M/lecular regulation of germ cell formation

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The molecular mechanism of germ cell formation, in particular spermatogenesis and oocyte maturation, has been investigated, using fish as a primary study model.1) The cDNAs encoding 5 steroidogenic enzymes responsible for 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17a, 20b-DP, a common maturation-inducing hormone in fish) biosynthesis have been isolated and sequenced. We have shown that gonadotropin-induced 20beta-HSD mRNA expression is a prerequisite for 17a, 20b-DP production by granulosa cells. 2) We characterized specific binding of [^3H]17a, 20b-DP to plasma membranes from defolliculated oocytes of rainbow trout. A gonadotropin-induced increase in 17a, 20b-DP receptor concentrations is responsible for the development of oocyte maturational competence. Carp maturation-promoting factor (MPF) (cdc2 kinase and cyclin B) was purified and characterized. 17a, 20b-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting cdc2 kinase through threonine phosphorylation (161) of cdc2 kinase by a threonine kinase (p40^<M015>). We discovered that cyclin degradation is triggered by limited proteolysis of cyclin B (Lys57) by active proteasome. 3) An organ culture system has been used to demonstrate that in the eel 11-ketotestosterone can induce all stages of spermatogenesis in vitro within 21 days. Our findings suggest that gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone, which in turn activates the Sertoli cells to produce activin B.Activin B then acts on spermatogonia to induce mitosis leading to the formation of spermatocytes.