| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
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.
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