|Budget Amount *help
¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥2,900,000 (Direct Cost : ¥2,900,000)
Fish oocyte maturation is induced by maturation-inducing hormone, 17alpha, 20beta-dihydroxy-4-pregnen-3-one (l7alpha, 2Obeta-DP) acting upon an oocyte surface receptor.We evidenced that a pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) was involved in the signal transduction pathway of l7alpha, 2Obeta-DP in rainbow trout oocytes.We cloned two Gialpha subunit cDNAs, Gialpha1 and Gialpha2, from Medaka (Oryzias latipes) oocyte cDNA library. Three rabbit polyclonal antibodies, A2, C4, and M1, were generated against deduced internal amino acid sequences of Gialpha1 and Gialpha2, and a common C-terminal sequence, respectively.Ml antibody successfully immunoprecipitated Gialphal and Gialpha2 subunit proteins.However, l7alpha, 2Obeta-DP-binding acitivity was almost not detected in the immunoprecipitate.
l7alpha, 2Obeta-DP receptor activity was solubilized from membrane preparations of goldfish oocytes by sucrose monolaurate. Solubilized activity was serially separated by following four-step chromatography ; gel-filtration, anion-exchange, hydrophobic interaction, and lectin affinity chromatography.Major seven proteineous bands on SDS-PAGE were detected by silver staining.
In medaka (Oryzias latipes) oocytes, the amount of l7alpha, 2Obeta-DP binding to the oocyte cortices was shown to increase with maturational competence of oocytes and reach a maximum one day before spawning.When oocyte maturation was triggered with l7alpha, 2Obeta-DP, 17alpha, 2Obeta-DP binding and Gi decreased quickly, suggesting a close relation between Gi and the l7alpha, 2Obeta-DP receptor.