Physiological Mechanisms Underlying Heightened Responsiveness of Gonadotropes to

描述（由申请人提供）：排卵是生殖过程中最基本的事件之一，需要垂体前叶释放大量促黄体生成素（LH）进入外周循环。下丘脑神经肽、促性腺激素释放激素（GnRH）引导垂体分泌LH。因此，下丘脑神经元分泌GnRH及其通过垂体促性腺激素的解释构成了生殖能力的基本中心事件。因此，了解并最终控制排卵前黄体生成素激增的产生机制仍然是生殖内分泌学基础和临床研究的主要焦点。从排卵前卵泡分泌的雌二醇- 172 （E2）通过两个作用点作为LH激增的最接近的调节剂，这已经成为几乎不言自明的事实。首先，E2通过增加GnRH受体的表达来增强垂体的敏感性。第二和暂时延迟，它刺激增加下丘脑分泌GnRH。由于这两种E2升高的生理后遗症已经知道了几十年，令人不安的是，我们对GnRH分泌水平（下丘脑）和GnRH反应性（垂体）的潜在机制的理解仍然很不完善。对于后者，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的反应性增加。相反，利用转基因小鼠模型，我们已经证明了羊GnRH受体基因的近端启动子是高度E2响应的。这种E2应答型没有；然而，似乎涉及到激活雌激素受体（ER）与绵羊基因的直接结合。相反，我们发现E2的膜不渗透偶联物调节绵羊的LH分泌并增加GnRH受体的表达。因此，在排卵前黄体生长素激增之前，垂体的高反应性可能通过膜作用部位反映E2信号——这是我们提出的研究的核心生物学问题。因此，通过体内和体外启动子功能测试以及内源性绵羊GnRH受体基因的平行分析，我们将确定影响GnRH受体表达的E2响应性的ER亚型的等级，E2引起GnRH受体表达增强的初始细胞作用位点，以及介导GnRHR基因对E2转录反应的调节元件的身份。了解GnRH及其受体的调控对生育控制具有重要意义；然而，GnRH激动剂和拮抗剂被用于治疗肌瘤、子宫内膜异位症和生殖组织癌，因此强调需要充分了解GnRH和GnRH受体在健康和疾病中的作用。

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.