Circadian Regulation of Gonadotropin-Releasing Hormone

描述（由申请人提供）：正常哺乳动物生殖依赖于下丘脑的促性腺激素释放激素（GnRH）脉冲式分泌，雌性排卵需要GnRH的强劲激增。虽然众所周知，但尚不清楚肽释放定时的两种模式的细胞和分子机制。虽然先前的研究暗示24小时生物钟在生殖激素分泌的两种机制中，但尚不清楚昼夜节律钟如何在GnRH神经元内起作用以调节同步GnRH分泌释放的幅度或频率来调节生殖。在上一个供资期间，使用GnRH分泌GT 1 -7细胞系和转基因小鼠模型进行的研究表明，GnRH特异性时钟的扰动可以调节分泌，可能导致生殖异常。此外，下文描述的初步研究表明，GnRH特异性时钟中断改变体内生殖能力，内源性振荡器可能通过潜在地改变能够改变GnRH细胞兴奋性的调节剂的时间模式，导致浪涌释放所需的神经元活性变化，在雌激素对GnRH神经元的正反馈效应的转导中发挥作用。为了进一步研究这一点，以下建议将1）评估两种新的转基因小鼠模型，以确定多种下丘脑分子钟在构建正常生殖所需的体内GnRH释放模式方面的各自贡献，2）探索内源性GnRH时钟对雌激素刺激的激增分泌和神经元活性变化的需求，在体外和体内进行评估，研究雌激素对体外培养的GnRH神经元中两种重要的神经调质Kiss-1受体GPR 54和BK离子通道转录调控的影响，并探讨内源性时钟在这些过程中的作用。该提案的结果解决了有关GnRH“脉冲发生器”的性质以及分泌模式如何被细胞-细胞和激素通信改变以产生排卵前激增的基本问题。此外，这些研究提供了对内分泌神经分泌更广泛机制的深入了解，并通过探索转录振荡如何控制同步多细胞事件来调节许多生物过程甚至协调复杂行为来推进昼夜节律生物学。潜在的应用可能会导致治疗一系列生殖生理疾病的新方向，这些疾病是由下丘脑神经分泌功能障碍引起的，包括多囊卵巢综合征（PCOS）和原发性特发性性腺功能减退症，这两种疾病都与非典型的激素释放模式有关。 公共卫生相关性：该提案产生的结果将解决有关生殖神经分泌模式如何通过细胞间通讯改变以产生排卵所需的激素激增的基本问题。此外，这些研究可能会通过展示转录振荡如何在多细胞水平上控制同步事件来推进昼夜节律生物学领域，以调节许多生物过程，甚至协调复杂的行为。潜在的应用可能会导致治疗一系列生殖生理疾病的新方向，这些疾病是由下丘脑神经分泌功能障碍引起的，包括多囊卵巢综合征（PCOS）和原发性特发性性腺功能减退症，这两种疾病都与非典型的激素释放模式有关。

DESCRIPTION (provided by applicant): Normal mammalian reproduction depends on pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, and ovulation in females requires a robust surge of GnRH. While well known, it is unclear what cellular and molecular mechanisms underlie both modes of peptide release timing. While previous studies implicate 24-hour biological clocks in both mechanisms of reproductive hormone secretion, it is unknown how circadian clocks might act within GnRH neurons to modulate amplitude or frequency of synchronous GnRH secretory release to regulate reproduction. Studies in the previous funding period, using GnRH-secreting GT1-7 cell lines and transgenic mouse models, demonstrated that perturbations in GnRH-specific clocks can modulate secretion, potentially leading to abnormalities in reproduction. Also, preliminary studies described below demonstrate that GnRHspecific clock disruption alters reproductive capacity in vivo, and that endogenous oscillators may play a role in transducing positive feedback effects of estrogen on GnRH neurons, by potentially altering temporal patterns of modulators capable of altering GnRH cell excitability, resulting in neuronal activity changes required for surge release. To investigate this further, the following proposal will 1) evaluate two novel transgenic mouse models to determine the respective contribution of multiple hypothalamic molecular clocks on constructing in vivo GnRH release patterns required for proper reproduction, 2) explore the requirement of endogenous GnRH clocks for estrogen-stimulated surge secretion and changes in neuronal activity, evaluated both in vitro and in vivo, and 3) examine the effects of estrogen on transcriptional regulation of two important neuromodulators, the Kiss-1 receptor GPR54, and the BK ion channel in GnRH neurons in vitro, and explore the involvement of endogenous clocks in these processes. Results from this proposal address fundamental questions about the nature of the GnRH "pulse generator", and how secretion patterns are altered by cell-cell and hormonal communication to produce preovulatory surges. Additionally, these studies provide insight into broader mechanisms of endocrine neurosecretion, and advance circadian biology by exploring how transcriptional oscillations can control synchronous multi-cellular events to regulate numerous biological processes and even orchestrate complex behaviors. Potential applications could lead to new directions in treating a range of reproductive physiological disorders that result from malfunction of hypothalamic neurosecretion, including polycystic ovarian syndrome (PCOS) and primary idiopathic hypogonadism, both of which have been associated with atypical hormone release patterns. PUBLIC HEALTH RELEVANCE: Results generated by this proposal will address fundamental questions about how reproductive neurosecretion patterns are altered by cell-cell communication to produce hormonal surges required for ovulation. Additionally, these studies may advance the circadian biological field by demonstrating how transcriptional oscillations can control synchronous events at the multi-cellular level, to regulate numerous biological processes and even orchestrate complex behaviors. Potential applications could lead to new directions in treating a range of reproductive physiological disorders that result from malfunction of hypothalamic neurosecretion, including polycystic ovarian syndrome (PCOS) and primary idiopathic hypogonadism, both of which have been associated with atypical hormone release patterns.