two genes are functional in COS-1 cells and they can use androstenedione and testosterone equally efficiently. The presence of two functional cyp19 in zebrafish has its evolutionary and physiological importance.     

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。       

赤点石斑鱼两种芳香化酶cDNA的克隆及其表达的组织特异性

以赤点石斑鱼 (Epinephelusakaara)脑垂体中提取的RNA为模板 ,根据芳香化酶的保守序列设计引物 ,利用GeneRacerTM 技术 ,克隆出两种芳香化酶即脑芳香化酶 (P4 5 0aromB)和性腺芳香化酶 (P4 5 0aromA)的cDNA ,其全长分别为 190 1bp (编码 5 0 9aa)和 1833bp (编码 5 18aa)。序列分析结果表明 ,赤点石斑鱼两种芳香化酶cDNA序列的同源性为 5 1 6 % ,氨基酸序列之间同源性为 6 2 5 % ,与斜带石斑鱼两种芳香化酶氨基酸同源性分别为 94 7%和 97 9%。对 8个科的 10种鱼进行了分子系统进化树分析 ,结果与根据传统的形态学和生化特征分类进化地位基本一致。以特异性引物扩增雌、雄赤点石斑鱼各种组织 (垂体、嗅球、端脑、下丘脑、中脑、后脑、延脑、心脏、肾脏、肝脏、脾脏、性腺、鳃、胃、肠、皮肤、脂肪、肌肉、头肾、胸腺、鳔 ) ,以β actin作内标比较各组织芳香化酶基因表达量的差异 ,结果表明 ,赤点石斑鱼脑芳香化酶 (P4 5 0aromB)有广泛的组织分布 ,脑和垂体的表达量很高 ,各组织表达量有明显的雌、雄差异 ;而性腺芳香化酶 (P4 5 0aromA)表达主要集中于垂体和性腺 ,且不论雌雄 ,其性腺表达量均高于脑垂体 ,和P4 5 0aromB的表达模式明显不同 ,表现为在脑部 ,P4 5 0aromB表达量高于P4 5 0aromA ,而在性腺 ,     

Phylogeny, expression and enzyme activity of zebrafish cyp19 (P450 aromatase) genes

The cyp19 encodes P450 aromatase, the enzyme catalyzing the conversion of estrogens from androgens. Estrogens affect the dimorphic, anatomical, functional and behavioral aspects of development of both males and females. In zebrafish, two cyp19 genes, cyp19a and cyp19b were found. They are expressed in ovary and brain, respectively. Expression of cyp19b can be detected by 11 days post-fertilization (dpf) by in situ hybridization in the olfactory bulbs, ventral telencephalic region and the hypothalamus of the brain in both male and female, where it is generally known to be affecting the reproductive function and sexual behavior. COS-1 clones permanently expressing the enzymes have been isolated. Both aromatase enzymes encoded by these two genes are functional in COS-1 cells and they can use androstenedione and testosterone equally efficiently. The presence of two functional cyp19 in zebrafish has its evolutionary and physiological importance.     

Time required for temperature-induced changes in gonadal aromatase activity and related gonadal structure in turtle embryos

Abstract. In the turtle Emys orbicularis , sexual differentiation of gonads is temperature-dependent. Oestrogens have been shown to be involved in this phenomenon and temperature has been expected to act, directly or indirectly, on regulation of synthesis or activity of cytochrome P-450 aromatase (P-450 arom). We have studied the effects of temperature shifts and of exposure at female- or male-producing temperatures for different times on gonadal aromatase activity and gonadal structure. In a first series of experiments, eggs were incubated at 25°C (masculinizing temperature) up to stage 18 and then exposed for 1 to 8 days at 35°C, a highly feminizing temperature. The response was exponential: aromatase activity increased clearly only after 4 day exposure at 35°C, then it was considerably enhanced. After 1 and 2 days at 35°C, the structure of gonads was not modified. With longer exposures at 35°C, gonads were progressively feminized: medullary epithelial cords disappeared, whereas an ovarian cortex was forming. In another type of experiment, eggs incubated at 30°C (feminizing temperature) until stage 19 were transferred at 25°C for 6 days. In embryos of these shifted eggs, gonadal aromatase activity was about ninefold lower than that in control embryos (maintained at 30°C). However, this activity did not fall to the level measured in embryos of the same stage incubated at 25°C from egg-laying and was about twofold higher than that measured at the time of transfer. Gonads exhibited a cortex anlage but the medulla was more voluminous than that of controls and epithelial cords were beginning to form within. Together these results show that changes in gonadal aromatase activity and in gonadal structure are correlated, and that temperature acts on regulation of P-450 arom synthesis. Amplification of this synthesis during the thermosensitive period at higher temperatures could reflect amplification of expression of the P-450 arom gene.     

Sex change strategy and the aromatase genes

Sequential hermaphroditism is a common reproductive strategy in many teleosts. Steroid production is known to mediate both the natural and induced sex change, yet beyond this the physiology directing this process has received little attention. Cytochrome P450 aromatase is a key enzyme in the hormonal pathway catalysing the conversion of sex steroids, androgens to oestrogens, and thus is highly relevant to the process of sex change. This study reports the isolation of cDNA sequences for aromatase isoforms CYP19A1 and CYP19A2 from teleost species representing three forms of sexual hermaphroditism: Lates calcarifer (protandry), Cromileptes altivelis (protogyny), and Gobiodon histrio (bi-directional). Deduced amino acid analysis of these isoforms with other reported isoforms from gonochoristic (single sex) teleosts revealed 56–95% identity within the same isoform while only 48–65% identity between isoforms irrespective of species and sexual strategy. Phylogenetic analysis supported this result separating sequences into isoform exclusive clades in spite of species apparent evolutionary distance. Furthermore, this study isolates 5′ flanking regions of all above genes and describes putative cis-acting elements therein. Elements identified include steroidogenic factor 1 binding site (SF-1), oestrogen response element (ERE), progesterone response element (PRE), androgen response element (ARE), glucocorticoid response elements (GRE), peroxisome proliferator-activated receptor /retinoid X receptor heterodimer responsive element (PPAR/RXR), nuclear factor kappaβ (NF-kappaβ), SOX 5, SOX 9, and Wilms tumor suppressor (WTI). A hypothetical in vivo model was constructed for both isoforms highlighting potential roles of these putative cis-acting elements with reference to normal function and sexual hermaphroditism.     

Neurological effects of aromatase deficiency in the mouse

In the brain, the conversion from androgen into estrogen is an important process for the differentiation of the brain function in male rodents. The aromatase is expressed in some nucleus of the brain. To assess the functional significance of the aromatase gene in development and activation of sex-specific behavior, we analyzed behavioral phenotypes of the aromatase knockout (ArKO) male mice. ArKO males obviously decreased their fertility and showed deficits in male sexual behavior including mount, intromission and ejaculation. Noncontact penile erection was not significantly affected by defect of the aromatase gene. A reduction of aggressive behavior against male intruders was also observed in ArKO males, while they tend to exhibit aggression toward estrous females during male copulatory tests. Moreover, the infanticide toward the pups was observed in the ArKO males, whereas characteristic parental behavior, but not infanticide was observed in wild-type males. These results indicate that aromatase gene expression is a critical step not only for motivational and consummatory aspects of male sexual behavior, but also for aggressive and parental behaviors in male mice.     

Aromatase immunolocalization and activity in the lizard's brain: Dynamic changes during the reproductive cycle

The purpose of the present study is to highlight the role of aromatase in the neuroendocrine control of the reproductive cycle of the male lizard Podarcis sicula during the three significant phases, i.e. the pre-reproductive, reproductive, and post-reproductive stages. Using immunohistochemical, biochemical, and hormonal tools, we have determined the localization and the activity of P450 aromatase (P450 aro) in the lizard's brain together with the determination of hormonal profile of sex steroids, i.e. testosterone and 17β-estradiol. The present data demonstrated that the localization of P450 is shown in brain regions involved in the regulation of the reproductive behavior (medial septum, preoptic area, and hypothalamus). Its activity, as well as the intensity of the signal, is modified according to the period of reproduction, resulting in functional dynamic changes. P450 aro activity and signal intensity decrease in the pre-reproductive period and progressively increase during the reproductive stage until it reaches the maximum peak level at the post-reproductive phase. P450 aro determines a local estrogen synthesis, balancing the testosterone and estradiol levels, and therefore its role is crucial, since it plays an important role in the neuroendocrine/behavioral regulation of the reproductive processes in the male lizard P. sicula.     

Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance

In adult male primates, estrogens play a role in both gonadotropin feedback and sexual behavior. Inhibition of aromatization in intact male monkeys acutely elevates serum levels of luteinizing hormone, an effect mediated, at least partially, within the brain. High levels of aromatase (CYP19) are present in the monkey brain and regulated by androgens in regions thought to be involved in the central regulation of reproduction. Androgens regulate aromatase pretranslationally and androgen receptor activation is correlated with the induction of aromatase activity. Aromatase and androgen receptor mRNAs display both unique and overlapping distributions within the hypothalamus and limbic system suggesting that androgens and androgen-derived estrogens regulate complimentary and interacting genes within many neural networks. Long-term castrated monkeys, like men, exhibit an estrogen-dependent neural deficit that could be an underlying cause of the insensitivity to testosterone that develops in states of chronic androgen deficiency. Future studies of in situ estrogen formation in brain in the primate model are important for understanding the importance of aromatase not only for reproduction, but also for neural functions such as memory and cognition that appear to be modulated by estrogens.     

Rat Sertoli cell aromatase cytochrome P450: Regulation by cell culture conditions and relationship to the state of cell differentiation

Summary Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions. No differences were found in P450arom enzymatic activity measured by the³H₂O release method in microsomes prepared from Sertoli cells cultured under the various conditions. Similarly, no differences were observed in the amount of protein detected by immunoblot analysis of Sertoli cell extracts using an antiserum raised against the human placental enzyme. Recombination experiments using microsomes from cells cultured on plastic or in the chambers and cytosol from control or FSH-treated cells cultured on plastic also proved inadequate in inducing P450arom activity. These data suggest that: a) P450arom activity could be used as a specific marker for Sertoli cell differentiation, and b) the differentiation process in Sertoli cells is associated with specific changes in the microenvironment or the regulation of P450arom, or both, that rendered the enzyme insensitive to FSH or cAMP induction.     

Efficacy of exemestane,a new generation of aromatase inhibitor,on sex differentiation in a gonochoristic fish

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 µg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 µg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17β level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3β-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.     

Brain aromatase: roles in reproduction and neuroprotection

It is well established that aromatization constitutes an essential part of testosterone's signaling pathway in brain and that estrogen metabolites, often together with testosterone, organize and activate masculine neural circuits. This paper summarizes the current understanding regarding the distribution, regulation and function of brain aromatase in mammals. Data from our laboratory are presented that highlight the important function of aromatase in the regulation of androgen feedback sensitivity in non-human primates and the possible role that aromatase plays in determining the brain structure and sexual partner preferences of rams. In addition, new data is presented indicating that the capacity for aromatization in cortical astrocytes is associated with cell survival and may be important for neuroprotection. It is anticipated that a better appreciation of the physiological and pathophysiological functions of aromatase will lead to important clinical insights.     

Embryological ontogeny of aromatase gene expression in Chrysemys picta and Apalone mutica turtles: comparative patterns within and across temperature-dependent and genotypic sex-determining mechanisms

Although the role of aromatase in many estrogen-dependent reproductive and metabolic functions is well documented in vertebrates, its involvement in the ovarian development of species exhibiting temperature-dependent sex determination (TSD) is incompletely understood. This is partly due to the conflicting temporal and spatial pattern of aromatase expression and activity across taxa. To help resolve this ongoing debate, we compared for the first time the embryological ontogeny of aromatase expression in turtles possessing genotypic sex determination (GSD) (Apalone mutica) and TSD (Chrysemys picta) incubated under identical conditions. As anticipated, we found no significant thermal differences in aromatase expression at any stage examined (prior to until the end of the thermosensitive period) in A. mutica. Surprisingly, the same was true for C. picta. When placed in a phylogenetic context, our results suggest that aromatase expression is evolutionarily plastic with respect to sex determination in reptiles, and that differences between reptilian TSD and GSD are not aromatase-driven. Further research across TSD and GSD species is warranted to fully decipher the evolution of functional differences among sex-determining mechanisms.     

泽蛙的性腺分化及温度对性别决定的影响

通过组织学方法观察了泽蛙(Rana limnocharis)原始生殖细胞(PGCs)的迁移、原始性腺的形成和性腺分化,并且探讨在不同的培育温度条件下性腺分化的差异。泽蛙的性腺分化有其特殊性:生殖嵴形成时,其中既有体细胞,又有原生殖细胞;无论原始性腺是分化成为精巢还是卵巢,其中都出现一个初生性腔。蝌蚪孵化后的17-34d(Gosner 26-38期)为性腺分化的敏感时期。在蝌蚪孵化后的第2d(Gosner 25期),分别用不同水温18℃±1℃、30℃±1℃、32℃±1℃、34℃±1℃培育蝌蚪,直至完成变态幼蛙(Gosner 46期)形成。自然水温23℃-25℃为对照。对照组的雌、雄性比接近1∶1(1∶1.06);18℃±1℃实验组的雌、雄比例为1.83∶1,雄性率仅35.1%(P<0.01);从30℃±1℃实验组起,雄性率提高,34℃±1℃实验组的雄性率达74.0%(P<0.01)。较高的培育温度可使泽蛙蝌蚪性别分化趋向雄性,而较低的培育温度则使蝌蚪雌性化。泽蛙的性别分化属于温度依赖型性决定(TSD)。当前全球性气候变暖对两栖类性比的稳定存在着威胁。     

Conditional knockout of the mouse NADPH-cytochrome p450 reductase gene

NADPH-cytochrome P450 reductase (CPR or POR) is the obligatory electron donor for all microsomal cytochrome P450 (CYP or P450)-catalyzed monooxygenase reactions. Disruption of the mouse Cpr gene has been reported to cause prenatal developmental defects and embryonic lethality. In this study, we generated a mouse model with a floxed Cpr allele (termed Cpr(lox)). Homozygous Cpr(lox) mice are fertile and without any histological abnormality or any change in CPR expression. The floxed Cpr allele was subsequently deleted efficiently by crossing Cpr(lox) mice with transgenic mice having liver-specific Cre expression (Alb-Cre); the result was a decrease in the level of CPR protein in liver microsomes. The Cpr(lox) strain will be valuable for conditional Cpr gene deletion and subsequent determination of the impact of CPR loss on the metabolism of endogenous and xenobiotic compounds, as well as on postnatal development and other biological functions.