Gene regulations of fish gonadotropin receptor, unity and diversity

作为阐明鱼类促性腺激素受体(GTH-R)统一性和多样性的第一步，对GTH-R的分子结构和基因表达谱进行了比较研究。首先，我们根据已知的哺乳动物糖蛋白激素受体家族包括GTH-Rs的结构，建立了简并RT-PCR体系。虽然我们利用这个系统成功地从几个物种中克隆了cDNA，但它往往产生非特异性的产物，需要进一步复杂的过程才能获得受体cDNA。因此，我们在已知鱼类GTH-Rs保守序列的基础上，设计了两套新的引物。新的引物成功地从所有物种中扩增出187bp(介于ECDI和TMD2之间)和257bp(介于ECD3和TMD6之间)的cDNA片段。到目前为止，我们已经成功地从7个不同目的11个物种中克隆了两种类型的GTH-R(FSH-R和Lh-R)cDNA(包括一些合作的结果)。这些结果表明，该聚合酶链式反应克隆体系具有很强的适应性。在几个物种的配子发生过程中，研究了这两种受体的mRNA水平的变化。在Amago鲑鱼卵子发生过程中，FSH-R基因的表达在卵黄发生早期较高，成熟后期呈下降趋势，而黄体生成素受体基因的表达在成熟末期出现峰值。在罗非鱼领先卵母细胞的发育过程中也观察到了类似的情况。在青竹卵巢中，两种受体mRNAs在卵黄形成期均可检测到，而FSH-RmRNA在成熟期较早消失。在鲑鱼和大马哈鱼的雄鱼中，两种类型的卵子形态相似，均在受精时达到峰值。最后，从Amago鲑鱼中克隆了FSH-R(1.4kb)和Lh-R(2.2kb)基因的5‘侧翼区，以研究它们的调控元件。初步报告实验表明，雌激素和雄激素均不直接影响其启动子活性。

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.