ACTIONS OF ESTROGEN IN MATURING GRAAFIAN FOLLICLES

正常卵泡发育在生物合成中达到顶峰 有能力的黄体，这是正常生殖必不可少的 在雌性体内发挥作用。在各种哺乳动物身上进行了广泛的实验工作 物种已经表现出对某些关键的内分泌的显著调节 性腺类固醇合成和细胞内类固醇代谢的步骤。我们 使用了定义良好的单层细胞培养系统，该系统包括 激素反应性未转化的猪卵巢(颗粒)细胞 检查雌激素、卵泡刺激素和胰岛素样生长因子-I单独作用的特定酶 并结合在一起调节特定的类固醇代谢酶 体外实验控制的条件。在受监管的主要机构中 我们已经研究过并建议进一步研究的地点是：(I)低密度脂蛋白 受体；(Ii)胆固醇侧链裂解酶；(Iii)HMG 辅酶A还原酶；和(Iv)固醇载体蛋白(SCP-2)。至 到目前为止，我们的大多数研究和其他研究都评估了激素调节 甾醇处理对低密度脂蛋白受体数量、酶生化水平的影响 活性和/或合成的特定蛋白质的量。我们的更新 根据这些早期调查的逻辑继续提出建议，将重点放在 关于基因组表达的特定激素调节。这一强调 得到了在我的平行项目下取得的方法和进展的大力支持 例如，RCDA，在其中我们能够展示突出的(3-10 FSH或FSH)刺激特异性CSCC mRNA积累 IGF-I单独作用，但也通过雌二醇FSH和IGF-I作用 协同作用。相比之下，结构性活跃的基因表达 磷酸甘油醛脱氢酶不受这些物质的诱导。 荷尔蒙。因此，我们现在处于一个很好的位置来使用这一点 原代颗粒细胞培养系统进一步检测坐标 在信使核糖核酸水平上调节性腺细胞的固醇代谢。值得注意的是， 猪颗粒细胞体外系统几乎是独一无二的，因为它 对三类主要的类固醇激素表现出显著的激素性反应 单独或协同作用的激素：即类固醇激素，雌二醇； 促性腺激素FSH和胰岛素样生长因子IGF-I。因此， 荷尔蒙的作用有三条重要而独特的“途径”，以及 联合-在这个实验模型中。我们的目标包括调查 稳态信使核糖核酸浓度、基因转录速率和原位 分子杂交。关于个体和坐标的研究 这里提出的对甾醇代谢酶和基因的调节应该 有助于我们更好地理解 相关激素单独作用和协同作用的机制 促进所需的类固醇合成分化的显著增加 在卵泡成熟期间。前面的研究应该有助于 对生育率的重要新见解和进一步发展 对人类、家畜和濒临灭绝的野生物种的监管。

Normal ovarian follicular development culminates in a biosynthetically competent corpus luteum, which is essential for normal reproductive function in the female. Extensive experimental work in various mammalian species has demonstrated significant endocrine regulation of certain key steps in gonadal steroidogenesis and intracellular sterol metabolism. We have used a well-defined monolayer cell culture system consisting of hormonally responsive untransformed swine ovarian (granulosa) cells to examine the specific enzymes by which estradiol, FSH, and IGF-I act singly and in combination to regulate specific sterol-metabolizing enzymes under experimentally controlled conditions in vitro. Among the major regulated sites we have studied, and propose to study further are: (i) the LDL receptor; (ii) the cholesterol side-chain cleavage enzyme; (iii) the HMG CoA reductase enzyme; and (iv) the sterol-carrier protein (SCP-2). To date, most of our and other studies have evaluated hormonal regulation of sterol processing at the level of LDL receptor number, enzyme biochemical activity and/or the amount of specific protein synthesized. Our renewal proposal continues logically from these earlier investigations by focusing on the specific hormonal regulation of genomic expression. This emphasis is strongly supported by methods and progress achieved under my parallel RCDA, for example in which we have been able to demonstrate prominent (3-10 fold) stimulation of specific CSCC mRNA accumulation not only by FSH or IGF-I acting alone, but also by estradiol FSH and IGF-I acting synergistically. In contrast, gene expression of a constitutively active enzyme, phosphoglyceraldehyde dehydrogenase, is not induced by these hormones. Accordingly, we are now in an excellent position to use this primary granulosa-cell culture system further to examine the coordinate regulation of gonadal-cell sterol metabolism at the mRNA level. Notably, the swine granulosa cell in vitro system is almost unique in that it manifests prominent steroidogenic responses to three major classes of hormones acting-alone or in concert: viz., the steroid hormone, estradiol; the gonadotropin, FSH; and the insulin-like growth factor, IGF-I. Thus, three significant and distinct "pathways of hormone action can be in, and jointly-in this experimental model. Our aims encompass an investigation of steady-state mRNA concentrations, gene transcription rates, and in situ molecular hybridization. Studies of the individual and coordinate regulation of sterol-metabolizing enzymes and genes as proposed here should contribute significantly to our better understanding of the essential mechanisms by which relevant hormones act both singly and in concert to promote the remarkable increases in steroidogenic differentiation required during follicular maturation. The preceding studies should contribute important new insights into and further developments in fertility regulation in man, domestic animals, and endangered wild species.