| Budget Amount *help |
¥11,800,000 (Direct Cost: ¥11,800,000)
Fiscal Year 1999: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 1998: ¥6,400,000 (Direct Cost: ¥6,400,000)
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| Research Abstract |
To investigate the gene regulatory mechanisms of gonadotropin-induced synthesis of gamete maturation-inducing hormone (17α, 20β-dihydroxy-4-pregnen-3-one, 17α, 2Oβ-DP) in fish, we have analyzed the 20β-hydroxysteroid dehydrogenase (20β-HSD, the enzyme which converts 17α-hydroxyprogesterone to 17α, 20β-DP) genes isolated from rainbow trout follicles. Two closely related 2Oβ-HSD cDNAs which encode two proteins with only three amino acid differences were isolated. Analysis of activity of recombinant proteins resulting from expression in E.coli showed that one type I had 20β-HSD enzyme activitv. but the other (tyae II) did not possess 20β-HSD activity. Northern blot analysis showed a marked 2Oβ-HSD gene expression in granulosa cells during final oocyte maturation.
The 5' flanking region (-750 bp) of the type I gene contained multiple cis-element motifs including GC-rich region, CRE, Ad4 sites with the presence of two TATA-box like sequences. Conversely, the type II gene did not possess either such cis-element motifs or TATA-box at least up to -750 bp. Comparative analysis of luciferase activity revealed the presence of promoter activity only in type I 20β-HSD gene. Analysis of series of 5'-deletion mutants showed that the 5'-flanking bases -477 to -467 (putative TATA-box) and -50 to -30 (a functional CRE with GC-rich region) are responsible for the promoter activity in type I 20β-HSD gene. Ad4 consensus motif did not show any promoter activity. In this study, we have also cloned and characterized, for the first time in fish, two different gonadotropin receptors from rainbow trout and tilapia ovaries. Temporal and spatial expression patterns suggest that LH receptors are involved in the gonadotropin (LH)-induced transcription of 2Oβ-HSD gene during final maturation of female and male gametes in these two teleost species.
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