Shibutani Makoto, Lee Kyoung-Youl, Igarashi Katsuhide, Woo Gye-Hyeong, Inoue Kaoru, Nishimura Tetsuji, Hirose Masao
Division of Pathology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan.
Dev Neurobiol. 2007 Feb 15;67(3):253-69. doi: 10.1002/dneu.20349.
To identify genes linked to early stages of disruption of brain sexual differentiation, hypothalamic region-specific microarray analyses were performed using a microdissection technique with neonatal rats exposed to endocrine-acting drugs. To validate the methodology, the expression fidelity of microarrays was first examined with two-round amplified antisense RNAs (aRNAs) from methacarn-fixed paraffin-embedded tissue (PET) in comparison with expression in unfixed frozen tissue (UFT). Decline of expression fidelity when compared with the 1x-amplified aRNAs from UFTs was found as a result of the preferential amplification of the 3' side of mRNAs in the second round in vitro transcription. However, expression patterns for the 2x-amplified aRNAs were mostly identical between methacarn-fixed PET and UFT, suggesting no obvious influence of methacarn fixation and subsequent paraffin embedding on expression levels. Next, in the main experiment, neonatal rats at birth were treated subcutaneously either with estradiol benzoate (EB; 10 microg/pup) or flutamide (FA; 250 microg/pup), and medial preoptic area (MPOA)-specific microarray analysis was performed 24 h later using 2x-amplified aRNAs from methacarn-fixed PET. Numbers of genes showing constitutively high expression in the MPOA predominated in males, implying a link with male-type growth supported by perinatal testosterone. Around 60% of genes showing sex differences in expression demonstrated altered levels after EB treatment in females, suggesting an involvement of genes necessary for brain sexual differentiation. When compared with EB, FA affected a rather small number of genes, but fluctuation was mostly observed in females, as with EB. Moreover, many selected genes common to EB and FA showed down-regulation in females with both drugs, suggesting a common mechanism for endocrine center disruption in females, at least at early stages of post-natal development.
为了鉴定与脑性别分化破坏早期阶段相关的基因，利用显微切割技术对暴露于内分泌作用药物的新生大鼠进行下丘脑区域特异性微阵列分析。为了验证该方法，首先用来自甲醇-氯仿固定石蜡包埋组织（PET）的两轮扩增反义RNA（aRNA）与未固定冷冻组织（UFT）中的表达进行比较，检测微阵列的表达保真度。与来自UFT的1倍扩增aRNA相比，由于第二轮体外转录中mRNA 3'端的优先扩增，发现表达保真度下降。然而，甲醇-氯仿固定的PET和UFT之间2倍扩增aRNA的表达模式大多相同，表明甲醇-氯仿固定和随后的石蜡包埋对表达水平没有明显影响。接下来，在主要实验中，新生大鼠出生时皮下注射苯甲酸雌二醇（EB；10μg/幼崽）或氟他胺（FA；250μg/幼崽），24小时后使用来自甲醇-氯仿固定PET的2倍扩增aRNA进行内侧视前区（MPOA）特异性微阵列分析。在MPOA中显示组成性高表达的基因数量在雄性中占主导，这意味着与围产期睾酮支持的雄性型生长有关。约60%在表达上显示性别差异的基因在雌性EB处理后表达水平发生改变，表明参与脑性别分化的基因发挥了作用。与EB相比，FA影响的基因数量较少，但与EB一样，波动大多在雌性中观察到。此外，许多EB和FA共有的选定基因在两种药物处理的雌性中均显示下调，表明至少在出生后发育早期，雌性内分泌中心破坏存在共同机制。
