| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Gonadotropin-releasing hormone(GnRH) is a highly conserved molecule, which is important for reproduction in vertebrate species. The present study was undertaken to identify molecules that control GnRH neuronal differentiation, migration and gene expression. To address this issue, we successfully coupled the anatomic specificity of immunofluorescently defined laser microdissection with the sensitivity of real-time quantitative RT-PCR (RT-Q-RT-PCR), which enabled us to examine the presence and quantity of novel and GnRH mRNAs in individual neurons harvested from the brain of mature and immature males of tilapia, Oreochromis niloticus. Here, we present evidence that shows preoptic GnRH1 is important for gonadal maturation, whereas GnRH2 and GnRH3 might have supplementary roles in reproductive behaviors or nonreproductive functions. Further, we isolated and cloned GPR54 for the first time in a non-mammalian vertebrate. GPR54, a novel G protein-coupled receptor, is speculated to be essential for sexual development. The tilapia GPR54 cDNA contains an open reading frame of 1131 bp encoding 377 amino acids and exhibits 56% identity to human GPR54. At the single-cell level, only in mature males, GnRH1 mRNA levels were inversely related to GPR54 mRNA (P<0.002). GPR54 was expressed in a significantly high percentage (45.0-60.0%) of mature GnRH1,GnRH2,and GnRH3 neurons and in immature GnRH3 neurons, which had migrated to the vicinity of their final locations in the brain : on the contrary, only 5.0% of immature GnRH1 and GnRH2 neurons had GPR54 transcripts (P<0.001). Thus, using a novel innovative single-cell gene profiling technique, we provide evidence that the expression of GPR54 is a "stop signal" for GnRH1,GnRH2,and GnRH3 neuronal migration, leading to suppression of cell growth and modulation of GnRH secretion, which is important for normal sexual development.
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