LHRH messenger RNA in neurons in the intact and castrate male rat forebrain, studied by in situ hybridization.
LHRH messenger RNA in neurons in the intact and castrate male rat forebrain, studied by in situ hybridization.
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通过原位杂交研究完整和去势雄性大鼠前脑神经元中的 LHRH 信使 RNA。
DOI：
10.1007/bf00269459
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发表时间：
1987
影响因子：
2
通讯作者：
Pfaff,DW
中科院分区：
文献类型：
--
作者：
Rothfeld,JM;Hejtmancik,JF;Conn,PM;Pfaff,DW
本研究的目的是定位促黄体生成素释放激素（LHRH）mRNA在雄性大鼠前脑内使用原位杂交方法。比较了去势和完整男性LHRH mRNA的表达，以探讨循环睾丸类固醇对肽基因表达的慢性影响。从完整和去势后2周的成年雄性大鼠前脑区域获得在多聚甲醛中固定的冷冻10 μm切片。使用与编码人LHRH前激素原的氨基酸-5至15的mRNA互补的寡聚体（59聚体）放射自显影检测LHRH mRNA。将单个脑切片在预杂交缓冲液中孵育2小时以减少非特异性结合。此后，将含有65，000 - 120，000 cpm的59聚体的20 μ 1杂交缓冲液施加到切片上，并在37° C下杂交3天。然后将切片冲洗48小时，脱水，浸入Kodak NTB 2液体乳剂中并暴露22天。显影放射自显影图并用固绿色和甲苯紫复染。雌性大鼠的LHRH信号细胞分布于腹隔区、Broca斜角带、视前区和下丘脑前部。有时，发现LHRH基因表达细胞以松散的簇出现。在用缺乏59聚体的杂交缓冲液处理的对照切片中从未发现标记的细胞。LHRH mRNA的细胞定位在完整的大鼠的总数没有显着差异去势组。发现完整组（30.1±1.2）和去势组（24±1.1）的每个细胞的平均颗粒计数（±SEM）不同，这两个群体的直方图分布也不同。这些结果与基于负反馈效应的预期结果相反，相反，表明长期暴露于睾丸类固醇实际上可以增加单个神经元内LHRH mRNA的含量。
The purpose of the present study was to localize luteinizing hormone-releasing hormone (LHRH) mRNA within the male rat forebrain using an in situ hybridization approach. The expression of LHRH mRNA was compared in castrate and intact males to approach questions on the chronic influences of circulating testicular steroids on the gene expression of the peptide. Frozen 10 μm sections fixed in paraformaldehyde were obtained from the forebrain region of intact and 2 week post-castrate adult male rats. LHRH mRNA was autoradiographically detected using an oligomer (59mer) complementary to the mRNA coding for amino acids-5 to 15 of the human LHRH preprohormone. Individual brain sections were incubated in prehybridization buffer for 2 h to reduce nonspecific binding. Following this, 20 μl of hybridization buffer containing 65,000–120,000 cpm of the 59mer were applied to sections and hybridized at 37° C for 3 days. The sections were then rinsed over a 48 h period, dehydrated, dipped in Kodak NTB2 liquid emulsion and exposed for 22 days. Autoradiograms were developed and counterstained with fast green and cresyl violet. As reported in the female, LHRH message-containing cells were localized in ventral septal regions, the diagonal bands of Broca, preoptic area and anterior hypothalamus. On occasion, LHRH gene expressing cells were found to appear in loose clusters. Labeled cells were never found in control sections treated with hybridization buffer lacking the 59mer. The total number of LHRH mRNA-containing cells localized in intact rats did not differ significantly from the castrate group. The mean grain counts per cell (±SEM) for the intact (30.1±1.2) and castrate (24±1.1) groups were found to differ, as did the histogram distribution of these two populations. These results are in contrast to those expected on the basis of a negative feedback effect, and instead suggest that long term exposure to testicular steroids can actually increase the content of LHRH mRNA within individual neurons.