Enrichment of a Gonadotrope Population for Cell Specific Study

用于细胞特异性研究的促性腺激素群体富集

• 批准号: 9130037
• 负责人: Colin M Clay
• 金额: $ 7.48万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-19 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9130037
• 关键词: Acute; Animals; Anterior Pituitary Gland; Area; Binding; Blood; Brain; Cell Count; Cells; Complement; Cues; Disease; Endocrine; Estradiol; Estrogen Antagonists; Estrogens; Event; Exclusion; Female; Fertility; Fluorescence; Fluorescence-Activated Cell Sorting; Foundations; Future; Gene Expression; Gene Targeting; Gene Transfer; Genomics; Goals; Gonadotrope Cell; Gonadotropin Hormone Releasing Hormone; Gonadotropin-Releasing Hormone Receptor; Gonadotropins; Graafian Follicles; Health; Heterogeneity; Hormones; Human; Hypothalamic structure; Infection; Knowledge; Luteinizing Hormone; Malignant Neoplasms; Mediating; Mediator of activation protein; Metabolic; Methodology; Modeling; Molecular; Mus; Neuropeptides; Noise; Nuclear Envelope; Ovarian; Ovary; Ovulation; Phenotype; Pituitary Gland; Population; Postmenopause; Primates; Production; Proteins; Receptor Gene; Regulation; Rodent; Sheep; Signal Transduction; Site; Sorting - Cell Movement; Therapeutic; Transgenic Organisms; Woman; Work; adenoviral-mediated; analog; base; efficacy testing; hormone therapy; hypothalamic pituitary gonadal axis; insight; mouse model; non-genomic; protein expression; public health relevance; receptor expression; reproductive; response

 DESCRIPTION (provided by applicant): Estradiol-17β (E2) is the key endocrine signal that initiates the pre-ovulatory surge of LH by acting at both hypothalamic and pituitary sites of action. The secretion of LH from the pituitary is ultimately cued by the hypothalamic neuropeptide, gonadotropin-releasing hormone (GnRH), which binds to GnRH receptors located on gonadotrope cells in the pituitary gland. Thus, gonadotropes must integrate both hypothalamic and ovarian signals to mount the LH surge. Despite the key role of E2 in regulating pituitary LH secretion and enhancing pituitary sensitivity to GnRH, our understanding of the mechanisms by which E2 exerts its actions at the level of the anterior pituitary gland remains limited. For example, while it is unequivocal that E2 stimulates expression of the GnRH receptor (GnRHR) gene, the underlying mechanisms within the gonadotrope remain undefined and may involve both nuclear and membrane signaling events, as well as genotropic and non- genotropic responses. Unfortunately, since gonadotropes comprise approximately 10% of the total pituitary endocrine cell population, whole pituitary interrogation of gonadotrope specific molecular events becomes problematic due to an almost insurmountable signal to noise ratio. The goal of this proposal is to develop a robust methodology based on adenoviral mediated gene transfer and fluorescence activated cell sorting (FACS) to isolate an enriched population of gonadotropes from the ovine pituitary that will allow for both directed and non-biased analytics of gene and protein expression in response to key mediators of gonadotrope function including E2 and GnRH. Our choice of the sheep model is both theoretical and technical. In regard to the former, we believe that a more complete, relevant, and informed molecular platform for understanding hypothalamic and ovarian regulation of gonadotrope function can be defined using sheep -- not to the exclusion of mouse models, but as an important complement. In regard to the latter, the sheer numbers of gonadotropes that could be isolated from a sheep pituitary will allow robust coverage and replication of both genomic and non-genomic responses in a single animal. Thus, we propose to fuse adenoviral mediated gene transfer and targeted expression of fluorescent proteins to yield a reliable approach for isolating an enriched population of ovine gondadotrope cells. If successful, this work will establish the technical foundation for future studies that fully integrate parallel and complementary approaches to identify the genotropic and non-genotropic events induced by hypothalamic, ovarian and metabolic inputs to gonadotropes. Given the widespread therapeutic application of E2 or E2 analogs, a thorough understanding of the molecular events underlying E2 actions throughout the body is highly relevant to human fertility and human health and disease.

 描述（由适用提供）：雌二醇-17β（E2）是关键内分泌信号，通过在下丘脑和垂体作用位点作用，从而启动LH的卵石前浪涌。 LH从垂体中的分泌最终由下丘脑神经肽，促性腺激素释放的马酮（GNRH）引起，该马酮（GNRH）与位于垂体腺体中促性腺肿瘤细胞上的GnRH受体结合。这是，促性腺激素必须同时整合下丘脑和卵巢信号以安装LH激增。尽管E2在控制垂体LH分泌和增强对GnRH的垂体敏感性方面的关键作用，但我们对E2在垂体前垂体水平上施加其作用的机制的理解仍然有限。例如，尽管E2刺激GNRH受体（GNRHR）基因的表达是明确的，但促性腺导体内的潜在机制仍然不确定，并且可能涉及核和膜信号事件，以及基因因和非基因质反应。不幸的是，由于促性腺激素约占垂体内分泌细胞总数的10％，因此由于几乎无法无法克服的信号与噪声比，促性腺支原体特异性分子事件的整个垂体询问变得有问题。该提案的目的是基于腺病毒介导的基因转移和荧光激活细胞分选（FACS）开发一种可靠的方法，以将丰富的促性腺激素种群与氧垂体中的富含腺泡群体隔离，从而允许对基因和蛋白质表达的指导和非偏置分析，并响应GONADOPROPE的基因和蛋白质表达，包括GONADOPROPE的基因和蛋白质的表达。我们对绵羊模型的选择既是理论和技术性的。关于前者，我们认为，可以使用绵羊定义一个更完整，相关，知情的分子平台，用于理解促性腺激素功能的下丘脑和卵巢调节，而不是排除小鼠模型，而是重要的补充。关于后期，可以从绵羊垂体中隔离的大量促性腺体数量将允许单个动物中基因组和非基因组反应的稳健覆盖范围和复制。这是我们建议将腺病毒介导的基因转移和靶向荧光蛋白的靶向表达融合，以产生一种可靠的方法，用于分离富集的氧化氧基细胞。如果成功的话，这项工作将为未来的研究建立技术基础，以完全整合平行和互补的方法，以鉴定下丘脑，卵巢和代谢输入引起的基因因和非促性事件。鉴于E2或E2类似物的宽度治疗应用，对整个人体E2作用的分子事件的透彻理解与人类的生育能力以及人类健康和疾病高度相关。