CELL BIOLOGY OF OVARIAN APOLIPOPROTEIN E

载脂蛋白（apo）E是一种充分表征的蛋白质， 公认的功能是介导的运输和摄取 富含胆固醇的血浆脂蛋白。 与这一角色不协调， 胆固醇稳态是反复观察载脂蛋白E也是 在多个肝外部位合成，包括卵巢颗粒 细胞 在研究卵巢对脂蛋白的需求时， 雄激素产生细胞，我发现载脂蛋白E是一种特异性抑制剂， 雄激素的合成。 在载脂蛋白E的存在下，培养的LH刺激的 卵泡膜/间质（T/I）细胞产生孕酮，但不能将其转化 雄激素 另外，我最近发现了一种合成肽 能够模拟完整apo E的apo E类似物。颗粒 细胞和卵泡膜细胞相互依赖， 雌激素的产生是控制卵泡发育的关键 发展 为了验证颗粒细胞载脂蛋白E是一种 卵巢内T/I细胞雄激素产生调节因子，5种特异性 目标将实现。 第一个是证明颗粒细胞 细胞衍生的apoE抑制T/I细胞雄激素的产生。 Apo E将是 从颗粒细胞培养上清液中分离并测试生物活性， 活动 第二个具体目标是描述 卵巢载脂蛋白E含量，载脂蛋白E合成和载脂蛋白E结合， 卵泡发育 用免疫组织化学和放射自显影 技术，载脂蛋白E及其结合位点将确定在卵泡 在不同的发展阶段。 此外，表达载脂蛋白E的细胞 将通过原位杂交鉴定mRNA。 第三个具体目标 是表征载脂蛋白E与培养的T/I细胞的相互作用， 以确定细胞结合位点的分子性质。 使用 颗粒细胞衍生的载脂蛋白E和合成的载脂蛋白E肽类似物， 将表征与T/I细胞的结合。 第四个具体目标是 确定apo E的结构特征，这些结构特征负责其 对T/I细胞的结合和生物学效应。 化学修饰和 将在apoE合成肽中进行氨基酸取代， 定义绑定和功能的结构要求。 的 第五个具体目标是确定抑制活性的位点， apo E在cAMP介导膜/间质细胞之前和之后 分化 根据从这一点上获得的信息， 调查，将有可能评估的生理作用， 颗粒细胞载脂蛋白E在卵巢功能中的作用

Apoprotein (apo) E is a well-characterized protein whose acknowledged function is to mediate the transport and uptake of cholesterol-rich plasma lipoproteins. Incongruous with this role in cholesterol homeostasis is the repeated observation that apo E is also synthesized in multiple extrahepatic sites including ovarian granulosa cells. While investigating the lipoprotein requirements of ovary androgen producing cells, I discovered that apo E is a specific inhibitor of androgen synthesis. In the presence of apo E, cultured LH-stimulated theca/interstitial (T/I) cells make progesterone, but cannot convert it to androgen. Furthermore, I have recently identified a synthetic peptide analogue of apo E that is capable of mimicking intact apo E. Granulosa cells and theca cells are dependent on one another to coordinate the production of estrogen that is critical in controlling follicular development. To test the hypothesis that granulosa cell apo E is an intraovarian regulator of T/I cell androgen production, five Specific Aims will be accomplished. The first is to demonstrate that granulosa cell-derived apo E inhibits T/I cell androgen production. Apo E will be isolated from granulosa cell culture supernatants and tested for biologic activity. The second Specific Aim is to characterize the changes in ovarian apo E content, apo E synthesis, and apo E binding during follicular development. Using immunohistochemical and autoradiographic techniques, apo E and its binding sites will be identified in follicles at different stages of development. In addition, cells expressing apo E mRNA will be identified by In situ hybridization. The third Specific Aim is to characterize the interaction of apo E with cultured T/I cells and to identify the molecular nature of the cellular binding sites. Using both granulosa cell-derived apo E and a synthetic apo E peptide analogue, binding to T/I cells will be characterized. The fourth Specific Aim is to define the structural features of apo E that are responsible for its binding to and biologic effect on T/I cells. Chemical modifications and amino acid substitutions will be made in the apo E synthetic peptide to define the structural requirements for both binding and function. The fifth Specific Aim is to identify the site of the inhibitory activity of apo E pre- and post-cAMP mediation of theca/interstitial cell differentiation. Based on the information obtained from this investigation, it will be possible to assess the physiologic role of granulosa cell apo E in ovarian function.