TRANSGENIC ANALYSIS OF LH HYPERSECRETION

正常的性腺功能需要调节LH的分泌。分泌过多 与女性生育能力受损相关，包括 卵巢功能和维持妊娠。了解 然而，LH分泌过多的原因和影响受到以下因素的阻碍： 下丘脑-垂体-性腺轴的复杂性和缺乏 基因定义的动物模型。 最近，我们使用α亚基启动子靶向表达一种 含有羧基末端肽（CTP）的LH β嵌合基因 在转基因小鼠中hCG β特异性结合促性腺激素。女性 转基因（alphaLHbetaCTP）超分泌嵌合LH，不育， 血清性类固醇水平升高，伴有一系列 性腺异常，包括多囊卵巢和颗粒细胞 肿瘤的在性类固醇升高的情况下发生LH升高， 女性提示alphaLHbetaCTP转基因的表达 至少逃脱了一些类固醇负反馈的成分。在 相反，转基因雄性动物具有正常LH水平的生育能力， 这表明性类固醇对嵌合体发挥了适当的控制作用， alphaLH β CTP转基因。 这一新的建议建立在这些调查结果的基础上，确定了三个 相互关联的特定目标，模拟与LH相关的事件 分泌过多 第一个目的考虑LH分泌过多的影响， 假设其发展模式有助于程度和 性腺异常的可逆性。 这一目标涉及操纵 我们现有的转基因模型包括卵巢移植 非转基因动物和转基因动物之间的差异。我们还建议发展 一种新的可诱导的转基因模型，用于控制LH的时间发作 分泌过多第二个目标是解决LH的潜在原因 促性腺激素细胞中的类固醇受体 对防止LH分泌增加至关重要。验证这个假设 包括构建新的转基因动物， 雄激素受体（AR）或雌激素受体的过表达或消除 (ER)特别是在促性腺激素细胞中。这些转基因模型将利用 AR或ER转基因以及促性腺激素特异性核酶。 我们的最后一个目标集中在转录机制所需的 适当调节LH分泌。最初的实验范式 将集中在AR调节亚基启动子活性。测试这个 目的是用截短的和位点特异性的AR进行转染试验， 突变体、AR和ER交换嵌合体以及酵母双杂交测定的应用 用于鉴定与AR特异性相互作用的转录因子。 最终，这一战略将扩展到ER。 总的来说，我们的工作将产生几个新的小鼠模型， 可用于：1）检查LH高分泌对卵巢的影响， 功能障碍; 2）提供进一步了解的潜在应用 多囊卵巢综合征（PCOS）和先天性 肾上腺增生（CAH）;和3）阐明特异性转录 防止LH分泌过多所需的机制。

Normal gonadal function requires regulated secretion of LH. Hypersecretion correlates with impaired fertility in women that includes disruptions in both ovarian function and maintenance of pregnancy. Understanding the causes and effects of LH hypersecretion, however, has been hampered by the complexity of the hypothalamic-pituitary-gonadal axis and the lack of genetically defined animal models. Recently, we used the alpha subunit promoter to target expression of an LHbeta chimeric gene containing the carboxyl terminal peptide (CTP) of hCGbeta specifically to gonadotropes in transgenic mice. Female transgenics (alphaLHbetaCTP) hypersecrete chimeric LH, are infertile, and have elevated serum levels of sex steroids accompanied by a range of gonadal abnormalities including polycystic ovaries and granulosa cell tumors. Elevated LH occurring in the presence of elevated sex steroids in females suggests that expression of the alphaLHbetaCTP transgene has escaped at least some component of negative steroidal feedback. In contrast, transgenic males are fertile with normal levels of LH, suggesting that sex steroids exerted appropriate control over the chimeric alphaLHbetaCTP transgene. This new proposal builds on these findings by identifying three interrelated Specific Aims that model events associated with LH hypersecretion. The first aim considers effects of LH hypersecretion and postulates that its developmental pattern contributes to the extent and reversibility of gonadal abnormalities. This aim involves manipulation of our existing transgenic models and includes ovarian transplantation between non-transgenic and transgenic animals. We also propose development of a new inducible transgenic model for controlling temporal onset of LH hypersecretion. The second aim addresses potential causes of LH hypersecretion by proposing that steroid receptors in gonadotropes are critical for preventing elevated secretion of LH. Testing this hypothesis involves construction of new transgenic animals with selective overexpression or ablation of androgen receptor (AR) or estrogen receptor (ER) specifically in gonadotropes. These transgenic models will utilize either AR- or ER-transgenes as well as gonadotrope-specific ribozymes. Our last aim focuses on transcriptional mechanisms required for appropriate regulation of LH secretion. The initial experimental paradigm will focus on AR regulation of a subunit promoter activity. Testing this aim involves transfection assays with truncated and site-specific AR mutants, AR and ER exchange chimeras, and use of a yeast two-hybrid assay for identifying transcription factors that interact specifically with AR. Ultimately, this strategy will be extended to ER. Collectively, our work will generate several new mouse models that should be useful for: 1) examining the effects of LH hypersecretion on ovarian dysfunction; 2) providing potential applications for further understanding clinical syndromes such as polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH); and 3) elucidating specific transcriptional mechanisms required for preventing hypersecretion of LH.