Callard G V, Drygas M, Gelinas D
Department of Biology, Boston University, MA 02215.
J Steroid Biochem Mol Biol. 1993 Mar;44(4-6):541-7. doi: 10.1016/0960-0760(93)90257-w.
Due to exceptionally high brain aromatase activity, teleost fish are advantageous for studying neural aromatase regulation, localization, and physiology. To determine the molecular mechanism of enhanced expression, we have isolated, cloned and sequenced a 3 kb full-length aromatase cDNA from a goldfish (Carassius auratus) brain library using a human placental aromatase cDNA as probe. The deduced sequence of goldfish aromatase is 510 amino acids (predicted Mw, 58 kDa) with 69% overall sequence similarity, when compared to human placental aromatase, and higher homologies in presumptive functional domains. A major 3 kb mRNA species was abundant in brain and low or non-detectable in non-neural tissues, reflecting the order of enzyme activities. To determine the cellular basis of high enzyme activity in goldfish brain, a human placental aromatase antibody was used to immunolocalize labeled cells. This antibody immunoprecipitated a single 56 kDa in vitro translation product of goldfish brain poly(A+)RNA and revealed discrete clusters of intensely stained neurons, processes, and terminals concentrated in, but not limited to, reproductive brain centers. Close proximity of aromatase- and androgen receptor-positive neurons in certain regions provides anatomic evidence of a functional relationship between direct and indirect pathways of neural androgen action. Aromatase-positive neurons and fibers formed interconnected networks in novel loci (e.g. retina-->optic tract-->optic tectum), and catalytic activity was confirmed biochemically in these tissues, indicating that neuroestrogen may have a role in visual input and integration. Availability of goldfish-specific nucleotide and antibody probes will facilitate further studies using this model.
由于硬骨鱼的脑芳香化酶活性极高，因此它们在研究神经芳香化酶的调节、定位及生理学方面具有优势。为了确定其表达增强的分子机制，我们以人胎盘芳香化酶cDNA为探针，从金鱼（Carassius auratus）脑文库中分离、克隆并测序了一个3 kb的全长芳香化酶cDNA。与人类胎盘芳香化酶相比，金鱼芳香化酶的推导序列为510个氨基酸（预测分子量为58 kDa），总体序列相似性为69%，在假定的功能域中具有更高的同源性。一种主要的3 kb mRNA在脑中含量丰富，在非神经组织中含量低或无法检测到，这反映了酶活性的顺序。为了确定金鱼脑中高酶活性的细胞基础，我们使用人胎盘芳香化酶抗体对标记细胞进行免疫定位。该抗体免疫沉淀了金鱼脑poly(A+)RNA的一种单一体外翻译产物，分子量为56 kDa，并揭示了密集染色的神经元、突起和终末的离散簇，这些主要集中在但不限于生殖脑区。在某些区域，芳香化酶阳性神经元和雄激素受体阳性神经元紧密相邻，为神经雄激素作用的直接和间接途径之间的功能关系提供了解剖学证据。芳香化酶阳性神经元和纤维在新的位点（如视网膜→视束→视顶盖）形成了相互连接的网络，并且在这些组织中通过生化方法证实了催化活性，这表明神经雌激素可能在视觉输入和整合中发挥作用。金鱼特异性核苷酸和抗体探针的可用性将有助于利用该模型进行进一步的研究。
