Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development
Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development
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DOI：
10.1210/en.142.2.740
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发表时间：
2001-02-01
期刊：
影响因子：
4.8
通讯作者：
Callard, GV
中科院分区：
文献类型：
--
作者：
Kishida, M;Callard, GV
作为了解雌激素在神经发育中的作用的第一步，使用PCR克隆策略分离编码两种不同的细胞色素P450芳香化酶异构体在成年斑马鱼（Danio rerio）脑和卵巢（分别称为P450 aromB和P450 aromA）的互补DNA。序列和系统发育分析表明，斑马鱼P450 arom形式是以前确定的cyp 19 b和cyp 19 a的直系同源物。金鱼的基因在北方印迹上，在脑中鉴定出P450 aromB亚型的单个4.4-kb转录物，在卵巢中鉴定出P450 aromA亚型的2.1-kb转录物，但RT-PCR显示一定程度的重叠表达。这两种信使RNA（mRNA）的形式在未受精卵和1.5 hpf（卵裂期）胚胎中检测到，但下降了12 hpf，表明母体转移。在12-24 hpf之间的mRNA的二次上升表明胚胎cyp 19 b和α转录的开始。这两种mRNA逐渐积累到120 hpf（早期幼虫阶段），但相对的变化幅度和模式是亚型特异性的。雌激素（E-2，1 μ M）促进和扩增P450 aromB mRNA的发育程序性积累，ICI 164.384降低表达水平，这意味着阻断内源性雌激素介导的调节成分。相反，E-2没有影响或降低P450 aromA mRNA。P450 aromB和P450 aromA亚型的早期胚胎表达，以及发育编程和雌激素调节的差异，意味着在主要的形态发生和分化事件中独立的调节机制和独特的功能。
As a first step toward understanding estrogen's role in neurodevelopment, a PCR cloning strategy was used to isolate complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms in adult zebrafish (Danio rerio) brain and ovary (termed P450aromB and P450aromA, respectively). Sequence and phylogenetic analysis showed that the zebrafish P450arom forms are orthologs of previously identified cyp19b and cyp19a. genes in goldfish. On Northern blots, a single 4.4-kb transcript of the P450aromB subtype was identified in brain, and a 2.1-kb transcript of the P450aromA subtype in ovary, but RT-PCR showed a degree of overlapping expression. Both messenger RNA (mRNA) forms were detected in unfertilized eggs and 1.5 hpf (cleavage stage) embryos but declined by 12 hpf, indicating maternal transfer. A secondary rise in mRNAs between 12-24 hpf indicated the onset of embryonic cyp19b and -alpha transcription. Both mRNA species accumulated progressively to 120 hpf (early larval stage), but the relative magnitude and pattern of change was isoform specific. Estradiol (E-2,1 muM) advanced and amplified the developmentally programmed accumulation of P450aromB mRNA, and ICI164.384 decreased expressed levels, implying blockade of an endogenous estrogen mediated regulatory component. Conversely, E-2 had no effect or decreased P450aromA mRNA. The early embryonic expression of P450aromB and P450aromA isoforms, and differences in developmental programming and estrogen regulation, imply independent regulatory mechanisms and unique functions during major morphogenetic and differentiative events.