Enrichment of a Gonadotrope Population for Cell Specific Study

点击翻译按钮获取中文摘要

 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.