Physiological Mechanisms Underlying Heightened Responsiveness of Gonadotropes to

说明(申请人提供)：排卵是生殖过程中最基本的事件之一，需要脑下垂体前叶释放大量的黄体生成素(LH)进入外周循环。促性腺激素释放激素(GnRH)是由下丘脑神经肽(GnRH)调节的。因此，下丘脑神经元促性腺激素释放激素的分泌和垂体促性腺激素的解释构成了生殖能力的基本中枢事件。因此，了解并最终控制排卵前黄体生成素高峰的发生机制仍然是基础和临床水平生殖内分泌学研究的主要重点。从排卵前卵泡分泌的雌二醇-172(E_2)是通过两个作用部位调节黄体生成素峰值的最接近的调节剂，这一点几乎是不言而喻的。首先，E2通过增加GnRH受体的表达来增强垂体的敏感性。其次，它会刺激下丘脑GnRH分泌增加，而且是暂时延迟的。由于这两种雌激素升高的生理后遗症已经知道几十年了，令人担忧的是，我们对GnRH分泌水平(下丘脑)和GnRH反应性(脑下垂体)的潜在机制的了解仍然很不发达。在后者方面，缺乏进展反映了体外模型(细胞系)和启动子(小鼠GnRH受体基因)无法概括E2诱导的垂体对GnRH反应性的增加。相反，使用转基因小鼠模型，我们已经证明了绵羊GnRH受体基因的近端启动子是高度E2响应的。这种对E2有反应的表型并不存在；然而，似乎涉及到激活的雌激素受体(ER)与绵羊基因的直接结合。相反，我们发现E2的膜不透性结合物调节绵羊的黄体生成素分泌，并增加促性腺激素释放激素受体的表达。因此，在排卵前促黄体生成素高峰之前，脑垂体的高反应性可能通过膜作用部位反映了E2信号--这是作为我们提议的研究的核心而存在的中心生物学问题。因此，利用体内和体外的启动子功能测试和对内源性绵羊GnRH受体基因的平行分析，我们将确定构成GnRH受体表达的E2反应性的ER亚型的层次结构，E2诱导GnRH受体表达增强的初始细胞作用部位，以及介导GnRH受体基因转录反应的调节元件(S)的身份。了解GnRH及其受体的调节对于生育控制很重要；然而，GnRH激动剂和拮抗剂用于治疗子宫肌瘤、子宫内膜异位症和生殖组织癌，因此强调了在健康和疾病中充分了解GnRH和GnRH受体的必要性。 与公共健康相关：这些研究将加深我们对垂体前叶(主要的内分泌腺)中雌激素信号的理解，以及雌激素调节全身重要生理靶点的新机制。了解这些机制对于开发新一代的促雌激素和抗雌激素样药物至关重要，这些药物对所需的生物效应越来越特异。了解生殖激素的生理调节，如促性腺激素释放激素(GnRH)和GnRH受体，对于生育调节很重要；然而，强大的GnRH激动剂和拮抗剂在治疗子宫肌瘤、子宫内膜异位症和生殖组织癌中的应用突显了这一主题在健康和疾病中的重要性。

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. PUBLIC HEALTH RELEVANCE: These studies will refine our understanding of estrogen signaling in the anterior pituitary gland (the "master" endocrine gland) as well novel mechanisms underlying estrogen regulation of important physiological targets throughout the body. Understanding these mechanisms is critical to the development of new generations of both pro and anti-estrogen-like pharmaceuticals that are increasingly specific for the desired biological effect. Understanding physiological regulation of reproductive hormones like gonadotropin releasing hormone (GnRH) and the GnRH receptor is important to fertility regulation; however, the application of potent GnRH agonists and antagonists in the treatment of uterine fibroids, endometriosis and reproductive tissue carcinomas underscores the importance of this topic in both health and disease.