Chang Xiaotian, Kobayashi Thoru, Senthilkumaran Balasubramanian, Kobayashi-Kajura Hiroko, Sudhakumari Cheni Chery, Nagahama Yoshitaka
Department of Reproductive Biology, National Institute for Basic Biology, 444 Okazaki, Japan.
Gen Comp Endocrinol. 2005 Apr;141(2):101-15. doi: 10.1016/j.ygcen.2004.11.020. Epub 2005 Jan 26.
We isolated a novel type of aromatase cDNA from a Nile tilapia (Oreochromis niloticus) ovary cDNA library. Because this aromatase is phylogenetically related to brain aromatase (CYP19b) of goldfish, zebrafish and sea bass, we named it tilapia CYP19b (tCYP19b). tCYP19b encodes a protein that is predicted to consist of 495 residues and have 63.8% homology with the aromatase (tCYP19a) we previously isolated from the same source. In vitro transient transfection of cultured COS7 cells demonstrated that tCYP19b codes a functional protein to catalyze estrogen production from an androgen substrate. RT-PCR and Northern hybridization analysis showed that tCYP19b was expressed at a high level in the brain and at a low level in a wide variety of other tissues, whereas tCYP19a was mainly present in the ovary and its level significantly increased during the vitellogenic stage. RT-PCR also detected tCYP19b expression in brain and gonad tissues of both female and male tilapia during sex differentiation, but tCYP19a was only found in the ovary of the fry at that period. These results suggest that tCYP19a plays a key role in sex differentiation and ovarian development. We also isolated genes of two tilapia aromatases. Based on the location of the transcription initiation site, we predicted that there is one promoter for tCYP19a and three promoters for tCYP19b. Although the two aromatase isoforms have similar gene structures in the coding region, we found that the binding regions of SF-1/Ad4 BP region, WT1-KTS and SRY, which are sex-determining factors in mammals, are present in the 5' flank region of tCYP19a but not tCYP19b. A similar situation is present in promoters of zebrafish and goldfish aromatase isoforms. This data indicates that CYP19a plays a decisive role in sex differentiation of those species. The unique presence of the ERE motif in the tCYP19b promoter and the high expression of tCYP19b in the brain support that CYP19b is mainly involved in estrogen-mediated neural estrogen synthesis.
我们从尼罗罗非鱼（Oreochromis niloticus）卵巢cDNA文库中分离出一种新型芳香化酶cDNA。由于这种芳香化酶在系统发育上与金鱼、斑马鱼和海鲈的脑芳香化酶（CYP19b）相关，我们将其命名为罗非鱼CYP19b（tCYP19b）。tCYP19b编码一种预计由495个氨基酸残基组成的蛋白质，与我们之前从同一来源分离出的芳香化酶（tCYP19a）具有63.8%的同源性。培养的COS7细胞的体外瞬时转染表明，tCYP19b编码一种功能性蛋白质，可催化雄激素底物生成雌激素。RT-PCR和Northern杂交分析表明，tCYP19b在脑中高水平表达，在多种其他组织中低水平表达，而tCYP19a主要存在于卵巢中，并且在卵黄生成期其水平显著增加。RT-PCR还检测到在性别分化期间，雌性和雄性罗非鱼的脑和性腺组织中均有tCYP19b表达，但在那个时期仅在鱼苗的卵巢中发现tCYP19a。这些结果表明，tCYP19a在性别分化和卵巢发育中起关键作用。我们还分离了两种罗非鱼芳香化酶的基因。基于转录起始位点的位置，我们预测tCYP19a有一个启动子，tCYP19b有三个启动子。尽管这两种芳香化酶同工型在编码区具有相似的基因结构，但我们发现哺乳动物中的性别决定因子SF-1/Ad4 BP区域、WT1-KTS和SRY的结合区域存在于tCYP19a的5'侧翼区域，而不存在于tCYP19b中。斑马鱼和金鱼芳香化酶同工型的启动子中也存在类似情况。这些数据表明，CYP19a在这些物种的性别分化中起决定性作用。tCYP19b启动子中ERE基序的独特存在以及tCYP19b在脑中的高表达支持了CYP19b主要参与雌激素介导的神经雌激素合成。
