Gene regulations of fish gonadotropin receptor, unity and diversity

为了阐明鱼类促性腺激素受体（GTH-R）的统一性和多样性，对GTH-R的分子结构和基因表达谱进行了比较研究。首先，我们在已知哺乳动物糖蛋白激素受体家族（包括GTH受体）结构的基础上，建立了简并RT-PCR体系。虽然我们成功地克隆了几个物种的cDNA，但它往往产生非特异性产物，并且需要进一步复杂的过程来获得受体cDNA。因此，我们在已知鱼类GTH受体保守序列的基础上设计了两对引物。新引物成功地从所有物种中扩增出187 bp（ECDI和TMD 2之间）和257 bp（ECD 3和TMD 6之间）的cDNA片段。到目前为止，我们已经成功地克隆了两种类型的GTH-R（FSH-R和LH-R）cDNA来自11个物种属于7个不同的目的（包括一些合作的结果）。这些结果表明，该PCR克隆系统具有较高的适用性，广泛的鱼类物种。在配子发生过程中，这两种受体的mRNA水平的变化在几个物种进行了研究。在卵子发生过程中，FSH-R mRNA在卵黄发生早期较高，向成熟期逐渐下降，LH-R mRNA在成熟末期出现高峰。在罗非鱼主窝卵母细胞的发育过程中也观察到类似的变化。在青鳉卵巢中，两种受体mRNA在卵黄发生期均被检测到，而FSH-R mRNA在成熟期提前消失。在鲑鱼和杜父鱼的雄性中，两种类型都表现出相似的模式，在精子产生时达到高峰。最后，从大麻哈鱼中克隆了FSH-R基因（1.4kb）和LH-R基因（2.2kb）的5 '侧翼区，以表征其调控元件。初步的报告基因分析表明，雌激素和雄激素都不直接影响它们的启动子活性。

As a first step to elucidate the unity and diversity of fish gonadotropin receptor (GTH-R), comparative study for molecular structure and gene expression profiles were performed. Initially, we established a degenerate RT-PCR system on the basis of the structures of known mammalian glycoprotein hormone receptor family including GTH-Rs. Although we succeeded to clone cDNAs from several species using this system, it often gave nonspecific products and required further complicated process to obtain receptor cDNAs. Therefore, we newly designed two primer sets on the basis of conserved sequences of known fish GTH-Rs. The new primer sets successfully amplified cDNA fragments of 187bp (between ECDI and TMD2) and 257bp (between ECD3 and TMD6) from all species attempted. To date, we have succeeded to clone both types of GTH-R (FSH-R and LH-R) cDNAs from 11 species belonging 7 different orders (including results of some collaborations). These results suggest that this PCR cloning system has high applicability to wide range of fish species. Change of mRNA levels for the two types of receptor during gametogenesis were examined in several species. In amago salmon oogenesis, FSH-R mRNA was high at early vitellogenic stage and decreased toward the maturational stage, whereas LH-R mRNA showed a peak at the final maturation. A similar profile was observed in the process of oocyte development of the leading clutch of tilapia. In medaka ovary, both types of receptor mRNAs were detected at vitellogenic stage, while FSH-R mRNA was disappeared earlier toward the maturational stage. In male of salmon and sculpin, both types showed similar pattern with a peak at spermiation. Finally, 5'-flanking regions of genes for FSH-R (1.4kb) and LH-R (2.2kb) were cloned from amago salmon to characterize their regulatory elements. Preliminary reporter assay showed that neither estrogen nor androgen do not directly affect their promoter activities.