Post-transcriptional Pathways that Signal Leptin Regulation of Gonadotropes

项目摘要/摘要 GnRHR的表达在生殖过程中是一个关键的、限速的步骤，尽管很少 了解调节GnRHR蛋白翻译的机制。促性腺激素功能 可能在食物缺乏的时候受到限制，这是瘦素缺乏的信号，然而有一种 在了解瘦素如何调节促性腺激素方面存在根本差距。瘦素对促性腺激素的重要性是 在不育小鼠模型中，促性腺激素上的瘦素受体(LEPR)的所有异构体都是 事实上，突变的促性腺激素释放激素严重减少了GnRHR蛋白，但没有减少mRNA 级别。这些发现为瘦素如何将生育与代谢状态联系起来提供了重要线索，然而 瘦素控制促性腺激素的分子机制尚不清楚。我们的长期目标是填补 通过确定促性腺激素不育症背后的潜在机制来实现这一知识差距-零 突变的小鼠。有待检验的中心假设是促性腺激素中瘦素信号的丢失 阻止正常的促性腺激素释放激素受体表达上调，导致黄体生成素迟钝或缺失 激增和不孕不育。瘦素通过转录后转录发挥作用的第二假说 优化促性腺激素功能的机制。拟议的研究将集中于三个具体目标。 具体目标1将确定促性腺激素中瘦素信号的丢失是否通过预防 GnRHR蛋白水平的间歇性上调。这些研究将确定LEPR-NULL 促性腺激素处于持续发情间期，因为GnRHR蛋白低，如果血清中正常的激增 促性腺激素缺乏症。外源性GnRH在挽救突变型和突变型GnRHR水平中的作用 将测试周期性和生育力的恢复。特定的AIM 2研究将确定瘦素信号是否与 MRNA的翻译调控机制是促性腺激素功能所必需的。这些研究将在全球范围内 评估瘦素对促性腺激素信使核糖核酸翻译的调节和对促性腺激素受体信使核糖核酸的调控 翻译。识别的候选翻译抑制物的基因敲除将用于恢复GnRHR 在突变的促性腺激素细胞中的表达。特定目标3研究将确定食物匮乏是否会重演 瘦素信号缺失对促性腺激素的影响，包括抑制促性腺激素受体mRNA的翻译。这些输入 活体研究将验证这样的假设，即在食物剥夺过程中失去瘦素信号也可以防止发情 GnRHR上调。这些研究还将确定已识别的翻译基因的体外缺失是否 调控机制(如MSI、miRNAs)减轻了禁食诱导的Gnrhr mRNA翻译的抑制。 我们的研究具有创新性，因为它研究了瘦素可能调节促性腺激素这一新概念。 在转录后水平发挥作用。这些发现具有重要意义，因为临床方案用于 不孕症的治疗(如克罗米芬)取决于对内源性GnRH的正常反应，我们有 这取决于正常的瘦素信号。

PROJECT SUMMARY/ABSTRACT The expression of GnRHRs is a critical, rate-limiting step in the reproductive process, however little is known about the mechanism behind the regulation of translation of GnRHR proteins. Gonadotrope functions may be limited in times of food deprivation, which is signaled by leptin deficiency, however there is a fundamental gap in understanding how leptin regulates gonadotropes. Leptin's importance to gonadotropes is highlighted by the infertile mouse model in which all isoforms of leptin receptors (LEPR) on gonadotropes are ablated, and the fact that the mutant gonadotropes have severely reduced GnRHR protein, but not mRNA levels. These findings provide important clues as to how leptin links fertility to metabolic status, however the molecular mechanisms underlying leptin control of gonadotropes are unknown. Our long-term goal is to fill this knowledge gap by identifying underlying mechanisms behind the infertility in the gonadotrope Lepr-null mutant mice. The central hypothesis to be tested is that loss of leptin signaling in gonadotropes prevents the normal diestrous upregulation of GnRH receptors causing a blunted or absent LH surge and infertility. A secondary hypothesis that leptin acts through posttranscriptional mechanisms to optimize gonadotrope function. The proposed studies will focus on three specific aims. Specific Aim 1 will determine if loss of leptin signaling in gonadotropes reduces fertility through prevention of the diestrous upregulation of GnRHR protein levels. These studies will ascertain if the Lepr-null gonadotropes are held in persistent diestrus because of low GnRHR proteins and if the normal surge in serum gonadotropins is absent. The efficacy of exogenous GnRH in the rescue of GnRHR levels in mutants and restoration of cyclicity and fertility will be tested. Specific Aim 2 studies will determine if leptin signaling to mRNA translational control mechanisms is required for gonadotrope function. These studies will globally assess leptin regulation of gonadotrope mRNA translation and specifically address control of Gnrhr mRNA translation. Genetic knockdown of identified candidate translational repressors will be used to restore GnRHR expression in mutant gonadotropes. Specific Aim 3 studies will determine if food deprivation recapitulates the effect of loss of leptin signaling to gonadotropes, including repression of Gnrhr mRNA translation. These in vivo studies will test the hypothesis that loss of leptin signaling during food deprivation also prevents diestrous upregulation of GnRHR. The studies will also determine if the in vitro deletion of identified translational control mechanisms (e.g. MSI, miRNAs) alleviates the fasting-induced repression of Gnrhr mRNA translation. Our research is innovative because it investigates the novel concept that leptin may regulate gonadotrope function at post-transcriptional levels. These findings are significant because clinical protocols used in infertility treatments (e.g. clomiphene) depend on normal responses to endogenous GnRH, which we have shown depends on normal leptin signaling.