M/lecular regulation of germ cell formation

以鱼类为研究对象，对鱼类生殖细胞形成的分子机制，特别是精子发生和卵母细胞成熟的分子机制进行了研究。1）分离并测序了5种甾体合成酶的cDNAs，这些酶负责17 α，20 β-二羟基-4-生育酚-3-酮（17 a，20 b-DP，鱼类中常见的成熟诱导激素）的生物合成。我们已经证明，促性腺激素诱导的20 β-HSD mRNA表达是颗粒细胞产生17 a，20 b-DP的先决条件。2)我们表征了[^3H] 17 a，20 b-DP与虹鳟鱼去卵泡卵母细胞质膜的特异性结合。促性腺激素诱导的17 a，20 b-DP受体浓度的增加是负责卵母细胞成熟能力的发展。对鲤鱼促成熟因子（MPF）（cdc 2激酶和细胞周期蛋白B）进行了纯化和鉴定。17 a，20 b-DP诱导卵母细胞合成细胞周期蛋白B，细胞周期蛋白B又通过苏氨酸激酶（p40 β）对cdc 2激酶的苏氨酸磷酸化（161）激活预先存在的cdc 2激酶<M015>。我们发现，细胞周期蛋白降解是由活性蛋白酶体对细胞周期蛋白B（Lys 57）的有限蛋白水解触发的。3)器官培养系统已被用来证明，在鳗鱼11-酮睾酮可以诱导精子发生的所有阶段在体外21天内。我们的研究结果表明，促性腺激素刺激睾丸间质细胞产生11-酮睾酮，这反过来又激活支持细胞产生激活素B。激活素B然后作用于精原细胞，诱导有丝分裂，导致精母细胞的形成。

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.