Colin M. Clay - US grants

The binding of gonadotropin-releasing hormone (GnRH) to specific, high-affinity receptors located on gonadotrope cells of the anterior pituitary gland is central to reproduction. In the absence of GnRH input, synthesis and secretion of luteinzing hormone and, consequently, normal gonadal function ceases. Thus, the GnRH receptor (GnRHR) is the site that receives and mediates the primary stimulatory input to gonadotropes. We have found that expression of the murine GnRHR in gonadotrope-derived alphaT3-1 cells is mediated by a complex enhancer whose components include a binding site for steroidogenic factor-1 (SF- 1), an AP-1 element, and an element we have termed the GnRH receptor activating sequence (GRAS). This complex enhancer also integrates multiple endocrine inputs. First, we have recently found that GRAS co-localizes with activin regulation of the GnRHR promoter. Unresolved, however, is the identity of the protein(s) that integrate functional activity at GRAS. In Specific Aim 1, we propose to identify the protein(s) that regulate the functional activity, and activin responsiveness of GRAS. Second, AP-1 appears to be the operative element that mediates GnRH regulation; however, important questions remain as to the signal transduction cascades and downstream targets that ultimately lead to GnRH activation at the GnRHR AP-1 site. In Specific Aim 2, our goal is to define the molecular mechanisms underlying GnRH regulation of GnRHR gene expression. We have also found that 1900 bp of proximal promoter is sufficient for tissue-specific expression and GnRH responsiveness in transgenic mice. In Specific Aim 3, we propose to expand these studies to further explore the requirements for tissue/cell-specific expression and hormonal regulation of the GnRHR gene. Finally, we have generated cell lines that express intrinsically fluorescent forms of the GnRHR. These molecules provide a unique opportunity to study the GnRHR as both an occupied and unoccupied receptor in living cells. In Specific Aim 4, we will use fluorescence resonance energy transfer to test the hypothesis that an early event in GnRH signaling is agonist induced receptor self- association. In terms of fertility regulation, the relevance of investigating GnRH and its cognate receptor is clear. However, the use of potent agonists and antagonists of GnRH in the treatment of fibroid tumors, endometriosis, and carcinomas of the breast, prostate, testes, and pituitary underscores the need for a full understanding of GnRH and the GnRHR in both health and disease.

Ovulation is perhaps the most fundamental event in reproduction. It requires a dramatic 20-30-fold surge of luteinizing hormone (LH) released by the anterior pituitary into the peripheral circulation to induce the release of the egg from the ovary. The secretion of LH from the pituitary is cued by the hypothalamic neuropeptide, gonadotropin-releasing hormone (GnRH). The secretion of GnRH from the brain and its interpretation by gonadotropes in the pituitary therefore constitute the essential central events for reproductive competence in virtually all vertebrates. Key aspects of LH surge regulation must still be elucidated. The current dogma states that the pituitary is a relatively passive player receiving hypothalamic direction (GnRH) and turning out pituitary product (LH) that simply leaks into the peripheral circulation upon release. This SGER project seeks to establish that cells in the pituitary respond to GnRH by physically moving to maximize their access to release their LH. This finding will alter the prevailing dogma by proclaiming that the pituitary is no longer a passive player in the process, but rather plays a dynamic role in creating an ovulatory surge of LH. If this phenomenon were more generalized, this concept would fundamentally change current concepts of endocrine organ signaling. The broader impacts of the proposed research stretch from the involvement of students at all levels of the educational process (K-12, undergraduate, graduate, postdoctoral), to Colorado State University's commitment as a land grant institution to public service, to the novelty and biological significance of the proposed research.

DESCRIPTION (provided by applicant): Ovulation is one of the most fundamental events in reproduction and requires a dramatic surge of luteinizing hormone (LH) released by the anterior pituitary gland into the peripheral circulation. The secretion of LH from the pituitary is cued by the hypothalamic neuropeptide, gonadotropin-releasing hormone (GnRH). Thus, the secretion of GnRH from hypothalamic neurons and its interpretation by gonadotropes in the pituitary constitute the essential central events for reproductive competence. Thus, understanding and, ultimately, controlling the mechanisms underlying the generation of the preovulatory LH surge remains a primary focus of research in reproductive endocrinology at both the basic and clinical levels. It has become almost axiomatic that estradiol- 172 (E2) secreted from the preovulatory follicle serves as the most proximate regulator of the LH surge via two sites of action. First, E2 enhances sensitivity of the pituitary by increasing GnRH receptor expression. Second and temporally delayed, it stimulates increased hypothalamic secretion of GnRH. As these 2 physiological sequelae to elevated E2 have been known for decades, it is troubling that our understanding of the underlying mechanisms at both the level of GnRH secretion (hypothalamic) and GnRH responsiveness (pituitary) remains quite undeveloped. In regard to the latter, the lack of progress reflects the inability of in vitro models (cell lines) and promoters (mouse GnRH receptor gene) to recapitulate the increase in pituitary responsiveness to GnRH induced by E2. In contrast, using transgenic mouse models we have demonstrated that the proximal promoter of the ovine GnRH receptor gene is highly E2 responsive. This E2 responsive phenotype does not; however, appear to involve direct binding of activated estrogen receptor (ER) to the sheep gene. Rather, we find that membrane impermeable conjugates of E2 regulate LH secretion in sheep and increase GnRH receptor expression. As such, heightened responsiveness of the pituitary preceding the preovulatory LH surge may reflect E2 signaling through a membrane site of action - this is the central biological issue that exists as the core of our proposed research. Accordingly, using both in vivo and in vitro tests of promoter function and parallel analyses of the endogenous ovine GnRH receptor gene we will determine the hierarchy of ER subtypes that underlie E2 responsiveness of GnRH receptor expression, the initial cellular site of action of E2 that elicits enhanced expression of GnRH receptors and the identity of the regulatory element(s) that mediate the transcriptional response of the GnRHR gene to E2. Understanding regulation of GnRH and its receptor is important for fertility control; however, GnRH agonists and antagonists are used to treat fibroid tumors, endometriosis, and reproductive tissue carcinomas thus underscoring the need for a full understanding of GnRH and the GnRH receptor in both health and disease.

? DESCRIPTION (provided by applicant): Estradiol-17? (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.